Compositions, systems, and methods for reducing low density lipoprotein through gene silencing targeting a target

Abstract

In some aspects, an epigenetic modification DNA targeting system, such as a CRISPR-Cas / guide RNA (gRNA) system for transcriptional repression of genes, is provided to promote a cell phenotype that results in a reduction of low density lipoprotein (LDL). In some embodiments, the epigenetic modification DNA targeting system binds to or targets a target site of at least one gene that regulates LDL or its regulatory element. In some embodiments, the system is a multiplexed system that binds to or targets target sites in at least two genes or their regulatory elements. Also provided herein are methods and uses related to the provided epigenetic modification DNA targeting system related to the treatment of cardiovascular disease and familial hypercholesterolemia.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority from U.S. Provisional Application No. 63 / 355,540, filed on June 24, 2022; U.S. Provisional Application No. 63 / 399,625, filed on August 19, 2022; U.S. Provisional Application No. 63 / 401,558, filed on August 26, 2022; U.S. Provisional Application No. 63 / 453,044, filed on March 17, 2023; U.S. Provisional Application No. 63 / 466,681, filed on May 15, 2023; and U.S. Provisional Application No. 63 / 472,224, filed on June 9, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] Incorporation by Reference of Sequence Listing This application has been filed in electronic format together with a sequence listing. The sequence listing is provided as a file named 224742001940SeqList.xml, created on June 22, 2023, with a size of 561,656 bytes. The electronic - format information of the sequence listing is hereby incorporated by reference in its entirety.

[0003] Field In some aspects, the present disclosure relates to epigenetic - modification DNA targeting systems, such as CRISPR - Cas / guide RNA (gRNA) systems for transcriptional repression of genes to promote cell phenotypes that result in reduction of low - density lipoprotein (LDL). In some embodiments, the epigenetic - modification DNA targeting system binds to or targets a target site of at least one gene that regulates LDL or its regulatory element. In some embodiments, the system is a multiplexed system that binds to or targets target sites in at least two genes or their regulatory elements. In some aspects, the present disclosure also provides methods and uses related to the epigenetic - modification DNA targeting systems provided in connection with the treatment of cardiovascular diseases and familial hypercholesterolemia.

Background Art

[0004] Background Familial hypercholesterolemia (FH) is a genetic condition that results in elevated levels of LDL cholesterol in the bloodstream, leading to an increased risk of cardiovascular disease. Current treatments for FH, including statin administration and antibody therapy, face challenges including toxicity and high costs associated with repeated drug administration. There is a need for new and improved methods to overcome these challenges. The present disclosure addresses these and other needs. SUMMARY OF THE INVENTION

[0005] Summary In some embodiments, provided herein is an epigenetic modification DNA targeting system comprising a plurality of DNA targeting modules for suppressing the transcription of a plurality of genes that regulate low density lipoprotein (LDL), the plurality of DNA targeting modules comprising a first DNA targeting module for suppressing the transcription of a first gene among the plurality of genes and a second DNA targeting module for suppressing the transcription of a second gene among the plurality of genes, each DNA targeting module comprising a fusion protein comprising (a) a DNA binding domain for targeting one target site among the plurality of genes and (b) at least one transcriptional repressor domain. In some of any of the provided embodiments, the DNA targeting system does not introduce gene disruption or DNA cleavage. In some of any of the provided embodiments, the fusion protein of each DNA targeting module comprises a DNA binding domain selected from a clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or an I-SceI enzyme or a variant thereof, and optionally, the DNA binding domain comprises a catalytically inactive variant of any of the foregoing. In some of any of the provided embodiments, the fusion protein of the first DNA targeting module comprises a DNA binding domain for targeting the target site of the first gene or its regulatory DNA element and at least one transcriptional repressor domain, and the fusion protein of the second DNA targeting module comprises a DNA binding domain for targeting the target site of the second gene or its regulatory DNA element and at least one transcriptional repressor domain. In some of any of the provided embodiments, any two or more of the DNA targeting modules comprise the same fusion protein. In some of any of the provided embodiments, the first and second DNA targeting modules comprise the same fusion protein. In some of any of the provided embodiments, any two or more of the DNA targeting modules comprise different fusion proteins.In some of the provided embodiments, the first and second DNA targeting modules comprise different fusion proteins.

[0006] In some aspects, provided herein is an epigenetic modification DNA targeting system comprising a plurality of DNA targeting modules for suppressing the transcription of a plurality of genes that regulate low density lipoprotein (LDL), the system comprising: (1) a first DNA targeting module that reduces the transcription of a first gene that regulates low density lipoprotein (LDL), the first DNA targeting module comprising a first fusion protein comprising (a) a DNA binding domain for targeting a target site of the first gene or its regulatory DNA element, and (b) at least one transcriptional repressor domain; and (2) a second DNA targeting module that reduces the transcription of a second gene that regulates LDL, the second DNA targeting module comprising a second fusion protein comprising (a) a DNA binding domain for targeting a target site of the second gene or its regulatory DNA element, and (b) at least one transcriptional repressor domain. In some of the provided embodiments, the first DNA targeting module comprises a first targeting polynucleotide for targeting a target site of the first gene, the second DNA targeting module comprises a second targeting polynucleotide for targeting a target site of the second gene, and the first and second targeting polynucleotides complex with the DNA binding domain of the fusion protein. In some of the provided embodiments, the DNA binding domain is a clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof, and the first and second targeting polynucleotides each comprise a first gRNA and a second gRNA, respectively.

[0007] In some aspects, provided herein is an epigenetic modification DNA targeting system comprising: (a) a fusion protein comprising a clustered regularly interspaced short palindromic repeat associated (Cas) protein or variant thereof and at least one transcriptional repressor domain; and (b) a plurality of guide RNAs (gRNAs) comprising at least a first gRNA and a second gRNA, wherein the first gRNA targets a target site of a first gene that regulates low density lipoprotein (LDL), and the second gRNA targets a target site of a second gene that regulates LDL.

[0008] In some of any of the provided embodiments, the system further comprises a third DNA targeting module for suppressing the transcription of a third gene that regulates low density lipoprotein (LDL). In some of any of the provided embodiments, the system further comprises a third gRNA that targets a target site of a third gene that regulates LDL, and optionally, the system further comprises a fourth gRNA that targets a target site of a fourth gene that regulates LDL, optionally, a fifth gRNA that targets a target site of a fifth gene that regulates LDL, and / or optionally, a sixth gRNA that targets a target site of a sixth gene that regulates LDL.

[0009] In some of any of the provided embodiments, the first gene and the second gene are each independently selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3. In some of any of the provided embodiments, the first gene and the second gene are different. In some of any of the provided embodiments, the first gene and the second gene are PCSK9 and LPA, PCSK9 and MYLIP, PCSK9 and ANGPTL3, PCSK9 and APOC3, PCSK9 and APOB, LPA and MYLIP, LPA and ANGPTL3, LPA and APOC3, LPA and APOB, MYLIP and ANGPTL3, MYLIP and APOC3, MYLIP and APOB, ANGPTL3 and APOC3, ANGPTL3 and APOB, or APOC3 and APOB. In some of any of the provided embodiments, the first gene, the second gene, and the third gene are each independently selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB. In some of any of the provided embodiments, the first gene, the second gene, and the third gene are different. In some of any of the provided embodiments, the first gene, the second gene, and the third gene are PCSK9, LPA, and MYLIP; PCSK9, LPA, and ANGPTL3; PCSK9, LPA, and APOC3; PCSK9, LPA, and APOB; PCSK9, MYLIP, and ANGPTL3; PCSK9, MYLIP, and APOC3; PCSK9, MYLIP, and APOB; PCSK9, ANGPTL3, and APOC3; PCSK9, ANGPTL3, and APOB; PCSK9, APOC3, and APOB; LPA, MYLIP, and ANGPTL3; LPA, MYLIP, and APOC3; LPA, MYLIP, and APOB; LPA, ANGPTL3, and APOC3; LPA, ANGPTL3, and APOB; LPA, APOC3, and APOB; MYLIP, ANGPTL3, and APOC3; MYLIP, ANGPTL3, and APOB; MYLIP, APOC3, and APOB; or ANGPTL3, APOC3, and APOB.In some of any of the provided embodiments, at least one gene is PCSK9. In some of any of the provided embodiments, at least two genes are PCSK9 and LPA. In some of any of the provided embodiments, at least three genes are PCSK9, LPA, and MYLIP. In some of any of the provided embodiments, at least three genes are PCSK9, MYLIP, and APOB.

[0010] In some of any of the provided embodiments, the target site of each of the plurality of genes is within the gene or its regulatory DNA element. In some of any of the provided embodiments, the regulatory DNA element is an enhancer or a promoter. In some of any of the provided embodiments, the target sites of the first gene and the second gene are selected from two different members of the group consisting of (a)-(f): (a) the target site of PCSK9 located within 500 bp from the human genome assembly GRCh38 (hg38) genomic coordinate chr1:55,039,548, (b) the target site of LPA located within 500 bp from the hg38 genomic coordinate chr6:160,664,275, (c) the target site of MYLIP located within 500 bp from the hg38 genomic coordinate chr6:16,129,086, (d) the target site of ANGPTL3 located within 500 bp from the hg38 genomic coordinate chr1:62,597,520, (e) the target site of APOC3 located within 500 bp from the hg38 genomic coordinate chr11:116,829,907, and (f) the target site of APOB located within 500 bp from the hg38 genomic coordinate chr2:21,044,073. In some of any of the provided embodiments, the target sites of the first gene and the second gene are selected from two different members of the group consisting of (a)-(f): (a) the target site located within 500 bp from the transcription start site of PCSK9, (b) the target site located within 500 bp from the transcription start site of LPA, (c) the target site located within 500 bp from the transcription start site of MYLIP, (d) the target site located within 500 bp from the transcription start site of ANGPTL3, (e) the target site located within 500 bp from the transcription start site of APOC3, and (f) the target site located within 500 bp from the transcription start site of APOB.In some of the provided embodiments, the target sites of the first gene, the second gene, and the third gene are selected from three different members of the group consisting of (a) to (f): (a) the target site of PCSK9 located within 500 bp from the human genome assembly GRCh38 (hg38) genomic coordinate chr1:55,039,548, (b) the target site of LPA located within 500 bp from the hg38 genomic coordinate chr6:160,664,275, (c) the target site of MYLIP located within 500 bp from the hg38 genomic coordinate chr6:16,129,086, (d) the target site of ANGPTL3 located within 500 bp from the hg38 genomic coordinate chr1:62,597,520, (e) the target site of APOC3 located within 500 bp from the hg38 genomic coordinate chr11:116,829,907, and (f) the target site of APOB located within 500 bp from the hg38 genomic coordinate chr2:21,044,073. In some of the provided embodiments, the target sites of the first gene, the second gene, and the third gene are selected from three different members of the group consisting of (a) to (f): (a) the target site located within 500 bp from the transcription start site of PCSK9, (b) the target site located within 500 bp from the transcription start site of LPA, (c) the target site located within 500 bp from the transcription start site of MYLIP, (d) the target site located within 500 bp from the transcription start site of ANGPTL3, (e) the target site located within 500 bp from the transcription start site of APOC3, and (f) the target site located within 500 bp from the transcription start site of APOB.In some of any of the provided embodiments, the target sites of the first gene and the second gene are (a) to (f): (a) a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, at least 14 nucleotides (nt) of its adjacent portion, or a target site of PCSK9 having a complementary sequence of any of the foregoing; (b) a sequence shown in any one of SEQ ID NOs: 14 to 23, at least 14 nucleotides (nt) of its adjacent portion, or a target site of LPA having a complementary sequence of any of the foregoing; (c) a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351, at least 14 nucleotides (nt) of its adjacent portion, or a target site of MYLIP having a complementary sequence of any of the foregoing; (d) a sequence shown in any one of SEQ ID NOs: 34 to 43, at least 14 nucleotides (nt) of its adjacent portion, or a target site of ANGPTL3 having a complementary sequence of any of the foregoing; (e) a sequence shown in any one of SEQ ID NOs: 44 to 53, at least 14 nucleotides (nt) of its adjacent portion, or a target site of APOC3 having a complementary sequence of any of the foregoing; and (f) a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377, at least 14 nucleotides (nt) of its adjacent portion, or a target site of APOB having a complementary sequence of any of the foregoing, and are selected from two different members of the group consisting of. In some embodiments, the target site of PCSK9 is (i) shown in SEQ ID NO: 3, (ii) has at least 14 nucleotides of the adjacent portion of SEQ ID NO: 3, or (iii) is a complementary sequence of (i) or (ii).In some of the provided embodiments, the target sites of the first gene and the second gene are selected from two different members of the group consisting of (a) to (f): (a) the target site of PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317; (b) the target site of LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23; (c) the target site of MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351; (d) the target site of ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43; (e) the target site of APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53; and (f) the target site of APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377. In some embodiments, the target site of PCSK9 is shown in SEQ ID NO: 3.In some of the provided embodiments, the target sites of the first gene, the second gene, and the third gene are (a) to (f): (a) a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, at least 14 nucleotides (nt) of its adjacent portion, or a target site of PCSK9 having a complementary sequence of any of the foregoing; (b) a sequence shown in any one of SEQ ID NOs: 14 to 23, at least 14 nucleotides (nt) of its adjacent portion, or a target site of LPA having a complementary sequence of any of the foregoing; (c) a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351, at least 14 nucleotides (nt) of its adjacent portion, or a target site of MYLIP having a complementary sequence of any of the foregoing; (d) a sequence shown in any one of SEQ ID NOs: 34 to 43, at least 14 nucleotides (nt) of its adjacent portion, or a target site of ANGPTL3 having a complementary sequence of any of the foregoing; (e) a sequence shown in any one of SEQ ID NOs: 44 to 53, at least 14 nucleotides (nt) of its adjacent portion, or a target site of APOC3 having a complementary sequence of any of the foregoing; and (f) a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377, at least 14 nucleotides (nt) of its adjacent portion, or a target site of APOB having a complementary sequence of any of the foregoing, and are selected from three different members of the group consisting thereof. In some embodiments, the target site of PCSK9 is (i) shown in SEQ ID NO: 3, (ii) has at least 14 nucleotides of the adjacent portion of SEQ ID NO: 3, or (iii) is a complementary sequence of (i) or (ii).In some of the provided embodiments, the target sites of the first gene, the second gene, or the third gene are selected from three different members of the group consisting of (a)-(f): (a) the target site of PCSK9 having a sequence shown in any one of SEQ ID NOs: 1-13 or 306-317; (b) the target site of LPA having a sequence shown in any one of SEQ ID NOs: 14-23; (c) the target site of MYLIP having a sequence shown in any one of SEQ ID NOs: 24-33 or 342-351; (d) the target site of ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34-43; (e) the target site of APOC3 having a sequence shown in any one of SEQ ID NOs: 44-53; and (f) the target site of APOB having a sequence shown in any one of SEQ ID NOs: 54-63 or 372-377. In some embodiments, the target site of PCSK9 is shown in SEQ ID NO: 3.

[0011] In some of the provided embodiments, the Cas protein or its variant is a variant Cas protein that is a dead (dCas) protein. In some of the provided embodiments, the dCas protein lacks nuclease activity. In some of the provided embodiments, the dCas protein is a dCas9 protein. In some of the provided embodiments, the dCas protein is a dCas12 protein.

[0012] In some of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of the provided embodiments, dSaCas9 includes at least one amino acid mutation selected from D10A and N580A with reference to the numbering at position of SEQ ID NO: 204. In some of the provided embodiments, the dSaCas9 protein includes the sequence shown in SEQ ID NO: 205, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of the provided embodiments, dSaCas9 is shown in SEQ ID NO: 205.

[0013] In some of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of the provided embodiments, the dSpCas9 protein includes at least one amino acid mutation selected from D10A and H840A with reference to the numbering at position of SEQ ID NO: 206. In some of the provided embodiments, dSpCas9 includes the sequence shown in SEQ ID NO: 207, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of the provided embodiments, dSpCas9 is shown in SEQ ID NO: 207.

[0014] In some of the provided embodiments, each gRNA comprises a gRNA spacer sequence that is complementary to a target site of a respective gene. In some of the provided embodiments, the first gRNA and the second gRNA are selected from two different members of the group consisting of (a)-(f): (a) a gRNA targeting a target site of PCSK9, comprising a sequence shown in any one of SEQ ID NOs: 64-76 or 318-329, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion; (b) a gRNA targeting a target site of LPA, comprising a sequence shown in any one of SEQ ID NOs: 77-86, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion; (c) a gRNA targeting a target site of MYLIP, comprising a sequence shown in any one of SEQ ID NOs: 87-96 or 352-361, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion; (d) a gRNA targeting a target site of ANGPTL3, comprising a sequence shown in any one of SEQ ID NOs: 97-106, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion; (e) a gRNA targeting a target site of APOC3, comprising a sequence shown in any one of SEQ ID NOs: 107-116, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion; and (f) a gRNA targeting a target site of APOB, comprising a sequence shown in any one of SEQ ID NOs: 117-126 or 378-383, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion. In some embodiments, the gRNA spacer sequence targeting the target site of PCSK9 comprises the sequence shown in SEQ ID NO: 66 or at least 14 nt of the adjacent portion.In some of the provided embodiments, the first gRNA, the second gRNA, and the third gRNA are selected from three different members of the group consisting of (a)-(f): (a) a gRNA targeting a target site of PCSK9, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 64-76 or 318-329, or at least 14 nt of its adjacent portion; (b) a gRNA targeting a target site of LPA, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 77-86, or at least 14 nt of its adjacent portion; (c) a gRNA targeting a target site of MYLIP, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 87-96 or 352-361, or at least 14 nt of its adjacent portion; (d) a gRNA targeting a target site of ANGPTL3, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 97-106, or at least 14 nt of its adjacent portion; (e) a gRNA targeting a target site of APOC3, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 107-116, or at least 14 nt of its adjacent portion; and (f) a gRNA targeting a target site of APOB, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 117-126 or 378-383, or at least 14 nt of its adjacent portion. In some embodiments, the gRNA spacer sequence targeting the target site of PCSK9 comprises the sequence shown in SEQ ID NO: 66 or at least 14 nt of its adjacent portion. In some of the provided embodiments, each gRNA independently comprises a spacer sequence having a length of 14 nt to 24 nt, or 16 nt to 22 nt. In some of the provided embodiments, each gRNA independently comprises a spacer sequence having a length of 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt.In some of the provided embodiments, the first gRNA and the second gRNA are selected from two different members of the group consisting of (a)-(f): (a) a gRNA targeting a target site of PCSK9 comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 64-76 or 318-329; (b) a gRNA targeting a target site of LPA comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 77-86; (c) a gRNA targeting a target site of MYLIP comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 87-96 or 352-361; (d) a gRNA targeting a target site of ANGPTL3 comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 97-106; (e) a gRNA targeting a target site of APOC3 comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 107-116; and (f) a gRNA targeting a target site of APOB comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 117-126 or 378-383. In some embodiments, the gRNA spacer sequence targeting the target site of PCSK9 comprises the sequence shown in SEQ ID NO: 66. In some of the provided embodiments, each gRNA further comprises a scaffold sequence shown in SEQ ID NO: 191.In some of the provided embodiments, the first gRNA and the second gRNA are selected from two different members of the group consisting of (a)-(f): (a) a gRNA targeting a target site of PCSK9 comprising a sequence shown in any one of SEQ ID NOs: 127-139 or 330-341, (b) a gRNA targeting a target site of LPA comprising a sequence shown in any one of SEQ ID NOs: 140-149, (c) a gRNA targeting a target site of MYLIP comprising a sequence shown in any one of SEQ ID NOs: 150-159 or 362-371, (d) a gRNA targeting a target site of ANGPTL3 comprising a sequence shown in any one of SEQ ID NOs: 160-169, (e) a gRNA targeting a target site of APOC3 comprising a sequence shown in any one of SEQ ID NOs: 170-179, and (f) a gRNA targeting a target site of APOB comprising a sequence shown in any one of SEQ ID NOs: 180-189 or 384-389. In some embodiments, the gRNA targeting the target site of PCSK9 comprises the sequence shown in SEQ ID NO: 129. In some of the provided embodiments, the first gRNA and the second gRNA are selected from two different members of the group consisting of (a)-(f): (a) a gRNA targeting a target site of PCSK9 shown in any one of SEQ ID NOs: 127-139 or 330-341, (b) a gRNA targeting a target site of LPA shown in any one of SEQ ID NOs: 140-149, (c) a gRNA targeting a target site of MYLIP shown in any one of SEQ ID NOs: 150-159 or 362-371, (d) a gRNA targeting a target site of ANGPTL3 shown in any one of SEQ ID NOs: 160-169, (e) a gRNA targeting a target site of APOC3 shown in any one of SEQ ID NOs: 170-179, and (f) a gRNA targeting a target site of APOB shown in any one of SEQ ID NOs: 180-189 or 384-389. In some embodiments, the gRNA targeting the target site of PCSK9 is shown in SEQ ID NO: 129.In some of the provided embodiments, the first gRNA, the second gRNA, and the third gRNA are selected from three different members of the group consisting of (a)-(f): (a) a gRNA targeting a target site of PCSK9 comprising a sequence shown in any one of SEQ ID NOs: 127-139 or 330-341; (b) a gRNA targeting a target site of LPA comprising a sequence shown in any one of SEQ ID NOs: 140-149; (c) a gRNA targeting a target site of MYLIP comprising a sequence shown in any one of SEQ ID NOs: 150-159 or 362-371; (d) a gRNA targeting a target site of ANGPTL3 comprising a sequence shown in any one of SEQ ID NOs: 160-169; (e) a gRNA targeting a target site of APOC3 comprising a sequence shown in any one of SEQ ID NOs: 170-179; and (f) a gRNA targeting a target site of APOB comprising a sequence shown in any one of SEQ ID NOs: 180-189 or 384-389. In some embodiments, the gRNA targeting the target site of PCSK9 comprises the sequence shown in SEQ ID NO: 129. In some of the provided embodiments, the first gRNA, the second gRNA, and the third gRNA are selected from three different members of the group consisting of (a)-(f): (a) a gRNA targeting a target site of PCSK9 shown in any one of SEQ ID NOs: 127-139 or 330-341; (b) a gRNA targeting a target site of LPA shown in any one of SEQ ID NOs: 140-149; (c) a gRNA targeting a target site of MYLIP shown in any one of SEQ ID NOs: 150-159 or 362-371; (d) a gRNA targeting a target site of ANGPTL3 shown in any one of SEQ ID NOs: 160-169; (e) a gRNA targeting a target site of APOC3 shown in any one of SEQ ID NOs: 170-179; and (f) a gRNA targeting a target site of APOB shown in any one of SEQ ID NOs: 180-189 or 384-389. In some embodiments, the gRNA targeting the target site of PCSK9 is shown in SEQ ID NO: 129.In some of the provided embodiments, at least one gRNA comprises modified nucleotides for improving stability.

[0015] In some of the provided embodiments, at least one transcriptional repressor domain can reduce the transcription of a gene. In some of the provided embodiments, the transcriptional repressor domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3A-DNMT3L fusion protein domain, an ERF repressor domain, an Mxi1 repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD1 repressor domain, an EZH2 repressor domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing. In some of the provided embodiments, the transcriptional repressor domain is a KRAB domain, a DNMT3A domain, or a DNMT3L domain, or a combination of any of the foregoing. In some of the provided embodiments, at least one transcriptional repressor domain comprises a sequence selected from any one of SEQ ID NOs: 193, 195, 197, 199, 201, 220-226, 283, 284, 285, 286, 287, 289, and 290, or a domain, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 193, 195, 197, 199, 201, 220-226, 283, 284, 285, 286, 287, 289, and 290.

[0016] In some of any of the provided embodiments, at least one transcriptional repressor domain comprises a KRAB domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 193, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 193. In some of any of the provided embodiments, at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 290, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 290. In some of any of the above embodiments, the KRAB domain, a portion or variant thereof is a sequence that exhibits transcriptional repressor activity.

[0017] In some of any of the provided embodiments, at least one transcriptional repressor domain comprises a DNMT3A domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some embodiments, the DNMT3A domain, or a portion thereof, is derived from human DNMT3A. In some of any of the provided embodiments, at least one effector domain comprises the sequence shown in SEQ ID NO: 284, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 284. In some embodiments, the portion is a catalytically active portion that exhibits transcriptional repressor activity. In some of any of the provided embodiments, at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 195, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 195. In some embodiments, at least one effector domain is or comprises the catalytically active portion of DNMT3A shown in SEQ ID NO: 195. In some of any of the provided embodiments, at least one effector domain comprises the sequence shown in SEQ ID NO: 285, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 285. In some embodiments, at least one effector domain is or comprises the catalytically active portion of DNMT3A shown in SEQ ID NO: 285.

[0018] In some of the provided embodiments, at least one transcriptional repressor domain comprises a DNMT3L domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of the provided embodiments, at least one transcriptional repressor domain is a fusion domain that comprises a DNMT3A domain, such as any DNMT3A domain, or the C-terminal portion or variant described above, and a DNMT3L domain, or a variant or portion thereof, and the fusion domain exhibits transcriptional repressor activity. In some embodiments, the DNMT3L domain, or a variant or C-terminal portion of the DNMT3L domain, stimulates enhancement of the transcriptional repressor activity of DNMT3A. In some embodiments, the DNMT3L domain, or a variant or C-terminal portion of the DNMT3L domain, interacts with DNMT3A. In some embodiments, the DNMT3L domain, or its C-terminal portion, is derived from murine DNMT3L. In some embodiments, the DNMT3L domain, or its C-terminal portion, is human or humanized DNMT3L. In some of the provided embodiments, the DNTML domain comprises the sequence shown in SEQ ID NO: 289, its C-terminal portion, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 289. In some of the provided embodiments, DNMT3L comprises the sequence shown in SEQ ID NO: 286, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 286. In some embodiments, the DNMT3L domain is or comprises the C-terminal portion of DNMT3L shown in SEQ ID NO: 286. In some of the provided embodiments, the DNMT3L domain comprises the sequence shown in SEQ ID NO: 197, its C-terminal portion, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 197.In some of the provided embodiments, DNMT3L comprises the sequence shown in SEQ ID NO: 287, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 287. In some embodiments, the DNMT3L domain is, or comprises, the C-terminal portion of DNMT3L shown in SEQ ID NO: 287.

[0019] In some of the provided embodiments, at least one transcriptional repressor domain is a DNMT3A-DNMT3L fusion protein domain or a variant thereof that exhibits transcriptional repressor activity. In some embodiments, the DNMT3A domain is shown in SEQ ID NO: 285 and the DNMT3L domain is shown in SEQ ID NO: 286, which may be present in any order. In some embodiments, a linker is present between the DNMT3A domain and the DNMT3L domain. In some of the provided embodiments, at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 199 or SEQ ID NO: 201, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 199 or SEQ ID NO: 201.

[0020] In some of the provided embodiments, at least one transcriptional repressor domain is fused to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the DNA binding domain. In some of the provided embodiments, the fusion protein further comprises one or more nuclear localization signals (NLSs). In some of the provided embodiments, the fusion protein further comprises one or more linkers that connect two or more of the DNA binding domain, at least one transcriptional repressor domain, and one or more nuclear localization signals.

[0021] In some embodiments, the fusion protein is dCas9-KRAB or a variant thereof.

[0022] In some of the provided embodiments, the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NO: 209, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 209. In some embodiments, the fusion protein has the sequence shown in SEQ ID NO: 209.

[0023] In any of the embodiments herein, the fusion protein is DNMT3A / 3L-dCas9-KRAB domain or a variant thereof.

[0024] In any of the embodiments herein, the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NO: 278, 280, or 282, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 278, 280, or 282. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 280. In some embodiments, the fusion protein has the sequence shown in SEQ ID NO: 278. In some embodiments, the fusion protein has the sequence shown in SEQ ID NO: 280. In some embodiments, the fusion protein has the sequence shown in SEQ ID NO: 282.

[0025] In some of the provided embodiments, each of the DNA targeting modules reduces the expression of each of the respective genes with a log2 fold change of -1.0 or less.

[0026] In some of any of the provided embodiments, reduced transcription of multiple genes in a cell or cell population results in a reduction in low density lipoprotein (LDL). In some of any of the provided embodiments, the reduction in LDL is greater than the reduction in LDL resulting from the corresponding reduced transcription of any one of the individual genes in the multiple genes. In some of any of the provided embodiments, the reduction in LDL occurs extracellularly. In some of any of the provided embodiments, the cell or cell population is a hepatocyte or includes hepatocytes. In some of any of the provided embodiments, the cell or cell population is within a subject. In some of any of the provided embodiments, the reduction in LDL occurs within the subject or its fluids, tissues, or organs. In some of any of the provided embodiments, the reduction in LDL occurs in the blood of the subject.

[0027] In some embodiments, provided herein is an epigenetic modification DNA targeting system for suppressing the transcription of a gene that regulates low density lipoprotein, comprising a fusion protein comprising (a) a DNA binding domain for targeting a target site of a gene and (b) at least one transcriptional repressor domain. In some of any of the provided embodiments, the target site of the gene is within the gene or its regulatory DNA element. In some of any of the provided embodiments, the DNA targeting system does not introduce gene disruption or DNA cleavage. In some of any of the provided embodiments, the DNA binding domain is selected from a clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or an I-SceI enzyme or a variant thereof, and optionally, the DNA binding domain comprises a catalytically inactive variant of any of the foregoing. In some of any of the provided embodiments, the DNA binding domain is a clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof, and the system further comprises a gRNA for targeting the DNA binding domain to the target site of the gene.

[0028] In some embodiments, provided herein is an epigenetic modification DNA targeting system comprising (a) a fusion protein comprising a clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof and at least one transcriptional repressor domain, and (b) a gRNA targeting a target site of a gene that regulates low density lipoprotein (LDL).

[0029] In some of any of the provided embodiments, the target site of the gene is within the gene and / or its regulatory DNA element. In some of any of the provided embodiments, the regulatory DNA element is a promoter or an enhancer. In some of any of the provided embodiments, the gene is selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3. In some of any of the provided embodiments, the target site is selected from: (a) the target site of PCSK9 located within 500 bp from the human genome assembly GRCh38 (hg38) genomic coordinate chr1:55,039,548; (b) the target site of LPA located within 500 bp from the hg38 genomic coordinate chr6:160,664,275; (c) the target site of MYLIP located within 500 bp from the hg38 genomic coordinate chr6:16,129,086; (d) the target site of ANGPTL3 located within 500 bp from the hg38 genomic coordinate chr1:62,597,520; (e) the target site of APOC3 located within 500 bp from the hg38 genomic coordinate chr11:116,829,907; and (f) the target site of APOB located within 500 bp from the hg38 genomic coordinate chr2:21,044,073. In some of any of the provided embodiments, the target site is selected from: (a) the target site located within 500 bp from the transcription start site of PCSK9; (b) the target site located within 500 bp from the transcription start site of LPA; (c) the target site located within 500 bp from the transcription start site of MYLIP; (d) the target site located within 500 bp from the transcription start site of ANGPTL3; (e) the target site located within 500 bp from the transcription start site of APOC3; and (f) the target site located within 500 bp from the transcription start site of APOB.In some of the provided embodiments, the target site is selected from: (a) a target site of PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; (b) a target site of LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; (c) a target site of MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; (d) a target site of ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; (e) a target site of APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; and (f) a target site of APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing. In some embodiments, the target site of PCSK9 has: (i) the sequence shown in SEQ ID NO: 3; (ii) at least 14 nucleotides of the adjacent portion shown in SEQ ID NO: 3, or (iii) a complementary sequence of (i) or (ii).In some of the embodiments provided, the target site is selected from: (a) the target site of PCSK9 having the sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317; (b) the target site of LPA having the sequence shown in any one of SEQ ID NOs: 14 to 23; (c) the target site of MYLIP having the sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351; (d) the target site of ANGPTL3 having the sequence shown in any one of SEQ ID NOs: 34 to 43; (e) the target site of APOC3 having the sequence shown in any one of SEQ ID NOs: 44 to 53; and (f) the target site of APOB having the sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377. In some embodiments, the target site of PCSK9 has the sequence shown in SEQ ID NO: 3.

[0030] In some of the provided embodiments, the Cas protein or its variant is a variant Cas protein that is an inactivated (dCas) protein. In some of the provided embodiments, the dCas protein lacks nuclease activity. In some of the provided embodiments, the dCas protein is a dCas9 protein. In some of the provided embodiments, the dCas protein is a dCas12 protein. In some of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of the provided embodiments, dSaCas9 includes at least one amino acid mutation selected from D10A and N580A with reference to the numbering at the position of SEQ ID NO: 204. In some of the provided embodiments, the dSaCas9 protein includes the sequence shown in SEQ ID NO: 205, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of the provided embodiments, dSaCas9 is shown in SEQ ID NO: 205. In some of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of the provided embodiments, the dSpCas9 protein includes at least one amino acid mutation selected from D10A and H840A with reference to the numbering at the position of SEQ ID NO: 206. In some of the provided embodiments, dSpCas9 includes the sequence shown in SEQ ID NO: 207, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of the provided embodiments, dSpCas9 is shown in SEQ ID NO: 207.

[0031] In some of the provided embodiments, the gRNA comprises a gRNA spacer that is complementary to the target site of a gene. In some of the provided embodiments, the gRNA is a gRNA targeting the target site of PCSK9, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 64-76 or 318-329, or at least 14 nt of its adjacent portion; (b) a gRNA targeting the target site of LPA, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 77-86, or at least 14 nt of its adjacent portion; (c) a gRNA targeting the target site of MYLIP, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 87-96 or 352-361, or at least 14 nt of its adjacent portion; (d) a gRNA targeting the target site of ANGPTL3, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 97-106, or at least 14 nt of its adjacent portion; (e) a gRNA targeting the target site of APOC3, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 107-116, or at least 14 nt of its adjacent portion; and (f) a gRNA targeting the target site of APOB, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 117-126 or 378-383, or at least 14 nt of its adjacent portion, and is selected from these. In some embodiments, the gRNA targeting the target site of PCSK9 comprises the gRNA spacer sequence shown in SEQ ID NO: 66 or at least 14 nt of its adjacent portion. In some of the provided embodiments, the gRNA comprises a spacer sequence with a length of 14 nt to 24 nt, or 16 nt to 22 nt. In some of the provided embodiments, the gRNA comprises a spacer sequence with a length of 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt.In some of the provided embodiments, the gRNA is selected from: (a) a gRNA targeting a target site of PCSK9 that includes a gRNA spacer sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329; (b) a gRNA targeting a target site of LPA that includes a gRNA spacer sequence shown in any one of SEQ ID NOs: 77 to 86; (c) a gRNA targeting a target site of MYLIP that includes a gRNA spacer sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361; (d) a gRNA targeting a target site of ANGPTL3 that includes a gRNA spacer sequence shown in any one of SEQ ID NOs: 97 to 106; (e) a gRNA targeting a target site of APOC3 that includes a gRNA spacer sequence shown in any one of SEQ ID NOs: 107 to 116; and (f) a gRNA targeting a target site of APOB that includes a gRNA spacer sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383. In some embodiments, the gRNA targeting the target site of PCSK9 includes the gRNA spacer sequence shown in SEQ ID NO: 66. In some of the provided embodiments, the gRNA further includes a scaffold sequence shown in SEQ ID NO: 191. In some of the provided embodiments, the gRNA is selected from: (a) a gRNA targeting a target site of PCSK9 that includes a sequence shown in any one of SEQ ID NOs: 127 to 139 or 330 to 341; (b) a gRNA targeting a target site of LPA that includes a sequence shown in any one of SEQ ID NOs: 140 to 149; (c) a gRNA targeting a target site of MYLIP that includes a sequence shown in any one of SEQ ID NOs: 150 to 159 or 362 to 371; (d) a gRNA targeting a target site of ANGPTL3 that includes a sequence shown in any one of SEQ ID NOs: 160 to 169; (e) a gRNA targeting a target site of APOC3 that includes a sequence shown in any one of SEQ ID NOs: 170 to 179; and (f) a gRNA targeting a target site of APOB that includes a sequence shown in any one of SEQ ID NOs: 180 to 189 or 384 to 389.In some embodiments, the gRNA targeting the target site of PCSK9 comprises the sequence shown in SEQ ID NO: 129. In some of any of the provided embodiments, the gRNA is (a) a gRNA targeting the target site of PCSK9 shown in any one of SEQ ID NOs: 127-139 or 330-341, (b) a gRNA targeting the target site of LPA shown in any one of SEQ ID NOs: 140-149, (c) a gRNA targeting the target site of MYLIP shown in any one of SEQ ID NOs: 150-159 or 362-371, (d) a gRNA targeting the target site of ANGPTL3 shown in any one of SEQ ID NOs: 160-169, (e) a gRNA targeting the target site of APOC3 shown in any one of SEQ ID NOs: 170-179, and (f) a gRNA targeting the target site of APOB shown in any one of SEQ ID NOs: 180-189 or 384-389. In some embodiments, the gRNA targeting the target site of PCSK9 is shown in SEQ ID NO: 129. In some of any of the provided embodiments, the gRNA comprises modified nucleotides for improving stability.

[0032] In some of any of the provided embodiments, at least one transcriptional repressor domain can reduce the transcription of a gene. In some of any of the provided embodiments, the transcriptional repressor domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3A-DNMT3L fusion protein domain, an ERF repressor domain, an Mxi1 repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD1 repressor domain, an EZH2 repressor domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing. In some of any of the provided embodiments, the transcriptional repressor domain is a KRAB domain, a DNMT3A domain, or a DNMT3L domain, or a combination of any of the foregoing. In some of any of the provided embodiments, at least one transcriptional repressor domain comprises a sequence selected from any one of SEQ ID NOs: 193, 195, 197, 199, 201, 220-226, 283, 284, 285, 286, 287, 289, and 290, or a domain, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 193, 195, 197, 199, 201, 220-226, 283, 284, 285, 286, 287, 289, and 290.

[0033] In some of any of the provided embodiments, at least one transcriptional repressor domain comprises a KRAB domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 193, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 193. In some of any of the provided embodiments, at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 290, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 290. In some of any of the above embodiments, the KRAB domain, a portion or variant thereof is a sequence that exhibits transcriptional repressor activity.

[0034] In some of any of the provided embodiments, at least one transcriptional repressor domain comprises a DNMT3A domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some embodiments, the DNMT3A domain, or a portion thereof, is derived from human DNMT3A. In some of any of the provided embodiments, at least one effector domain comprises the sequence shown in SEQ ID NO: 284, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 284. In some embodiments, the portion is a catalytically active portion that exhibits transcriptional repressor activity. In some of any of the provided embodiments, at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 195, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 195. In some embodiments, at least one effector domain is, or comprises, the catalytically active portion of DNMT3A shown in SEQ ID NO: 195. In some of any of the provided embodiments, at least one effector domain comprises the sequence shown in SEQ ID NO: 285, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 285. In some embodiments, at least one effector domain is, or comprises, the catalytically active portion of DNMT3A shown in SEQ ID NO: 285.

[0035] In some of the provided embodiments, at least one transcriptional repressor domain comprises a DNMT3L domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of the provided embodiments, at least one transcriptional repressor domain is a fusion domain that comprises a DNMT3A domain, such as any DNMT3A domain, or the C-terminal portion or variant described above, and a DNMT3L domain, or a variant or portion thereof, and the fusion domain exhibits transcriptional repressor activity. In some embodiments, the DNMT3L domain, or a variant or C-terminal portion of the DNMT3L domain, stimulates the enhancement of the transcriptional repressor activity of DNMT3A. In some embodiments, the DNMT3L domain, or a variant or C-terminal portion of the DNMT3L domain, interacts with DNMT3A. In some embodiments, the DNMT3L domain, or its C-terminal portion, is derived from murine DNMT3L. In some embodiments, the DNMT3L domain, or its C-terminal portion, is human or humanized DNMT3L. In some of the provided embodiments, the DNTML domain comprises the sequence shown in SEQ ID NO: 289, its C-terminal portion, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 289. In some of the provided embodiments, DNMT3L comprises the sequence shown in SEQ ID NO: 286, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 286. In some embodiments, the DNMT3L domain is or comprises the C-terminal portion of DNMT3L shown in SEQ ID NO: 286. In some of the provided embodiments, the DNMT3L domain comprises the sequence shown in SEQ ID NO: 197, its C-terminal portion, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 197.In some of any of the provided embodiments, DNMT3L comprises the sequence shown in SEQ ID NO: 287, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 287. In some embodiments, the DNMT3L domain is or comprises the C-terminal portion of DNMT3L shown in SEQ ID NO: 287.

[0036] In some of any of the provided embodiments, at least one transcriptional repressor domain is a DNMT3A-DNMT3L fusion protein domain or a variant thereof that exhibits transcriptional repressor activity. In some embodiments, the DNMT3A domain is shown in SEQ ID NO: 285 and the DNMT3L domain is shown in SEQ ID NO: 286, which may be present in any order. In some embodiments, a linker is present between the DNMT3A domain and the DNMT3L domain. In some of any of the provided embodiments, at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 199 or SEQ ID NO: 201, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 199 or SEQ ID NO: 201.

[0037] In some of any of the provided embodiments, at least one transcriptional repressor domain is fused to the N-terminus, C-terminus, or both the N-terminus and C-terminus of a DNA-binding domain. In some of any of the provided embodiments, the fusion protein further comprises one or more nuclear localization signals (NLSs). In some of any of the provided embodiments, the fusion protein further comprises one or more linkers that connect two or more of the DNA-binding domain, at least one transcriptional repressor domain, and one or more nuclear localization signals.

[0038] In some embodiments, the fusion protein is dCas9-KRAB or a variant thereof.

[0039] In some of the provided embodiments, the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NO: 209, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 209. In some embodiments, the fusion protein has the sequence shown in SEQ ID NO: 209.

[0040] In any of the embodiments herein, the fusion protein is a DNMT3A / 3L-dCas9-KRAB domain or a variant thereof.

[0041] In any of the embodiments herein, the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NO: 278, 280, or 282, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 278, 280, or 282. In some embodiments, the fusion protein has the sequence shown in SEQ ID NO: 278. In some embodiments, the fusion protein has the sequence shown in SEQ ID NO: 280. In some embodiments, the fusion protein has the sequence shown in SEQ ID NO: 282.

[0042] In any of the embodiments herein, the fusion protein comprises a gRNA targeting a target site of PCSK9, the gRNA comprising an amino acid sequence shown in SEQ ID NO: 280, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 280, and a spacer sequence shown in SEQ ID NO: 66. In some embodiments, the gRNA has the sequence shown in SEQ ID NO: 129.

[0043] In some of any of the provided embodiments, the epigenetic modification DNA targeting system reduces gene expression with a log2 fold change of -1.0 or less. In some of any of the provided embodiments, the suppression of gene transcription in a cell or cell population results in a reduction of low density lipoprotein (LDL). In some of any of the provided embodiments, the reduction of LDL occurs extracellularly. In some of any of the provided embodiments, the cell or cell population is a liver cell or includes liver cells. In some of any of the provided embodiments, the cell or cell population is within a subject. In some of any of the provided embodiments, the reduction of LDL occurs within the subject or its fluid, tissue, or organ. In some of any of the provided embodiments, the reduction of LDL occurs in the blood of the subject.

[0044] In some aspects, provided herein is a combination of epigenetic modification DNA targeting systems comprising at least two of the DNA targeting systems provided herein, wherein each DNA targeting system suppresses the transcription of a different gene. In some of any of the provided embodiments, each DNA targeting system suppresses the transcription of a different gene.

[0045] In some aspects, provided herein is a guide RNA (gRNA) that targets a target site of a gene that regulates low density lipoprotein (LDL). In some of any of the provided embodiments, the gene is selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3. In some of any of the provided embodiments, the target site of the gene is within the gene or its regulatory DNA element. In some of any of the provided embodiments, the regulatory DNA element is an enhancer or a promoter. In some of any of the provided embodiments, the target site is selected from the group consisting of: (a) a target site of PCSK9 located within 500 bp of genomic coordinates chr1:55,039,548 of the human genome assembly GRCh38 (hg38); (b) a target site of LPA located within 500 bp of hg38 genomic coordinates chr6:160,664,275; (c) a target site of MYLIP located within 500 bp of hg38 genomic coordinates chr6:16,129,086; (d) a target site of ANGPTL3 located within 500 bp of hg38 genomic coordinates chr1:62,597,520; (e) a target site of APOC3 located within 500 bp of hg38 genomic coordinates chr11:116,829,907; and (f) a target site of APOB located within 500 bp of hg38 genomic coordinates chr2:21,044,073. In some of any of the provided embodiments, the target site is selected from the group consisting of: (a) a target site located within 500 bp of the transcription start site of PCSK9; (b) a target site located within 500 bp of the transcription start site of LPA; (c) a target site located within 500 bp of the transcription start site of MYLIP; (d) a target site located within 500 bp of the transcription start site of ANGPTL3; (e) a target site located within 500 bp of the transcription start site of APOC3; and (f) a target site located within 500 bp of the transcription start site of APOB.In some of the provided embodiments, the target site is selected from: (a) a target site of PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; (b) a target site of LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; (c) a target site of MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; (d) a target site of ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; (e) a target site of APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing; and (f) a target site of APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing. In some embodiments, the target site has (i) the sequence shown in SEQ ID NO: 3, (ii) at least 14 nt of the adjacent portion of SEQ ID NO: 3, or (iii) a complementary sequence of (i) or (ii).In some of the provided embodiments, the target site is selected from: (a) a target site of PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317; (b) a target site of LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23; (c) a target site of MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351; (d) a target site of ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43; (e) a target site of APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53; and (f) a target site of APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377. In some embodiments, the target site has the sequence shown in SEQ ID NO: 3. In some of the provided embodiments, the gRNA is: (a) a gRNA targeting the target site of PCSK9, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329, or at least 14 nt of its adjacent portion; (b) a gRNA targeting the target site of LPA, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 77 to 86, or at least 14 nt of its adjacent portion; (c) a gRNA targeting the target site of MYLIP, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361, or at least 14 nt of its adjacent portion; (d) a gRNA targeting the target site of ANGPTL3, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 97 to 106, or at least 14 nt of its adjacent portion; (e) a gRNA targeting the target site of APOC3, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 107 to 116, or at least 14 nt of its adjacent portion; and (f) a gRNA targeting the target site of APOB, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383, or at least 14 nt of its adjacent portion.In some embodiments, the gRNA targeting the target site of PCSK9 comprises the sequence shown in SEQ ID NO: 66, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion. In some of any of the provided embodiments, the gRNA comprises a spacer sequence having a length of 14 nt to 24 nt, or 16 nt to 22 nt. In some of any of the provided embodiments, the gRNA comprises a spacer sequence having a length of 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt. In some of any of the provided embodiments, the gRNA is (a) a gRNA targeting the target site of PCSK9 comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329, (b) a gRNA targeting the target site of LPA comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 77 to 86, (c) a gRNA targeting the target site of MYLIP comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361, (d) a gRNA targeting the target site of ANGPTL3 comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 97 to 106, (e) a gRNA targeting the target site of APOC3 comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 107 to 116, and (f) a gRNA targeting the target site of APOB comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383. In some embodiments, the gRNA targeting the target site of PCSK9 comprises the gRNA spacer sequence shown in SEQ ID NO: 66. In some of any of the provided embodiments, the gRNA further comprises the scaffold sequence shown in SEQ ID NO: 191.In some of the provided embodiments, the gRNA is selected from: (a) a gRNA targeting a target site of PCSK9 comprising a sequence shown in any one of SEQ ID NOs: 127 - 139 or 330 - 341; (b) a gRNA targeting a target site of LPA comprising a sequence shown in any one of SEQ ID NOs: 140 - 149; (c) a gRNA targeting a target site of MYLIP comprising a sequence shown in any one of SEQ ID NOs: 150 - 159 or 362 - 371; (d) a gRNA targeting a target site of ANGPTL3 comprising a sequence shown in any one of SEQ ID NOs: 160 - 169; (e) a gRNA targeting a target site of APOC3 comprising a sequence shown in any one of SEQ ID NOs: 170 - 179; or (f) a gRNA targeting a target site of APOB comprising a sequence shown in any one of SEQ ID NOs: 180 - 189 or 384 - 389. In some embodiments, the gRNA targeting the target site of PCSK9 comprises the sequence shown in SEQ ID NO: 129. In some of the provided embodiments, the gRNA is selected from: (a) a gRNA targeting a target site of PCSK9 shown in any one of SEQ ID NOs: 127 - 139 or 330 - 341; (b) a gRNA targeting a target site of LPA shown in any one of SEQ ID NOs: 140 - 149; (c) a gRNA targeting a target site of MYLIP shown in any one of SEQ ID NOs: 150 - 159 or 362 - 371; (d) a gRNA targeting a target site of ANGPTL3 shown in any one of SEQ ID NOs: 160 - 169; (e) a gRNA targeting a target site of APOC3 shown in any one of SEQ ID NOs: 170 - 179; or (f) a gRNA targeting a target site of APOB shown in any one of SEQ ID NOs: 180 - 189 or 384 - 389. In some embodiments, the gRNA targeting the target site of PCSK9 is shown in SEQ ID NO: 129. In some of the provided embodiments, the gRNA comprises modified nucleotides for improving stability.

[0046] In some embodiments, provided herein is a plurality of gRNAs comprising at least a first gRNA and a second gRNA, wherein the first gRNA targets a target site of a first gene that regulates low density lipoprotein (LDL), and the second gRNA targets a target site of a second gene that regulates LDL. In some of any of the provided embodiments, the plurality of gRNAs comprises a third gRNA that targets a target site of a third gene that regulates LDL, and optionally, the plurality of gRNAs further comprises a fourth gRNA that targets a target site of a fourth gene that regulates LDL, optionally a fifth gRNA that targets a target site of a fifth gene that regulates LDL, and / or optionally a sixth gRNA that targets a target site of a sixth gene that regulates LDL. In some of any of the provided embodiments, each gRNA is selected from any of the gRNAs provided herein. In some of any of the provided embodiments, the first gene and the second gene are independently selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3. In some of any of the provided embodiments, the first gene and the second gene are different. In some of any of the provided embodiments, the first gene and the second gene are PCSK9 and LPA, PCSK9 and MYLIP, PCSK9 and ANGPTL3, PCSK9 and APOC3, PCSK9 and APOB, LPA and MYLIP, LPA and ANGPTL3, LPA and APOC3, LPA and APOB, MYLIP and ANGPTL3, MYLIP and APOC3, MYLIP and APOB, ANGPTL3 and APOC3, ANGPTL3 and APOB, or APOC3 and APOB. In some of any of the provided embodiments, the first gene, the second gene, and the third gene are each independently selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB. In some of any of the provided embodiments, the first gene, the second gene, and the third gene are different.In some of the provided embodiments, the first gene, the second gene, and the third gene are PCSK9, LPA, and MYLIP; PCSK9, LPA, and ANGPTL3; PCSK9, LPA, and APOC3; PCSK9, LPA, and APOB; PCSK9, MYLIP, and ANGPTL3; PCSK9, MYLIP, and APOC3; PCSK9, MYLIP, and APOB; PCSK9, ANGPTL3, and APOC3; PCSK9, ANGPTL3, and APOB; PCSK9, APOC3, and APOB; LPA, MYLIP, and ANGPTL3; LPA, MYLIP, and APOC3; LPA, MYLIP, and APOB; LPA, ANGPTL3, and APOC3; LPA, ANGPTL3, and APOB; LPA, APOC3, and APOB; MYLIP, ANGPTL3, and APOC3; MYLIP, ANGPTL3, and APOB; MYLIP, APOC3, and APOB; or ANGPTL3, APOC3, and APOB. In some of the provided embodiments, at least one gene is PCSK9. In some of the provided embodiments, at least two genes are PCSK9 and LPA. In some of the provided embodiments, at least three genes are PCSK9, LPA, and MYLIP. In some of the provided embodiments, the target site of each of the plurality of genes is within the gene or its regulatory DNA element.

[0047] In some aspects, provided herein is a combination of a clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof (a), and any gRNA or plurality of gRNAs provided herein (b), a Cas guide RNA (gRNA). In some of any of the provided embodiments, the Cas protein or variant thereof is a variant Cas protein that is a dead (dCas) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dCas protein is a dCas9 protein. In some of any of the provided embodiments, the dCas protein is a dCas12 protein. In some of any of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of any of the provided embodiments, dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to the numbering of the positions of SEQ ID NO: 204. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence shown in SEQ ID NO: 205, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, dSaCas9 is shown in SEQ ID NO: 205. In some of any of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to the numbering of the positions of SEQ ID NO: 206.In some of any of the provided embodiments, dSpCas9 comprises the sequence shown in SEQ ID NO: 207, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, dSpCas9 is shown in SEQ ID NO: 207.

[0048] In some aspects, provided herein is a polynucleotide encoding any of the epigenetic modification DNA targeting systems provided herein, a combination of epigenetic modification DNA targeting systems, a gRNA, multiple gRNAs, or a combination of Cas-gRNAs, or a portion or component of any of the foregoing. In some aspects, provided herein is a polynucleotide encoding any of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein is a polynucleotide encoding at least one DNA targeting module of any of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein is a polynucleotide encoding a fusion protein of any of the epigenetic modification DNA targeting systems provided herein, and at least a first gRNA and a second gRNA. In some aspects, provided herein is a polynucleotide encoding a fusion protein of any of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein is a polynucleotide encoding a fusion protein and a gRNA of any of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein is a polynucleotide encoding any combination of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein is a polynucleotide encoding any gRNA provided herein. In some aspects, provided herein is a polynucleotide encoding any multiple gRNAs provided herein. In some aspects, provided herein is a polynucleotide encoding any combination of Cas-gRNAs provided herein.

[0049] In some aspects, provided herein are polynucleotides encoding any of the epigenetic modification DNA targeting systems provided herein, combinations of epigenetic modification DNA targeting systems, multiple gRNAs, or combinations of Cas-gRNAs, or any part or component of any of the foregoing. In some aspects, provided herein are polynucleotides encoding any of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein are polynucleotides encoding at least one DNA targeting module of any of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein are polynucleotides encoding a fusion protein of any of the epigenetic modification DNA targeting systems provided herein, and multiple polynucleotides encoding at least a first gRNA and a second gRNA. In some aspects, provided herein are polynucleotides encoding a fusion protein of any of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein are polynucleotides encoding a fusion protein and gRNA of any of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein are polynucleotides encoding any combination of the epigenetic modification DNA targeting systems provided herein. In some aspects, provided herein are polynucleotides encoding any multiple gRNAs provided herein. In some aspects, provided herein are polynucleotides encoding any combination of Cas-gRNAs provided herein.

[0050] In some embodiments, provided herein is a vector comprising any polynucleotide provided herein. In some embodiments, provided herein is a vector comprising any plurality of polynucleotides provided herein. In some of any of the provided embodiments, the vector is a viral vector. In some of any of the provided embodiments, the vector is an adeno-associated virus (AAV) vector. In some of any of the provided embodiments, the vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some of any of the provided embodiments, the vector is a lentiviral vector. In some of any of the provided embodiments, the vector is a non-viral vector. In some of any of the provided embodiments, the non-viral vector is selected from lipid nanoparticles, liposomes, exosomes, or cell-penetrating peptides. In some of any of the provided embodiments, the non-viral vector is a lipid nanoparticle. In some of any of the provided embodiments, the vector exhibits tropism for hepatocytes. In some of any of the provided embodiments, the vector comprises one vector, or two or more vectors. In some embodiments, provided herein is a lipid nanoparticle comprising any polynucleotide or plurality of polynucleotides provided herein.

[0051] In some embodiments, provided herein is a method of reducing the transcription of at least two genes in a cell or cell population, the method comprising administering to the cell or cell population any epigenetic modification DNA targeting system, any combination of epigenetic modification DNA targeting systems, any gRNA, any plurality of gRNAs, any combination of Cas-gRNA, any polynucleotide, any plurality of polynucleotides, or any vector or lipid nanoparticle provided herein, or any portion or component of any of the foregoing. In some of any of the provided embodiments, the at least two genes are epigenetically modified. In some of any of the provided embodiments, the transcription of each of the at least two genes is reduced as compared to an equivalent cell or cell population not subjected to the method. In some of any of the provided embodiments, the transcription of each of the at least two genes is reduced by at least about 1.2-fold, 1.25-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.75-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold. In some of any of the provided embodiments, the reduced transcription of each of the at least two genes results in a reduction in low density lipoprotein (LDL). In some of any of the provided embodiments, the reduction in LDL resulting from the reduced transcription of each of the at least two genes is greater than the reduction in LDL resulting from the corresponding reduced transcription of any individual one of the at least two genes alone.

[0052] In some aspects, provided herein is a method of reducing LDL, the method comprising introducing into a cell or cell population any epigenetic modification DNA targeting system, any combination of epigenetic modification DNA targeting systems, any gRNA, any plurality of gRNAs, any combination of Cas-gRNA, any polynucleotide, any plurality of polynucleotides, or any vector or lipid nanoparticle provided herein, or any portion or component of any of the foregoing. In some of any of the provided embodiments, the cell or cell population is a liver cell or comprises liver cells. In some of any of the provided embodiments, the cell or cell population is in a subject and the method is performed in vivo. In some of any of the provided embodiments, LDL is reduced in the subject or in its fluids, tissues, or organs. In some of any of the provided embodiments, LDL is reduced in the blood of the subject. In some of any of the provided embodiments, the subject is human. In some of any of the provided embodiments, the subject has or is suspected of having a disease, condition, or disorder. In some embodiments, the disease, condition, or disorder is cardiovascular disease. In some of any of the provided embodiments, the subject has or is suspected of having one or more of an elevated level of low density lipoprotein in the blood, an increased risk of cardiovascular disease, an increased risk of premature onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in the low density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia. In some of any of the provided embodiments, the subject has or is suspected of having familial hypercholesterolemia.

[0053] In some embodiments, provided herein is a pharmaceutical composition comprising any epigenetic modification DNA targeting system, any combination of epigenetic modification DNA targeting systems, any gRNA, any plurality of gRNAs, any combination of Cas-gRNAs, any polynucleotide, any plurality of polynucleotides, or any vector or lipid nanoparticle provided herein, or any part or component of any of the foregoing. In some of any of the provided embodiments, the pharmaceutical composition is for use in the treatment of a disease, condition, or disorder in a subject. In some embodiments, the disease, condition, or disorder is a cardiovascular disease. In some of any of the provided embodiments, the use of the pharmaceutical composition is for the manufacture of a medicament for treating a disease, condition, or disorder in a subject. In some embodiments, the disease, condition, or disorder is a cardiovascular disease. In some of any of the provided embodiments, the subject has or is suspected of having a disease, condition, or disorder. In some embodiments, the disease, condition, or disorder is a cardiovascular disease. In some of any of the provided embodiments, the subject has or is suspected of having one or more of an elevated level of low density lipoprotein in the blood, an increased risk of cardiovascular disease, an increased risk of premature onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in the low density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia. In some of any of the provided embodiments, the subject has or is suspected of having familial hypercholesterolemia. In some of any of the provided embodiments, the pharmaceutical composition will be administered to the subject in vivo. In some of any of the provided embodiments, the pharmaceutical composition targets or will be administered to the liver of the subject. In some of any of the provided embodiments, after administration of the pharmaceutical composition, the expression of at least two genes is reduced in the cells of the subject.In some of any of the provided embodiments, after administration of the pharmaceutical composition, the expression of at least two genes is reduced in the liver cells of the subject. In some of any of the provided embodiments, the at least two genes are selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3.

[0054] In some aspects, provided herein is a method for doing so in a subject in need of treatment of a disease, condition, or disorder associated with elevated low-density lipoprotein (LDL), the method comprising administering to the subject any epigenetic modification DNA targeting system, any combination of epigenetic modification DNA targeting systems, any gRNA, any plurality of gRNAs, any combination of Cas-gRNA, any polynucleotide, any plurality of polynucleotides, any vector or lipid nanoparticle, or any pharmaceutical composition provided herein, or any portion or component of any of the foregoing. In some of any of the provided embodiments, the subject has, or is suspected of having, one or more of an increase in the level of low-density lipoprotein in the blood, an increased risk of cardiovascular disease, an increased risk of premature-onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in the low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia. In some aspects, the disease, condition, or disorder associated with elevated LDL is cardiovascular disease.

[0055] In some embodiments, provided herein is a method for treating familial hypercholesterolemia in a subject, comprising administering to the subject any epigenetic modification DNA targeting system, any combination of epigenetic modification DNA targeting systems, any gRNA, any plurality of gRNAs, any combination of Cas-gRNA, any polynucleotide, any plurality of polynucleotides, any vector or lipid nanoparticle, or any pharmaceutical composition provided herein, or any portion or component of any of the foregoing. In some of any of the provided embodiments, the administration is a single-dose injection to the subject. In some of any of the provided embodiments, the administration is repeated at least once. In some embodiments, the administration is repeated multiple times at regular intervals. In some embodiments, the administration is a multiple-dose administration comprising at least a first dose and a second dose. In some embodiments, the first dose and the second dose are the same. In some embodiments, the second dose is lower than the first dose. In some embodiments, the second dose is 25% to 75% of the first dose. In some embodiments, the second dose is about 30%, about 40%, about 50%, about 60% or about 70% of the first dose, or a percentage between any of the foregoing. In some embodiments, the second dose is higher than the first dose. In some embodiments, the second dose is 150% to 500% of the first dose. In some embodiments, the second dose is about 200%, about 300%, about 400% or about 500% of the first dose, or a percentage between any of the foregoing. In some of any of the embodiments, any lipid nanoparticle provided herein is administered to the subject.

[0056] In some of any of the embodiments, the pharmaceutical composition is for single-dose injection into a subject. In some of any of the embodiments, the pharmaceutical composition is for repeated-dose administration. In some embodiments, the pharmaceutical composition is for multiple repeated-dose administrations at regular intervals. In some embodiments, the administration is a multiple-dose administration including at least a first dose and a second dose. In some embodiments, the first dose and the second dose are the same. In some embodiments, the second dose is lower than the first dose. In some embodiments, the second dose is 25% to 75% of the first dose. In some embodiments, the second dose is about 30%, about 40%, about 50%, about 60% or about 70% of the first dose, or a percentage between any of the foregoing. In some embodiments, the second dose is higher than the first dose. In some embodiments, the second dose is 150% to 500% of the first dose. In some embodiments, the second dose is about 200%, about 300%, about 400% or about 500% of the first dose, or a percentage between any of the foregoing. In some of any of the embodiments, the pharmaceutical composition comprises any of the lipid nanoparticles provided herein.

Brief Description of the Drawings

[0057]

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Mode for Carrying Out the Invention

[0058] Detailed Description Provided herein is an epigenetic modification DNA targeting system comprising one or more, e.g., a plurality of DNA targeting modules for suppressing the transcription of one or more, e.g., a plurality of genes that regulate low density lipoprotein (LDL). In some embodiments, the provided epigenetic modification DNA targeting system for suppressing the transcription of genes that regulate LDL comprises a fusion protein comprising (a) a DNA binding domain for targeting a target site of a gene that regulates LDL, and (b) at least one transcriptional repressor domain. In some embodiments, the genes associated with the regulation of LDL include PCSK9, LPA, MYLIP, ANGLPTL3, APOC3, and APOB, or any combination thereof. Also provided herein is an epigenetic modification DNA targeting system multiplexed with a plurality of targeting modules such that the system can target such combinations of genes. In some embodiments, each module of the DNA targeting system suppresses the transcription of a different gene. Also provided herein is a method of using an epigenetic modification DNA targeting system for reducing LDL in a subject. In some embodiments, the method can be used in therapies for reducing LDL, such as the treatment of familial hypercholesterolemia (FH).

[0059] FH is a genetic disorder of cholesterol metabolism characterized by abnormally elevated serum levels of low-density lipoprotein (LDL) cholesterol, which can lead to early-onset cardiovascular disease and death. Unless otherwise stated, references to LDL throughout this disclosure refer to LDL cholesterol (LDL-C) as this form is the standard measure of the risk of LDL and LDL-related cardiovascular disease. FH is a relatively common disorder, affecting approximately 1 in 250 people worldwide. FH can be caused by mutations in several genes, some of which play a role in LDL cholesterol metabolism (Konstantina Valanti, E. et al., Metabolism 116:154461(2020), Defesche, J.C. et al. Nat.Rev.Dis.Primers 3:17093(2017), Bouhairie, V.E. et al. Cardiol.Clin. 33(2):169-179(2015)).

[0060] The most common form of FH is an autosomal dominant disorder caused by loss-of-function mutations in the gene encoding the LDL receptor (LDLR), accounting for 85 - 90% of all FH cases. The LDLR is a cell surface receptor that mediates the endocytosis of LDL, including in liver cells, and thus reduces the concentration of circulating LDL. Loss-of-function LDLR mutations decrease the ability to remove LDL from the bloodstream, resulting in elevated levels of circulating LDL.

[0061] Loss-of-function mutations in apolipoprotein B (ApoB) are also associated with FH and account for approximately 5 - 10% of FH cases. ApoB functions as the protein component of LDL and binds to the LDLR to facilitate the endocytosis of LDL. ApoB mutations that cause FH generally disrupt the ability of ApoB to bind to the LDLR and thus reduce the rate of LDL clearance from the bloodstream.

[0062] Rare gain-of-function mutations in proprotein convertase subtilisin / kexin type 9 (PCSK9) have also been associated with FH. PCSK9 binds to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR and negatively regulates the cell surface expression of the LDLR by targeting it for lysosomal degradation. Thus, mutations that result in increased PCSK9 activity reduce the ability of cells to express the LDLR at the cell surface, leading to elevated circulating LDL. Conversely, loss-of-function PCSK9 mutations are associated with reduced LDL levels and protection from cardiovascular disease (Lagace, T. A. Curr. Opin. Lipidol. 25(5):387-393(2014), Peterson, A. S. et al. J. Lipid Res. 49(6):1152-1156(2008), Bouhairie, V. E. et al. Cardiol. Clin. 33(2):169-179(2015)).

[0063] Lipoprotein(a) [Lp(a)] is an LDL-like particle that has been identified as an independent risk factor for FH and cardiovascular disease. Elevated Lp(a) affects approximately 20% of the general population and is more common in individuals with FH (30-50% of individuals with FH have elevated Lp(a)), increasing the risk of cardiovascular disease.

[0064] Understanding FH as a genetic disorder that affects cholesterol metabolism has led to several approaches for treating this disease (Bouhairie et al.). FH is typically first treated with statins, a class of lipid-lowering drugs also known as HMG-CoA reductase inhibitors that reduce cholesterol synthesis. Statin therapy can reduce LDL in FH patients. However, in some FH patients, statins do not sufficiently reduce the risk of LDL and / or negative cardiovascular events, including in patients with additional independent risk factors for cardiovascular disease, including elevated Lp(a) levels. However, statins alone may not be effective in certain FH patients and can be associated with a range of toxic side effects, including statin-related muscle symptoms (Ward, N.C. et al. Circ. Res. 124:328-350 (2019)).

[0065] To sufficiently lower LDL in FH patients who are not effectively treated with statins alone, combination therapy typically including statins and secondary non-statin therapies is required (Bouhairie, V.E. et al. Cardiol. Clin. 33(2):169-179 (2015)). If statins and secondary therapies are not effective, some patients may additionally require a third therapy. Secondary non-statin therapies can include ezetimibe, bile acid sequestrants (such as colesevelam, cholestyramine, and cholestyramine), niacin, and fibrates. Such therapies are associated with several side effects, including gastrointestinal and metabolic dysregulation, as well as interference with other drugs, including statins themselves.

[0066] LDL apheresis, in which LDL is removed from a patient's blood in a clinical setting, is another potential treatment for FH patients, particularly those with homozygous loss-of-function LDLR mutations, in whom the above treatments are not effective. The cost and short-term intensive nature of LDL apheresis are disadvantages, and LDL apheresis typically requires 3-hour treatment sessions every 1-2 weeks.

[0067] PCSK9 monoclonal antibodies have emerged as a promising secondary non-statin therapy to complement statin therapy for FH. PCSK9 antibodies inhibit the binding of PCSK9 to the LDLR, thus reducing LDLR degradation and increasing LDL clearance. However, PCSK9 antibodies entail a significant time commitment and cost, requiring injections once every 2 - 4 weeks.

[0068] In summary, current therapies for FH face several challenges. Combination therapies are complicated by toxic side effects, high costs, or inconvenient treatment schedules, while first-line treatment with statins alone can be ineffective for many patients. Alternative FH therapies are needed that effectively reduce LDL and exhibit reduced treatment outcomes such as higher or more sustained efficacy.

[0069] The embodiments provided herein relate to targeting genetic mechanisms that contribute to the regulation of LDL, including the treatment of cardiovascular disease and FH, using engineered DNA binding systems. Understanding the genetic risk factors for FH, in combination with new technologies for targeted regulation of gene expression in vivo, provides new opportunities for treating the disease. Naturally occurring sequence-specific DNA binding systems such as zinc fingers, transcription activator-like effectors, and CRISPR / Cas systems can be engineered to target one or more genes for activation or repression in vivo (Adli, M. Nat. Commun. 9, 1911 (2018)). The provided embodiments also relate to engineering DNA binding systems to target two or more genes, thus eliminating the need for combination therapies that would previously have been required, as well as the costs and inconveniences associated with such combination therapies.

[0070] The provided embodiments relate to the targeted repression of one or more genes in liver cells to promote a phenotype that reduces LDL in the blood, such as increased LDLR expression and / or increased LDL uptake. In aspects, the provided embodiments include introducing epigenetic modifications in liver cells using an effector domain that is a transcriptional repressor (i.e., a transcriptional repressor domain), which can be directed to regions of a target gene (e.g., regulatory elements such as a promoter or enhancer) for transcriptional repression and reduced expression of the target gene. For example, provided herein is an epigenetic modification DNA-binding system that combines a DNA-binding domain (e.g., a combination of dCas and gRNA) with an effector domain, where the effector domain can target a target site of a gene or its regulatory element to precisely repress or reduce the transcription of the gene through epigenetic regulation. Transcriptional repression leading to reduced gene expression increases the ability of cells to remove LDL from the blood. Additionally, epigenetic modification of cells does not modify DNA at the sequence level, thereby avoiding concerns about the safety of gene editing approaches.

[0071] The ability to epigenetically control the ability of hepatocytes to remove LDL provides an advantageous approach for treating FH and cardiovascular disease, eliminating the need for other therapies or combinations of therapies that can be ineffective, toxic, expensive, or inconvenient. Additionally, the ability to target multiple genes for repression allows for reducing LDL through multiple mechanisms with a single treatment, thereby avoiding the complex schedules and toxicity requirements associated with some combination therapies. Epigenetic repression of genes may provide a long-term solution for reducing LDL and have the potential for a less short-term intensive dosing schedule than current therapies.

[0072] In particular, among the embodiments provided herein are epigenetic modification DNA targeting systems for suppressing the transcription of a gene or multiple genes that regulate LDL, such as any of those described herein. The epigenetic modification DNA targeting system includes a fusion protein comprising (a) at least one DNA binding domain for targeting a target site of a gene and (b) at least one transcription repressor domain capable of suppressing or reducing the transcription of the gene. In some embodiments, the provided epigenetic modification DNA targeting system is for multiplexed target suppression of multiple different genes that regulate LDL, and the system includes multiple DNA targeting modules, each for suppressing the transcription of a different gene that regulates LDL. In some embodiments, the epigenetic modification DNA targeting system includes multiple DNA targeting modules for suppressing the transcription of multiple genes that regulate LDL, and each DNA targeting module includes a fusion protein comprising (a) a DNA binding domain for targeting a target site of one of the multiple genes and (b) at least one transcription repressor domain. In some embodiments, at least two different genes that regulate LDL are targeted for suppression, and the multiple DNA targeting modules include a first DNA targeting module for suppressing the transcription of a first gene among the multiple genes and a second DNA targeting module for suppressing the transcription of a second gene among the multiple genes. Also provided herein are polynucleotides, vectors, and compositions containing them that encode the DNA targeting system or the fusion protein of the DNA targeting system.

[0073] In some embodiments of the provided epigenetic modification DNA targeting system, the DNA binding domain is a nuclease-inactive clustered regularly interspaced short palindromic repeat associated (Cas) protein such as dead Cas (dCas, e.g., dCas9) or a variant thereof, and the DNA targeting system further comprises at least one gRNA that can complex with the Cas. In some embodiments, the DNA binding domain is a nuclease-inactive clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof complexed with a guide RNA (gRNA). In such a system, the gRNA has a spacer sequence that can hybridize to the target site of a gene. Also provided herein are related gRNAs, including compositions and methods comprising or related to a Cas / gRNA combination, polynucleotide, epigenetic modification DNA targeting system.

[0074] In some embodiments of the provided epigenetic modification DNA targeting system, the DNA binding domain is a protein domain engineered for sequence-specific binding to a target site. For example, in some embodiments, the DNA binding domain is a zinc finger (ZFN)-based DNA binding domain as described herein, or a transcription activator-like effector DNA binding domain.

[0075] Also provided herein are methods of using an epigenetic modification DNA targeting system to regulate the transcription or phenotype of liver cells. Also provided herein are methods of using an epigenetic modification DNA targeting system to reduce LDL in a subject. In some embodiments, the method can be used in a therapy for reducing LDL, such as the treatment of FH.

[0076] Accordingly, in some embodiments, the DNA targeting system includes synthetic transcription factors that can regulate, e.g., reduce or suppress, gene transcription in a target manner. In the provided embodiments, the provided epigenetic modification DNA targeting system reduces the transcription of a gene or genes, thereby promoting a phenotype that reduces LDL. The provided embodiments can be used to target multiple genetic mechanisms for reducing LDL in patients with FH or cardiovascular disease while avoiding the toxicity, cost, and inconvenience of current combination therapies. For example, atherosclerotic cardiovascular disease (ASCVD) is a chronic inflammatory disease caused by the accumulation of LDL plaques in arteries. ASCVD is involved in one in four deaths in the United States and hundreds of millions of deaths worldwide and is the underlying cause of approximately 50% of all deaths in Westernized societies. In addition, 75% of acute heart attacks are due to atherosclerotic plaque rupture. Statins can be used to stabilize atherosclerotic plaques but not reverse them. However, the need for repeated dosing and side effects of statin therapy limits uptake and compliance. Combination therapies including statins and PCSK9 antibodies are a promising alternative, but in many patients, LDL levels remain too high. Gene editing approaches targeting PCSK9 can generate DNA mutations and have off-target effects, while RNAi-based methods have not proven durable. Accordingly, improved therapies are needed.

[0077] Thus, the approach provided herein provides a substantial clinical solution for treating cardiovascular diseases such as FH and atherosclerotic cardiovascular disease (ASCVD) by reducing circulating LDL while avoiding problems associated with current therapies. In particular, the provided embodiments relate to methods for targeted inhibition of PCSK9 by the provided DNA targeting systems. The results herein demonstrate a surprisingly high and persistent level of suppression of PCSK9 in serum for over 22 weeks after a single transient dose to an animal model equipped with a DNA targeting inhibitory factor system specific to the PCSK9 promoter. The results demonstrate that silencing PCSK9 results in a reduction of LDL cholesterol and thus is a therapeutic target for the prevention or treatment of cardiovascular disease and FH. Currently approved PCSK9 inhibitors require repeated dosing to maintain efficacy, whereas the DNA targeting systems provided for targeted inhibition of PCSK9 offer the possibility of persistently silencing PCSK9 without altering the gene sequence.

[0078] All publications, including patent documents, scientific papers, and databases referred to herein, are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, including patents, patent applications, or scientific papers, was specifically and individually indicated to be incorporated by reference. If the definitions set forth herein conflict with or are otherwise inconsistent with the definitions set forth in patents, applications, publications, and other publications incorporated by reference herein, the definitions set forth herein shall control over the definitions incorporated by reference herein.

[0079] The section headings used herein are for purposes of organization only and are not to be construed as limiting the subject matter described.

[0080] I. DNA Targeting Systems In some embodiments, provided is a DNA targeting system that specifically targets a target site of at least one gene (also referred to herein as a target gene) or its DNA regulatory element and can reduce the transcription of at least one gene, wherein each of the at least one gene encodes a gene product that regulates LDL. In the provided embodiments, for each target gene to be targeted, the DNA targeting system includes a DNA binding domain that binds to the target site of the gene or its regulatory DNA element. In some embodiments, the DNA targeting system further includes at least one effector domain capable of epigenetically modifying one or more DNA bases of the gene or its regulatory element, and the epigenetic modification results in a reduction in the transcription of the gene (e.g., inhibits or reduces the transcription of the gene as compared to the absence of the DNA targeting system). Thus, the terms DNA targeting system and epigenetic modification DNA targeting system can be used synonymously herein. In some embodiments, the DNA targeting system includes a fusion protein comprising (a) at least one DNA binding domain capable of targeting a target site and (b) at least one effector domain capable of reducing the transcription of the gene. For example, the at least one effector domain is a transcriptional repressor domain.

[0081] In some embodiments, the DNA targeting system contains at least one DNA targeting module, and each DNA targeting module of the system is, as provided, a component of the DNA targeting system that can independently target one target site of a target gene. In some embodiments, each DNA targeting module includes (a) a DNA binding domain capable of targeting a target site of a target gene that regulates LDL and (b) an effector domain capable of reducing the transcription of the gene.

[0082] In some embodiments, the DNA targeting system comprises a single DNA targeting module for targeting the repression of a single gene. In some embodiments, the DNA targeting module can target (a) a target site of the target gene or a regulatory element encoding a gene product that regulates LDL of the target gene, and (b) an effector domain capable of reducing the transcription of the gene.

[0083] In some embodiments, the DNA targeting system comprises a plurality of DNA targeting modules, each DNA targeting module being for targeting the repression of a different gene. In some embodiments, the DNA targeting system is a multiplexed DNA targeting system, i.e., targeting target sites of multiple genes. Thus, the term DNA targeting system may include a multiplexed epigenetic modification DNA targeting system comprising a plurality of DNA targeting modules. The multiplexed epigenetic modification DNA targeting system includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30 DNA targeting modules, or any value between any of the foregoing.

[0084] In some embodiments, any two DNA targeting modules of the DNA targeting system comprise distinct (i.e., non-overlapping) components. In some embodiments, each DNA targeting module of the DNA targeting system comprises distinct (i.e., non-overlapping) components. For example, the DNA targeting system may include a first DNA targeting module comprising a first fusion protein comprising a DNA binding domain (e.g., a ZFN or TALE-based DNA binding domain) targeting a first target site, and a second DNA targeting module comprising a second fusion protein comprising a second DNA binding domain (e.g., a ZFN or TALE-based DNA binding domain) targeting a second target site.

[0085] In some embodiments, any two DNA targeting modules of a DNA targeting system may include shared (i.e., overlapping) components. In some embodiments, each DNA targeting module of a DNA targeting system includes shared (i.e., overlapping) components. For example, a DNA targeting system may include a first DNA targeting module that includes (a) a fusion protein comprising a Cas protein and a transcriptional repressor domain, and (b) a first gRNA that complexes with the Cas protein to target a first target site, and a second DNA targeting module that includes (a) the fusion protein of the first DNA targeting module, and (b) a second gRNA that complexes with the Cas protein to target a second target site. It will be appreciated that providing two or more different gRNAs for a given Cas protein enables different molecules of the same Cas protein to be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g., SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs, as described herein. Thus, it is possible to engineer a single DNA targeting system that includes multiple non-overlapping CRISPR / Cas-based DNA targeting modules.

[0086] In some aspects, provided herein is an epigenetic modification DNA targeting system that includes a plurality of DNA targeting modules for suppressing the transcription of a plurality of genes that regulate low density lipoprotein (LDL). In some embodiments, the plurality of DNA targeting modules includes a first DNA targeting module for suppressing the transcription of a first gene of the plurality of genes, and a second DNA targeting module for suppressing the transcription of a second gene of the plurality of genes. In some embodiments, each DNA targeting module includes a fusion protein that includes (a) a DNA binding domain for targeting one of the target sites of the plurality of genes, and (b) at least one transcriptional repressor domain.

[0087] In some of the provided embodiments, the gene(s) is selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3. In some embodiments, the gene is PCSK9.

[0088] In some aspects, provided herein is an epigenetic modification DNA targeting system comprising a plurality of DNA targeting modules for suppressing the transcription of a plurality of genes that regulate low density lipoprotein (LDL), the (1) first DNA targeting module that reduces the transcription of a first gene that regulates low density lipoprotein (LDL), the first DNA targeting module comprising a first fusion protein comprising (a) a DNA binding domain for targeting a target site of the first gene or its regulatory DNA element, and (b) at least one transcriptional repressor domain, and the (2) second DNA targeting module that reduces the transcription of a second gene that regulates LDL, the second DNA targeting module comprising a second fusion protein comprising (a) a DNA binding domain for targeting a target site of the second gene or its regulatory DNA element, and (b) at least one transcriptional repressor domain.

[0089] In some aspects, provided herein is an epigenetic modification DNA targeting system comprising (a) a fusion protein comprising a clustered regularly interspaced short palindromic repeat (Cas) protein or a variant thereof and at least one transcriptional repressor domain, and (b) a plurality of guide RNAs (gRNAs) comprising at least a first gRNA and a second gRNA, wherein the first gRNA targets a target site of a first gene that regulates low density lipoprotein (LDL), and the second gRNA targets a target site of a second gene that regulates LDL.

[0090] In some embodiments, provided herein is an epigenetic modification DNA targeting system for suppressing the transcription of a gene that regulates low density lipoprotein, the system comprising a fusion protein comprising (a) a DNA binding domain for targeting a target site of a gene and (b) at least one transcriptional repressor domain.

[0091] In some embodiments, provided herein is an epigenetic modification DNA targeting system comprising (a) a fusion protein comprising a clustered regularly interspaced short palindromic repeat (Cas) protein or a variant thereof and at least one transcriptional repressor domain, and (b) a guide RNA (gRNA) targeting a target site of a gene that regulates low density lipoprotein (LDL).

[0092] In aspects of the provided embodiments, the DNA targeting system provided herein targets a combination of genes or their regulatory elements to reduce the transcription of genes in liver cells, such as hepatocytes, and the reduced transcription regulates one or more activities or functions of the liver cells, such as the phenotype of the liver cells. In some embodiments, the reduced transcription of the gene results in reduced gene expression in the liver cells, i.e., reduced gene expression. In some embodiments, the reduced transcription of the gene, such as reduced gene expression, promotes a phenotype that results in, for example, a reduction of LDL in a subject (i.e., an enhanced LDL-reducing phenotype).

[0093] In some embodiments, the cell is a liver cell, such as a hepatocyte. In some embodiments, the cell is a hepatocyte. For example, provided herein is a DNA targeting system that targets a combination of genes or their regulatory elements to reduce the transcription of genes in hepatocytes, and the reduced transcription regulates one or more activities or functions of the hepatocytes, such as the phenotype of the hepatocytes. In some embodiments, the reduced transcription of the gene results in reduced gene expression in the hepatocytes, i.e., reduced gene expression.

[0094] In some embodiments, the cells are derived from a human subject. In some embodiments, the cells are cells in a subject (i.e., in vivo cells).

[0095] In some embodiments, the DNA binding domain comprises or is derived from a CRISPR-associated (Cas) protein, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, the I-SceI enzyme, or variants thereof. In some embodiments, the DNA binding domain comprises a catalytically inactive (e.g., nuclease-inactive or nuclease-inactivated) variant of any of the foregoing. In some embodiments, the DNA binding domain comprises an inactivated Cas9 (dCas9) protein or a variant thereof that is inactive against nuclease activity and is catalytically inactivated so as not to be able to cleave DNA.

[0096] In some embodiments, the DNA binding domain comprises or is derived from a Cas protein such as nuclease-inactive Cas or dCas (e.g., dCas9) or a variant thereof, and the DNA targeting system comprises one or more guide RNAs (gRNAs) such as a combination of gRNAs (e.g., two gRNAs or three gRNAs). In some embodiments, the gRNA comprises a spacer sequence that can target and / or hybridize to a target site. In some embodiments, the gRNA can complex with a Cas protein or a variant thereof. In some embodiments, the gRNA directs or recruits a Cas protein or a variant thereof to a target site.

[0097] In some embodiments, the effector domain comprises a transcriptional repressor domain and / or can reduce the transcription of a gene. In some embodiments, the effector domain directly or indirectly results in reduced gene transcription. In some embodiments, the effector domain induces, catalyzes, or causes transcriptional repression. In some embodiments, the effector domain induces transcriptional repression. In some aspects, the effector domain is selected from a KRAB domain, an ERF repressor domain, an MXI1 domain, a SID4X domain, a MAD-SID domain, a DNMT family protein domain (e.g., DNMT3A or DNMT3B), a fusion of one or more DNMT family proteins or their domains (e.g., DNMT3A / L including a fusion of DNMT3A and DNMT3L domains), LSD1, EZH2, a SunTag domain, a partially or fully functional fragment or domain of any of the foregoing, or any combination of the foregoing. In some embodiments, the effector domain is KRAB. In some embodiments, the effector domain is DNMT3A / L.

[0098] In some embodiments, the fusion protein of the DNA targeting system comprises a dCas9-KRAB fusion protein. In some embodiments, the fusion protein of the DNA targeting system comprises a dCas9-KRAB-DNMT3A / L fusion protein. For the purposes of this specification, unless a specific sequence number is specified or a specific order is designated, the term dCas9-KRAB-DNMT3A / L can refer to any orientation of the fusion protein as described above. In some embodiments, the fusion protein of the DNA targeting system comprises a DNMT3A / L-dCas9-KRAB-fusion protein in order from the N-terminus to the C-terminus. In some embodiments, the fusion protein of the DNA targeting system comprises a KRAB-dCas9-DNMT3A / L-fusion protein in order from the N-terminus to the C-terminus.

[0099] Exemplary components and features of the DNA targeting system are provided in the following subsections.

[0100] A. Target gene and target site In some embodiments, the target gene is a gene in which reduced gene expression regulates a cellular phenotype. In some embodiments, the target gene can regulate a phenotype in hepatocytes. In some embodiments, the target gene can regulate the ability of hepatocytes to reduce LDL levels in the blood of a subject. In some embodiments, reduced gene transcription, such as reduced gene expression, promotes a phenotype within hepatocytes (i.e., an enhanced LDL-reducing phenotype) that leads to a reduction in LDL.

[0101] In some aspects, the phenotype is characterized by the cell surface phenotype of the cell. In some embodiments, the phenotype includes increased expression of the low density lipoprotein receptor (LDL-R).

[0102] Cells that are positive (+) for a particular cell surface marker are understood to be cells that express the marker on their surface at a detectable level. Similarly, cells that are negative (-) for a particular cell surface marker are understood to be cells that express the marker on their surface at an undetectable level. Antibodies and other binding entities can be used to detect the expression level of a marker protein in order to identify or detect a given cell surface marker. Suitable antibodies can include polyclonal, monoclonal, fragments (such as Fab fragments), single-chain antibodies, and other forms of specific binding molecules. Antibody reagents for the above cell surface markers are readily known to those skilled in the art. Some well-known methods for assessing the expression level of a surface marker or protein can be used, for example, affinity-based methods in the setting of surface markers such as flow cytometry, detection by immunoa ff inity-based methods, etc. In some embodiments, the label is a fluorophore, and the method for detection or identification of a cell surface marker on a cell (e.g., a hepatocyte) is by flow cytometry. In some embodiments, different labels are used for each of the different markers by multicolor flow cytometry. In some embodiments, surface expression can be determined by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the binding of the antibody to the marker.

[0103] In some embodiments, a cell (e.g., a hepatocyte) is positive (pos or +) for a particular marker if there is a detectable presence of the particular marker, which can be an intracellular or surface marker, on or within the cell. In some embodiments, surface expression is positive when staining by flow cytometry is detectable at a level substantially above the detected staining, under otherwise identical conditions, and / or at a level substantially similar to, or in some cases higher than, cells known to be positive for the marker, and / or at a level higher than cells known to be negative for the marker, when performing the same procedure using an isotype-matched control. In some embodiments, a cell (e.g., a hepatocyte) contacted by the DNA targeting system described herein has increased expression of a particular marker (e.g., LDL-R) if the staining is substantially greater than that of similar cells not contacted by the DNA targeting system.

[0104] In some embodiments, a cell (e.g., a hepatocyte) is negative (neg or -) for a particular marker if there is no detectable presence of the particular marker, which can be an intracellular or surface marker, on or within the cell. In some embodiments, surface expression is negative when staining by flow cytometry is not detectable at a level substantially above the detected staining, under otherwise identical conditions, and / or at a level substantially lower than cells known to be positive for the marker, and / or at a level substantially similar to cells known to be negative for the marker, when performing the same procedure using an isotype-matched control.

[0105] In some embodiments, the phenotype is functionally characterized. In some embodiments, the phenotype may be characterized by one or more functions of the cell. In some embodiments, the phenotype includes increased uptake of LDL by the cell. In some embodiments, the phenotype includes reduced expression of lipoprotein(a) (Lp(a)), an LDL variant, by the cell. In some embodiments, the phenotype includes reduced LDL in one or more tissues of a subject comprising the cell. In some embodiments, the phenotype includes reduced LDL in the blood of a subject comprising the cell.

[0106] Target genes for regulation by the multiplexed epigenetic modification DNA targeting system provided herein include those whose transcription and expression are reduced in cells having a particular or desired function or activity, such as a cellular phenotype (e.g., a phenotype for reducing LDL). After the provided DNA targeting system has contacted or been introduced into the cell and been selected for a desired activity or function such as a cellular phenotype, various methods may be utilized to characterize the transcription or expression level of a gene within the cell (e.g., a hepatocyte). In some embodiments, the phenotype can be a phenotype that includes one or more cell surface markers as described above. In some embodiments, the phenotype is increased LDL-R expression. In some embodiments, analysis of gene transcriptional activity or expression can be by RNA analysis. In some embodiments, RNA analysis includes RNA quantification. In some embodiments, RNA quantification is performed by reverse transcription quantitative PCR (RT-qPCR), multiplexed qRT-PCR, fluorescence in situ hybridization (FISH), FlowFISH, RNA sequencing (RNA-seq), or combinations thereof.

[0107] In some embodiments, the gene is a gene whose expression in a cell (e.g., a hepatocyte) is reduced after contacting or introducing it into the provided multiplexed epigenetic DNA targeting system. In some embodiments, the reduction of gene expression in a cell (e.g., a hepatocyte) is a log2 fold change of less than about -1.0. For example, the log2 fold change is less than about -1.5, less than about -2.0, less than about -2.5, less than about -3.0, less than about -4.0, less than about -5.0, less than about -6.0, less than about -7.0, less than about -8.0, less than about -9.0, less than about -10.0, or less than any value between any of the foregoing.

[0108] In some embodiments, the gene is selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB. In some embodiments, the target site is a sequence selected from any one of SEQ ID NOs: 1-63, 306-317, 342-351, or 372-377, an adjacent portion of at least 14 nucleotides of any one of SEQ ID NOs: 1-63, 306-317, 342-351, or 372-377, or a complementary sequence of any of the foregoing. In some embodiments, the target site is an adjacent portion of any one of SEQ ID NOs: 1-63, 306-317, 342-351, or 372-377 having a length of 15, 16, 17, 18, or 19 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site has a sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% to all or an adjacent portion of the target site sequences described hereinabove. In some embodiments, the target site is a sequence shown in any one of SEQ ID NOs: 1-63, 306-317, 342-351, or 372-377.

[0109] In some embodiments, the gene is PCSK9. PCSK9 is a gene encoding a proprotein convertase subtilisin / kexin type 9 (also known as FH3, HCHOLA3, LDLCQ1, NARC-1, NARC1, PC9, FHCL3). PCSK9 plays a role in the regulation of plasma cholesterol homeostasis, binds to members of the low-density lipoprotein receptor family, and promotes their degradation (Poirier et al., J. Biol. Chem. 283:2363-2372 (2008)). In some embodiments, the target site of PCSK9 is located within 500 bp (e.g., +500 of 55,039,548 or -500 of 55,039,548 or a position in between the aforementioned) from the genomic coordinate chr1:55,039,548 of the human genome assembly GRCh38 (hg38). In some embodiments, the target site is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of the genomic coordinate chr1:55,039,548. In some embodiments, the target site is located within approximately 80 bp of the genomic coordinate chr1:55,039,548. In some embodiments, the target site is within the region of -40 to +40 of the genomic coordinate chr1:55,039,548. In some embodiments, the target site is located within 20 bp of the genomic coordinate chr1:55,039,548. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of the genomic coordinate chr1:55,039,548. In some embodiments, any of such target sites includes or spans the genomic coordinate chr1:55,039,548 which is the PCSK9 transcription start site (TSS). In some embodiments, the target site is within or overlaps with the range of coordinates chr1:55,039,538-55,039,557. In some embodiments, the target site is or includes the coordinates chr1:55,039,538-55,039,557.In some embodiments, the target site comprises a sequence selected from any one of SEQ ID NOs: 1-13 or 306-317, an adjacent portion of at least 14 nucleotides of any one of SEQ ID NOs: 1-13 or 306-317, or a complementary sequence of any of the foregoing. In some embodiments, the target site is an adjacent portion of any one of SEQ ID NOs: 1-13 or 306-317 that is 15, 16, 17, 18, or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or an adjacent portion of the target site sequence described hereinabove. In some embodiments, the target site is a sequence shown in any one of SEQ ID NOs: 1-13 or 306-317. In some embodiments, the target site is the sequence shown in SEQ ID NO: 3, or an adjacent portion of at least 14 nucleotides of SEQ ID NO: 3. In some embodiments, the target site is the sequence shown in SEQ ID NO: 3.

[0110] In some embodiments, the gene is LPA. LPA is a gene that encodes apolipoprotein(a) (also known as AK38, APOA, LP, lipoprotein(a), Lp(a)). Apolipoprotein(a) is a component of lipoprotein(a), which has been identified as a risk factor for cardiovascular disease. In some embodiments, the target site of LPA is located within 500 bp from the hg38 genomic coordinate chr6:160,664,275. In some embodiments, the target site comprises a sequence selected from any one of SEQ ID NOs: 14-23, an adjacent portion of at least 14 nucleotides of any one of SEQ ID NOs: 14-23, or a complementary sequence of any of the foregoing. In some embodiments, the target site is an adjacent portion of any one of SEQ ID NOs: 14-23 having a length of 15, 16, 17, 18, or 19 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or an adjacent portion of the target site sequence described hereinabove. In some embodiments, the target site is a sequence shown in any one of SEQ ID NOs: 14-23.

[0111] In some embodiments, the gene is MYLIP. MYLIP is a gene encoding myosin regulatory light chain interacting protein (IDOL, MIR, also known as myosin regulatory light chain interacting protein). MYLIP is an E3 ubiquitin-protein ligase that mediates the ubiquitination and subsequent proteasomal degradation of myosin regulatory light chain (MRLC), LDLR, VLDLR, and LRP8. MYLIP functions as a sterol-dependent inhibitor of cellular cholesterol uptake by mediating ubiquitination and subsequent degradation of LDLR. In some embodiments, the target site of MYLIP is located within 500 bp from the hg38 genomic coordinate chr6:16,129,086. In some embodiments, the target site is a sequence selected from any one of SEQ ID NOs: 24-33 or 342-351, an adjacent portion of at least 14 nucleotides of any one of SEQ ID NOs: 24-33 or 342-351, or a complementary sequence of any of the foregoing. In some embodiments, the target site is an adjacent portion of any one of SEQ ID NOs: 24-33 or 342-351 having a length of 15, 16, 17, 18, or 19 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or an adjacent portion of the target site sequences described hereinabove. In some embodiments, the target site is a sequence shown in any one of SEQ ID NOs: 24-33 or 342-351.

[0112] In some embodiments, the gene is ANGPTL3. ANGPTL3 is a gene encoding angiopoietin-related protein 3 (also known as ANG-5, ANGPT5, ANL3, FHBL2, angiopoietin-like 3). ANGPTL3 is a member of the angiopoietin-like family of secreted factors and is involved in the regulation of lipid and glucose metabolism. In some embodiments, the target site of ANGPTL3 is located within 500 bp from the hg38 genomic coordinates chr1:62,597,520. In some embodiments, the target site comprises a sequence selected from any one of SEQ ID NOs: 34-43, an adjacent portion of at least 14 nucleotides of any one of SEQ ID NOs: 34-43, or a complementary sequence of any of the foregoing. In some embodiments, the target site is an adjacent portion of any one of SEQ ID NOs: 34-43 having a length of 15, 16, 17, 18, or 19 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or an adjacent portion of the target site sequences described hereinabove. In some embodiments, the target site is a sequence shown in any one of SEQ ID NOs: 34-43.

[0113] In some embodiments, the gene is APOC3. APOC3 is a gene encoding apolipoprotein C-III (also known as APOCIII, HALP2, apolipoprotein C3, Apo-C3, ApoC-3). APOC3 is a component of triglyceride-rich very low density lipoprotein (VLDL) and high density lipoprotein (HDL) in plasma and plays a role in triglyceride homeostasis. In some embodiments, the target site of APOC3 is located within 500 bp from the hg38 genomic coordinate chr11:116,829,907. In some embodiments, the target site comprises a sequence selected from any one of SEQ ID NOs: 44 to 53, an adjacent portion of at least 14 nucleotides of any one of SEQ ID NOs: 44 to 53, or a complementary sequence of any of the foregoing. In some embodiments, the target site is an adjacent portion of any one of SEQ ID NOs: 44 to 53 having a length of 15, 16, 17, 18 or 19 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or an adjacent portion of the target site sequences described hereinabove. In some embodiments, the target site is a sequence shown in any one of SEQ ID NOs: 44 to 53.

[0114] In some embodiments, the gene is APOB. APOB is a gene that encodes apolipoprotein B-100 (also known as FLDB, LDLCQ4, apoB-100, apoB-48, apolipoprotein B, FCHL2). APOB is a component of low-density lipoprotein and functions as a recognition signal for cell binding and internalization of LDL particles by the apoB / E receptor. In some embodiments, the target site of APOB is located within 500 bp from the hg38 genomic coordinate chr2:21,044,073. In some embodiments, the target site is a sequence selected from any one of SEQ ID NOs: 54-63 or 372-377, an adjacent portion of at least 14 nucleotides of any one of SEQ ID NOs: 54-63 or 372-377, or a complementary sequence of any of the foregoing. In some embodiments, the target site is an adjacent portion of any one of SEQ ID NOs: 54-63 or 372-377 having a length of 15, 16, 17, 18, or 19 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or an adjacent portion of the target site sequences described hereinabove. In some embodiments, the target site is a sequence shown in any one of SEQ ID NOs: 54-63 or 372-377.

[0115] In some embodiments, the multiplexed epigenetic modification DNA targeting system targets or binds to a target site within a gene, such as any of the above. In some embodiments, the target site is located in a regulatory DNA element of a gene in a cell (e.g., a hepatocyte). In some embodiments, the regulatory DNA element is a sequence to which a gene regulatory protein can bind and affect the transcription of the gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of the gene. In some embodiments, the regulatory DNA element is a promoter or enhancer of the gene. In some embodiments, the target site is located within a promoter, enhancer, exon, intron, untranslated region (UTR), 5’UTR, or 3’UTR of the gene. In some embodiments, the promoter is a nucleotide sequence to which RNA polymerase binds to initiate transcription of the gene. In some embodiments, the promoter is a nucleotide sequence located within about 100 bp, about 500 bp, about 1000 bp, or more of the transcription start site of the gene. In some embodiments, the target site is located within a sequence of unknown or known function suspected of being able to control the expression of the gene.

[0116] In some embodiments, provided herein is a multiplexed epigenetic modification DNA targeting system that targets a combination of at least two target genes or their regulatory DNA elements described herein.

[0117] In some embodiments, provided herein is a multiplexed epigenetic modification DNA targeting system that targets a first gene and a second gene. In some embodiments, the first gene is selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, the second gene is selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, and the first and second genes are different. In some embodiments, the first gene is PCSK9 and the second gene is selected from the list consisting of LPA, MYLIP, ANGPTL3, APOC3, and APOB. In some embodiments, the first gene is PCSK9 and the second gene is LPA.

[0118] In some embodiments, the first gene and the second gene are selected from the combinations listed in Table 1.

[0119] (Table 1) Combinations of the first gene and the second gene targeted by the multiplexed epigenetic modification DNA targeting system provided herein TIFF2025524469000002.tif88128

[0120] In some embodiments, provided herein is a multiplexed epigenetic modification DNA targeting system that targets a first gene, a second gene, and a third gene. In some embodiments, the first gene is selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, the second gene is selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, the third gene is selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, and the first, second, and third genes are different. In some embodiments, the first gene is PCSK9, the second gene is selected from the list consisting of LPA, MYLIP, ANGPTL3, APOC3, and APOB, the third gene is selected from the list consisting of LPA, MYLIP, ANGPTL3, APOC3, and APOB, and the second gene and the third gene are different. In some embodiments, the first gene is PCSK9, the second gene is LPA, and the third gene is selected from the list consisting of MYLIP, ANGPTL3, APOC3, and APOB.

[0121] In some embodiments, the first gene, the second gene, and the third gene are selected from the combinations listed in Table 2.

[0122] (Table 2) Combinations of the first gene, the second gene, and the third gene targeted by the multiplexed epigenetic modification DNA targeting system provided herein TIFF2025524469000003.tif116128

[0123] B. CRISPR-based DNA targeting system Provided herein is a multiplexed epigenetic targeting DNA targeting system based on a CRISPR / Cas system, i.e., a CRISPR / Cas-based DNA targeting system, that can bind to a target site in a target gene or a target site in a combination of target genes. In some embodiments, the CRISPR / Cas DNA binding domain is nuclease-inactive, such as including dCas (e.g., dCas9), so that the system binds to the target site in the target gene without mediating nucleic acid cleavage at the target site. The CRISPR / Cas-based DNA targeting system can be used to regulate the expression of a target gene in a cell, such as a hepatocyte. In some embodiments, the target gene may include those described herein, including those described above in Section I.A. In some embodiments, the target site of the target gene may include those described herein, including those described above in Section I.A. In some embodiments, the CRISPR / Cas-based DNA targeting system may include any known Cas enzyme and generally include nuclease-inactive or dCas. In some embodiments, the CRISPR / Cas-based DNA targeting system includes a fusion protein of a nuclease-inactive Cas protein or a variant thereof, an effector domain (e.g., a transcriptional repressor) that reduces gene transcription, and at least one gRNA.

[0124] The CRISPR system (also known as the CRISPR / Cas system or CRISPR-Cas system) refers to a conserved microbial nuclease system found in the genomes of bacteria and archaea that provides a form of acquired immunity against invading phages and plasmids. Clustered regularly interspaced short palindromic repeats (CRISPR) refer to loci containing multiple repetitive DNA elements separated by non-repetitive DNA sequences called spacers. Spacers are short sequences of foreign DNA that are integrated into the genome between CRISPR repeats and function as a "memory" of past exposure. Spacers encode the DNA target portion of the RNA molecules that confer the specificity of nucleic acid cleavage by the CRISPR system. The CRISPR locus contains or is adjacent to one or more CRISPR-associated (Cas) genes that can function as RNA-guided nucleases to mediate cleavage, as well as non-protein-coding DNA elements that encode RNA molecules that can program the specificity of CRISPR-mediated nucleic acid cleavage.

[0125] In type II CRISPR / Cas systems with the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex that directs Cas9 nuclease activity. The CRISPR RNA (crRNA) is complementary to the target nucleic acid sequence (target site) and contains the spacer sequence that encodes the sequence specificity of the complex. The trans-activating crRNA (tracrRNA) bases pair with a portion of the crRNA, form a complex with the Cas9 protein, and form a structure that forms the Cas / RNA RNP complex.

[0126] Naturally occurring CRISPR / Cas systems, such as those having Cas9, have been engineered to enable efficient programming of Cas / RNA RNPs to target desired sequences within a cell of interest for both gene editing and regulation of gene expression. The tracrRNA and crRNA are engineered to form a single chimeric guide RNA molecule, commonly referred to as guide RNA (gRNA), as described, for example, in WO2013 / 176772, WO2014 / 093661, WO2014 / 093655, Jinek, M. et al. Science 337(6096):816-21(2012), or Cong, L. et al. Science 339(6121):819-23(2013). The spacer sequence of the gRNA can be selected by the user to target the Cas / gRNA RNP complex to a desired locus, e.g., a desired target site within a target gene.

[0127] Cas proteins are also engineered to be catalytically inactivated or nuclease-inactivated such that they enable targeting of the Cas / gRNA RNP without inducing cleavage at the target site. Mutations in the Cas protein can reduce or abolish the nuclease activity of the Cas protein, rendering the Cas protein catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to herein interchangeably as inactivated Cas (dCas), or nuclease-inactive Cas (iCas) proteins. For example, as described in WO2013 / 176772, WO2014 / 093661, Jinek, M. et al. Science 337(6096):816-21(2012), and Qi, L. et al. Cell 152(5):1173-83(2013), an exemplary inactivated Cas9 (dCas9) derived from S. pyogenes contains silencing mutations in the RuvC and HNH nuclease domains (D10A and H840A). Exemplary dCas variants derived from the Cas12 family (i.e., Cpf1) are described, for example, in WO2017 / 189308 and Zetsche, B. et al. Cell 163(3):759-71(2015). Conserved domains that mediate nucleic acid cleavage, such as the RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologs and can be mutated to produce inactive variants, as described, for example, in Zetsche, B. et al. Cell 163(3):759-71(2015).

[0128] dCas fusion proteins with transcription and / or epigenetic regulators have been used as a highly versatile platform for ectopically regulating gene expression in target cells. These include fusions of Cas with effector domains such as transcriptional activators or transcriptional repressors. For example, fusing dCas9 with a transcriptional activator such as VP64 (a polypeptide composed of four tandem copies of VP16, the 16-amino acid transactivation domain of herpes simplex virus) can result in robust induction of gene expression. Alternatively, fusing dCas9 with a transcriptional repressor such as KRAB (Krueppel-associated box) can result in potent repression of gene expression. A variety of dCas fusion proteins with transcription and epigenetic regulators can be engineered for the regulation of gene expression, as described, for example, in WO2014 / 197748, WO2016 / 130600, WO2017 / 180915, WO2021 / 226555, WO2013 / 176772, WO2014 / 152432, WO2014 / 093661, WO2021 / 247570, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013), Maeder, M. L. et al. Nat. Methods 10, 977-979 (2013), Gilbert, L. A. et al. Cell 154(2):442-451 (2013), and Nunez, J. K. et al. Cell 184(9):2503-2519 (2021).

[0129] In some embodiments, provided is a DNA targeting system comprising a DNA binding domain comprising a nuclease-inactive Cas protein or a variant thereof, and an effector domain (i.e., a transcriptional repressor) for reducing transcription or inducing transcriptional repression when targeting a target gene in a cell (e.g., a hepatocyte). In such embodiments, the DNA targeting system also includes one or more gRNAs provided in combination with or as a complex with the dCas protein or a variant thereof to target the DNA targeting system to a target site of the target gene. In some embodiments, the fusion protein is directed to a specific target site sequence of the target gene by a guide RNA, and the effector domain mediates a target epigenetic modification to reduce or suppress transcription of the target gene. In some embodiments, a combination of gRNAs directs the fusion protein to a combination of target site sequences of a combination of genes, and the effector domain mediates a target epigenetic modification to reduce or suppress transcription of the combination of target genes. Any of the various effector domains for reducing or suppressing transcription can be used as further described below.

[0130] i. CRISPR-based DNA binding domain In some embodiments, the DNA binding domain comprises or is derived from a CRISPR-associated (Cas) protein or a variant thereof. In certain embodiments herein, the Cas protein is nuclease-inactive (i.e., a dCas protein).

[0131] In some embodiments, the Cas protein is derived from a Class 1 CRISPR system (i.e., a multi-Cas protein system), e.g., a type I, III, or IV CRISPR system. In some embodiments, the Cas protein is derived from a Class 2 CRISPR system (i.e., a single Cas protein system), e.g., a type II, V, or VI CRISPR system. In some embodiments, the Cas protein is derived from a type V CRISPR system. In some embodiments, the Cas protein is derived from a Cas12 protein (i.e., Cpf1) or a variant thereof, as described, for example, in WO2017 / 189308 and Zetsche, B. et al. Cell. 163(3):759-71(2015). In some embodiments, the Cas protein is derived from a type II CRISPR system. In some embodiments, the Cas protein is derived from a Cas9 protein or a variant thereof as described, for example, in WO2013 / 176772, WO2014 / 152432, WO2014 / 093661, WO2014 / 093655, Jinek, M. et al. Science 337(6096):816-21(2012), Mali, P. et al. Science 339(6121):823-6(2013), Cong, L. et al. Science 339(6121):819-23(2013), Perez-Pinera, P. et al. Nat.Methods 10,973-976(2013), or Mali, P. et al. Nat.Biotechnol.31,833-838(2013). Various CRISPR / Cas systems and related Cas proteins for use in gene editing and regulation are described, for example, in Moon, S.B. et al. Exp.Mol.Med. 51,1-11(2019), Zhang, F.Q. Rev.Biophys. 52,E6(2019), and Makarova K.S. et al. Methods Mol.Biol. 1311:47-75(2015).

[0132] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule or a variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis, or a variant thereof. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. aureus. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes.

[0133] Non-limiting examples of Cas9 orthologs from other bacterial strains include Acaryochloris marina MBIC11017, Acetohalobium arabaticum DSM5501, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans ATCC23270, Alicyclobacillus acidocaldarius LAA1, Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM446, Allochromatium vinosum DSM180, Ammonifex degensii KC4, Anabaena variabilis ATCC29413, Arthrospira maxima CS-328, Arthrospira platensis str. Paraca, Arthrospira sp. PCC8005, Bacillus pseudomycoides DSM12442, Bacillus selenitireducens MLS10, Burkholderiales bacterium1_1_47, Caldicelulosiruptor becscii DSM6725, Candidatus Desulforudis audaxviator MP104C, Caldicellulosiruptor hydrothermalis108, Clostridium phage c-st, Clostridium botulinum A3 str. Loch Maree, Clostridium botulinum Ba4 str. 657, Clostridium difficile QCD-63q42, Crocosphaera watsonii WH8501, Cyanothece sp. ATCC51142, Cyanothece sp. CCY0110, Cyanothece sp. PCC7424, Cyanothece sp.PCC7822, Exiguobacterium sibiricum 255-15, Finegoldia magna ATCC29328, Ktedonobacter racemifer DSM44963, Lactobacillus delbrueckii subsp. bulgaricus PB2003 / 044-T3-4, Lactobacillus salivarius ATCC11741, Listeria innocua, Lyngbya sp. PCC8106, Marinobacter sp. ELB17, Methanohalobium evestigatum Z-7303, Microcystis phage Ma-LMM01, Microcystis aeruginosa NIES-843, Microscilla marina ATCC23134, Microcoleus chthonoplastes PCC7420, Neisseria meningitidis, Nitrosococcus halophilus Nc4, Nocardiopsis dassonvillei subsp. dassonvillei DSM43111, Nodularia spumigena CCY9414, Nostoc sp. PCC7120, Oscillatoria sp. PCC6506, Pelotomaculum_thermopropionicum SI, Petrotoga mobilis SJ95, Polaromonas naphthalenivorans CJ2, Polaromonas sp. JS666, Pseudoalteromonas haloplanktis TAC125, Streptomyces pristinaespiralis ATCC25486, Streptomyces pristinaespiralis ATCC25486, Streptococcus thermophilus, Streptomyces viridochromogenes DSM40736, Streptosporangium roseum DSM43021, Synechococcus sp. PCC7335, and Thermosipho africanus TCF52B (Chylinski et al.Examples include, but are not limited to, the Cas proteins identified in RNA Biol., 2013;10(5):726-737).

[0134] In some embodiments, the Cas protein is a variant lacking nuclease activity (i.e., a dCas protein). In some embodiments, the Cas protein is mutated such that its nuclease activity is reduced or eliminated. Such Cas proteins are referred to herein interchangeably as inactivated Cas or dead Cas (dCas) or nuclease-inactivated Cas (iCas) proteins. In some embodiments, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or is an inactivated Cas9 (dCas9, or iCas9) protein.

[0135] In some embodiments, the Cas9 protein or a variant thereof is derived from the Staphylococcus aureus Cas9 (SaCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Staphylococcus aureus dCas9 protein (dSaCas9) comprising at least one amino acid mutation selected from D10A and N580A, with reference to the numbering at the position of SEQ ID NO: 204. In some embodiments, the variant Cas9 protein comprises the sequence shown in SEQ ID NO: 205, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0136] In some embodiments, the Cas9 protein or a variant thereof is derived from the Streptococcus pyogenes Cas9 (SpCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Streptococcus pyogenes dCas9 protein (dSpCas9) comprising at least one amino acid mutation selected from D10A and H840A, with reference to the numbering at the position of SEQ ID NO: 206. In some embodiments, the variant Cas9 protein comprises the sequence shown in SEQ ID NO: 207, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0137] ii. Guide RNA In some embodiments, the Cas protein (e.g., dCas9) is provided in combination with or as a complex with one or more guide RNAs (gRNAs). In some aspects, the gRNA is a nucleic acid that facilitates specific targeting or homing of the gRNA / Cas RNP complex to a target site of a target gene (such as those described above). In some embodiments, the target site of the gRNA can be referred to as a protospacer.

[0138] Provided herein are gRNAs, e.g., gRNAs that target or bind to a target gene or its DNA regulatory element as described above in Section I.A. In some embodiments, the gRNA can complex with or bind to a Cas protein or a variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e., a spacer sequence or a guide sequence) that can hybridize to a target site or is complementary to a target site such as any target site described in Section I.A or further below. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to a Cas protein.

[0139] In some embodiments, the gRNAs provided herein are chimeric gRNAs. Generally, a gRNA can be a single molecule (i.e., composed of a single RNA molecule) or modular (including two or more, typically two separate RNA molecules). Modular gRNAs can be engineered to be a single molecule, and the sequences from separate modular RNA molecules are included in a single gRNA molecule, sometimes called a chimeric gRNA, a synthetic gRNA, or a single gRNA. In some embodiments, the chimeric gRNA is a fusion of two non-coding RNA sequences: a crRNA sequence and a tracrRNA sequence, such as those described in, for example, WO2013 / 176772 or Jinek, M. et al. Science 337(6096):816-21 (2012). In some embodiments, the chimeric gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the type II effector system, and the naturally occurring crRNA:tracrRNA duplex functions as a guide for the Cas9 protein.

[0140] In some aspects, the spacer sequence of the gRNA hybridizes to a target site of a target gene and comprises at least a portion having sufficient complementarity with the target gene or its DNA regulatory element (e.g., those described in Section I.A) to direct sequence-specific binding of the CRISPR complex to the sequence of the target site. Complete complementarity is not necessarily required, so long as there is sufficient complementarity to cause hybridization and promote formation of the CRISPR complex. In some embodiments, the gRNA comprises a spacer sequence that is, for example, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% (e.g., completely complementary) to the target site. The strand of the target nucleic acid that contains the target site sequence can be referred to as the "complementary strand" of the target nucleic acid.

[0141] In some embodiments, the gRNA spacer sequence is about 14 nucleotides (nt) to about 26 nt, or 16 nt to 22 nt in length. In some embodiments, the gRNA spacer sequence is 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt or 22 nt, 23 nt, 24 nt, 25 nt, or 26 nt in length. In some embodiments, the gRNA spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some embodiments, the gRNA spacer sequence is 19 nt in length.

[0142] The target site of the gRNA can be referred to as a protospacer. In some embodiments, the gRNA spacer is designed to target a protospacer having a specific protospacer adjacent motif (PAM), i.e., a sequence that directly flanks the protospacer and contributes to and / or is required for Cas binding specificity. Different CRISPR / Cas systems have different PAM requirements for targeting. For example, in some embodiments, S. pyogenes Cas9 uses the PAM 5'-NGG-3' (SEQ ID NO: 202), where N is any nucleotide. S. aureus Cas9 uses the PAM 5'-NNGRRT-3' (SEQ ID NO: 203), where N is any nucleotide and R is G or A. N. meningitidis Cas9 uses the PAM 5'-NNNNGATT-3' (SEQ ID NO: 210), where N is any nucleotide. C. jejuni Cas9 uses the PAM 5'-NNNNRYAC-3' (SEQ ID NO: 211), where N is any nucleotide, R is G or A, and Y is C or T. S. thermophilus uses the PAM 5'-NNAGAAW-3' (SEQ ID NO: 212), where N is any nucleotide and W is A or T. F. Novicida Cas9 uses the PAM 5'-NGG-3' (SEQ ID NO: 213), where N is any nucleotide. T. denticola Cas9 uses the PAM 5'-NAAAAC-3' (SEQ ID NO: 214), where N is any nucleotide. Cas12a (also known as Cpf1) from various species uses the PAM 5'-TTTV-3' (SEQ ID NO: 215). The Cas protein may use a PAM different from those listed above or may be engineered to use one. For example, variant SpCas9 proteins may use a PAM selected from 5'-NGG-3' (SEQ ID NO: 202), 5'-NGAN-3' (SEQ ID NO: 216), 5'-NGNG-3' (SEQ ID NO: 217), 5'-NGAG-3' (SEQ ID NO: 218), or 5'-NGCG-3' (SEQ ID NO: 219).In some embodiments, the PAM sequence for complexing with S.pyogenes Cas9 or its variant is shown in SEQ ID NO: 202. In some embodiments, the PAM sequence for complexing with S.aureus Cas9 or its variant is shown in SEQ ID NO: 203.

[0143] The gRNA spacer sequence may be selected to reduce the degree of secondary structure within the spacer sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm.

[0144] In some embodiments, the gRNA (including the guide sequence) contains the base uracil (U), while the DNA encoding the gRNA molecule will contain the base thymine (T). Without wishing to be bound by theory, in some embodiments, the complementarity between the guide sequence and the target sequence is thought to contribute to the specificity of the interaction between the gRNA molecule / Cas molecule complex and the target nucleic acid. In the guide sequence and target sequence pair, it is understood that the uracil base in the guide sequence pairs with the adenine base in the target sequence. The gRNA spacer sequence herein may be defined by the DNA sequence encoding the gRNA spacer, and / or the RNA sequence of the spacer.

[0145] In some embodiments, one, two or more, or all of the nucleotides of the gRNA can have modifications, for example, to make the gRNA less susceptible to degradation and / or to improve biocompatibility. As a non-limiting example, the backbone of the gRNA can be modified with phosphorothioate, or other modification(s). In some cases, the nucleotides of the gRNA can include 2'-modifications, for example, 2'-acetylation, for example, 2'-methylation, or other modification(s).

[0146] Methods for designing gRNAs and exemplary targeting domains can include, for example, those described in International PCT Publications WO2014 / 197748, WO2016 / 130600, WO2017 / 180915, WO2021 / 226555, WO2013 / 176772, WO2014 / 152432, WO2014 / 093661, WO2014 / 093655, WO2015 / 089427, WO2016 / 049258, WO2016 / 123578, WO2021 / 076744, WO2014 / 191128, WO2015 / 161276, WO2017 / 193107, and WO2017 / 093969.

[0147] In some embodiments, the gRNAs provided herein target target sites in genes or their DNA regulatory elements within a cell (e.g., a hepatocyte), and the gene is selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB.

[0148] In some embodiments, the gRNA targets a target site comprising a sequence selected from any one of SEQ ID NOs: 1-63, 306-317, 342-351, or 372-377, an adjacent portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is an adjacent portion of any one of SEQ ID NOs: 1-63, 306-317, 342-351, or 372-377 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is shown in any one of SEQ ID NOs: 1-63, 306-317, 342-351, or 372-377. In some embodiments, the gRNA targets a target site in the PCSK9 promoter. In some embodiments, the gRNA comprises SEQ ID NO: 3 or targets the target site shown in SEQ ID NO: 3 or an adjacent portion thereof of at least 14 nucleotides. In some embodiments, the gRNA comprises SEQ ID NO: 3 or targets the target site shown in SEQ ID NO: 3.

[0149] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOs: 64-126, 318-329, 352-361, or 378-383, as shown in Table 3, or an adjacent portion thereof of at least 14 nt, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is an adjacent portion of any one of SEQ ID NOs: 64-126, 318-329, 352-361, or 378-383, which is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is shown in any one of SEQ ID NOs: 64-126, 318-329, 352-361, or 378-383. In some embodiments, the spacer sequence of the gRNA comprises or is shown in an adjacent portion of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides) of SEQ ID NO: 66, or in SEQ ID NO: 66. In some embodiments, the spacer sequence of the gRNA is shown in SEQ ID NO: 66.

[0150] In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence shown in SEQ ID NO: 191 (GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC), or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a portion thereof. In some embodiments, the scaffold sequence is shown in SEQ ID NO: 191. In some embodiments, the scaffold sequence comprises the DNA sequence shown in SEQ ID NO: 190.

[0151] In some embodiments, the gRNA provided herein comprises a spacer sequence selected from any one of SEQ ID NOs: 64-126, 318-329, 352-361, or 378-383, as shown in Table 3. In some embodiments, the gRNA further comprises a scaffold sequence shown in SEQ ID NO: 191. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 127-189, 330-341, 362-371, or 384-389, as shown in Table 4, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 127-189, 330-341, 362-371, or 384-389. In some embodiments, the gRNA is shown by any one of SEQ ID NOs: 127-189, 330-341, 362-371, or 384-389. In some embodiments, any of the provided gRNA sequences is complexed with Cas9 or provided in combination with Cas9. In some embodiments, Cas9 is dCas9. In some embodiments, dCas9 is dSpCas9 such as dSpCas9 shown in SEQ ID NO: 207, or a variant and / or fusion thereof.

[0152] (Table 3) Genes, Target Site Sequences, and gRNA Spacer Sequences TIFF2025524469000004.tif64160TIFF2025524469000005.tif239160TIFF2025524469000006.tif239160TIFF2025524469000007.tif212160

[0153] (Table 4) Genes and Gene-Targeting gRNAs TIFF2025524469000008.tif228166TIFF2025524469000009.tif241166TIFF2025524469000010.tif241166TIFF2025524469000011.tif241166TIFF2025524469000012.tif204166

[0154] In some embodiments, the gRNAs provided herein target PCSK9 or its DNA regulatory elements. In some embodiments, the gRNA targets a target site located within 500 bp (e.g., +500 of 55,039,548, or -500 of 55,039,548, or a position in between the foregoing) from the genomic locus chr1:55,039,548 of the human genome assembly GRCh38 (hg38). In some embodiments, the gRNA targets a target site that is within 400 bp, 300 bp, 200 bp, 100 bp, 80 bp, 60 bp, 50 bp, 40 bp, 30 bp, or 20 bp of the genomic locus chr1:55,039,548. In some embodiments, the gRNA targets a target site located within approximately 80 bp of the genomic locus chr1:55,039,548. In some embodiments, the gRNA targets a target site within the region of -40 to +40 of the genomic locus chr1:55,039,548. In some embodiments, the gRNA targets a target site located within 20 bp of the genomic locus chr1:55,039,548. In some embodiments, the gRNA targets a target site within the region of -10 to +10 of the genomic locus chr1:55,039,548. In some embodiments, any of such target sites includes or spans the genomic locus chr1:55,039,548 which is the PCSK9 transcription start site (TSS). In some embodiments, the gRNA targets a target site that is within or overlaps coordinates chr1:55,039,538-55,039,557. In some embodiments, the target site is or includes coordinates chr1:55,039,538-55,039,557. In some embodiments, an epigenetic modification DNA targeting system provided for epigenetic modification of PCSK9, or a multiplexed epigenetic modification DNA targeting system for epigenetic modification of at least two genes including PCSK9, includes any of the foregoing gRNAs complexed with a Cas protein such as Cas9 protein. In some embodiments, the Cas9 is dCas9.In some embodiments, dCas9 is dSpCas9 such as the dSpCas9 shown in SEQ ID NO: 207, or a variant and / or fusion thereof.

[0155] In some embodiments, the gRNA targets a target site in a DNA regulatory element comprising a sequence selected from PCSK9, or any one of SEQ ID NOs: 1-13 or 306-317, an adjacent portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOs: 64-76 or 318-329, an adjacent portion thereof of at least 14 nt (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence shown in SEQ ID NO: 191, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 191. In some embodiments, the gRNA comprising the spacer sequence and the scaffold sequence is selected from any one of SEQ ID NOs: 127-139 or 330-341, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a portion thereof. In some embodiments, the gRNA targeting PCSK9 or its DNA regulatory element is shown in a sequence selected from any one of SEQ ID NOs: 127-139 or 330-341.In some embodiments, the epigenetic modification DNA targeting system provided for epigenetic modification of PCSK9, or the multiplexed epigenetic modification DNA targeting system for epigenetic modification of at least two genes including PCSK9, comprises any of the aforementioned gRNAs complexed with a Cas protein such as Cas9 protein. In some embodiments, the Cas9 is dCas9. In some embodiments, the dCas9 is dSpCas9 such as dSpCas9 shown in SEQ ID NO: 207, or a variant and / or fusion thereof.

[0156] In certain embodiments, the gRNA targets a target site in the PCSK9 promoter. In some embodiments, the target site is or comprises the sequence shown in SEQ ID NO: 3. In some embodiments, the gRNA provided herein comprises the spacer sequence shown in SEQ ID NO: 66. In some embodiments, the gRNA further comprises the scaffold sequence shown in SEQ ID NO: 191. In some embodiments, the gRNA comprises the sequence shown in SEQ ID NO: 129, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 129. In some embodiments, the gRNA is shown in SEQ ID NO: 129. In some embodiments, the gRNA is a gRNA designated as PCSK9-C. In some embodiments, the gRNA or its DNA regulatory element targeting PCSK9 is shown in a sequence selected from any one of SEQ ID NOS: 127-139 or 330-341. In some embodiments, the epigenetic modification DNA targeting system provided for epigenetic modification of PCSK9, or the multiplexed epigenetic modification DNA targeting system for epigenetic modification of at least two genes including PCSK9, comprises any one of the aforementioned gRNAs complexed with a Cas protein such as Cas9 protein. In some embodiments, the Cas9 is dCas9. In some embodiments, the dCas9 is dSpCas9 such as dSpCas9 shown in SEQ ID NO: 207, or a variant and / or fusion thereof.

[0157] In some embodiments, the gRNAs provided herein target LPA or its DNA regulatory elements. In some embodiments, the gRNA targets a target site located within 500 bp from the hg38 genomic coordinate chr6:160,664,275. In some embodiments, the gRNA targets LPA, or a sequence selected from any one of SEQ ID NOs: 14-23, an adjacent portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing in its DNA regulatory element containing the target site. In some embodiments, the gRNA contains a spacer sequence selected from any one of SEQ ID NOs: 77-86, an adjacent portion thereof of at least 14 nt (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA further contains a scaffold sequence. In some embodiments, the scaffold sequence contains the sequence shown in SEQ ID NO: 191, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 191. In some embodiments, the gRNA containing the spacer sequence and the scaffold sequence contains a sequence selected from any one of SEQ ID NOs: 140-149, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a portion thereof. In some embodiments, the gRNA targeting LPA or its DNA regulatory element is shown in a sequence selected from any one of 140-149.In some embodiments, the epigenetic modification DNA targeting system provided for epigenetic modification of LPA, or the multiplexed epigenetic modification DNA targeting system for epigenetic modification of at least two genes including LPA, comprises any of the aforementioned gRNAs complexed with a Cas protein such as Cas9 protein. In some embodiments, Cas9 is dCas9. In some embodiments, dCas9 is dSpCas9 such as dSpCas9 shown in SEQ ID NO: 207, or a variant and / or fusion thereof.

[0158] In some embodiments, the gRNAs provided herein target MYLIP or its DNA regulatory elements. In some embodiments, the gRNA targets a target site located within 500 bp from the hg38 genomic coordinate chr6:16,129,086. In some embodiments, the gRNA targets MYLIP, or a sequence selected from any one of SEQ ID NOs: 24-33 or 342-351, an adjacent portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing in a DNA regulatory element containing the target site. In some embodiments, the gRNA contains a spacer sequence selected from any one of SEQ ID NOs: 87-96 or 352-361, an adjacent portion thereof of at least 14 nt (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA further contains a scaffold sequence. In some embodiments, the scaffold sequence contains the sequence shown in SEQ ID NO: 191, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 191. In some embodiments, the gRNA containing the spacer sequence and the scaffold sequence is a sequence selected from any one of SEQ ID NOs: 150-159 or 362-371, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or part of any of the foregoing.In some embodiments, the gRNA targeting MYLIP or its DNA regulatory element is shown in a sequence selected from any one of SEQ ID NOs: 150 to 159 or 362 to 371. In some embodiments, the epigenetic modification DNA targeting system provided for epigenetic modification of MYLIP, or the multiplexed epigenetic modification DNA targeting system for epigenetic modification of at least two genes including MYLIP, comprises any one of the aforementioned gRNAs complexed with a Cas protein such as Cas9 protein. In some embodiments, the Cas9 is dCas9. In some embodiments, the dCas9 is dSpCas9 such as the dSpCas9 shown in SEQ ID NO: 207, or a variant and / or fusion thereof.

[0159] In some embodiments, the gRNAs provided herein target ANGPTL3 or its DNA regulatory elements. In some embodiments, the gRNA targets a target site located within 500 bp from the hg38 genomic coordinate chr1:62,597,520. In some embodiments, the gRNA targets ANGPTL3, or a sequence selected from any one of SEQ ID NOs: 34-43, an adjacent portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing in the DNA regulatory element containing the target site. In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOs: 97-106, an adjacent portion thereof of at least 14 nt (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence shown in SEQ ID NO: 191, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 191. In some embodiments, the gRNA comprising the spacer sequence and the scaffold sequence is a sequence selected from any one of SEQ ID NOs: 160-169, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a portion thereof.In some embodiments, the gRNA targeting ANGPTL3 or its DNA regulatory element is shown in a sequence selected from any one of SEQ ID NOs: 160 to 169. In some embodiments, the epigenetic modification DNA targeting system provided for the epigenetic modification of ANGPTL3, or the multiplexed epigenetic modification DNA targeting system for the epigenetic modification of at least two genes including ANGPTL3, comprises any one of the aforementioned gRNAs complexed with a Cas protein such as Cas9 protein. In some embodiments, the Cas9 is dCas9. In some embodiments, the dCas9 is dSpCas9 such as dSpCas9 shown in SEQ ID NO: 207, or a variant and / or fusion thereof.

[0160] In some embodiments, the gRNAs provided herein target APOC3 or its DNA regulatory elements. In some embodiments, the gRNA targets a target site located within 500 bp from the hg38 genomic coordinate chr11:116,829,907. In some embodiments, the gRNA targets APOC3, or a sequence selected from any one of SEQ ID NOs: 44-53, an adjacent portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing in its DNA regulatory element containing the target site. In some embodiments, the gRNA contains a spacer sequence selected from any one of SEQ ID NOs: 107-116, an adjacent portion thereof of at least 14 nt (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA further contains a scaffold sequence. In some embodiments, the scaffold sequence contains the sequence shown in SEQ ID NO: 191, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 191. In some embodiments, the gRNA containing the spacer sequence and the scaffold sequence contains a sequence selected from any one of SEQ ID NOs: 170-179, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or part of any of the foregoing.In some embodiments, the gRNA targeting APOC3 or its DNA regulatory element is shown in a sequence selected from any one of SEQ ID NOs: 170 to 179. In some embodiments, the epigenetic modification DNA targeting system provided for epigenetic modification of APOC3, or the multiplexed epigenetic modification DNA targeting system for epigenetic modification of at least two genes including APOC3, comprises any one of the aforementioned gRNAs complexed with a Cas protein such as Cas9 protein. In some embodiments, Cas9 is dCas9. In some embodiments, dCas9 is dSpCas9 such as dSpCas9 shown in SEQ ID NO: 207, or a variant and / or fusion thereof.

[0161] In some embodiments, the gRNA provided herein targets APOB or its DNA regulatory element. In some embodiments, the gRNA targets a target site located within 500 bp from the hg38 genomic coordinate chr2:21,044,073. In some embodiments, the gRNA targets APOB, or a sequence selected from any one of SEQ ID NOs: 54-63 or 372-377, an adjacent portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), a complementary sequence of any of the foregoing, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing in its DNA regulatory element. In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOs: 117-126 or 378-383, an adjacent portion thereof of at least 14 nt (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence shown in SEQ ID NO: 191, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 191. In some embodiments, the gRNA comprising the spacer sequence and the scaffold sequence is selected from any one of SEQ ID NOs: 180-189 or 384-389, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a portion thereof.In some embodiments, the gRNA targeting APOB or its DNA regulatory element is shown in a sequence selected from any one of SEQ ID NOs: 180-189 or 384-389. In some embodiments, the epigenetic modification DNA targeting system provided for epigenetic modification of APOB, or the multiplexed epigenetic modification DNA targeting system for epigenetic modification of at least two genes including APOB, comprises any one of the aforementioned gRNAs complexed with a Cas protein such as Cas9 protein. In some embodiments, Cas9 is dCas9. In some embodiments, dCas9 is dSpCas9 such as dSpCas9 shown in SEQ ID NO: 207, or a variant and / or fusion thereof.

[0162] In some embodiments, provided herein is a combination of gRNAs. In some embodiments, provided herein is a multiplexed epigenetic modification DNA targeting system comprising a combination of gRNAs.

[0163] In some embodiments, the combination of gRNAs comprises at least two gRNAs targeting at least two different genes. In some embodiments, the combination of gRNAs comprises a first gRNA targeting a first gene and a second gRNA targeting a second gene. In some embodiments, the first gRNA targets a gene selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, and the second gRNA targets a gene selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, and the first and second gRNAs target different genes. In some embodiments, the first gRNA targets PCSK9 and the second gRNA targets a gene selected from the list consisting of LPA, MYLIP, ANGPTL3, APOC3, and APOB. In some embodiments, the first gRNA targets PCSK9 and the second gRNA targets LPA. In some embodiments, the first gRNA and the second gRNA target a combination of two genes selected from the combinations of genes listed in Table 1. In some embodiments, the first gRNA and the second gRNA are each independently selected from any of the gRNAs described herein.

[0164] In some embodiments, the combination of gRNAs comprises at least three gRNAs targeting at least three different genes. In some embodiments, the combination of gRNAs comprises a first gRNA targeting a first gene, a second gRNA targeting a second gene, and a third gRNA targeting a third gene. In some embodiments, the first gRNA targets a gene selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, the second gRNA targets a gene selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, the third gRNA targets a gene selected from the list consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB, and the first, second, and third gRNAs each target a different gene. In some embodiments, the first gRNA targets PCSK9, the second gRNA targets a gene selected from the list consisting of LPA, MYLIP, ANGPTL3, APOC3, and APOB, the third gRNA targets a gene selected from the list consisting of LPA, MYLIP, ANGPTL3, APOC3, and APOB, and the second and third gRNAs target different genes. In some embodiments, the first gRNA targets PCSK9, the second gRNA targets LPA, and the third gRNA targets a gene selected from the list consisting of MYLIP, ANGPTL3, APOC3, and APOB. In some embodiments, the first gRNA, the second gRNA, and the third gRNA target a combination of three genes selected from the combinations of genes listed in Table 2. In some embodiments, the first gRNA, the second gRNA, and the third gRNA are each independently selected from any of the gRNAs described herein.

[0165] C. Other DNA Binding Domains In some of the provided embodiments, the DNA binding domain includes a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or an I-SceI enzyme or variant thereof. In some embodiments, the DNA binding domain includes a catalytically inactive variant of any of the foregoing.

[0166] In some embodiments, a ZFP, zinc finger DNA binding protein, or zinc finger DNA binding domain is a protein that binds DNA sequence-specifically via one or more zinc fingers, or a domain within a larger protein, where one or more zinc fingers are regions of amino acid sequence within a binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among ZFPs, there are artificial or engineered ZFPs that contain a ZFP domain that targets a specific DNA sequence, typically 9 to 18 nucleotides in length, generated by the assembly of individual fingers. A ZFP contains an alpha helix with a single finger domain approximately 30 amino acids in length and containing two invariant histidine residues coordinated via two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, the sequence specificity of a ZFP may be altered by making amino acid substitutions at four helix positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, for example, a ZFP or ZFP-containing molecule may be engineered to bind to a non-naturally occurring, e.g., selected target site.

[0167] In some embodiments, the zinc fingers are custom-designed (i.e., designed by the user) or obtained from a commercial source. Various methods for designing zinc finger proteins are available. For example, methods for designing zinc finger proteins to bind to a target DNA sequence of interest are described, for example, in Liu, Q. et al., PNAS, 94(11):5525-30 (1997), Wright, D. A. et al., Nat. Protoc., 1(3):1637-52 (2006), Gersbach, C. A. et al., Acc. Chem. Res., 47(8):2309-18 (2014), Bhakta M. S. et al., Methods Mol. Biol., 649:3-30 (2010), and Gaj et al., Trends Biotechnol, 31(7):397-405 (2013). In addition, various web-based tools for designing zinc finger proteins to bind to a target DNA sequence of interest are publicly available. See, for example, the Scripps Zinc Finger Tools design website available on the World Wide Web at scripps.edu / barbas / zfdesign / zfdesignhome.php. Various commercial services for designing zinc finger proteins to bind to a target DNA sequence of interest are also available.See, for example, commercially available services or kits provided by Creative Biolabs (world wide web at creative-biolabs.com / Design-and-Synthesis-of-Artificial-Zinc-Finger-Proteins.html), the Zinc Finger Consortium modular assembly kit available from Addgene (world wide web at addgene.org / kits / zfc-modular-assembly / ), or the CompoZr Custom ZFN Service from Sigma Aldrich (world wide web at sigmaaldrich.com / life-science / zinc-finger-nuclease-technology / custom-zfn.html).

[0168] Transcription activator-like effector (TALE) is a protein naturally found in Xanthomonas bacteria. TALE contains multiple repetitive amino acid sequences, and each repeat has binding specificity for one base within the target sequence. Each repeat contains a pair of variable residues at positions 12 and 13 (repeat variable diresidue; RVD) that determine the nucleotide specificity of the repeat. In some embodiments, the RVDs associated with the recognition of different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G, or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A, YG for recognizing T, TL for recognizing A, VT for recognizing A or G, and SW for recognizing A. In some embodiments, the RVDs can be mutated towards other amino acid residues to modulate, particularly to enhance, their specificity for nucleotides A, T, C, and G. Binding domains with similar modular base-by-base nucleic acid binding properties can also be derived from different bacterial species. These alternative modular proteins may exhibit more sequence variability than the TALE repeats.

[0169] In some embodiments, a "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains / units. Each repeat domain, which contains a repeat variable diresidue (RVD), is involved in the binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33 - 35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. TALE proteins may be designed to bind to a target site using canonical or non-canonical RVDs within the repeat unit. See, for example, U.S. Patent Nos. 8,586,526 and 9,458,205.

[0170] In some embodiments, the TALE is a fusion protein comprising a nucleic acid binding domain and an effector domain derived from a TALE.

[0171] Zinc finger and TALE DNA binding domains can be engineered to bind to a given nucleotide sequence, for example, by engineering the recognition helix region of a naturally occurring zinc finger protein (modifying one or more amino acids), by engineering the amino acids in the TALE repeats involved in DNA binding (the repeat variable diresidue or RVD region), or by the systematic ordering of modular DNA binding domains such as TALE repeats or ZFP domains. Thus, engineered zinc finger proteins or TALE proteins are non-natural proteins. Non-limiting examples of methods for engineering zinc finger proteins and TALEs are design and selection. Engineered proteins are proteins that do not exist in nature, whose design / composition results primarily from rational criteria. Rational criteria for design include the application of substitution rules and computerized algorithms for processing information in databases storing information on existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U.S. Pat. Nos. 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261. See also WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536; and WO03 / 016496.

[0172] D. Effector Domain In some embodiments, the DNA targeting systems provided herein further include one or more effector domains. In some embodiments, provided herein is a DNA targeting system comprising: (a) a DNA binding domain capable of targeting a target site in a gene such as any of those described above or its regulatory DNA element, and (b) a fusion protein comprising at least one effector domain. In some embodiments, the effector domain is capable of reducing gene transcription or a combination of genes. In some embodiments, the effector domain comprises a transcriptional repressor domain.

[0173] In some embodiments, the effector domain induces, catalyzes, or effects reduced and / or repressed transcription of a gene when ectopically recruited to a gene or its DNA regulatory element.

[0174] In some embodiments, the effector domain induces, catalyzes, or effects transcriptional repression, transcriptional corepression, histone modification, histone acetylation, histone deacetylation, nucleosome remodeling, chromatin remodeling, heterochromatin formation, proteolysis, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, splicing, DNA methylation, DNA demethylation, histone methylation, histone demethylation, or DNA base oxidation. In some embodiments, the effector domain induces, catalyzes, or effects transcriptional repression or transcriptional corepression. In some embodiments, the effector domain induces transcriptional repression. In some embodiments, the effector domain has (i.e., directly acts on) one of the aforementioned activities itself. In some embodiments, the effector domain recruits and / or interacts with a protein or polypeptide domain having (i.e., indirectly acts on) one of the aforementioned activities.

[0175] The gene expression of endogenous mammalian genes such as human genes can be achieved by targeting a fusion protein containing a DNA binding domain such as dCas9 and an effector domain such as a transcriptional repression domain to a mammalian gene or its regulatory DNA element (e.g., promoter or enhancer) via one or more gRNAs. Any of various effector domains (e.g., transcriptional repression domain) for transcriptional repression is known and can be used according to the provided embodiments. The transcriptional repression domain, and the transcriptional repression of the target gene using a Cas fusion protein having the transcriptional repression domain are described, for example, in WO2014 / 197748, WO2017 / 180915, WO2021 / 226077, WO2013 / 176772, WO2014 / 152432, WO2014 / 093661, Adli, M. Nat. Commun. 9, 1911 (2018), and Gilbert, L. A. et al. Cell 154(2):442 - 451(2013).

[0176] In some embodiments, the effector domain can include a KRAB domain, an ERF repressor domain, an MXI1 domain, an SID4X domain, a MAD - SID domain, a DNMT family protein domain (e.g., DNMT3A or DNMT3B), a fusion of one or more DNMT family proteins or their domains (e.g., DNMT3A / L including a fusion of DNMT3A and DNMT3L domains), LSD1, EZH2, a SunTag domain, a partially or fully functional fragment or domain of any of the foregoing, or any combination of the foregoing. For example, the fusion protein may be dCas9 - KRAB, or dCas9 - KRAB - DNMT3A / L. In some embodiments, the fusion protein may be dCas9 - KRAB. In some embodiments, the fusion protein may be DNMT3A / L - dCas9 - KRAB. In some embodiments, the fusion protein may be KRAB - dCas9 - DNMT3A / L.

[0177] In some embodiments, the effector domain comprises a transcriptional repressor domain as described in WO2021 / 226077.

[0178] In some embodiments, the effector domain comprises a KRAB domain, or a variant thereof. KRAB-containing zinc finger proteins constitute the largest family of transcriptional repressors in mammals. The Krueppel-associated box (KRAB) domain is a transcriptional repressor domain present in many zinc finger protein-based transcription factors. The KRAB domain contains charged amino acids and can be divided into subdomains A and B. The KRAB domain recruits epigenetic readers such as the co-repressor KAP1 (KRAB-associated protein-1), heterochromatin protein 1 (HP1), and other chromatin regulators that induce transcriptional repression through heterochromatin formation. KRAB-mediated gene repression is associated with the loss of histone H3-acetylation and the increase in H3 lysine 9 trimethylation (H3K9me3) at the repressed gene promoter. The KRAB domain, including in dCas fusion proteins, is described, for example, in WO2017 / 180915, WO2014 / 197748, US2019 / 0127713, WO2013 / 176772, Urrutia R. et al. Genome Biol. 4, 231 (2003), Groner A.C. et al. PLoS Genet. 6, e1000869 (2010). In some embodiments, the effector domain comprises at least one KRAB domain or a variant thereof. In some embodiments, an exemplary KRAB domain is shown in SEQ ID NO: 193. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 193, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 193. In some embodiments, an exemplary KRAB domain is shown in SEQ ID NO: 290. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 290, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 290.

[0179] In some embodiments, the effector domain comprises at least one ERF repressor domain, or a variant thereof. ERF (ETS2 repressor factor) contains a conserved ets-DNA binding domain and is a potent transcriptional repressor that suppresses transcription via a separate domain at the carboxyl terminus of the protein. ERF repressor domains, including dCas fusion proteins, are described, for example, in WO2017180915, WO2014197748, WO2013176772, Mavrothalassitis, G., Ghysdael, J. Proteins of the ETS family with transcriptional repressor activity. Oncogene 19, 6524-6532 (2000). In some embodiments, the effector domain comprises at least one ERF repressor domain or a variant thereof. An exemplary ERF repressor domain is shown in SEQ ID NO: 220. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 220, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0180] In some embodiments, the effector domain comprises at least one MXI1 domain or a variant thereof. The MXI1 domain functions by antagonizing myc transcriptional activity by competing for binding to myc-related factor x (MAX). The MXI1 domain comprising the dCas fusion protein is described, for example, in WO2017180915, WO2014197748, US20190127713. In some embodiments, the effector domain comprises at least one MXI1 domain or a variant thereof. An exemplary MXI1 domain is shown in SEQ ID NO: 221. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 221, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0181] In some embodiments, the effector domain comprises at least one SID4X domain, or a variant thereof. The mSin3 interaction domain (SID) is present on different transcriptional repressor proteins. This interacts with the paired amphipathic alpha-helix 2 (PAH2) domain of mSin3, which is a transcriptional repressor domain that binds to transcriptional repressor proteins such as the mSin3A corepressor. The dCas9 molecule can be fused to four linked mSin3 interaction domains (SID4X). The SID domain comprising the dCas fusion protein is described, for example, in WO2017180915, WO2014197748, WO2014093655. In some embodiments, the effector domain comprises at least one SID domain or a variant thereof. An exemplary SID domain is shown in SEQ ID NO: 222. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 222, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0182] In some embodiments, the effector domain comprises at least one MAD domain or a variant thereof. Mad1, Mxi1, Mad3, and Mad4, which are MAD family proteins, belong to the basic helix-loop-helix-zip class and contain a conserved N-terminal region (referred to as the Sin3 interaction domain (SID)) required for repression activity. MAD-SID domains, including dCas fusion proteins, are described, for example, in WO2017180915, WO2014197748, WO2013176772. In some embodiments, the effector domain comprises at least one MAD-SID domain or a variant thereof. An exemplary MAD-SID domain is shown in SEQ ID NO: 223. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 223, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0183] In some embodiments, the effector domain comprises at least one DNMT3 domain, or a variant thereof. In some embodiments, the at least one DNMT3 domain or a variant thereof is derived from DNMT3 or a portion thereof having DNA methyltransferase activity or a functionally active variant. DNMT3A and DNMT3B are two DNA methyltransferases that catalyze de novo methylation and may be associated with transcriptional repression depending on the site. DNMTs such as DNMT3 mediate the transfer of a methyl group from the universal methyl donor S-adenosyl-L-methionine (SAM) to the 5-position of cytosine residues. In some aspects, these DNMT3 DNA methyltransferases direct de novo methylation of cytosine bases to 5-methylcytosine. DNMT3 comprising the dCas fusion protein is described, for example, in US20190127713, Liu, X.S. et al. Cell 167, 233-247.e17 (2016), Lei, Y. et al. Nat. Commun. 8, 16026 (2017). DNMT3 proteins such as DNMT3A and DNMT3B include an N-terminal portion that is naturally involved in regulatory activity and targeting, and a C-terminal catalytic domain called the MTase C5-type domain. In some embodiments, the effector domain in the embodiments provided herein comprises the catalytically active portion of DNMT3A or DNMT3B containing the catalytically active C-terminal domain. In particular, the isolated catalytic domains of DNMT3a and DNMT3b are catalytically active (see, e.g., Gowher and Jeltsch (2002) J. Biol. Chem., 277:20409).

[0184] In some embodiments, the effector domain comprises at least one DNMT3 domain or a variant thereof. In some embodiments, the DNMT3 domain may be the effector domain of DNMT3A or DNMT3B that is catalytically active. In some embodiments, the effector domain may be full-length DNMT3A or DNMT3B, or the catalytically active portion thereof. In some embodiments, the effector domain is a catalytically active portion that is less than the full-length sequence of DNMT3A or DNMT3B. In some embodiments, the catalytically active portion is, for example, an adjacent sequence of amino acids that confers DNA methyltransferase activity by mediating the methylation of cytosine bases to 5-methylcytosine. In some embodiments, the adjacent sequence of amino acids is the adjacent C-terminal portion of a DNMT3 protein such as DNMT3A or DNMT3B that is 280 to 330 amino acids in length. In some embodiments, the adjacent portion is 280 amino acids, 290 amino acids, 300 amino acids, 310 amino acids, 320 amino acids, or 330 amino acids in length, or any value in between the foregoing. In some embodiments, the catalytically active portion of a DNMT such as DNMT3 comprises a SAM-dependent MTase C5-type domain. In some embodiments, the DNMT3 domain such as the domain of DNMT3A or DNMT3B is of human origin.

[0185] Exemplary DNMT3A domains are shown in SEQ ID NO: 195 or 285. Exemplary DNMT3B domains are shown in SEQ ID NO: 224. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 195, SEQ ID NO: 285, or SEQ ID NO: 224, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0186] In some embodiments, the DNMT3A domain is an amino acid sequence that is shown in SEQ ID NO: 195, or is its catalytically active portion, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 195 or its catalytically active portion that exhibits DNA methyltransferase activity. In some embodiments, the DNMT3A domain is shown in SEQ ID NO: 195. In some embodiments, the DNMT3A domain is encoded by the nucleotide sequence shown in SEQ ID NO: 194.

[0187] In some embodiments, the DNMT3A domain is an amino acid sequence that is shown in SEQ ID NO: 285, or is its catalytically active portion, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 285 or its catalytically active portion that exhibits DNA methyltransferase activity. In some embodiments, the DNMT3A domain is shown in SEQ ID NO: 285.

[0188] In some embodiments, the effector domain is derived from DNMT3B, or its catalytically active portion or variant that exhibits DNA methyltransferase activity. Exemplary DNMT3B domains are shown in SEQ ID NO: 224, or its catalytically active portion, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 224 or its catalytically active portion that exhibits DNA methyltransferase activity. In some embodiments, the catalytically active portion is an adjacent portion of the amino acids of SEQ ID NO: 224 that includes the SAM-dependent MTase C5-type domain (e.g., corresponding to amino acids 575-853 of SEQ ID NO: 224). In some embodiments, the adjacent sequence of the amino acids of SEQ ID NO: 224 includes at least 250 amino acids, 275 amino acids, 300 amino acids or 325 amino acids, or any value between any of the foregoing. In some embodiments, the adjacent sequence of amino acids is an adjacent portion of SEQ ID NO: 224 that includes amino acids 575-853 and has a length of 280 amino acids to 330 amino acids. In some embodiments, the adjacent portion is 280 amino acids, 290 amino acids, 300 amino acids, 310 amino acids, 320 amino acids, or 330 amino acids in length, or any value in length between any of the foregoing.

[0189] Any of a variety of assays is known to evaluate or monitor methyltransferase (MTase) activity. In some embodiments, exemplary assays for evaluating DNA methyltransferase activity include, but are not limited to, radioactive DNA MTase assay, colorimetric DNA MTase activity assay, fluorescent DNA MTase activity assay, chemiluminescent / bioluminescent DNA MTase activity assay, electrochemical DNA MTase activity assay, and electrochemiluminescence (ECL) DNA MTase activity assay. Exemplary assays are described in Poh et al. Theranostics, 2016, 6:369-391, Li et al., Methods Appl. Fluoresc., 2017, 5:012002, Deng et al., Anal Chem., 2014, 86:2117-23, and Ma et al. J Mater Chem B., 2020, 8:3488-3501.

[0190] In some embodiments, the effector domain comprises at least one DNMT3L domain or a variant thereof. The DNMT3L domain or a variant thereof can be DNMT3L or a portion of DNMT3L, or a variant of DNMT3L or a portion thereof. DNMT3L (DNA (cytosine-5)-methyltransferase 3-like) is a catalytically inactive regulator of DNA methyltransferases that can promote or inhibit DNA methylation depending on the setting. DNMT3L is essential for the function of DNMT3A and DNMT3B, and DNMT3L interacts with DNMT3A and DNMT3B and significantly enhances their catalytic activities. For example, DNMT3L interacts with the catalytic domain of DNMT3A to form a heterodimer, demonstrating that DNMT3L has a dual function of binding to the unmethylated histone tail and activating DNA methyltransferase. In some embodiments, the reference to a portion or variant of DNMT3L for the purposes herein refers to a sufficient C-terminal sequence portion of DNMT3L that can interact with the catalytic domain of DNMT3A or DNMT3B and stimulate or promote the DNA methyltransferase activity of DNMT3A or DNMT3B (see, for example, Jia et al. Nature, 2007, 449:248-251, Gowher et al. J. Biol. Chem., 2005, 280:13341-13348). In some embodiments, DNMT3L or a portion thereof is of animal origin. In some embodiments, the domain derived from DNMT3L is of mouse origin. In some embodiments, the domain derived from DNMT3L is of human origin.

[0191] In some embodiments, the DNMT3L domain is DNMT3L, or its C-terminal portion or variant, that interacts with the catalytic domain of DNMT3A to form a heterodimer and provides a more active DNA methyltransferase. In some embodiments, the effector domain is a fusion domain of the DNMT3A domain and the DNMT3L domain (DNMT3A / 3L).

[0192] In some embodiments, the DNMT3L domain is DNMT3L, or a C-terminal portion or variant thereof, that interacts with the catalytic domain of DNMT3B to form a heterodimer and provides a more active DNA methyltransferase. In some embodiments, the effector domain is a fusion domain of the DNMT3B domain and the DNMT3L domain (DNMT3B / 3L).

[0193] In some embodiments, the DNMT3L domain is the C-terminal portion of DNMT3L that is composed of the adjacent C-terminal portion of full-length DNMT3L that does not include the N-terminal cysteine-rich ATRX-Dnmt3-Dnmt3L (ADD) domain (e.g., corresponding to residues 41-73 of SEQ ID NO: 197 or 75-207 of the sequence shown in SEQ ID NO: 289). In some embodiments, the DNMT3L domain is the adjacent C-terminal portion of DNMT3L that is less than 220 amino acids in length, e.g., 100-215 amino acids, e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 215 amino acids in length, or a length between any of the foregoing values. In some embodiments, the DNMT3L domain is the adjacent C-terminal portion of DNMT3L that is 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 or 215 amino acids in length.

[0194] Exemplary DNMT3L domains are those shown in SEQ ID NO: 289 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 289 or a portion thereof. In some embodiments, the DNMT3L domain is the adjacent C-terminal portion of the full-length DNMT3L shown in SEQ ID NO: 289 that does not include the N-terminal cysteine-rich ATRX-Dnmt3-Dnmt3L (ADD) domain (corresponding to residues 75-207 of the sequence shown in SEQ ID NO: 289). In some embodiments, the DNMT3L domain is the adjacent C-terminal portion of the full-length DNMT3L shown in SEQ ID NO: 289 that is less than 220 amino acids in length, e.g., 100-215 amino acids, e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 215 amino acids in length, or a length between any of the foregoing values. In some embodiments, the DNMT3L domain is the adjacent C-terminal portion of the full-length DNMT3L shown in SEQ ID NO: 289 and is 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, or 215 amino acids in length.

[0195] In some embodiments, the DNMT3L domain is those shown in SEQ ID NO: 286 or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 286. In some embodiments, the DNMT3L domain is as shown in SEQ ID NO: 286. In some embodiments, the DNMT3L domain does not contain an N-terminal methionine as shown in SEQ ID NO: 286.

[0196] In some embodiments, the DNMT3L domain is human or humanized DNMT3L. The corresponding sequence of human DNMT3L is highly homologous to mouse-derived DNMT3L and has at least 90% sequence identity with the mouse sequence. Humanizing non-human sequences of DNMT3L domains, such as the domain of mouse DNMT3L, is within the level of those skilled in the art. In some embodiments, the effector domain comprises a DNMT3L domain that is a humanized variant of mouse DMT3L shown in SEQ ID NO: 289, or a portion thereof that can interact with DNMT3A or DNMT3A. In some embodiments, the effector domain comprises a DNMT3L domain that is a humanized variant of the mouse C-terminal portion of DNMT3L shown in SEQ ID NO: 286.

[0197] Exemplary DNMT3L domains of human origin are shown in SEQ ID NO: 197, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 197, or a portion thereof. In some embodiments, the DNMT3L domain is the adjacent C-terminal portion of the full-length DNMT3L shown in SEQ ID NO: 197 that does not include the N-terminal cysteine-rich ATRX-Dnmt3-Dnmt3L (ADD) domain (corresponding to residues 41-73 of the sequence shown in SEQ ID NO: 197). In some embodiments, the DNMT3L domain is the adjacent C-terminal portion of the full-length DNMT3L shown in SEQ ID NO: 197 that is less than 220 amino acids in length, e.g., 100-215 amino acids, e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 215 amino acids in length, or a length between any of the foregoing values. In some embodiments, the DNMT3L domain is the adjacent C-terminal portion of the full-length DNMT3L shown in SEQ ID NO: 197 and is 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, or 215 amino acids in length.

[0198] Exemplary DNMT3L domains are shown in SEQ ID NO: 197. In some embodiments, the DNMT3L domain comprises the sequence shown in SEQ ID NO: 197, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 197. In some embodiments, the DNMT3L domain is encoded by the nucleotide sequence shown in SEQ ID NO: 196.

[0199] In some embodiments, the DNMT3L domain comprises the sequence shown in SEQ ID NO: 287, or a portion thereof, or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 287. In some embodiments, the DNMT3L domain is shown in SEQ ID NO: 287. In some embodiments, the DNMT3L domain contains an N-terminal methionine.

[0200] In some embodiments, the effector domain comprises a fusion of DNMT3A and DNMT3L (DNMT3A / L). The fusion protein contains the DNMT3A and DNMT3L domains, which can be any of those as described above. In some embodiments, the fusion protein comprises the DNMT3A domain shown in SEQ ID NO: 195 and the DNMT3L domain shown in SEQ ID NO: 289, arranged in any order. In some embodiments, the fusion protein comprises the DNMT3A domain shown in SEQ ID NO: 195 and the DNMT3L domain shown in SEQ ID NO: 286, arranged in any order. In some embodiments, the fusion protein comprises the DNMT3A domain shown in SEQ ID NO: 195 and the DNMT3L domain shown in SEQ ID NO: 287, arranged in any order. In some embodiments, the fusion protein comprises the DNMT3A domain shown in SEQ ID NO: 285 and the DNMT3L domain shown in SEQ ID NO: 289, arranged in any order. In some embodiments, the fusion protein comprises the DNMT3A domain shown in SEQ ID NO: 285 and the DNMT3L domain shown in SEQ ID NO: 286, arranged in any order. In some embodiments, the fusion protein comprises the DNMT3A domain shown in SEQ ID NO: 285 and the DNMT3L domain shown in SEQ ID NO: 287, arranged in any order. In some embodiments, the DNMT3A and DNMT3L domains present in the provided fusion protein are separated from each other within the fusion protein by intervening sequences such as a DNA binding domain, another effector domain or a linker. In some embodiments, the domains are directly linked to each other or are linked via a linker such as a peptide linker. In some embodiments, the DNMT3A domain and the DNMT3L domain are connected as a fusion domain via a linker that connects the DNMT3A domain and the DNMT3L domain. Exemplary linkers are described herein. In some embodiments, the linker is the linker shown in SEQ ID NO: 288.

[0201] An exemplary DNMT3A / L fusion domain is shown in SEQ ID NO: 199. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 199, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 199. In some embodiments, the DNMT3A / L fusion domain is encoded by the nucleotide sequence shown in SEQ ID NO: 198.

[0202] An exemplary DNMT3A / L fusion domain is shown in SEQ ID NO: 201. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 201, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 201. In some embodiments, the DNMT3A / L fusion domain is encoded by the nucleotide sequence shown in SEQ ID NO: 200.

[0203] In some embodiments, the effector domain may comprise an LSD1 domain. LSD1 (also known as lysine-specific histone demethylase 1A) is a histone demethylase that can demethylate lysine residues of histone H3 and thereby acts as a coactivator or a corepressor depending on the context. LSD1-containing dCas fusion proteins are described, for example, in WO2013 / 176772, WO2014 / 152432, and Kearns, N. A. et al. Nat. Methods. 12(5):401-403 (2015). An exemplary LSD1 polypeptide is shown in SEQ ID NO: 225. In some embodiments, the effector domain comprises the sequence shown in SEQ ID NO: 225, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0204] In some embodiments, the effector domain may include an EZH2 domain. EZH2 (also known as histone-lysine N-methyltransferase EZH2) is the catalytic subunit of the PRC2 / EED-EZH2 complex, which methylates "Lys-9" (H3K9me) and "Lys-27" (H3K27me) of histone H3 and, in some aspects, leads to transcriptional repression of the diseased target gene. EZH2 (including dCas fusion proteins) is described, for example, in O’Geen, H., et al., Epigenetics Chromatin. 12(1):26 (2019). An exemplary EZH2 polypeptide is shown in SEQ ID NO: 283. In some embodiments, the effector domain includes the sequence shown in SEQ ID NO: 283, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0205] In some embodiments, the effector domain may include a SunTag domain. SunTag is a repetitive peptide array that can recruit multiple copies of an antibody fusion protein that binds to the repetitive peptide. The antibody fusion protein may include additional effector domains, such as a transcriptional repression domain (e.g., KRAB), to reduce transcription of the target gene. SunTag, including the dCas fusion protein for gene regulation, is described, for example, in WO2016 / 011070 and Tanenbaum, M. et al. Cell. 159(3):635 - 646(2014). An exemplary SunTag effector domain includes a repetitive GCN4 peptide having the amino acid sequence LLPKNYHLENEVARLKKLVGER (SEQ ID NO: 226) separated by a linker having the amino acid sequence GGSGG (SEQ ID NO: 227). In some embodiments, the effector domain includes at least one copy of the sequence shown in SEQ ID NO: 226, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some embodiments, the SunTag effector domain recruits an antibody fusion protein containing KRAB and binds to the GCN4 peptide.

[0206] E. Fusion Protein In some embodiments, the DNA targeting systems provided herein include fusion proteins. In some embodiments, provided herein is a DNA targeting system that is a fusion protein comprising (a) a DNA binding domain capable of targeting a target site in a gene or its regulatory DNA element encoding a gene product that regulates LDL, and (b) at least one effector domain. In some embodiments, provided herein is a DNA targeting system comprising at least one DNA targeting module, each of the at least one DNA targeting module comprising a fusion protein comprising (a) a DNA binding domain capable of targeting a target site in a gene or its regulatory DNA element encoding a gene product that regulates LDL, and (b) at least one effector domain. In some embodiments, the fusion protein comprises at least one of any of the DNA binding domains described herein and at least one of any of the effector domains described herein. For example, in some embodiments, the fusion protein comprises a CRISPR-Cas DNA binding domain as described in Section II.B and at least one effector domain described herein. In some embodiments, the effector domain of the provided fusion protein is a transcriptional repressor domain as described in Section I.D. In some embodiments, the fusion protein targets a target site in a gene or its regulatory element and results in reduced or suppressed transcription of the gene. In some embodiments, the fusion protein targets target sites in a combination of genes or their regulatory elements and results in reduced or suppressed transcription of each of the genes.

[0207] In some embodiments, the DNA binding domain and the effector domain of the fusion protein are heterologous, i.e., the domains are from different species or at least one of the domains is not found in nature. In some embodiments, the fusion protein is an engineered fusion protein, i.e., the fusion protein is not found in nature.

[0208] In some embodiments, at least one effector domain is fused to the N-terminus, C-terminus, or both the N-terminus and C-terminus of a DNA binding domain or a component thereof. The at least one effector domain may be fused directly to the DNA binding domain or may be fused via any intervening amino acid sequence such as a linker sequence or a nuclear localization sequence (NLS).

[0209] In some embodiments, the fusion protein of the provided DNA binding system, or its DNA targeting module, contains, in order from the N-terminus to the C-terminus, a transcriptional repressor effector domain and a DNA binding domain. In some embodiments, the fusion protein of the provided DNA binding system, or its DNA targeting module, contains, in order from the N-terminus to the C-terminus, a DNA binding domain and a transcriptional repressor effector domain.

[0210] In some embodiments, at least one effector domain of the fusion protein contains two or more effector domains. In some embodiments, the fusion protein contains two, three, or four effector domains. In some embodiments, at least two of the effector domains of the fusion protein are different. In some embodiments, each of the effector domains of the fusion protein is different. In some embodiments, at least one effector domain contains two effector domains that are different. In some embodiments, the effector domain and the DNA binding domain can be arranged in any order.

[0211] In some embodiments, at least one effector domain of the fusion protein comprises two different effector domains. The two different effector domains and the DNA binding domain can be arranged in any order. In some embodiments, each of the effector domains is N-terminal to the DNA binding domain, the first effector domain is fused to the N-terminus of the second effector domain, and the second effector domain is fused to the N-terminus of the DNA binding domain. In some embodiments, the fusion protein of the provided DNA binding system, or its DNA targeting module, comprises, in order from N-terminus to C-terminus, a first transcriptional repressor effector domain, a second transcriptional repressor effector domain, and a DNA binding domain. In some embodiments, each of the effector domains is C-terminal to the DNA binding domain, the first effector domain is fused to the C-terminus of the DNA binding domain, and the second effector domain is fused to the C-terminus of the first effector domain. In some embodiments, the fusion protein of the provided DNA binding system, or its DNA targeting module, comprises, in order from N-terminus to C-terminus, a DNA binding domain, a first transcriptional repressor effector domain, and a second transcriptional repressor effector domain. In some embodiments, the DNA binding domain is between the effector domains, one effector domain is fused to the N-terminus of the DNA binding domain, and the other effector domain is fused to the C-terminus of the DNA binding domain. In some embodiments, the fusion protein of the provided DNA binding system, or its DNA targeting module, comprises, in order from N-terminus to C-terminus, a first transcriptional effector domain, a DNA binding domain, and a second transcriptional repressor effector domain. In some embodiments, one or more of the components may be fused to each other directly or via any intervening amino acid sequence, e.g., via a linker sequence or a nuclear localization sequence (NLS).

[0212] In some embodiments, the fusion protein comprises one or more linkers. In some embodiments, the linker is a peptide linker. In some embodiments, one or more linkers connect a DNA binding domain or a component thereof to at least one effector domain. The linker can be included anywhere in the polypeptide sequence of the fusion protein, for example, between the effector domain and the DNA binding domain or a component thereof. The linker can be of any length and can be designed to promote or restrict the mobility of the components in the fusion protein. The linker can comprise any amino acid sequence of about 2 to about 100, about 5 to about 80, about 10 to about 60, or about 20 to about 50 amino acids. The linker can comprise an amino acid sequence of at least about 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids. The linker can comprise an amino acid sequence of less than about 100, 90, 80, 70, 60, 50, or 40 amino acids. One skilled in the art can readily select an appropriate linker for connecting two domains. In some embodiments, the linker is a flexible linker. Flexible linkers generally consist of small, non-polar or polar residues such as glycine, serine or threonine. In some embodiments, the linker is Gly4Ser (n) is a linker, and n is an integer from 1 to 10. The linker may comprise a continuous or tandem repeat of an amino acid sequence that is 2 to 20 amino acids in length. The linker may be rich in the amino acids glycine (G), serine (S), and / or alanine (A). The linker may include, for example, a GS linker. An exemplary GS linker is represented by the sequence GGGGS (SEQ ID NO: 228). The linker may include a repeat of a sequence such as represented by the formula (GGGGS)n, wherein n is an integer representing the number of times the GGGGS sequence is repeated (e.g., 1 to 10 times). The number of times the linker sequence is repeated can be adjusted to optimize the linker length and achieve proper separation of the functional domains. Other examples of linkers can include, for example, GGGGG (SEQ ID NO: 229), GGAGG (SEQ ID NO: 230), GGGGSSS (SEQ ID NO: 231), or GGGAAA (SEQ ID NO: 232).

[0213] In some embodiments, an artificial linker sequence can be used. In some embodiments, the linker is EASGSGRASPGIPGSTR (SEQ ID NO: 293). In some embodiments, the linker is GIHGVPAA (SEQ ID NO: 294). In some embodiments, the linker is SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 288). In some embodiments, the linker is KRPAATKKAGQAKKKKASDAKSLTAWS (SEQ ID NO: 298).

[0214] In some embodiments, including a SunTag linker in the fusion protein enhances the suppression of the target gene. In some embodiments, the SunTag linker comprises the sequence shown in SEQ ID NO: 226, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0215] In some embodiments, the linker is an XTEN linker. In some aspects, the XTEN linker is a recombinant polypeptide lacking hydrophobic amino acid residues (e.g., an unstructured recombinant peptide). Exemplary XTEN linkers are described, for example, in Schellenberger et al. Nature Biotechnology 27, 1186-1190 (2009) or WO2021 / 247570. In some embodiments, including a linker in the fusion protein enhances the suppression of the target gene. In some embodiments, the linker comprises the sequence shown in SEQ ID NO: 233, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 233. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 233, or an adjacent portion of SEQ ID NO: 233 of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 amino acids. In some aspects, the linker consists of the sequence shown in SEQ ID NO: 233, or an adjacent portion of SEQ ID NO: 233 of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 amino acids. In some embodiments, the linker comprises the sequence shown in SEQ ID NO: 233. In some embodiments, the linker consists of the sequence shown in SEQ ID NO: 233. In some embodiments, the linker comprises the sequence shown in SEQ ID NO: 299, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some aspects, the linker comprises the sequence shown in SEQ ID NO: 299, or an adjacent portion of SEQ ID NO: 299 of at least 5, 10, or 15 amino acids. In some aspects, the linker consists of the sequence shown in SEQ ID NO: 299, or an adjacent portion of SEQ ID NO: 299 of at least 5, 10, or 15 amino acids. In some embodiments, the linker comprises the sequence shown in SEQ ID NO: 299. In some embodiments, the linker consists of the sequence shown in SEQ ID NO: 299.A suitable linker can be selected or designed based on reasonable criteria known in the art, for example, as described in Chen et al. Adv. Drug Deliv. Rev. 65(10):1357-1369(2013). In some embodiments, the linker comprises a linker described in WO2021 / 247570.

[0216] In some embodiments, the fusion protein of the DNA targeting system, or its DNA targeting module, comprises one or more nuclear localization signals (NLSs). In some embodiments, the fusion proteins described herein comprise one or more nuclear localization sequences (NLSs), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. When multiple NLSs are present, each can be selected independently of the others such that a single NLS can be present in multiple copies and / or can be present in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLSs include the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 234), the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 249)), the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 235) or RQRRNELKRSP (SEQ ID NO: 236), the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 237), the sequence of the IBB domain from importin-alpha, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 238), the sequences of the myoma T protein, VSRKRPRP (SEQ ID NO: 239) and PPKKARED (SEQ ID NO: 240), the sequence of human p53, PQPKKKPL (SEQ ID NO: 241), the sequence of mouse c-abl IV, SALIKKKKKMAP (SEQ ID NO: 242), the sequences of influenza virus NS1, DRLRR (SEQ ID NO: 243) and PKQKKRK (SEQ ID NO: 244), the sequence of hepatitis delta antigen, RKLKKKIKKL (SEQ ID NO: 245), the sequence of mouse Mx1 protein, REKKKFLKRR (SEQ ID NO: 246), the sequence of human poly(ADP-ribose) polymerase, KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 247), and NLS sequences derived from the sequence of the steroid hormone receptor (human) glucocorticoid, RKCLQAGMNLEARKTKK (SEQ ID NO: 248). An NLS can comprise any portion of any of the foregoing.In general, one or more NLSs are of sufficient strength to promote the accumulation of detectable amounts of the fusion protein within the nucleus of a eukaryotic cell. In general, the strength of the nuclear localization activity can be derived from the number of NLSs in the fusion protein, the particular NLS(s) used, or a combination of these factors. Detection of accumulation within the nucleus can be carried out by any suitable technique. For example, a detectable marker may be fused to the fusion protein such that the intracellular location can be visualized in combination with means for detecting the location of the nucleus (e.g., staining specific for the nucleus such as DAPI). The cell nucleus may also be isolated from the cell, and its contents may then be analyzed by any suitable process for detecting proteins such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation within the nucleus may be determined indirectly, for example, by assay of the effect of the fusion protein (e.g., assay of altered gene expression activity in cells transformed with a DNA targeting system containing the fusion protein) compared to control conditions (e.g., untransformed cells).

[0217] In some embodiments, the NLS is linked via a linker to the N-terminus or C-terminus of the DNA binding domain. In some embodiments, the NLS is linked via a linker to the N-terminus or C-terminus of the effector domain. The linker may be any linker as described above. In some embodiments, the linker is GIHGVPAA (SEQ ID NO: 294). In some embodiments, the NLS and the linker have the sequence PKKKRKVGIHGVPAA (SEQ ID NO: 291).

[0218] In some configurations, the N-terminus or C-terminus of the fusion protein may be linked to a moiety for detection and / or purification. In some aspects, this moiety is a His tag such as Flag tag DYKDDDDK (SEQ ID NO: 292), 3×Flag tag MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 295), HA tag YPYDVPDYA (SEQ ID NO: 296), or HHHHHH (SEQ ID NO: 297), or includes them.

[0219] 1. Split fusion protein In some embodiments, the fusion protein is a split protein, i.e., it comprises two or more distinct polypeptide domains that interact or self-organize to form a functional fusion protein. In some aspects, the split fusion protein comprises dCas9 and an effector domain. In some aspects, the fusion protein comprises a split dCas9-effector domain fusion protein.

[0220] In some embodiments, the split fusion protein is assembled from distinct polypeptide domains that include trans-splicing inteins. Inteins are internal protein elements that self-excise from their host proteins and catalyze the ligation of adjacent sequences with peptide bonds. In some embodiments, the split fusion protein is assembled from a first polypeptide that includes an N-terminal intein and a second polypeptide that includes a C-terminal intein. In some embodiments, the N-terminal intein is the N-terminal Npu intein shown in SEQ ID NO: 300. In some embodiments, the C-terminal intein is the C-terminal Npu intein shown in SEQ ID NO: 302. In some embodiments, the N-terminal intein is the N-terminal Npu intein encoded by the nucleotide sequence shown in SEQ ID NO: 305. In some embodiments, the C-terminal intein is the C-terminal Npu intein encoded by the nucleotide sequence shown in SEQ ID NO: 301.

[0221] In some embodiments, the split fusion protein comprises a split dCas9-effector domain fusion protein assembled from two polypeptides. In an exemplary embodiment, the first polypeptide comprises an effector catalytic domain and an N-terminal fragment of dSpCas9, followed by an N-terminal Npu intein (effector domain-dSpCas9-573N), and the second polypeptide comprises a C-terminal Npu intein, followed by a C-terminal fragment of dSpCas9 (dSpCas9-573C). In some embodiments, the C-terminal Npu intein, followed by the C-terminal fragment of dSpCas9 (dSpCas9-573C), is shown in SEQ ID NO: 304. In some embodiments, the C-terminal Npu intein, followed by the C-terminal fragment of dSpCas9 (dSpCas9-573C), is encoded by the nucleotide sequence shown in SEQ ID NO: 303. The N-terminal and C-terminal fragments of the fusion protein are split at position 573Glu of the dSpCas9 molecule, with reference to SEQ ID NO: 206 (corresponding to residue 572Glu of the dSpCas9 molecule shown in SEQ ID NO: 207). In some aspects, the N-terminal Npu intein (SEQ ID NO: 300) and the C-terminal Npu intein (shown in SEQ ID NO: 302) can self-excise and ligate the two fragments, thereby forming a full-length dSpCas9 effector domain fusion protein when expressed intracellularly.

[0222] In some embodiments, the polypeptides of the split protein can interact non-covalently to form a complex that recapitulates the activity of the non-split protein. For example, two domains of a Cas enzyme expressed as separate polypeptides can be recruited by a gRNA, as described, for example, in Wright et al. PNAS 112(10):2984-2989 (2015), to form a ternary complex that recapitulates the activity of the full-length Cas enzyme in a complex with the gRNA. In some embodiments, the assembly of the split protein is inducible (e.g., photoinducible, chemically inducible, small molecule inducible).

[0223] In some embodiments, the two polypeptides of the split fusion protein can be delivered and / or expressed from separate vectors such as any of the vectors described herein. In some embodiments, the two polypeptides of the split fusion protein may be delivered to cells and / or expressed from two separate AAV vectors, for example, using a split AAV-based approach as described in WO2017 / 197238.

[0224] Approaches for the rational design of split proteins and their delivery, including Cas proteins and their fusions, are described, for example, in WO2016 / 114972, WO2017 / 197238, Zetsche.et al.Nat.Biotechnol.33(2):139-42(2015), Wright et al.PNAS 112(10):2984-2989(2015), Truong.et al.Nucleic Acids Res.43,6450-6458(2015), and Fine et al.Sci.Rep.5,10777(2015).

[0225] 2. Exemplary fusion proteins In some embodiments, the fusion protein of the provided DNA targeting system or its DNA targeting module comprises a DNA binding domain that targets a target site in a gene encoding a gene product that regulates LDL or its regulatory elements, and at least one transcriptional repressor effector domain. The DNA binding domain and the transcriptional repressor domain include any of the above. Exemplary fusion proteins are further described below.

[0226] In some embodiments, unless a specific SEQ ID NO is specified or a specific order is designated, the fusion proteins named herein include the elements of the named fusion proteins in any configuration or order. For example, the dCas9-KRAB fusion protein may include a KRAB domain fused to the N-terminus or C-terminus of the dSpCas9 molecule. In another example, the dSpCas9-KRAB-DNMT3A / L fusion protein may include dSpCas9, KRAB, and DNMT3A / L in any order. For example, the dSpCas9-KRAB-DNMT3A / L fusion protein may include DNMT3A / L, dSpCas9, and KRAB from the N-terminus to the C-terminus. In some embodiments, the fusion protein of the DNA targeting system includes dSpCas9-KRAB-DNMT3A / L in the order from the N-terminus to the C-terminus. In some embodiments, the fusion protein of the DNA targeting system includes DNMT3A / L-dSpCas9-KRAB in the order from the N-terminus to the C-terminus. The fusion proteins named herein may include additional elements. For example, the dSpCas9-KRAB-DNMT3A / L fusion protein may include one or more linkers, NLS sequences, or other sequences in any combination or order. Similar fusion proteins with the same orientation are provided where a different dCa is used as the DNA binding domain instead of the exemplary dSpCas9. Any dCas is contemplated to include those described herein. In some embodiments, the DNA binding domain is dCas. In some embodiments, dCas is dCas9. In some embodiments, dCas9 is dSpCas9.

[0227] In some embodiments, the exemplary linker or NLS sequence can be any of those described herein. In some embodiments, the exemplary linker or NLS sequence can be positioned in any order between the DNA binding domain (e.g., dSpCas9) and one or more effector domains (e.g., KRAB and DNMT3A / L). In any of the above embodiments, the NLS can be any of those described, for example, as shown in Section I.E. In any of the above embodiments, the linker can be any of those described, for example, as shown in Section I.E.

[0228] In some embodiments, the fusion protein of the DNA targeting system provided herein, or its DNA targeting module, comprises a DNA binding domain and a KRAB domain. In some embodiments, the DNA binding domain is a catalytically inactive Cas enzyme (dCas). In some embodiments, the Cas is a dCas9 such as dSaCas9 or dSpCas9. In some embodiments, the DNA binding domain is dSpCas9. In some embodiments, the DNA binding domain is the dSpCas9 shown in SEQ ID NO: 207, and the KRAB domain is shown in SEQ ID NO: 193. In some embodiments, the DNA binding domain is the dSpCas9 shown in SEQ ID NO: 207, and the KRAB domain is shown in SEQ ID NO: 290. In some embodiments, the fusion protein may include one or more linker or NLS sequences, such as at the N-terminus or C-terminus of the fusion protein, or between the Cas and the KRAB domain. The linker or NLS can be any of those described herein.

[0229] In some embodiments, the fusion protein of the DNA targeting system, or its DNA targeting module, contains, in order from the N-terminus to the C-terminus, an NLS and / or a linker, dSpCas9 shown in SEQ ID NO: 207, a linker and / or an NLS, and a KRAB domain shown in SEQ ID NO: 193. In some embodiments, the fusion protein of the DNA targeting system, or its DNA targeting module, contains, in order from the N-terminus to the C-terminus, an NLS and / or a linker, dSpCas9 shown in SEQ ID NO: 207, a linker and / or an NLS, and a KRAB domain shown in SEQ ID NO: 290.

[0230] In some embodiments, the fusion protein provided herein contains dCas9 and KRAB. In some embodiments, the fusion protein provided herein contains dSpCas9-KRAB. In some embodiments, the fusion protein provided herein contains the sequence shown in SEQ ID NO: 209, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the fusion protein contains the sequence shown in SEQ ID NO: 209.

[0231] In some embodiments, the fusion protein of the DNA targeting system provided herein, or its DNA targeting module, comprises a DNA binding domain, a DNMT3A domain, and a DNMT3L domain. In some embodiments, the effector domain comprises a fusion domain of DNMT3A and DNMT3L (DNMT3A / L domain). In some embodiments, the DNA binding domain is a catalytically inactive Cas enzyme (dCas). In some embodiments, Cas is a dCas9 such as dSaCas9 or dSpCas9. In some embodiments, the DNA binding domain is dSpCas9. In some embodiments, the DNA binding domain is the dSpCas9 shown in SEQ ID NO: 207. In some embodiments, a linker or NLS connects one of the DNMT3A domain or the DNMT3L domain to the DNA binding domain. In some embodiments, the DNA binding domain is Cas, and the linker or NLS may be present between Cas and one or both of the DNMT3A domain and the DNMT3L domain. In some embodiments, the fusion protein comprises the DNA binding domain, the DNMT3A domain, and the DNMT3L domain in any order. In some embodiments, the linker connects the DNMT3A domain to the DNMT3L domain. In some embodiments, the DNMT3A domain and the DNMT3L domain are connected as a fusion domain. In some embodiments, the fusion domain is the DNMT3A / L domain shown in SEQ ID NO: 199. In some embodiments, the fusion domain is the DNMT3A / L domain shown in SEQ ID NO: 201.

[0232] In some embodiments, the DNA targeting system or fusion protein comprises, in order from the N-terminus to the C-terminus, an NLS and / or linker, dSpCas9 shown in SEQ ID NO: 207, a linker and / or NLS, and the DNMT3A / L domain shown in SEQ ID NO: 199. In some embodiments, the DNA targeting system or fusion protein comprises, in order from the N-terminus to the C-terminus, an NLS and / or linker, dSpCas9 shown in SEQ ID NO: 207, a linker and / or NLS, and the DNMT3A / L domain shown in SEQ ID NO: 201.

[0233] In some embodiments, the DNA targeting system or fusion protein comprises, in order from the N-terminus to the C-terminus, an NLS and / or linker, dSpCas9 shown in SEQ ID NO: 207, a linker and / or NLS, the DNMT3A / L domain shown in SEQ ID NO: 199, and a linker and / or NLS. In some embodiments, the DNA targeting system or fusion protein comprises, in order from the N-terminus to the C-terminus, an NLS and / or linker, dSpCas9 shown in SEQ ID NO: 207, a linker and / or NLS, the DNMT3A / L domain shown in SEQ ID NO: 201, and a linker and / or NLS.

[0234] In some embodiments, the fusion protein of the DNA targeting system provided herein, or its DNA targeting module, comprises a DNA binding domain and two effector domains, one of which is a KRAB domain and the other is a DNMT3A domain and a DNMT3L domain. In some embodiments, the effector domain composed of the DNMT3A domain and the DNMT3L domain is a fusion domain of DNMT3A and DNMT3L (DNMT3A / L domain). In some embodiments, the DNA binding domain is a catalytically inactive Cas enzyme (dCas). In some embodiments, Cas is a dCas9 such as dSaCas9 or dSpCas9. In some embodiments, the DNA binding domain is dSpCas9. In some embodiments, each of the KRAB domain, the DNMT3A domain, and the DNMT3L domain is N-terminal to the DNA binding domain. In some embodiments, each of the KRAB domain, the DNMT3A domain, and the DNMT3L domain is C-terminal to the DNA binding domain. In some embodiments, the DNA binding domain is between the KRAB domain and one of the DNMT3A or DNMT3L domains. In some embodiments, the fusion domain is the DNMT3A / L domain shown in SEQ ID NO: 199 or 201. In some embodiments, the KRAB domain is shown in SEQ ID NO: 193. In some embodiments, the KRAB domain is shown in SEQ ID NO: 290. In some embodiments, the DNA binding domain is dSpCas9 shown in SEQ ID NO: 207. In some embodiments, the fusion protein may include one or more linkers or NLSs, such as at the N-terminus or C-terminus of the fusion protein, or between Cas and the KRAB domain or the DNMT3A / L domain. The linker or NLS can be any of those described herein. In some embodiments, the linker or NLS can be present between the DNMT3A / L domain and Cas. In some embodiments, the linker or NLS can be present between the Cas domain and the KRAB domain.

[0235] In some embodiments, the fusion protein comprises, as the first and second effector domains, a DNA binding domain, and a KRAB domain and a DNMT3A / 3L fusion domain. In some embodiments, the first effector domain is fused to the N-terminus of the second effector domain, and the second effector domain is fused to the N-terminus of the DNA binding domain. In some embodiments, the fusion protein provided herein comprises DNMT3A / L-KRAB-dSpCas9 in that order. In some embodiments, the fusion protein provided herein comprises KRAB-DNMT3A / L-dSpCas9 in that order. In some embodiments, each of the KRAB domain and the DNMT3A / 3L domain is C-terminal to the DNA binding domain, the first effector domain is fused to the C-terminus of the DNA binding domain, and the second effector domain is fused to the C-terminus of the first effector domain. In some embodiments, the fusion protein provided herein comprises dSpCas9-DNMT3A / L-KRAB in that order. In some embodiments, the fusion protein provided herein comprises dSpCas9-KRAB-DNMT3A / L in that order. In some embodiments, the DNA binding domain is between the KRAB domain and the DNMT3A / 3L domain, one effector domain is fused to the N-terminus of the DNA binding domain, and the other effector domain is fused to the C-terminus of the DNA binding domain. In some embodiments, the fusion protein provided by the present invention comprises KRAB-dSpCas9-DNMT3A / L in that order. In some embodiments, the fusion protein provided by the present invention comprises DNMT3A / L-dSpCas9-KRAB in that order.

[0236] In some embodiments, the fusion protein of the DNA targeting system, or its DNA targeting module, contains, in order from the N-terminus to the C-terminus, the DNMT3A / L fusion domain shown in SEQ ID NO: 199, an NLS and / or linker, the dSpCas9 shown in SEQ ID NO: 207, a linker and / or NLS, and the KRAB domain shown in SEQ ID NO: 193. In some embodiments, the fusion protein of the DNA targeting system, or its DNA targeting module, contains, in order from the N-terminus to the C-terminus, the DNMT3A / L fusion domain shown in SEQ ID NO: 199, an NLS and / or linker, the dSpCas9 shown in SEQ ID NO: 207, a linker and / or NLS, and the KRAB domain shown in SEQ ID NO: 290.

[0237] In some embodiments, the fusion protein of the DNA targeting system, or its DNA targeting module, contains, in order from the N-terminus to the C-terminus, the DNMT3A / L fusion domain shown in SEQ ID NO: 201, an NLS and / or linker, the dSpCas9 shown in SEQ ID NO: 207, a linker and / or NLS, and the KRAB domain shown in SEQ ID NO: 193. In some embodiments, the fusion protein of the DNA targeting system, or its DNA targeting module, contains, in order from the N-terminus to the C-terminus, the DNMT3A / L fusion domain shown in SEQ ID NO: 201, an NLS and / or linker, the dSpCas9 shown in SEQ ID NO: 207, a linker and / or NLS, and the KRAB domain shown in SEQ ID NO: 290.

[0238] In some embodiments, the fusion protein provided herein contains dCas9, KRAB, and DNMT3A / L. In some embodiments, the fusion protein provided herein contains dSpCas9-KRAB-DNMT3A / L.

[0239] In some embodiments, the fusion proteins provided herein include the sequence set forth in SEQ ID NO: 278, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 278. In some embodiments, the fusion protein includes the sequence set forth in SEQ ID NO: 278.

[0240] In some embodiments, the fusion proteins provided herein include the sequence set forth in SEQ ID NO: 280, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 280. In some embodiments, the fusion protein includes the sequence set forth in SEQ ID NO: 280.

[0241] In some embodiments, the fusion proteins provided herein include the sequence set forth in SEQ ID NO: 282, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the fusion protein includes the sequence set forth in SEQ ID NO: 282.

[0242] II. Polynucleotides, Vectors, and Related Methods for Delivery In some aspects, polynucleotides encoding any of the DNA targeting systems described herein, or any part or component of any of the foregoing, are provided. In some aspects, the polynucleotide can encode any of the components of the DNA targeting system and / or any nucleic acid or proteinaceous molecule necessary to carry out aspects of the methods of the present disclosure. In certain embodiments, provided is a polynucleotide encoding any of the fusion proteins described herein. Also provided herein is a polynucleotide encoding any of the gRNAs or combinations of gRNAs described herein.

[0243] In some embodiments, provided is a polynucleotide comprising the gRNA described herein. In some embodiments, the gRNA is transcribed from a gene construct (i.e., a vector or plasmid) within the target cell. In some embodiments, the gRNA is produced by in vitro transcription and delivered to the target cell. In some embodiments, the gRNA comprises one or more modified nucleotides to improve stability. In some embodiments, the gRNA is delivered to the target cell pre-complexed as a fusion protein and RNP.

[0244] In some embodiments, the provided polynucleotide encodes a fusion protein described herein that comprises (a) a DNA binding domain capable of targeting a target site of the described target gene, and (b) at least one effector domain capable of reducing transcription of the gene. In some embodiments, the fusion protein comprises a fusion protein of a Cas protein or a variant thereof, and at least one effector domain capable of reducing transcription of the gene. In certain examples, the Cas is a dCas such as dCas9. In some embodiments, the dCas9 is a dSpCas9 such as a polynucleotide encoding dSpCas9 shown in SEQ ID NO: 207. Examples of such domains and fusion proteins include any of those described in Section I.

[0245] In some embodiments, the polynucleotide comprises a sequence encoding a dCas9-KRAB fusion protein. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO: 208, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide is set forth in SEQ ID NO: 208. In some embodiments, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 209, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide encodes the amino acid sequence set forth in SEQ ID NO: 209.

[0246] In some embodiments, the polynucleotide comprises a sequence encoding a dCas9-KRAB-DNMT3A / L fusion protein. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO: 277, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide is set forth in SEQ ID NO: 277. In some embodiments, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 278, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide encodes the amino acid sequence set forth in SEQ ID NO: 278.

[0247] In some embodiments, the polynucleotide comprises a sequence encoding a dCas9-KRAB-DNMT3A / L fusion protein such as the sequence shown in SEQ ID NO: 279, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide is shown in SEQ ID NO: 279. In some embodiments, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 280, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 280.

[0248] In some embodiments, the polynucleotide comprises a sequence encoding a dCas9-KRAB-DNMT3A / L fusion protein such as the sequence shown in SEQ ID NO: 281, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide is shown in SEQ ID NO: 281. In some embodiments, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 282, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 282.

[0249] In some embodiments, the polynucleotide is an mRNA molecule encoding a dCas9-KRAB-DNMT3A / L fusion protein such as the sequence set forth in SEQ ID NO: 395, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the polynucleotide is set forth in SEQ ID NO: 395.

[0250] In some embodiments, the polynucleotide is RNA or DNA. In some embodiments, the polynucleotide, such as the polynucleotide encoding the provided fusion protein, is mRNA. In some embodiments, the gRNA is provided as RNA and the polynucleotide encoding the fusion protein is mRNA. The mRNA can be 5'-capped and / or 3'-polyadenylated. In another embodiment, the polynucleotides provided herein, such as the polynucleotide encoding the provided fusion protein, are DNA. The DNA can be present in a vector.

[0251] Also provided herein is a vector containing any of the provided polynucleotides. In some embodiments, the vector comprises a gene construct such as a plasmid or an expression vector.

[0252] In some embodiments, an expression vector comprising a sequence encoding a fusion protein of the DNA targeting system provided herein can further comprise a polynucleotide sequence encoding at least one gRNA. In some embodiments, the expression vector comprises a polynucleotide sequence encoding two gRNAs or a combination of polynucleotide sequences. In some embodiments, the expression vector comprises a polynucleotide sequence encoding three gRNAs or a combination of polynucleotide sequences. The sequence encoding the gRNA can be operably linked to at least one transcriptional control sequence for expression of the gRNA intracellularly. For example, the DNA encoding the gRNA can be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Examples of suitable Pol III promoters include, but are not limited to, mammalian U6, U3, H1, and 7SL RNA promoters.

[0253] An expression vector (DNA or RNA (e.g., mRNA) expression vector) can comprise any number of suitable transcriptional control sequences. For example, the transcriptional control sequences can include enhancers, promoters, or untranslated regions (UTRs) such as 3’UTR or 5’UTR. In some embodiments, the UTR is encoded by the expression vector (e.g., a DNA vector) and / or is present within the expression vector. In some embodiments, the mRNA encoding the fusion protein comprises a UTR. In some aspects, different transcriptional control sequences can be selected, for example, for use within an expression vector to achieve appropriate expression levels. For example, in some embodiments, a UTR that facilitates expression in a particular tissue or cell type (e.g., liver or hepatocytes) can be selected. In some embodiments, the 5’UTR of the expression vector encodes or comprises the sequence set forth in SEQ ID NO: 393. In some embodiments, the 3’UTR of the expression vector encodes or comprises the sequence set forth in SEQ ID NO: 394.

[0254] In some embodiments, provided is a vector containing a polynucleotide encoding a fusion protein comprising a DNA binding domain comprising dCas and at least one effector domain capable of increasing transcription of a gene, and a polynucleotide or combination of polynucleotides encoding a gRNA, or a combination of gRNAs such as two gRNAs or three gRNAs. In some embodiments, the dCas is a dCas9 such as dSpCas9. In some embodiments, the polynucleotide encodes a fusion protein comprising dSpCas9 set forth in SEQ ID NO: 207. In some embodiments, the polynucleotide(s) encodes a gRNA or combination of gRNAs described in Section II.B.ii. For example, the polynucleotide can encode a combination of gRNAs each comprising a spacer sequence selected from any one of SEQ ID NOs: 64-126, 318-329, 352-361, or 378-383, or at least a 14 nt adjacent portion thereof. In some embodiments, the polynucleotide(s) encodes a combination of gRNAs each comprising a sequence set forth in any one of SEQ ID NOs: 127-189, 330-341, 362-371, or 384-389.

[0255] In some embodiments, the effector domain is KRAB. In some embodiments, the effector domain is DNMT3A / L. In some embodiments, the vector comprises a polynucleotide comprising SEQ ID NO: 192, SEQ ID NO: 277, SEQ ID NO: 279, or SEQ ID NO: 281, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, and one or more polynucleotides encoding a gRNA or combination of gRNAs as described in Section I.B.ii. In some embodiments, the polynucleotide(s) encode a combination of gRNAs, each comprising a spacer sequence selected from any one of SEQ ID NOs: 64-126, 318-329, 352-361, or 378-383, or at least 14 nt of its adjacent portion. In some embodiments, each gRNA further comprises the sequence shown in SEQ ID NO: 191. In some embodiments, the polynucleotide(s) encode a combination of gRNAs, each comprising a sequence shown in any one of SEQ ID NOs: 127-189, 330-341, 362-371, or 384-389.

[0256] In some embodiments, the polynucleotide encodes a combination of a fusion protein and a gRNA.

[0257] In some embodiments, the polynucleotides provided herein can be codon-optimized for efficient translation into protein in a eukaryotic cell or animal of interest. For example, the codons can be optimized for expression in humans, mice, rats, hamsters, cows, pigs, cats, dogs, fish, amphibians, plants, yeast, insects, etc. Programs for codon optimization are available as freeware. Commercially available codon optimization programs are also available.

[0258] In some embodiments, the polynucleotides described herein can include one or more transcriptional and / or translational control elements. Depending on the host / vector system utilized, any of a plurality of suitable transcriptional and translational control elements (including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc.) can be used within the expression vector.

[0259] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters that function within eukaryotic cells) include those from the cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, the human elongation factor 1 promoter (EF1), hybrid constructs containing the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG), the mouse stem cell virus promoter (MSCV), the phosphoglycerate kinase-1 locus promoter (PGK), and the mouse metallothionein-I.

[0260] For expressing small RNAs, including guide RNAs used in DNA targeting systems, various promoters such as RNA polymerase III promoters including U6 and H1 can be advantageous. Descriptions and parameters for enhancing the use of such promoters are known in the art and additional information and approaches are regularly described (see, for example, Ma, H. et al., Molecular Therapy - Nucleic Acids 3, e161 (2014) doi:10.1038 / mtna.2014.12).

[0261] The expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector can also include an appropriate sequence for amplifying expression. The expression vector can also include a nucleotide sequence that encodes a non-native tag (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) and results in a fusion protein by fusing with a site-specific polypeptide.

[0262] The promoter can be an inducible promoter (e.g., heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.). The promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some cases, the promoter can be a spatially restricted promoter and / or a temporally restricted promoter (e.g., tissue-specific promoter, cell type-specific promoter (e.g., hepatocyte-specific promoter, etc.)).

[0263] Examples of the intended expression vectors include, but are not limited to, virus vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex, human immunodeficiency virus, retrovirus (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors. Other vectors intended for eukaryotic target cells include, but are not limited to, vectors of pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Other vectors can also be used as long as they are compatible with the host cell.

[0264] In some embodiments, the vector is a viral vector such as an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or a gammaretroviral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is selected from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 vectors. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a non-viral vector, such as a lipid nanoparticle, liposome, exosome, or cell-penetrating peptide. In some embodiments, the vector comprises one vector, or two or more vectors.

[0265] In some embodiments, the vectors described herein are, or comprise, lipid nanoparticles (LNPs). Among the provided embodiments are lipid nanoparticles containing any of the polynucleotides provided for delivery of an epigenetic DNA targeting system. In some embodiments, the LNP comprises a polynucleotide encoding a fusion protein provided herein that comprises (a) a DNA binding domain capable of targeting a target site of a target gene as described, and (b) at least one effector domain capable of reducing transcription of the gene. In some embodiments, the DNA binding domain is Cas (e.g., dCas), and the LNP further comprises a gRNA. In some embodiments, the polynucleotide encoding the fusion protein is an mRNA, and the gRNA is provided as an RNA.

[0266] In some embodiments, any of the epigenetic modification DNA targeting systems, gRNAs, Cas-gRNA combinations, polynucleotides, fusion proteins, or components thereof described herein are incorporated, for example, into lipid nanoparticles (LNPs) for delivery. In some embodiments, the lipid nanoparticles are vectors for delivery. In some embodiments, the nanoparticles may comprise at least one lipid. The lipid can be selected from, but not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, and pegylated lipids. In another aspect, the lipid can be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, and DODMA. Typically, the LNP is composed of two or more lipids, e.g., 3, 4, or 5 lipids. In some embodiments, at least the lipid is either ionizable cationic or cationic.

[0267] Lipid nanoparticles can be used for delivery of encapsulated or associated (e.g., complexed) therapeutic agents, including nucleic acids and proteins such as those encoding and / or containing the CRISPR / Cas system. See, for example, U.S. Patent No. 10,723,692, U.S. Patent No. 10,941,395, and WO2015 / 035136.

[0268] In some embodiments, the provided method involves the use of lipid nanoparticles (LNPs) containing mRNA encoding any of the protein components of any of the provided DNA targeting systems, such as any of the fusion proteins provided herein. In some embodiments, the mRNA can be produced using methods known in the art, such as in vitro transcription. In some embodiments of this method, the mRNA contains a 5' cap. In some embodiments, the 5' cap is an altered nucleotide on the 5' end of a primary transcript, such as messenger RNA. In some aspects, the 5' cap of the mRNA improves one or more of RNA stability and processing, mRNA metabolism, processing and maturation of nuclear RNA transcripts, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of mRNA into protein. In some embodiments, the 5' cap can be a naturally occurring 5' cap or different from the naturally occurring cap of the mRNA. The 5' cap can be any 5' cap known to those skilled in the art. In certain embodiments, the 5' cap is selected from the group consisting of an anti-reverse cap analog (ARCA) cap, a 7-methyl-guanosine (7mG) cap, a CleanCap® analog, a vaccinia cap, and analogs thereof. For example, the 5' cap can include, but is not limited to, an anti-reverse cap analog (ARCA) (US7074596), 7-methyl-guanosine, a CleanCap® analog such as a Cap1 analog (Trilink; San Diego, CA), or one enzymatically capped using, for example, a vaccinia capping enzyme. In some embodiments, the mRNA may be polyadenylated. The mRNA may contain various 5' and 3' untranslated sequence elements to enhance the expression of the encoded protein and / or the stability of the mRNA itself. Such elements can include, for example, post-transcriptional regulatory elements such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the mRNA contains at least one nucleoside modification.mRNA can contain modifications of naturally occurring nucleosides to nucleoside analogs. Any nucleoside analog known in the art is contemplated. Such nucleoside analogs can include, for example, those described in US8,278,036. In certain embodiments of the method, the nucleoside modification is selected from the group consisting of the modification from uridine to pseudouridine and the modification from uridine to NI-methylpseudouridine. In certain embodiments of this method, the nucleoside modification is from uridine to pseudouridine.

[0269] In some embodiments, the LNP composition comprises a PEG-lipid (e.g., a lipid containing a polyethylene glycol component). In some embodiments, the described LNP composition comprises two or more PEG-lipids. Exemplary PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, one or more PEG lipids can include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipid, or combinations thereof. In some embodiments, the PEG moiety is a linear or branched polymer optionally substituted with ethylene glycol or ethylene oxide. In some embodiments, the PEG moiety is substituted, for example, with one or more alkyl groups, alkoxy groups, acyl groups, hydroxy groups, or aryl groups. In some embodiments, the PEG moiety comprises a PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., j. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)). In some embodiments, the PEG-lipid is a PEG-lipid conjugate. In some embodiments, the PEG-lipid comprises from about 0.1 mol% to about 6 mol% of the total lipid content present in the nanoparticle composition. In some embodiments, the number average molecular weight of the PEG-lipid is from about 200 Da to about 5000 Da. In some embodiments, the LNP comprises and / or is conjugated to N-acetylgalactosamine (GalNAc), an amino sugar derivative of galactose. In some aspects, GalNAc is a sugar molecule that can recognize and bind to a cell surface protein, the asialoglycoprotein receptor (ASGPR). ASGPR is abundantly expressed on liver cells (hepatocytes). In some embodiments, conjugation of GalNAc to the LNP can improve delivery to hepatocytes.In some embodiments, the lipid nanoparticles comprise a GalNAC-conjugated lipid (e.g., GalNAc-PEG lipid). In some embodiments, the molar percentage of the GalNAC-conjugated lipid in the lipid nanoparticles is from about 0% to about 2%. In some embodiments, the molar percentage of the GalNAC-conjugated lipid in the lipid nanoparticles is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, or more, or a value between any of the foregoing.

[0270] In some embodiments, LNPs useful in the present method include cationic lipids selected from DLin-DMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLin-MC3-DMA (dilinoleylmethyl-4-dimethylaminobutyrate), DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), DODMA (1,2-dioleyloxy-N,N-dimethyl-3-aminopropane), SS-OP (bis[2-(4-{2-[4-(cis-9 octadecenooyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl] disulfide), and derivatives thereof. DLin-MC3-DMA and its derivatives are described, for example, in WO2010 / 144740. DODMA and its derivatives are described, for example, in US7,745,651 and Mok et al. (1999), Biochimica et Biophysica Acta, 1419(2):137-150. DLin-DMA and its derivatives are described, for example, in US7,799,565. DLin-KC2-DMA and its derivatives are described, for example, in US9,139,554. SS-OP (NOF America Corporation, White Plains, NY) is described, for example, at https: / / www.nofamerica.com / store / index.php?dispatch=products.view&product_id=962.Additional and non-limiting examples of cationic lipids include methylpyridyldialkyl acid (MPDACA), palmitoyl-oleoyl-nor-arginine (PONA), guanidino-dialkyl acid (GUADACA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), bis{2-[N-methyl-N-(α-D-tocopherol hemisuccinate propyl)amino]ethyl}disulfide (SS-33 / 3AP05), bis{2-[4-(α-D-tocopherol hemisuccinate ethyl)piperidyl]ethyl}disulfide (SS33 / 4PE15), bis{2-[4-(cis-9-octadecenoate ethyl)-1-piperidinyl]ethyl}disulfide (SS18 / 4PE16), and bis{2-[4-(cis,cis-9,12-octadecadienoate ethyl)-1-piperidinyl]ethyl}disulfide (SS18 / 4PE13). In further embodiments, the lipid nanoparticles also include one or more non-cationic lipids and lipid conjugates.

[0271] In some embodiments, the molar concentration of the cationic lipid is about 20% to about 80%, about 30% to about 70%, about 40% to about 60%, about 45% to about 55%, or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total lipid molar concentration, and the total lipid molar concentration is the sum of the cationic lipid, non-cationic lipid, and lipid conjugate molar concentrations. In certain embodiments, the lipid nanoparticles include a molar ratio of cationic lipid to any of about 1 to about 20, about 2 to about 16, about 4 to about 12, about 6 to about 10, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 polynucleotides.

[0272] In some embodiments, the lipid nanoparticles can include at least one non-cationic lipid. In certain embodiments, the molar concentration of the non-cationic lipid is about 20% to about 80%, about 30% to about 70%, about 40% to about 70%, about 40% to about ********%, about 46% to about 50%, or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 48.5%, about 50%, about 55%, about ******, about 65%, about 70%, about 75%, or about 80% of the total lipid molar concentration. In some embodiments, the non-cationic lipids include phospholipids and steroids.

[0273] Note: There seems to be an error in the original text where some numbers are cut off (******** and ******). I've translated it as accurately as possible with the given text.In some embodiments, phospholipids useful for the lipid nanoparticles described herein include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dierucoyl-sn-glycero-3-phosphate (sodium salt) (DEPA-NA), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-dierucoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)(DEPG-NA), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt) (DLPA-NA), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)(DLPG-NA), 1,2-dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)(DLPG-NH4), 1,2-dilauroyl-sn-glycero-3-phosphoserine (sodium salt) (DLPS-NA), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA-NA), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)(DMPG-NA), 1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)(DMPG-NH4), 1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium / ammonium salt)(DMPG-NH4 / NA), 1,2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt) (DMPS-NA), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA-NA), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)(sodium salt)(DOPG-NA), 1,2-Dioleoyl-sn-glycero-3-phosphoserine (sodium salt)(DOPS-NA), 1,2-Dipalmitoyl-sn-glycero-3-phosphate (sodium salt)(DPPA-NA), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Dipalmitoyl-sn-glycero-3[Phospho-rac-(1-glycerol)(sodium salt)(DPPG-NA), 1,2-Dipalmitoyl-sn-glycero-3[Phospho-rac-(1-glycerol)(ammonium salt)(DPPG-NH4), 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (sodium salt)(DPPS-NA), 1,2-Distearoyl-sn-glycero-3-phosphate (sodium salt)(DSPA-NA), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol)(sodium salt)(DSPG-NA), 1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol)(ammonium salt)(DSPG-NH4), 1,2-Distearoyl-sn-glycero-3-phosphoserine (sodium salt) (DSPS-NA), Egg-PC (EPC), hydrogenated Egg PC (HEPC), hydrogenated Soy PC (HSPC), 1-myristoyl-sn-glycero-3-phosphocholine (LYSOPCMYRISTIC), 1-palmitoyl-sn-glycero-3-phosphocholine (LYSOPCPALMITIC), 1-stearoyl-sn-glycero-3-phosphocholine (LYSOPCSTEARIC), 1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)] (sodium salt) (POPG-NA), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC) are mentioned, but not limited thereto. In certain embodiments, the phospholipid is DSPC. In certain embodiments, the phospholipid is DOPE. In certain embodiments, the phospholipid is DOPC.,

[0274] In some embodiments, the non-cationic lipid composed of lipid nanoparticles contains one or more steroids. Steroids useful for the lipid nanoparticles described herein include, but are not limited to, cholestanes such as cholesterol, cholans such as cholic acid, pregnans such as progesterone, androstanes such as testosterone, and estranes such as estradiol. Further steroids include cholesterol (sheep), cholesterol sulfate, desmosterol-d6, cholesterol-d7, lasosterol-d7, desmosterol, stigmasterol, lanosterol, dehydrocholesterol, dihydrolanosterol, dimosterol, lasosterol, dimosterol-d5, 14-demethyl-lanosterol, 14-demethyl-lanosterol-d6, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, diosgenin, DHEA sulfate, DHEA, lanosterol-d6, dihydrolanosterol-d7, campesterol-d6, sitosterol, lanosterol-95, dihydro FF-MAS-d6, dimostenol-d7, dimostenol, sitostanol, campestanol, campesterol, 7-dehydrodesmosterol, pregnenolone, sitosterol-d7, dihydro T-MAS, delta5-avenasterol, brassicasterol, dihydro FF-MAS, 24-methylenecholesterol, cholic acid derivatives, cholesteryl esters, and glycosylated steroids, but are not limited thereto. In certain embodiments, the lipid nanoparticles contain cholesterol.

[0275] In some embodiments, the lipid nanoparticles comprise a lipid conjugate. Such lipid conjugates include, but are not limited to, ceramide PEG derivatives such as C8 PEG2000 ceramide, C16 PEG2000 ceramide, C8 PEG5000 ceramide, C16 PEG5000 ceramide, C8 PEG750 ceramide, and C16 PEG750 ceramide; phosphoethanolamine PEG derivatives such as 16:0 PEG5000PE, 14:0 PEG5000PE, 18:0 PEG5000PE, 18:1 PEG5000PE, 16:0 PEG3000PE, 14:0 PEG3000PE, 18:0 PEG3000PE, 18:1 PEG3000PE, 16:0 PEG2000PE, 14:0 PEG2000PE, 18:0 PEG2000PE, 18:1 PEG2000 PE 16:0 PEG1000PE, 14:0 PEG1000PE, 18:0 PEG1000PE, 18:1 PEG 1000PE, 16:0 PEG750PE, 14:0 PEG750PE, 18:0 PEG750PE, 18:1 PEG750PE, 16:0 PEG550PE, 14:0 PEG550PE, 18:0 PEG550PE, 18:1 PEG550PE, 16:0 PEG350PE, 14:0 PEG350PE, 18:0 PEG350PE, and 18:1 PEG350; sterol PEG derivatives such as Chol-PEG600; and glycerol PEG derivatives such as DMG-PEG5000, DSG-PEG5000, DPG-PEG5000, DMG-PEG3000, DSG-PEG3000, DPG-PEG3000, DMG-PEG2000, DSG-PEG2000, DPG-PEG2000, DMG-PEG1000, DSG-PEG1000, DPG-PEG1000, DMG-PEG750, DSG-PEG750, DPG-PEG750, DMG-PEG550, DSG-PEG550, DPG-PEG550, DMG-PEG350, DSG-PEG350, and DPG-PEG350. In some embodiments, the lipid conjugate is DMG-PEG. In some particular embodiments, the lipid conjugate is DMG-PEG2000.In some specific embodiments, the lipid conjugate is DMG-PEG5000.

[0276] Based on characteristics of the selected lipid(s), the nature of delivery to the intended target cells, and the characteristics of the nucleic acid and / or protein to be delivered, etc., selecting a cationic lipid, non-cationic lipid, and / or lipid conjugate comprising lipid nanoparticles, as well as the relative molar ratios of such lipids to each other, is within the level of a person skilled in the art. Additional considerations include, for example, saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity, and toxicity of the selected lipid(s). Accordingly, the molar ratio of each individual component may be adjusted accordingly.

[0277] Lipid nanoparticles for use in the present method can be prepared by various techniques known to those skilled in the art. Nucleic acid-lipid particles and preparation methods are disclosed, for example, in U.S. Patent Publication Nos. 20040142025 and 20070042031.

[0278] In some embodiments, the lipid nanoparticles have a size in the range of about 25 to about 500 nm. In some embodiments, the lipid nanoparticles have a size of about 50 nm to about 300 nm, or about 60 nm to about 120 nm. The size of the lipid nanoparticles may be determined by quasi-elastic light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421A150 (1981). Various methods for producing a population of lipid nanoparticles within a specific size range, such as sonication or homogenization, are known in the art. One such method is described in U.S. Patent No. 4,737,323.

[0279] In some embodiments, the lipid nanoparticles include cell targeting molecules such as targeting ligands (e.g., antibodies, scFv proteins, DART molecules, peptides, aptamers, etc.) immobilized on the surface of the lipid nanoparticles that selectively bind the lipid nanoparticles to target cells such as any of the cells described herein, such as hepatocytes.

[0280] In some embodiments, the vector exhibits tropism for hepatocytes and / or liver cells.

[0281] In some aspects, provided herein is a plurality of vectors comprising any one of the vectors described herein, and any additional portion or additional component of any one of the DNA targeting systems described herein, any one of the gRNAs described herein, any one of the fusion proteins described herein, or one or more additional vectors encoding any portion or component of any of the foregoing.

[0282] Provided is a plurality of vectors comprising a first vector comprising any one of the polynucleotides described herein, a second vector comprising any one of the polynucleotides described herein, and optionally one or more additional vectors comprising any one of the polynucleotides described herein.

[0283] In some aspects, the vectors provided herein may be referred to as delivery vehicles. In some aspects, any of the DNA targeting systems, components thereof, or polynucleotides disclosed herein may be packaged within or on the surface of a delivery vehicle for delivery to a cell. Delivery vehicles contemplated include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. As described in the art, various targeting moieties can be used to improve the preferential interaction of such vehicles with a desired cell type or location.

[0284] Methods for introducing nucleic acids into host cells are known in the art, and any known method can be used to introduce nucleic acids (e.g., expression constructs) into cells. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc. In some embodiments, the composition may be delivered by mRNA delivery and ribonucleoprotein (RNP) complex delivery. Direct delivery of an RNP complex containing a DNA-binding domain complexed with sgRNA can eliminate the need for intracellular transcription and translation and provide a robust platform for host cells with low transcriptional and translational activity. The RNP complex can be introduced into host cells by any of the methods known in the art.

[0285] The nucleic acids or RNPs of the present disclosure can be incorporated into a host using virus-like particles (VLPs). VLPs contain normal viral vector components such as an envelope and a capsid but lack a viral genome. For example, nucleic acids expressing Cas and sgRNA can be fused to viral vector components such as gag and introduced into producer cells. The resulting virus-like particles containing the sgRNA expression vector can infect host cells for efficient editing.

[0286] Introduction of the complexes, polypeptides, and nucleic acids of the present disclosure can occur by protein transduction domains (PTDs). PTDs including human immunodeficiency virus-1 TAT, herpes simplex virus-1 VP22, Drsophila Antennapedia Antp, and polyarginine are peptide sequences that can cross the cell membrane, enter host cells, and deliver complexes, polypeptides, and nucleic acids into the cells.

[0287] The introduction of the complexes, polypeptides, and nucleic acids of the present disclosure into cells can occur, for example, by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, as described in WO2017 / 193107, WO2016 / 123578, WO2014 / 152432, WO2014 / 093661, WO2014 / 093655, or WO2021 / 226555.

[0288] Various methods for the introduction of polynucleotides are well known and can be used with the provided methods and compositions. Exemplary methods include methods for the transfer of polynucleotides encoding the DNA targeting systems provided herein, including via viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation.

[0289] In some embodiments, the polynucleotide can be cloned into a suitable vector, such as one or more expression vectors. The expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable cell. Suitable vectors include vectors designed for propagation and expansion, or for expression, or for both, such as plasmids and viruses.

[0290] In some embodiments, the vector can be a vector of the pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, La Jolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, viral vectors such as lentiviral vectors or retroviral vectors are used. In some embodiments, the recombinant expression vector can be prepared using standard recombinant DNA techniques. In some embodiments, the vector can contain regulatory sequences such as transcription and translation start and stop codons that are specific to the type of host into which the vector is introduced, taking into account whether the vector is DNA-based or RNA-based as needed. In some embodiments, the vector can contain a non-native promoter operably linked to a nucleotide sequence encoding a recombinant receptor. In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, and the promoter found in the long terminal repeat of murine stem cell virus. Other promoters known to those skilled in the art are also contemplated.

[0291] In some embodiments, the recombinant nucleic acid is transferred into cells using recombinant infectious viral particles such as vectors derived from, for example, simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is transferred into cells (e.g., hepatocytes) using a retroviral vector such as a recombinant lentiviral vector or a gamma-retroviral vector (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi:10.1038 / gt.2014.25, Carlens et al. (2000) Exp Hematol 28(10):1137-46, Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93, Park et al., Trends Biotechnol. 2011 November 29(11):550-557).

[0292] In some embodiments, the retroviral vector has a retroviral vector derived from a long terminal repeat (LTR), such as Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus-forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retrovirus includes those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning that they can infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Some exemplary retroviral systems are described (e.g., U.S. Patent Nos. 5,219,740, 6,207,453, 5,219,740, Miller and Rosman (1989) BioTechniques 7:980-990, Miller, A.D. (1990) Human Gene Therapy 1:5-14, Scarpa et al. (1991) Virology 180:849-852, Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037, and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0293] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector is an integrase-deficient lentiviral vector. In some embodiments, the lentiviral vector is a recombinant lentiviral vector. In some embodiments, the lentivirus is selected or engineered for a desired tropism (e.g., tropism for liver cells or hepatocytes). Methods for lentivirus production, transduction, and engineering are known, for example, as described in Kasaraneni, N. et al. Sci. Rep. 8(1):10990 (2018), Ghaleh, H. E. G. et al. Biomed. Pharmacother. 128:110276 (2020), and Milone, M. C. et al. Le...

Claims

1. An epigenetic modification DNA targeting system comprising a plurality of DNA targeting modules for suppressing the transcription of a plurality of genes that regulate low-density lipoprotein (LDL), wherein the plurality of DNA targeting modules include a first DNA targeting module for suppressing the transcription of a first gene among the plurality of genes, and a second DNA targeting module for suppressing the transcription of a second gene among the plurality of genes, each DNA targeting module comprising a fusion protein comprising (a) a DNA binding domain for targeting one target site among the plurality of genes, and (b) at least one transcription repressor domain, the epigenetic modification DNA targeting system.

2. The epigenetic modification DNA targeting system according to claim 1, which does not introduce gene disruption or DNA cleavage.

3. The fusion protein of each DNA targeting module comprises a DNA binding domain selected from a clustered regularly interspaced short palindromic repeat related (Cas) protein or a variant thereof, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or an I-SceI enzyme or a variant thereof, and optionally, the DNA binding domain comprises a catalytically inactive variant of any of the foregoing. The epigenetic modification DNA targeting system according to claim 1 or claim 2.

4. The fusion protein of the first DNA targeting module comprises a DNA binding domain for targeting a target site of the first gene or its regulatory DNA element, and at least one transcription repressor domain, The fusion protein of the second DNA targeting module comprises a DNA binding domain for targeting a target site of the second gene or its regulatory DNA element, and at least one transcription repressor domain, The epigenetic modification DNA targeting system according to any one of claims 1 to 3.

5. The epigenetic modification DNA targeting system according to any one of claims 1 to 4, wherein any two or more of the DNA targeting modules comprise the same fusion protein.

6. The epigenetic modification DNA targeting system according to any one of claims 1 to 5, wherein the first and second DNA targeting modules contain the same fusion protein. **Claim 7** The epigenetic modification DNA targeting system according to any one of claims 1 to 4, wherein any two or more of the DNA targeting modules contain different fusion proteins. **Claim 8** The epigenetic modification DNA targeting system according to any one of claims 1 to 4 and 7, wherein the first and second DNA targeting modules contain different fusion proteins. **Claim 9** (1) A first DNA targeting module that reduces the transcription of a first gene that regulates low-density lipoprotein (LDL), the first DNA targeting module comprising a first fusion protein comprising: (a) a DNA binding domain for targeting a target site of the first gene or its regulatory DNA element; and (b) at least one transcriptional repressor domain, and (2) A second DNA targeting module that reduces the transcription of a second gene that regulates LDL, the second DNA targeting module comprising a second fusion protein comprising: (a) a DNA binding domain for targeting a target site of the second gene or its regulatory DNA element; and (b) at least one transcriptional repressor domain An epigenetic modification DNA targeting system comprising a plurality of DNA targeting modules for suppressing the transcription of a plurality of genes that regulate low-density lipoprotein (LDL). **Claim 10** The epigenetic modification DNA targeting system according to claim 5 or 6, wherein the first DNA targeting module contains a first targeting polynucleotide for targeting the target site of the first gene, the second DNA targeting module contains a second targeting polynucleotide for targeting the target site of the second gene, and the first and second targeting polynucleotides complex with the DNA binding domain of the fusion protein. **Claim 11** The epigenetic modification DNA targeting system according to claim 10, wherein the DNA binding domain is a clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof, and the first and second targeting polynucleotides each contain a first gRNA and a second gRNA. **Claim 12** a fusion protein comprising a clustered regularly interspaced short palindromic repeat (Cas) protein or a variant thereof clustered and arranged regularly, and at least one transcriptional repressor domain, and b a plurality of guide RNAs (gRNAs) comprising at least a first gRNA and a second gRNA, wherein the first gRNA targets a target site of a first gene that regulates low density lipoprotein (LDL), and the second gRNA targets a target site of a second gene that regulates LDL, the plurality of gRNAs An epigenetic modification DNA targeting system comprising.

13. The epigenetic modification DNA targeting system according to any one of claims 1 to 12, further comprising a third DNA targeting module for suppressing the transcription of a third gene that regulates low density lipoprotein (LDL).

14. Further comprising a third gRNA targeting a target site of a third gene that regulates LDL, Optionally, a fourth gRNA targeting a target site of a fourth gene that regulates LDL, optionally, a fifth gRNA targeting a target site of a fifth gene that regulates LDL, and / or optionally, a sixth gRNA targeting a target site of a sixth gene that regulates LDL, further comprising The epigenetic modification DNA targeting system according to any one of claims 11 to 13.

15. The epigenetic modification DNA targeting system according to any one of claims 1 to 14, wherein the first gene and the second gene are independently selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3.

16. The epigenetic modification DNA targeting system according to any one of claims 1 to 15, wherein the first gene and the second gene are different.

17. The epigenetic modification DNA targeting system according to any one of claims 1 to 16, wherein the first gene and the second gene are PCSK9 and LPA, PCSK9 and MYLIP, PCSK9 and ANGPTL3, PCSK9 and APOC3, PCSK9 and APOB, LPA and MYLIP, LPA and ANGPTL3, LPA and APOC3, LPA and APOB, MYLIP and ANGPTL3, MYLIP and APOC3, MYLIP and APOB, ANGPTL3 and APOC3, ANGPTL3 and APOB, or APOC3 and APOB.

18. The epigenetic modification DNA targeting system according to any one of claims 1 to 17, wherein the first gene, the second gene, and the third gene are each independently selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB.

19. The epigenetic modification DNA targeting system according to any one of claims 13 to 18, wherein the first gene, the second gene, and the third gene are different.

20. The combination of the epigenetic modification DNA targeting system according to any one of claims 13 to 19, wherein the first gene, the second gene, and the third gene are PCSK9, LPA, and MYLIP; PCSK9, LPA, and ANGPTL3; PCSK9, LPA, and APOC3; PCSK9, LPA, and APOB; PCSK9, MYLIP, and ANGPTL3; PCSK9, MYLIP, and APOC3; PCSK9, MYLIP, and APOB; PCSK9, ANGPTL3, and APOC3; PCSK9, ANGPTL3, and APOB; PCSK9, APOC3, and APOB; LPA, MYLIP, and ANGPTL3; LPA, MYLIP, and APOC3; LPA, MYLIP, and APOB; LPA, ANGPTL3, and APOC3; LPA, ANGPTL3, and APOB; LPA, APOC3, and APOB; MYLIP, ANGPTL3, and APOC3; MYLIP, ANGPTL3, and APOB; MYLIP, APOC3, and APOB; or ANGPTL3, APOC3, and APOB.

21. The epigenetic modification DNA targeting system according to any one of claims 1 to 20, wherein at least one gene is PCSK9.

22. The epigenetic modification DNA targeting system according to any one of claims 1 to 21, wherein at least two genes are PCSK9 and LPA.

23. The epigenetic modification DNA targeting system according to any one of claims 13 to 22, wherein at least three genes are PCSK9, LPA, and MYLIP.

24. The epigenetic modification DNA targeting system according to any one of claims 1 to 23, wherein the target site of each of the plurality of genes is within the gene or its regulatory DNA element.

25. The epigenetic modification DNA targeting system according to claim 24, wherein the regulatory DNA element is an enhancer or a promoter.

26. The target sites of the first gene and the second gene are (a) to (f): (a) A target site for PCSK9 located within 500 bp from the human genome assembly GRCh38 (hg38) genomic coordinate chr1: 55,039,548. (b) A target site for LPA located within 500 bp from the hg38 genomic coordinate chr6: 160,664,275. (c) A target site for MYLIP located within 500 bp from the hg38 genomic coordinate chr6: 16,129,086. (d) A target site for ANGPTL3 located within 500 bp from the hg38 genomic coordinate chr1: 62,597,520. (e) A target site for APOC3 located within 500 bp from the hg38 genomic coordinate chr11: 116,829,907, and (f) A target site for APOB located within 500 bp from the hg38 genomic coordinate chr2: 21,044,073 The epigenetic modification DNA targeting system according to any one of claims 1 to 25, selected from two different members of the group consisting of.

27. The target sites of the first gene and the second gene are (a) to (f): (a) A target site located within 500 bp from the transcription start site of PCSK9. (b) A target site located within 500 bp from the transcription start site of LPA. (c) A target site located within 500 bp from the transcription start site of MYLIP. (d) A target site located within 500 bp from the transcription start site of ANGPTL3, (e) A target site located within 500 bp from the transcription start site of APOC3, and (f) A target site located within 500 bp from the transcription start site of APOB The epigenetic modification DNA targeting system according to any one of claims 1 to 26, which is selected from two different members of the group consisting of. **Claim 28** The target sites of the first gene, the second gene, and the third gene are (a) to (f): (a) A target site for PCSK9 located within 500 bp from the human genome assembly GRCh38 (hg38) genomic coordinate chr1: 55,039,548, (b) A target site for LPA located within 500 bp from the hg38 genomic coordinate chr6: 160,664,275, (c) A target site for MYLIP located within 500 bp from the hg38 genomic coordinate chr6: 16,129,086, (d) A target site for ANGPTL3 located within 500 bp from the hg38 genomic coordinate chr1: 62,597,520, (e) A target site for APOC3 located within 500 bp from the hg38 genomic coordinate chr11: 116,829,907, and (f) A target site for APOB located within 500 bp from the hg38 genomic coordinate chr2: 21,044,073 The epigenetic modification DNA targeting system according to any one of claims 13 to 27, which is selected from three different members of the group consisting of. **Claim 29** The target sites of the first gene, the second gene, and the third gene are (a) to (f): (a) A target site located within 500 bp from the transcription start site of PCSK9, (b) A target site located within 500 bp from the transcription start site of LPA, (c) A target site located within 500 bp from the transcription start site of MYLIP, (d) A target site located within 500 bp from the transcription start site of ANGPTL3, (e) A target site located within 500 bp from the transcription start site of APOC3, and (f) A target site located within 500 bp from the transcription start site of APOB The epigenetic modification DNA targeting system according to any one of claims 13 to 28, which is selected from three different members of the group consisting of. **Claim 30** The target sites of the first gene and the second gene are (a) to (f): A target site for PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing, optionally, (i) shown in SEQ ID NO: 3, (ii) having an adjacent portion of SEQ ID NO: 3 of at least 14 nucleotides, or (iii) a complementary sequence of (i) or (ii), the target site for said PCSK9 A target site for LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing A target site for MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing A target site for ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing A target site for APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing, and A target site for APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing The epigenetic modification DNA targeting system according to any one of claims 1 to 29, selected from two different members of the group consisting of

31. The target sites of the first gene and the second gene are (a) to (f): A target site for PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, optionally, the target site for said PCSK9 shown in SEQ ID NO: 3 A target site for LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23 A target site for MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351 (d) a target site of ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43, (e) a target site of APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53, and (f) a target site of APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377 The combination of the epigenetic modification DNA targeting system according to any one of claims 1 to 30, selected from two different members of the group consisting of.

32. The target sites of the first gene, the second gene, and the third gene are (a) to (f): (a) a target site of PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing, optionally, (i) shown in SEQ ID NO: 3, (ii) having at least 14 nucleotides of the adjacent portion of SEQ ID NO: 3, or (iii) a complementary sequence of (i) or (ii), the target site of the PCSK9, (b) a target site of LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing, (c) a target site of MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing, (d) a target site of ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing, (e) a target site of APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing, and (f) a target site of APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377, at least 14 nucleotides (nt) of its adjacent portion, or a complementary sequence of any of the foregoing A combination of the epigenetic modification DNA targeting systems according to any one of claims 13 to 31, selected from three different members of the group consisting of.

33. The target site of the first gene, the second gene, or the third gene is (a) to (f): (a) A target site of PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, and optionally, the target site of PCSK9 shown in SEQ ID NO: 3, (b) A target site of LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23, (c) A target site of MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351, (d) A target site of ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43, (e) A target site of APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53, and (f) A target site of APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377 A combination of the epigenetic modification DNA targeting systems according to any one of claims 13 to 32, selected from three different members of the group consisting of.

34. The epigenetic modification DNA targeting system according to any one of claims 11 to 33, wherein the Cas protein or a variant thereof is a variant Cas protein that is an inactivated (dCas) protein.

35. The epigenetic modification DNA targeting system according to claim 34, wherein the dCas protein lacks nuclease activity.

36. The epigenetic modification DNA targeting system according to claim 34 or claim 35, wherein the dCas protein is a dCas9 protein.

37. The epigenetic modification DNA targeting system according to claim 34 or claim 35, wherein the dCas protein is a dCas12 protein.

38. The epigenetic modification DNA targeting system according to claim 36, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

39. The epigenetic modification DNA targeting system according to claim 38, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A with reference to the numbering at the position of SEQ ID NO:

204.

40. The epigenetic modification DNA targeting system according to claim 38 or claim 39, wherein the dSaCas9 protein comprises the sequence shown in SEQ ID NO: 205, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

41. The epigenetic modification DNA targeting system according to any one of claims 38 to 40, wherein the dSaCas9 is the sequence shown in SEQ ID NO:

205.

42. The epigenetic modification DNA targeting system according to claim 36, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

43. The epigenetic modification DNA targeting system according to claim 42, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A with reference to the numbering at the position of SEQ ID NO:

206.

44. The epigenetic modification DNA targeting system according to claim 42 or claim 43, wherein the dSpCas9 comprises the sequence shown in SEQ ID NO: 207, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

45. The epigenetic modification DNA targeting system according to any one of claims 42 to 44, wherein the dSpCas9 is the sequence shown in SEQ ID NO:

207.

46. The epigenetic modification DNA targeting system according to any one of claims 11 to 45, wherein each gRNA comprises a gRNA spacer sequence complementary to the target site of each said gene.

47. The first gRNA and the second gRNA are (a) to (f): A gRNA targeting a target site of PCSK9, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329, or an adjacent portion thereof of at least 14 nt, optionally, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 66, or an adjacent portion thereof of at least 14 nt, said gRNA A gRNA targeting a target site of LPA, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 77 to 86, or an adjacent portion thereof of at least 14 nt A gRNA targeting a target site of MYLIP, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361, or an adjacent portion thereof of at least 14 nt A gRNA targeting a target site of ANGPTL3, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 97 to 106, or an adjacent portion thereof of at least 14 nt A gRNA targeting a target site of APOC3, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 107 to 116, or an adjacent portion thereof of at least 14 nt, and A gRNA targeting a target site of APOB, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383, or an adjacent portion thereof of at least 14 nt The epigenetic modification DNA targeting system according to any one of claims 11 to 46, selected from two different members of the group consisting of

48. The first gRNA, the second gRNA, and the third gRNA are (a) to (f): A gRNA targeting a target site of PCSK9, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329, or an adjacent portion thereof of at least 14 nt, optionally, said gRNA spacer sequence comprising the sequence shown in SEQ ID NO: 66, or an adjacent portion thereof of at least 14 nt, said gRNA A gRNA targeting a target site of LPA, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 77 to 86, or an adjacent portion thereof of at least 14 nt A gRNA targeting a target site of MYLIP, comprising a sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion, A gRNA targeting a target site of ANGPTL3, comprising a sequence shown in any one of SEQ ID NOs: 97 to 106, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion, A gRNA targeting a target site of APOC3, comprising a sequence shown in any one of SEQ ID NOs: 107 to 116, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion, and A gRNA targeting a target site of APOB, comprising a sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion The epigenetic modification DNA targeting system according to any one of claims 13 to 47, selected from three different members of the group consisting of

49. The epigenetic modification DNA targeting system according to any one of claims 11 to 48, wherein each gRNA independently comprises a spacer sequence having a length of 14 nt to 24 nt, or 16 nt to 22 nt.

50. The epigenetic modification DNA targeting system according to any one of claims 11 to 49, wherein each gRNA independently comprises a spacer sequence having a length of 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt.

51. The first gRNA and the second gRNA are (a) to (f): A gRNA targeting a target site of PCSK9, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329, and optionally, the gRNA spacer is the gRNA shown in SEQ ID NO: 66, A gRNA targeting a target site of LPA, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 77 to 86, A gRNA targeting a target site of MYLIP, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361, A gRNA targeting a target site of ANGPTL3, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 97 to 106, (e) A gRNA targeting the target site of APOC3, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 107 to 116, and (f) A gRNA targeting the target site of APOB, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383 The epigenetic modification DNA targeting system according to any one of claims 11 to 50, selected from two different members of the group consisting of

52. The epigenetic modification DNA targeting system according to any one of claims 43 to 45, wherein each gRNA further comprises a scaffold sequence shown in SEQ ID NO:

191.

53. The first gRNA and the second gRNA are (a) to (f): (a) A gRNA targeting the target site of PCSK9, comprising a sequence shown in any one of SEQ ID NOs: 127 to 139 or 330 to 341, and optionally, the gRNA comprising the sequence shown in SEQ ID NO: 129, (b) A gRNA targeting the target site of LPA, comprising a sequence shown in any one of SEQ ID NOs: 140 to 149, (c) A gRNA targeting the target site of MYLIP, comprising a sequence shown in any one of SEQ ID NOs: 150 to 159 or 362 to 371, (d) A gRNA targeting the target site of ANGPTL3, comprising a sequence shown in any one of SEQ ID NOs: 160 to 169, (e) A gRNA targeting the target site of APOC3, comprising a sequence shown in any one of SEQ ID NOs: 170 to 179, and (f) A gRNA targeting the target site of APOB, comprising a sequence shown in any one of SEQ ID NOs: 180 to 189 or 384 to 389 The epigenetic modification DNA targeting system according to any one of claims 15 to 52, selected from two different members of the group consisting of

54. The first gRNA and the second gRNA are (a) to (f): (a) A gRNA targeting the target site of PCSK9, shown in any one of SEQ ID NOs: 127 to 139 or 330 to 341, and optionally, the gRNA shown in SEQ ID NO: 129, (b) A gRNA targeting the target site of LPA, shown in any one of SEQ ID NOs: 140 to 149, (c) A gRNA targeting the target site of MYLIP, which is shown by any one of SEQ ID NOs: 150 to 159 or 362 to 371, (d) A gRNA targeting the target site of ANGPTL3, which is shown by any one of SEQ ID NOs: 160 to 169, (e) A gRNA targeting the target site of APOC3, which is shown by any one of SEQ ID NOs: 170 to 179, and (f) A gRNA targeting the target site of APOB, which is shown by any one of SEQ ID NOs: 180 to 189 or 384 to 389 The epigenetic modification DNA targeting system according to any one of claims 15 to 53, selected from two different members of the group consisting of

55. The first gRNA, the second gRNA, and the third gRNA are (a) to (f): (a) A gRNA targeting the target site of PCSK9, which contains a sequence shown by any one of SEQ ID NOs: 127 to 139 or 330 to 341, and optionally contains the sequence shown by SEQ ID NO: 129, the gRNA, (b) A gRNA targeting the target site of LPA, which contains a sequence shown by any one of SEQ ID NOs: 140 to 149, (c) A gRNA targeting the target site of MYLIP, which contains a sequence shown by any one of SEQ ID NOs: 150 to 159 or 362 to 371, (d) A gRNA targeting the target site of ANGPTL3, which contains a sequence shown by any one of SEQ ID NOs: 160 to 169, (e) A gRNA targeting the target site of APOC3, which contains a sequence shown by any one of SEQ ID NOs: 170 to 179, and (f) A gRNA targeting the target site of APOB, which contains a sequence shown by any one of SEQ ID NOs: 180 to 189 or 384 to 389 The combination of epigenetic modification DNA targeting according to any one of claims 15 to 54, selected from three different members of the group consisting of

56. The first gRNA, the second gRNA, and the third gRNA are (a) to (f): (a) A gRNA targeting the target site of PCSK9, which is shown by any one of SEQ ID NOs: 127 to 139 or 330 to 341, and optionally, the gRNA shown by SEQ ID NO: 129, (b) A gRNA targeting the target site of LPA, which is shown by any one of SEQ ID NOs: 140 to 149, (c) A gRNA targeting the target site of MYLIP, which is shown by any one of SEQ ID NOs: 150 to 159 or 362 to 371, (d) A gRNA targeting the target site of ANGPTL3, which is shown by any one of SEQ ID NOs: 160 to 169, (e) A gRNA targeting the target site of APOC3, which is shown by any one of SEQ ID NOs: 170 to 179, and (f) A gRNA targeting the target site of APOB, which is shown by any one of SEQ ID NOs: 180 to 189 or 384 to 389 The combination of epigenetic modification DNA targeting according to any one of claims 15 to 55, selected from three different members of the group consisting of

57. The epigenetic modification DNA targeting system according to any one of claims 11 to 56, wherein at least one gRNA contains a modified nucleotide for improving stability.

58. The combination of epigenetic modification DNA targeting according to any one of claims 1 to 57, wherein the at least one transcriptional repressor domain can reduce the transcription of the gene.

59. The epigenetic modification DNA targeting system according to any one of claims 1 to 58, wherein the at least one transcriptional repressor domain contains a DNA methyltransferase.

60. The epigenetic modification DNA targeting system according to any one of claims 1 to 59, wherein the at least one transcriptional repressor domain contains a histone methyltransferase or a repressor domain capable of mobilizing a heterochromatin inducer, and optionally, the heterochromatin inducer contains a histone methyltransferase.

61. The epigenetic modification DNA targeting system according to any one of claims 1 to 60, wherein the at least one transcriptional repressor domain contains a DNA methyltransferase and a repressor domain capable of mobilizing a heterochromatin inducer, and optionally, the heterochromatin inducer contains a histone methyltransferase.

62. The epigenetic modification DNA targeting system according to any one of claims 1 to 61, wherein the at least one transcriptional repressor domain contains a DNA methyltransferase and a histone methyltransferase.

63. One or more of the at least one transcriptional repressor domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3A-DNMT3L fusion protein domain, an ERF repressor domain, an Mxi1 repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD1 repressor domain, an EZH2 repressor domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing, the combination of epigenetic modification DNA targeting according to any one of claims 1 to 62.

64. The epigenetic modification DNA targeting system according to any one of claims 1 to 63, wherein the at least one transcriptional repressor domain comprises a KRAB domain, a DNMT3A domain, a DNMT3L domain, or a combination of any of the foregoing.

65. The epigenetic modification DNA targeting system according to any one of claims 1 to 64, wherein the at least one transcriptional repressor domain comprises a KRAB domain or a variant or portion thereof that exhibits transcriptional repressor activity.

66. The epigenetic modification DNA targeting system according to any one of claims 1 to 65, wherein the at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 193, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

67. The epigenetic modification DNA targeting system according to any one of claims 1 to 63, wherein the at least one transcriptional repressor domain comprises a DNMT3A domain or a variant or portion thereof that exhibits transcriptional repressor activity.

68. The epigenetic modification DNA targeting system according to any one of claims 1 to 63 and 67, wherein the at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 195, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

69. The epigenetic modification DNA targeting system according to any one of claims 1 to 63, wherein the at least one transcriptional repressor domain contains a DNMT3L domain exhibiting transcriptional repressor activity, or a variant or a portion thereof.

70. The epigenetic modification DNA targeting system according to any one of claims 1 to 63 and 69, wherein the at least one transcriptional repressor domain contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 197, a portion thereof, or any of the foregoing.

71. The epigenetic modification DNA targeting system according to any one of claims 1 to 63, wherein the at least one transcriptional repressor domain is a DNMT3A-DNMT3L fusion protein domain exhibiting transcriptional repressor activity or a variant thereof.

72. The epigenetic modification DNA targeting system according to any one of claims 1 to 63 and 71, wherein the at least one transcriptional repressor domain contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 199 or SEQ ID NO: 201, a portion thereof, or any of the foregoing.

73. The epigenetic modification DNA targeting system according to any one of claims 1 to 63, wherein the at least one transcriptional repressor domain contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from any one of SEQ ID NOs: 193, 195, 197, 199, 201, 220 to 226, and 283, or a domain thereof, a portion thereof, or any of the foregoing.

74. The epigenetic modification DNA targeting system according to any one of claims 1 to 73, wherein the at least one transcriptional repressor domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus of the DNA binding domain.

75. The epigenetic modification DNA targeting system according to any one of claims 1 to 74, wherein the fusion protein further contains one or more nuclear localization signals (NLSs).

76. The epigenetic modification DNA targeting system according to claim 75, wherein the fusion protein further comprises one or more linkers connecting two or more of the DNA binding domain, the at least one transcriptional repressor domain, and the one or more nuclear localization signals.

77. The epigenetic modification DNA targeting system according to any one of claims 1 to 76, wherein the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 209, 278, 280, or 282, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and optionally, the fusion protein comprises the sequence shown in SEQ ID NO:

280.

78. The epigenetic modification DNA targeting system according to any one of claims 1 to 77, wherein each of the DNA targeting modules reduces the expression of each of the respective genes with a log2 fold change of -1.0 or less.

79. The epigenetic modification DNA targeting system according to any one of claims 1 to 78, wherein the suppressed transcription of the plurality of genes in a cell or cell population results in a reduction of low density lipoprotein (LDL).

80. The epigenetic modification DNA targeting system according to claim 79, wherein the reduction of LDL is greater than the reduction of LDL resulting from the corresponding suppressed transcription of any one of the individual genes in the plurality of genes alone.

81. The epigenetic modification DNA targeting system according to claim 79 or 80, wherein the reduction of LDL occurs extracellularly.

82. The epigenetic modification DNA targeting system according to any one of claims 79 to 81, wherein the cell or cell population is a liver cell or comprises liver cells.

83. The epigenetic modification DNA targeting system according to any one of claims 79 to 82, wherein the cell or cell population is within a subject.

84. The epigenetic modification DNA targeting system according to claim 83, wherein the reduction of LDL occurs within the subject or its fluid, tissue, or organ.

85. The epigenetic modification DNA targeting system according to any one of claims 79 to 84, wherein the reduction of LDL occurs in the blood of the subject.

86. An epigenetic modification DNA targeting system for suppressing the transcription of a gene that regulates low-density lipoprotein, comprising a fusion protein containing (a) a DNA binding domain for targeting a target site of the gene and (b) at least one transcriptional repressor domain. [

87. ] The epigenetic modification DNA targeting system according to claim 86, wherein the target site of the gene is within the gene or its regulatory DNA element. [

88. ] The epigenetic modification DNA targeting system according to claim 86 or 87, which does not introduce gene disruption or DNA cleavage. [

89. ] The DNA binding domain is selected from clustered regularly interspaced short palindromic repeat associated (Cas) proteins or variants thereof, zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), meganucleases, homing endonucleases, or I-SceI enzymes or variants thereof, and optionally, the DNA binding domain comprises a catalytically inactive variant of any of the foregoing. The epigenetic modification DNA targeting system according to any one of claims 86 to 88. [

90. ] The DNA binding domain is a clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof, and the system further comprises a gRNA for targeting the DNA binding domain to the target site of the gene. The epigenetic modification DNA targeting system according to claim 89. [

91. ] An epigenetic modification DNA targeting system comprising (a) a fusion protein containing a clustered regularly interspaced short palindromic repeat associated (Cas) protein or a variant thereof and at least one transcriptional repressor domain, and (b) a gRNA targeting a target site of a gene that regulates low-density lipoprotein (LDL). [

92. ] The epigenetic modification DNA targeting system according to any one of claims 86 to 91, wherein the target site of the gene is within the gene and / or its regulatory DNA element. [

93. ] The epigenetic modification DNA targeting system according to claim 92, wherein the regulatory DNA element is a promoter or an enhancer. [

94. ] The epigenetic modification DNA targeting system according to any one of claims 86 to 93, wherein the gene is selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3.

95. The target site is (a) a target site for PCSK9 located within 500 bp from the genomic coordinate chr1:55,039,548 of the human genome assembly GRCh38 (hg38), (b) a target site for LPA located within 500 bp from the genomic coordinate chr6:160,664,275 of hg38, (c) a target site for MYLIP located within 500 bp from the genomic coordinate chr6:16,129,086 of hg38, (d) a target site for ANGPTL3 located within 500 bp from the genomic coordinate chr1:62,597,520 of hg38, (e) a target site for APOC3 located within 500 bp from the genomic coordinate chr11:116,829,907 of hg38, and (f) a target site for APOB located within 500 bp from the genomic coordinate chr2:21,044,073 of hg38 The epigenetic modification DNA targeting system according to any one of claims 86 to 94, which is selected from.

96. The target site is (a) a target site located within 500 bp from the transcription start site of PCSK9, (b) a target site located within 500 bp from the transcription start site of LPA, (c) a target site located within 500 bp from the transcription start site of MYLIP, (d) a target site located within 500 bp from the transcription start site of ANGPTL3, (e) a target site located within 500 bp from the transcription start site of APOC3, and (f) a target site located within 500 bp from the transcription start site of APOB The epigenetic modification DNA targeting system according to any one of claims 86 to 95, which is selected from.

97. The target site is (a) a target site for PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, an adjacent portion of at least 14 nucleotides (nt) thereof, or a complementary sequence of any of the foregoing, and optionally, (i) the sequence shown in SEQ ID NO: 3, (ii) an adjacent portion of at least 14 nucleotides shown in SEQ ID NO: 3, or (iii) a complementary sequence of (i) or (ii), the target site for PCSK9. A target site for LPA, having a sequence shown in any one of SEQ ID NOs: 14 to 23, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. A target site for MYLIP, having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. A target site for ANGPTL3, having a sequence shown in any one of SEQ ID NOs: 34 to 43, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. A target site for APOC3, having a sequence shown in any one of SEQ ID NOs: 44 to 53, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; and A target site for APOB, having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. The epigenetic modification DNA targeting system according to any one of claims 86 to 96, selected from the above.

98. The target site is (a) A target site for PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, and optionally having the sequence shown in SEQ ID NO: 3, the target site for PCSK9; (b) A target site for LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23; (c) A target site for MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351; (d) A target site for ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43; (e) A target site for APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53; and (f) A target site for APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377. The epigenetic modification DNA targeting system according to any one of claims 86 to 97, selected from the above.

99. The epigenetic modification DNA targeting system according to any one of claims 90 to 98, wherein the Cas protein or its variant is a variant Cas protein that is an inactivated (dCas) protein.

100. The epigenetic modification DNA targeting system according to claim 99, wherein the dCas protein lacks nuclease activity.

101. The epigenetic modification DNA targeting system according to claim 99 or 100, wherein the dCas protein is a dCas9 protein.

102. The epigenetic modification DNA targeting system according to claim 99 or 100, wherein the dCas protein is a dCas12 protein.

103. The epigenetic modification DNA targeting system according to claim 101, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

104. The epigenetic modification DNA targeting system according to claim 103, wherein the dSaCas9 contains at least one amino acid mutation selected from D10A and N580A with reference to the numbering at the position of SEQ ID NO:

204.

105. The epigenetic modification DNA targeting system according to claim 103 or 104, wherein the dSaCas9 protein contains the sequence shown in SEQ ID NO: 205, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

106. The epigenetic modification DNA targeting system according to any one of claims 103 to 105, wherein the dSaCas9 is the sequence shown in SEQ ID NO:

205.

107. The epigenetic modification DNA targeting system according to claim 101, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

108. The epigenetic modification DNA targeting system according to claim 107, wherein the dSpCas9 protein contains at least one amino acid mutation selected from D10A and H840A with reference to the numbering at the position of SEQ ID NO:

206.

109. The epi-genetic modification DNA targeting system according to claim 35 or 108, wherein the dSpCas9 comprises the sequence shown in SEQ ID NO: 207, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. **Claim 110** The epi-genetic modification DNA targeting system according to any one of claims 107 to 109, wherein the dSpCas9 is the sequence shown in SEQ ID NO:

207. **Claim 111** The epi-genetic modification DNA targeting system according to any one of claims 90 to 110, wherein the gRNA comprises a gRNA spacer complementary to the target site of the gene. **Claim 112** The gRNA is a gRNA targeting the target site of PCSK9, comprising: (a) a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329, or at least a 14-nt adjacent portion thereof, and optionally, the gRNA spacer comprises the sequence shown in SEQ ID NO: 66, or at least a 14-nt adjacent portion thereof; a gRNA targeting the target site of LPA, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 77 to 86, or at least a 14-nt adjacent portion thereof; a gRNA targeting the target site of MYLIP, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361, or at least a 14-nt adjacent portion thereof; a gRNA targeting the target site of ANGPTL3, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 97 to 106, or at least a 14-nt adjacent portion thereof; a gRNA targeting the target site of APOC3, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 107 to 116, or at least a 14-nt adjacent portion thereof; and a gRNA targeting the target site of APOB, comprising a gRNA spacer sequence comprising the sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383, or at least a 14-nt adjacent portion thereof selected from the epi-genetic modification DNA targeting systems according to any one of claims 90 to 111. **Claim 113** The epigenetic modification DNA targeting system according to any one of claims 90 to 112, wherein the gRNA comprises a spacer sequence having a length of 14 nt to 24 nt, or 16 nt to 22 nt.

114. The epigenetic modification DNA targeting system according to any one of claims 90 to 113, wherein the gRNA comprises a spacer sequence having a length of 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt.

115. The gRNA is a gRNA targeting a target site of PCSK9, which comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329, and optionally, the gRNA spacer sequence is shown in SEQ ID NO: 66, the gRNA; a gRNA targeting a target site of LPA, which comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 77 to 86; a gRNA targeting a target site of MYLIP, which comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361; a gRNA targeting a target site of ANGPTL3, which comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 97 to 106; a gRNA targeting a target site of APOC3, which comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 107 to 116, and a gRNA targeting a target site of APOB, which comprises a gRNA spacer sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383 selected from the epigenetic modification DNA targeting systems according to any one of claims 90 to 114.

116. The epigenetic modification DNA targeting system according to any one of claims 107 to 115, wherein the gRNA further comprises a scaffold sequence shown in SEQ ID NO:

191.

117. The gRNA is a gRNA targeting a target site of PCSK9, which comprises a sequence shown in any one of SEQ ID NOs: 127 to 139 or 330 to 341, and optionally, the gRNA comprises a sequence shown in SEQ ID NO: 129; a gRNA targeting a target site of LPA, which comprises a sequence shown in any one of SEQ ID NOs: 140 to 149; (c) A gRNA targeting the target site of MYLIP, comprising the sequence shown in any one of SEQ ID NOs: 150 to 159 or 362 to 371; (d) A gRNA targeting the target site of ANGPTL3, comprising the sequence shown in any one of SEQ ID NOs: 160 to 169; (e) A gRNA targeting the target site of APOC3, comprising the sequence shown in any one of SEQ ID NOs: 170 to 179, and (f) A gRNA targeting the target site of APOB, comprising the sequence shown in any one of SEQ ID NOs: 180 to 189 or 384 to 389 The epigenetic modification DNA targeting system according to any one of claims 107 to 116, selected from the above.

118. The gRNA is (a) A gRNA targeting the target site of PCSK9, shown in any one of SEQ ID NOs: 127 to 139 or 330 to 341, and optionally, the gRNA shown in SEQ ID NO: 129; (b) A gRNA targeting the target site of LPA, shown in any one of SEQ ID NOs: 140 to 149; (c) A gRNA targeting the target site of MYLIP, shown in any one of SEQ ID NOs: 150 to 159 or 362 to 371; (d) A gRNA targeting the target site of ANGPTL3, shown in any one of SEQ ID NOs: 160 to 169; (e) A gRNA targeting the target site of APOC3, shown in any one of SEQ ID NOs: 170 to 179, and (f) A gRNA targeting the target site of APOB, shown in any one of SEQ ID NOs: 180 to 189 or 384 to 389 The epigenetic modification DNA targeting system according to any one of claims 107 to 117, selected from the above.

119. The epigenetic modification DNA targeting system according to any one of claims 90 to 118, wherein the gRNA contains modified nucleotides for improving stability.

120. The combination of epigenetic modification DNA targeting according to any one of claims 86 to 119, wherein the at least one transcriptional repressor domain can reduce the transcription of the gene.

121. The combination for epigenetic modification DNA targeting according to any one of claims 86 to 120, wherein the transcription repressor domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3A-DNMT3L fusion protein domain, an ERF repressor domain, an Mxi1 repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD1 repressor domain, an EZH2 repressor domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing.

122. The epigenetic modification DNA targeting system according to any one of claims 86 to 121, wherein the transcription repressor domain is a KRAB domain, a DNMT3A domain, or a DNMT3L domain, or a combination of any of the foregoing.

123. The epigenetic modification DNA targeting system according to any one of claims 86 to 122, wherein the at least one transcription repressor domain comprises a KRAB domain or a variant or portion thereof that exhibits transcription repressor activity.

124. The epigenetic modification DNA targeting system according to any one of claims 86 to 123, wherein the at least one transcription repressor domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 193, a portion thereof, or any of the foregoing.

125. The epigenetic modification DNA targeting system according to any one of claims 86 to 122, wherein the at least one transcription repressor domain comprises a DNMT3A domain or a variant or portion thereof that exhibits transcription repressor activity.

126. The epigenetic modification DNA targeting system according to any one of claims 86 to 122 and 125, wherein the at least one transcription repressor domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence shown in SEQ ID NO: 195, a portion thereof, or any of the foregoing.

127. The epigenetic modification DNA targeting system according to any one of claims 86 to 122, wherein the at least one transcriptional repressor domain comprises a DNMT3L domain exhibiting transcriptional repressor activity, or a variant or a portion thereof.

128. The epigenetic modification DNA targeting system according to any one of claims 86 to 122 and 127, wherein the at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 197, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

129. The epigenetic modification DNA targeting system according to any one of claims 86 to 122, wherein the at least one transcriptional repressor domain is a DNMT3A-DNMT3L fusion protein domain exhibiting transcriptional repressor activity or a variant thereof.

130. The epigenetic modification DNA targeting system according to any one of claims 86 to 122 and 129, wherein the at least one transcriptional repressor domain comprises the sequence shown in SEQ ID NO: 199 or SEQ ID NO: 201, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

131. The epigenetic modification DNA targeting system according to any one of claims 86 to 121, wherein the at least one transcriptional repressor domain comprises a sequence selected from any one of SEQ ID NOs: 193, 195, 197, 199, 201, 220 to 226, and 283, or a domain thereof, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

132. The epigenetic modification DNA targeting system according to any one of claims 86 to 131, wherein the at least one transcriptional repressor domain is fused to the N-terminus, C-terminus, or both the N-terminus and the C-terminus of the DNA binding domain.

133. The epigenetic modification DNA targeting system according to any one of claims 86 to 132, wherein the fusion protein further comprises one or more nuclear localization signals (NLSs).

134. The epigenetic modification DNA targeting system according to claim 133, wherein the fusion protein further comprises one or more linkers connecting two or more of the DNA binding domain, the at least one transcriptional repressor domain, and the one or more nuclear localization signals.

135. The fusion protein comprises a sequence shown in any one of SEQ ID NOs: 209, 278, 280, or 282, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Optionally, the fusion protein is the one shown in SEQ ID NO:

280. The epigenetic modification DNA targeting system according to any one of claims 86 to 134.

136. The fusion protein comprises a sequence shown in SEQ ID NO: 280, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 280, and a spacer sequence shown in SEQ ID NO: 66, and a gRNA targeting a target site of PCSK9. Optionally, the gRNA has a sequence shown in SEQ ID NO:

129. The epigenetic modification DNA targeting system according to any one of claims 90 to 135.

137. In the epigenetic modification DNA targeting system according to any one of claims 86 to 136, each of the DNA targeting modules reduces the expression of each of the respective genes with a log2 fold change of -1.0 or less.

138. The repressed transcription of the gene in the cell or cell population results in a reduction in low density lipoprotein (LDL). Optionally, the reduction in LDL occurs extracellularly. The epigenetic modification DNA targeting system according to any one of claims 86 to 137.

139. The epigenetic modification DNA targeting system according to claim 138, wherein the cell or cell population is a liver cell or comprises liver cells.

140. The epigenetic modification DNA targeting system according to any one of claims 138 to 139, wherein the cell or cell population is within a subject. **Claim 141** The epigenetic modification DNA targeting system according to claim 140, wherein the reduction of LDL occurs within the subject or its fluid, tissue, or organ. **Claim 142** The epigenetic modification DNA targeting system according to any one of claims 138 to 141, wherein the reduction of LDL occurs in the blood of the subject. **Claim 143** A combination of epigenetic modification DNA targeting systems, comprising at least two of the DNA targeting systems according to any one of claims 86 to 142, wherein each DNA targeting system suppresses the transcription of a different gene. **Claim 144** The combination of epigenetic modification DNA targeting systems according to claim 143, wherein each DNA targeting system suppresses the transcription of a different gene. **Claim 145** A guide RNA (gRNA) that targets a target site of a gene that regulates low-density lipoprotein (LDL). **Claim 146** The gRNA according to claim 145, wherein the gene is selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3. **Claim 147** The gRNA according to claim 145 or 146, wherein the target site of the gene is within the gene or its regulatory DNA element. **Claim 148** The gRNA according to claim 147, wherein the regulatory DNA element is an enhancer or a promoter. **Claim 149** The target site is (a) a target site for PCSK9 located within 500 bp from the human genome assembly GRCh38 (hg38) genomic coordinate chr1:55,039,548, (b) a target site for LPA located within 500 bp from the hg38 genomic coordinate chr6:160,664,275, (c) a target site for MYLIP located within 500 bp from the hg38 genomic coordinate chr6:16,129,086, (d) a target site for ANGPTL3 located within 500 bp from the hg38 genomic coordinate chr1:62,597,520, (e) a target site for APOC3 located within 500 bp from the hg38 genomic coordinate chr11:116,829,907, and A target site for APOB located within 500 bp from the hg38 genomic coordinate chr2:21,044,073 The gRNA according to any one of claims 145 to 148, selected from the group consisting of **Claim 150** The target site is (a) A target site located within 500 bp from the transcription start site of PCSK9, (b) A target site located within 500 bp from the transcription start site of LPA, (c) A target site located within 500 bp from the transcription start site of MYLIP, (d) A target site located within 500 bp from the transcription start site of ANGPTL3, (e) A target site located within 500 bp from the transcription start site of APOC3, and (f) A target site located within 500 bp from the transcription start site of APOB The gRNA according to any one of claims 145 to 149, selected from the group consisting of **Claim 151** The target site is (a) A target site for PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing, optionally, (i) a sequence shown in SEQ ID NO: 3, (ii) an adjacent portion of SEQ ID NO: 3 of at least 14 nt, or (iii) a complementary sequence of (i) or (ii), the target site for PCSK9; (b) A target site for LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) A target site for MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) A target site for ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) A target site for APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing, and A target site for APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377, an adjacent portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing The gRNA according to any one of claims 145 to 150, selected from **Claim 152** wherein the target site is (a) A target site for PCSK9 having a sequence shown in any one of SEQ ID NOs: 1 to 13 or 306 to 317, optionally having the sequence shown in SEQ ID NO: 3, the target site for PCSK9 (b) A target site for LPA having a sequence shown in any one of SEQ ID NOs: 14 to 23 (c) A target site for MYLIP having a sequence shown in any one of SEQ ID NOs: 24 to 33 or 342 to 351 (d) A target site for ANGPTL3 having a sequence shown in any one of SEQ ID NOs: 34 to 43 (e) A target site for APOC3 having a sequence shown in any one of SEQ ID NOs: 44 to 53, and (f) A target site for APOB having a sequence shown in any one of SEQ ID NOs: 54 to 63 or 372 to 377 The gRNA according to any one of claims 145 to 151, selected from **Claim 153** (a) A gRNA targeting a target site for PCSK9, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329, or an adjacent portion thereof of at least 14 nt, optionally wherein the gRNA spacer sequence comprises the sequence shown in SEQ ID NO: 66, or an adjacent portion thereof of at least 14 nt, the gRNA (b) A gRNA targeting a target site for LPA, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 77 to 86, or an adjacent portion thereof of at least 14 nt (c) A gRNA targeting a target site for MYLIP, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361, or an adjacent portion thereof of at least 14 nt (d) A gRNA targeting a target site for ANGPTL3, comprising a gRNA spacer sequence comprising a sequence shown in any one of SEQ ID NOs: 97 to 106, or an adjacent portion thereof of at least 14 nt (e) A gRNA targeting the target site of APOC3, comprising a sequence shown in any one of SEQ ID NOs: 107 to 116, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion, and (f) A gRNA targeting the target site of APOB, comprising a sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383, or a gRNA spacer sequence comprising at least 14 nt of its adjacent portion The gRNA according to any one of claims 145 to 152, selected from

154. The gRNA according to any one of claims 145 to 153, comprising a spacer sequence having a length of 14 nt to 24 nt, or 16 nt to 22 nt.

155. The gRNA according to any one of claims 145 to 154, comprising a spacer sequence having a length of 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt.

156. (a) A gRNA targeting the target site of PCSK9, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 64 to 76 or 318 to 329, optionally, the gRNA spacer is the gRNA shown in SEQ ID NO: 66, (b) A gRNA targeting the target site of LPA, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 77 to 86. (c) A gRNA targeting the target site of MYLIP, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 87 to 96 or 352 to 361. (d) A gRNA targeting the target site of ANGPTL3, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 97 to 106. (e) A gRNA targeting the target site of APOC3, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 107 to 116, and (f) A gRNA targeting the target site of APOB, comprising a gRNA spacer sequence shown in any one of SEQ ID NOs: 117 to 126 or 378 to 383 The gRNA according to any one of claims 145 to 155, selected from

157. The gRNA according to any one of claims 145 to 156, further comprising a scaffold sequence shown in SEQ ID NO:

191.

158. A gRNA targeting a target site of PCSK9, comprising a sequence shown in any one of SEQ ID NOs: 127 to 139 or 330 to 341, and optionally comprising the sequence shown in SEQ ID NO: 129, said gRNA, A gRNA targeting a target site of LPA, comprising a sequence shown in any one of SEQ ID NOs: 140 to 149, A gRNA targeting a target site of MYLIP, comprising a sequence shown in any one of SEQ ID NOs: 150 to 159 or 362 to 371, A gRNA targeting a target site of ANGPTL3, comprising a sequence shown in any one of SEQ ID NOs: 160 to 169, A gRNA targeting a target site of APOC3, comprising a sequence shown in any one of SEQ ID NOs: 170 to 179, or A gRNA targeting a target site of APOB, comprising a sequence shown in any one of SEQ ID NOs: 180 to 189 or 384 to 389 The gRNA according to any one of claims 145 to 157, selected from

159. A gRNA targeting a target site of PCSK9, shown in any one of SEQ ID NOs: 127 to 139 or 330 to 341, and optionally, said gRNA shown in SEQ ID NO: 129, A gRNA targeting a target site of LPA, comprising a sequence shown in any one of SEQ ID NOs: 140 to 149, A gRNA targeting a target site of MYLIP, comprising a sequence shown in any one of SEQ ID NOs: 150 to 159 or 362 to 371, A gRNA targeting a target site of ANGPTL3, comprising a sequence shown in any one of SEQ ID NOs: 160 to 169, A gRNA targeting a target site of APOC3, comprising a sequence shown in any one of SEQ ID NOs: 170 to 179, or A gRNA targeting a target site of APOB, comprising a sequence shown in any one of SEQ ID NOs: 180 to 189 or 384 to 389 The gRNA according to any one of claims 145 to 158, selected from

160. The gRNA according to any one of claims 145 to 159, comprising a modified nucleotide for improving stability.

161. A plurality of gRNAs comprising at least a first gRNA and a second gRNA, wherein the first gRNA targets a target site of a first gene that regulates low-density lipoprotein (LDL), and the second gRNA targets a target site of a second gene that regulates LDL.

162. Comprising a third gRNA that targets a target site of a third gene that regulates LDL, Optionally, a fourth gRNA that targets a target site of a fourth gene that regulates LDL, optionally, a fifth gRNA that targets a target site of a fifth gene that regulates LDL, and / or optionally, a sixth gRNA that targets a target site of a sixth gene that regulates LDL. The plurality of gRNAs according to claim 161.

163. The plurality of gRNAs according to claim 161 or 162, wherein each gRNA is selected from the gRNAs according to any one of claims 141 to 156.

164. The plurality of gRNAs according to any one of claims 161 to 163, wherein the first gene and the second gene are independently selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3.

165. The plurality of gRNAs according to any one of claims 161 to 164, wherein the first gene and the second gene are different.

166. The plurality of gRNAs according to any one of claims 161 to 165, wherein the first gene and the second gene are PCSK9 and LPA, PCSK9 and MYLIP, PCSK9 and ANGPTL3, PCSK9 and APOC3, PCSK9 and APOB, LPA and MYLIP, LPA and ANGPTL3, LPA and APOC3, LPA and APOB, MYLIP and ANGPTL3, MYLIP and APOC3, MYLIP and APOB, ANGPTL3 and APOC3, ANGPTL3 and APOB, or APOC3 and APOB.

167. The plurality of gRNAs according to any one of claims 162 to 166, wherein the first gene, the second gene, and the third gene are each independently selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOC3, and APOB.

168. The plurality of gRNAs according to any one of claims 162 to 167, wherein the first gene, the second gene, and the third gene are different.

169. The first gene, the second gene, and the third gene are PCSK9, LPA, and MYLIP; PCSK9, LPA, and ANGPTL3; PCSK9, LPA, and APOC3; PCSK9, LPA, and APOB; PCSK9, MYLIP, and ANGPTL3; PCSK9, MYLIP, and APOC3; PCSK9, MYLIP, and APOB; PCSK9, ANGPTL3, and APOC3; PCSK9, ANGPTL3, and APOB; PCSK9, APOC3, and APOB; LPA, MYLIP, and ANGPTL3; LPA, MYLIP, and APOC3; LPA, MYLIP, and APOB; LPA, ANGPTL3, and APOC3; LPA, ANGPTL3, and APOB; LPA, APOC3, and APOB; MYLIP, ANGPTL3, and APOC3; MYLIP, ANGPTL3, and APOB; MYLIP, APOC3, and APOB; or ANGPTL3, APOC3, and APOB. The plurality of gRNAs according to any one of claims 162 to 168.

170. The plurality of gRNAs according to any one of claims 161 to 169, wherein at least one gene is PCSK9.

171. The plurality of gRNAs according to any one of claims 161 to 170, wherein at least two genes are PCSK9 and LPA.

172. The plurality of gRNAs according to any one of claims 161 to 171, wherein at least three genes are PCSK9, LPA, and MYLIP.

173. The plurality of gRNAs according to any one of claims 161 to 172, wherein each of the target sites of the plurality of genes is within the gene or its regulatory DNA element.

174. (a) A clustered regularly interspaced short palindromic repeat (Cas) protein or a variant thereof, and (b) The gRNA according to any one of claims 145 to 160, or the plurality of gRNAs according to any one of claims 161 to 173 A combination of Cas-guide RNA (gRNA) comprising.

175. The combination of Cas-gRNA according to claim 174, wherein the Cas protein or its variant is a variant Cas protein that is an inactivated (dCas) protein.

176. The combination of Cas-gRNA according to claim 175, wherein the dCas protein lacks nuclease activity.

177. The combination of Cas-gRNA according to claim 175 or claim 176, wherein the dCas protein is a dCas9 protein.

178. The combination of Cas-gRNA according to claim 175 or claim 176, wherein the dCas protein is a dCas12 protein.

179. The combination of Cas-gRNA according to claim 177, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

180. The combination of Cas-gRNA according to claim 179, wherein the dSaCas9 contains at least one amino acid mutation selected from D10A and N580A with reference to the numbering of the positions of SEQ ID NO:

204.

181. The combination of Cas-gRNA according to claim 179 or claim 180, wherein the dSaCas9 protein contains the sequence shown in SEQ ID NO: 205, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

182. The combination of Cas-gRNA according to any one of claims 179 to 181, wherein the dSaCas9 is the sequence shown in SEQ ID NO:

205.

183. The combination of Cas-gRNA according to claim 177, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

184. The combination of Cas-gRNA according to claim 183, wherein the dSpCas9 protein contains at least one amino acid mutation selected from D10A and H840A with reference to the numbering of the positions of SEQ ID NO:

206.

185. The combination of Cas-gRNA according to claim 183 or claim 184, wherein the dSpCas9 comprises the sequence shown in SEQ ID NO: 207, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

186. The combination of Cas-gRNA according to any one of claims 183 to 185, wherein the dSpCas9 is the sequence shown in SEQ ID NO:

207.

187. A polynucleotide encoding an epigenetic modification DNA targeting system according to any one of claims 1 to 142, a combination of epigenetic modification DNA targeting systems according to claim 143 or 144, a gRNA according to any one of claims 145 to 160, a plurality of gRNAs according to any one of claims 161 to 173, a combination of Cas-gRNA according to any one of claims 174 to 186, or any part or component thereof described above.

188. A polynucleotide encoding an epigenetic modification DNA targeting system according to any one of claims 1 to 142.

189. A polynucleotide encoding at least one DNA targeting module of an epigenetic modification DNA targeting system according to any one of claims 1 to 11 and 13 to 85.

190. A polynucleotide encoding the fusion protein of the epigenetic modification DNA targeting system according to any one of claims 11 to 85 and the at least first gRNA and second gRNA.

191. A polynucleotide encoding the fusion protein of an epigenetic modification DNA targeting system according to any one of claims 86 to 142.

192. A polynucleotide encoding the fusion protein and gRNA of an epigenetic modification DNA targeting system according to any one of claims 90 to 142.

193. A polynucleotide encoding a combination of epigenetic modification DNA targeting systems according to claim 143 or 144.

194. A polynucleotide encoding a gRNA according to any one of claims 145 to 160.

195. A polynucleotide encoding a plurality of gRNAs according to any one of claims 161 to 173.

196. A polynucleotide encoding a combination of Cas-gRNAs according to any one of claims 174 to 186.

197. A plurality of polynucleotides encoding an epigenetic modification DNA targeting system according to any one of claims 1 to 142, a combination of epigenetic modification DNA targeting systems according to claim 143 or 144, a plurality of gRNAs according to any one of claims 161 to 173, a combination of Cas-gRNAs according to any one of claims 174 to 186, or any part or component thereof described above.

198. A plurality of polynucleotides encoding an epigenetic modification DNA targeting system according to any one of claims 1 to 142.

199. A plurality of polynucleotides encoding at least one DNA targeting module of an epigenetic modification DNA targeting system according to any one of claims 1 to 11 and 13 to 85.

200. A plurality of polynucleotides encoding the fusion protein of the epigenetic modification DNA targeting system according to any one of claims 11 to 85 and the at least first gRNA and second gRNA.

201. A plurality of polynucleotides encoding the fusion protein of an epigenetic modification DNA targeting system according to any one of claims 86 to 142.

202. A plurality of polynucleotides encoding the fusion protein and gRNA of an epigenetic modification DNA targeting system according to any one of claims 90 to 142.

203. A plurality of polynucleotides encoding a combination of epigenetic modification DNA targeting systems according to claim 143 or 144.

204. A plurality of polynucleotides encoding a plurality of gRNAs according to any one of claims 161 to 173.

205. A plurality of polynucleotides encoding a combination of Cas-gRNAs according to any one of claims 174 to 186.

206. A vector comprising a polynucleotide according to any one of claims 187 to 196.

207. A vector comprising a plurality of polynucleotides according to any one of claims 197 to 205.

208. The vector according to claim 206 or claim 207, which is a viral vector.

209. The vector according to claim 208, which is an adeno-associated virus (AAV) vector.

210. The vector according to claim 209, which is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

211. The vector according to claim 208, which is a lentiviral vector.

212. The vector according to claim 206 or claim 207, which is a non-viral vector.

213. The vector according to claim 212, wherein the non-viral vector is selected from lipid nanoparticles, liposomes, exosomes, or cell-penetrating peptides.

214. The vector according to claim 212, wherein the non-viral vector is a lipid nanoparticle.

215. The vector according to claim 214, wherein the lipid nanoparticle contains an amino sugar derivative of galactose and optionally an N-acetylgalactosamine (GalNAc) moiety.

216. The vector according to any one of claims 206 to 215, which exhibits hepatocyte tropism.

217. A lipid nanoparticle comprising the polynucleotide according to any one of claims 187 to 196 or a plurality of polynucleotides according to any one of claims 197 to 205.

218. A method for reducing the transcription of at least two genes in a cell or cell population, the method comprising administering to the cell or cell population the epigenetic modification DNA targeting system according to any one of claims 1 to 142, the combination of epigenetic modification DNA targeting systems according to claim 143 or 144, the gRNA according to any one of claims 145 to 160, the plurality of gRNAs according to any one of claims 161 to 173, the combination of Cas-gRNA according to any one of claims 174 to 186, the polynucleotide according to any one of claims 187 to 196, the plurality of polynucleotides according to any one of claims 197 to 205, the vector according to any one of claims 206 to 216, or the lipid nanoparticle according to claim 217, or any part or component thereof.

219. The method according to claim 218, wherein the at least two genes are epigenetically modified.

220. The method according to claim 218 or claim 219, wherein the transcription of each of the at least two genes is reduced as compared to an equivalent cell or cell population not subjected to the method.

221. The method according to any one of claims 218 to 220, wherein the transcription of each of the at least two genes is reduced by at least about 1.2-fold, 1.25-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.75-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold.

222. The method according to any one of claims 218 to 221, wherein the reduced transcription of each of the at least two genes results in a reduction of low density lipoprotein (LDL).

223. The method according to claim 222, wherein the reduction of LDL resulting from the reduced transcription of each of the at least two genes is greater than the reduction of LDL resulting from the corresponding reduced transcription of any individual one of the at least two genes alone.

224. A method for reducing LDL, comprising introducing into a cell or cell population an epigenetic modification DNA targeting system according to any one of claims 1 to 142, a combination of epigenetic modification DNA targeting systems according to claim 143 or 144, a gRNA according to any one of claims 145 to 160, a plurality of gRNAs according to any one of claims 161 to 173, a combination of Cas-gRNAs according to any one of claims 174 to 186, a polynucleotide according to any one of claims 187 to 196, a plurality of polynucleotides according to any one of claims 197 to 205, a vector according to any one of claims 206 to 216, the lipid nanoparticle according to claim 217, or any part or component of the foregoing.

225. The method according to any one of claims 218 to 224, wherein the cell or cell population is a liver cell or comprises liver cells.

226. The method according to any one of claims 218 to 225, wherein the cell or cell population is in a subject and the method is performed in vivo.

227. The method according to claim 226, wherein LDL is reduced in the subject or its fluid, tissue, or organ.

228. The method according to any one of claims 218 to 227, wherein LDL is reduced in the blood of the subject.

229. The method according to any one of claims 226 to 228, wherein the subject is a human.

230. The method according to any one of claims 226 to 229, wherein the subject has or is suspected of having a disease, condition, or disorder, and optionally, the disease, condition, or disorder is a cardiovascular disease.

231. The method according to any one of claims 226 to 230, wherein the subject has or is suspected of having one or more of an increase in the level of low-density lipoprotein in the blood, an increased risk of cardiovascular disease, an increased risk of early-onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation of the low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation of APOB, a gain-of-function mutation of PCSK9, and familial hypercholesterolemia.

232. The method according to any one of claims 226 to 231, wherein the subject has or is suspected of having familial hypercholesterolemia.

233. A pharmaceutical composition comprising an epigenetic modification DNA targeting system according to any one of claims 1 to 142, a combination of epigenetic modification DNA targeting systems according to claim 143 or 144, a gRNA according to any one of claims 145 to 160, a plurality of gRNAs according to any one of claims 161 to 173, a combination of Cas-gRNA according to any one of claims 174 to 186, a polynucleotide according to any one of claims 187 to 196, a plurality of polynucleotides according to any one of claims 197 to 205, a vector according to any one of claims 206 to 216, a lipid nanoparticle according to claim 217, or any part or component of the foregoing.

234. The pharmaceutical composition according to claim 233, for use in the treatment of a disease, condition, or disorder in a subject, and optionally, the disease, condition, or disorder is a cardiovascular disease.

235. Use of the pharmaceutical composition according to claim 233 in the manufacture of a medicament for treating a disease, condition, or disorder in a subject, and optionally, the disease, condition, or disorder is a cardiovascular disease.

236. The pharmaceutical composition according to claim 234, or the use according to claim 235, wherein the subject has or is suspected of having a disease, condition, or disorder, and optionally, the disease, condition, or disorder is a cardiovascular disease.

237. The pharmaceutical composition or use according to any one of claims 234 to 236, wherein the subject has or is suspected of having one or more of an increase in the level of low-density lipoprotein in the blood, an increased risk of cardiovascular disease, an increased risk of early-onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation of the low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation of APOB, a gain-of-function mutation of PCSK9, and familial hypercholesterolemia.

238. The pharmaceutical composition or use according to any one of claims 234 to 237, wherein the subject has or is suspected of having familial hypercholesterolemia.

239. [[ID=I6]]The pharmaceutical composition or use according to any one of claims 234 to 238, wherein the pharmaceutical composition is to be administered to the subject in vivo.

240. The pharmaceutical composition or use according to claim 239, wherein the pharmaceutical composition targets or is to be administered to the liver of the subject.

241. The pharmaceutical composition or use according to claim 239 or 240, wherein the expression of at least two genes is reduced in the cells of the subject after administration of the pharmaceutical composition.

242. The pharmaceutical composition or use according to any one of claims 239 to 241, wherein the expression of at least two genes is reduced in the liver cells of the subject after administration of the pharmaceutical composition.

243. The pharmaceutical composition or use according to claim 241 or 242, wherein the at least two genes are selected from the group consisting of PCSK9, LPA, MYLIP, ANGPTL3, APOB, and APOC3.

244. A method for treating a disease, condition, or disorder associated with elevated low-density lipoprotein (LDL) in a subject in need thereof, comprising administering to the subject the epigenetic modification DNA targeting system according to any one of claims 1 to 142, the combination of epigenetic modification DNA targeting systems according to claim 143 or 144, the gRNA according to any one of claims 145 to 160, the plurality of gRNAs according to any one of claims 161 to 173, the combination of Cas-gRNA according to any one of claims 174 to 186, the polynucleotide according to any one of claims 187 to 196, the plurality of polynucleotides according to any one of claims 197 to 205, the vector according to any one of claims 206 to 216, the lipid nanoparticle according to claim 217, the pharmaceutical composition according to any one of claims 233 to 243, or any part or component of the foregoing.

245. The method according to claim 244, wherein the disease, condition, or disorder associated with elevated LDL is a cardiovascular disease.

246. The method according to claim 244 or claim 245, wherein the subject has or is suspected of having one or more of an increase in the level of low-density lipoprotein in the blood, an increased risk of cardiovascular disease, an increased risk of early-onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in the low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia.

247. A method for treating familial hypercholesterolemia in a subject, comprising administering to the subject the epigenetic modification DNA targeting system according to any one of claims 1 to 142, the combination of epigenetic modification DNA targeting systems according to claim 143 or 144, the gRNA according to any one of claims 145 to 160, the plurality of gRNAs according to any one of claims 161 to 173, the combination of Cas-gRNA according to any one of claims 174 to 186, the polynucleotide according to any one of claims 187 to 196, the plurality of polynucleotides according to any one of claims 197 to 205, the vector according to any one of claims 206 to 216, the lipid nanoparticle according to claim 217, the pharmaceutical composition according to any one of claims 233 to 243, or any part or component of the foregoing.

248. The method according to any one of claims 218 to 232 and 244 to 247, wherein the administration is a single-dose injection into the subject.

249. The method according to any one of claims 218 to 232 and 244 to 247, wherein the administration is repeated at least once, optionally a plurality of times, at regular intervals.

250. The method according to any one of claims 218 to 232, 244 to 247 and 249, wherein the administration is a multi-dose administration comprising at least a first dose and a second dose.

251. The method according to claim 250, wherein the first dose and the second dose are the same.

252. The method according to claim 250, wherein the second dose is lower than the first dose, and optionally, the second dose is 25% to 75% of the first dose (e.g., about 30%, about 40%, about 50%, about 60%, or about 70%, or a percentage between any of the foregoing).

253. The method according to claim 250, wherein the second dose is higher than the first dose, and optionally, the second dose is 150% to 500% of the first dose (e.g., about 200%, about 300%, about 400%, or about 500%, or a percentage between any of the foregoing).

254. The method according to any one of claims 218 to 232 and 244 to 253, wherein the lipid nanoparticle according to claim 212 is administered to the subject.

255. The pharmaceutical composition or use according to any one of claims 233 to 243, wherein the pharmaceutical composition is for single-dose injection into the subject.

256. The pharmaceutical composition or use according to any one of claims 233 to 243, wherein the pharmaceutical composition is for repeated-dose administration, optionally multiple administrations at regular intervals.

257. The pharmaceutical composition or use according to any one of claims 233 to 243 and 256, wherein the administration is a multiple-dose administration comprising at least a first dose and a second dose.

258. The pharmaceutical composition or use according to claim 257, wherein the first dose and the second dose are the same.

259. The pharmaceutical composition or use according to claim 257, wherein the second dose is lower than the first dose, and optionally, the second dose is 25% to 75% of the first dose (e.g., about 30%, about 40%, about 50%, about 60%, or about 70%, or a percentage between any of the foregoing).

260. The pharmaceutical composition or use according to claim 257, wherein the second dose is higher than the first dose, and optionally, the second dose is 150% to 500% of the first dose (e.g., about 200%, about 300%, about 400%, or about 500%, or a percentage between any of the foregoing).

261. The pharmaceutical composition or use according to any one of claims 233 to 243, and 255 to 260, wherein the pharmaceutical composition comprises the lipid nanoparticles according to claim 217.

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