In VIVO gene editing with crispr systems

Abstract

The present disclosure provides methods and compositions for therapeutic use, where the methods and compositions include the use of the Cascade effector complex. The Cascade effector complex is used to perform therapeutic genome editing in vivo in somatic cells of an organism.

Description

PATENT Docket No.: CB 1062.30 IN VIVO GENE EDITING WITH CRISPR SYSTEMSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to a U.S. provisional patent application Ser. No.63 / 763,519 filed on February 26, 2025 which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] Not applicable.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 2, 2025, is named CBI062_30_SL.xml and is 395,282 bytes in size. FIELD OF THE INVENTION

[0004] The present disclosure relates generally to the field of gene therapies such as somatic in vivo gene therapies utilizing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems.BACKGROUND OF THE INVENTION

[0005] Gene therapy holds the promise of treating a variety of genetic and acquired diseases associated with aberrant expression of genes in cells of an organ. Currently approved methods of gene therapy are limited to transient expression of a functional copy of the gene whose expression was diminished or abolished by a mutation. For example, ZOLGENSMA® is an AAV -based therapy for spinal muscular atrophy that involves delivery of a functional copy of the SMN1 gene. Similarly, ELEVYDIS® is an AAV-based therapy for Duchenne muscular dystrophy that involves delivery of the micro-dystrophin gene. AAV-based gene therapies are currently being developed for retinal diseases. In vivo gene delivery has also been attempted in the oncology space. Recent attempts at CAR-T cell therapy involve delivery of the chimeric antigen receptor (CAR) encoding mRNA directly to T cells in the patient’s body.

[0006] Without integration into the cellular genome, the genetic material delivered via viruses or lipid nanoparticles is at risk of attrition in both proliferating and not proliferating cells. Stable integration into the cellular genome is much preferrable but has so far been limitedPATENT Docket No.: CB 1062.30 to lentiviral vectors that integrate non-specifically and thus pose the risk of unintended consequences.

[0007] Precision gene editing with CRISPR endonucleases for therapeutic purposes has been successfully carried out only in vitro. CASGEVY® is an FDA-approved therapy for sickle cell anemia involving ex vivo gene editing in patient’s hematopoietic cells and reinfusion of the edited cells. Because the treatment is individualized, it is associated with substantial cost. In addition, the cell harvesting and reinfusion strategy successful with hematopoietic cells is unsuitable for other tissues and organs. There is a need for safe, effective, and long-lasting in vivo gene therapy for inherited and acquired diseases.SUMMARY OF THE INVENTION

[0008] In one embodiment, the invention is a composition for nucleic acid modification comprising: a nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex, a first guide polynucleotide comprising a first half-site capable of hybridizing to a first target site within the target nucleic acid, and a second guide polynucleotide comprising a second half-site capable of hybridizing to a second target site within the target nucleic acid, wherein the first and the second target sites in the nucleic acid are separated by an inter-nicking distance, and wherein the nCas3 is capable of nicking the first and the second target site. In some embodiments, the nCas3 is the Pseudomonas sp. S-6-2 nCas3, e.g., is selected from the group consisting of D448A nCas3, D448R nCas3, D448C nCas3, D448N nCas3, D448Q nCas3, D448G nCas3, D448K nCas3, D448M nCas3, D448S nCas3, D448T nCas3, and D448V nCas3. In some embodiments, the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for an nCas3 protein, a Cas7 protein, a Cas5 protein, a Cas8 protein, a Casl 1 protein, and a Cas6 protein. In some embodiments, the nucleic acid encoding the nCas3 Cascade effector complex in an mRNA having the SEQ ID NOs.: 1-6. In some embodiments, the mRNA comprises codon optimization for optimizing mRNA expression in mammalian cells. In some embodiments, the mRNA comprises modifications minimizing immunogenicity in mammalian recipients of the mRNA, e.g., a uridine modification or a cytidine modification: 5-methoxyuridine, 5-methyluridine, 5-carboxymethytl ester uridine, 2-thiouridine, pseudouridine, 2-methoxycytidine and derivatives thereof. In some embodiments, the mRNA comprises a 5’-cap having a formula selected from the group consisting of N7-(4-PATENT Docket No.: CB 1062.30 chlorophenoxyethyl)-m3'-OG(5')ppp(5')G, N7-(4-bromophenoxyethyl)-m3'-OG(5')ppp(5')G, N7mG(5’)ppp(5’)G, N7mG(3’OMe)(5’)ppp(5')m6A(2’OMe)pG, and N7mG(3’OMe)(5’)ppp(5’)m6G(2’OMe)pG, or the structure 3’-G(5’)PPP-5' wherein the G is a modified guanosine selected from the group consisting of N7-(4-chlorophenoxyethyl)-guanosine, N7-(4-chloro-phenoxy ethyl)- guanosine, N7-(4-chlorophenoxyethyl)-m3'-O-guanosine, and N7-(4-bromophenoxyethyl)-m3'-O-guanosine, or the structure N7mG(3’OMe)(5’)ppp(5')m6A(2’OMe)pG or the structure N7mG(5’)ppp(5’)G. In some embodiments, the mRNA is a single polycistronic mRNA encoding the nCas3 protein, the Cas7 protein, the Cas5 protein, the Cas8 protein, the Casll protein, and the Cas6 protein. In some embodiments, the mRNA consists of a group of mRNA molecules comprising an mRNA encoding the nCas3 protein, an mRNA encoding the Cas7 protein, an mRNA encoding the Cas5 protein, an mRNA encoding the Cas8 protein, an mRNA encoding the Casll protein, and an mRNA encoding the Cas6 protein. In some embodiments, the mRNA encoding the nCas3 protein comprises SEQ ID NO: 6, the mRNA encoding the Cas7 protein comprises SEQ ID NO: 3, the mRNA encoding the Cas5 protein comprises SEQ ID NO: 1, the mRNA encoding the Cas8 protein comprises SEQ ID NO: 4, the mRNA encoding the Casl 1 protein comprises SEQ ID NO: 5, and the mRNA encoding the Cas6 protein comprises SEQ ID NO: 2. In some embodiments, the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for a nuclear localization signal (NLS), e.g., the NLS selected from the group consisting of SV40 large T-antigen, nucleoplasmin, 53BP1, VACM-1 / CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrpl, c-Myc, Mouse c-able IV, Mata2 and MINIYO NLS. In some embodiments, the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for a tag selected from the group consisting of a FLAG-tag, a HA tag, a FC tag, a GFP tag, a HIS tag, a MYC tag. In some embodiments, the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for an effector selected from the group consisting of a nuclease, a phosphatase, a transcription factor, a histone acetyltransferase, a histone deacetylase, a kinase, a HUH endonuclease, and a methylase. In some embodiments, the first guide polynucleotide and the second guide polynucleotide are RNA comprising a chemical modification of one or more nucleotides at the termini of the RNA, e.g., 2’0-methyl ribose or a phsophorothioate linkage. In some embodiments, the first guidePATENT Docket No.: CB 1062.30 polynucleotide and the second guide polynucleotides comprise the structure 5’-2’OMe-PS-2’OMe-PS-2’OMe-RN-2’OMe-PS-2’OMe-PS-2’OMe-3’ wherein 2’OMe is the 2’-O-methyl ribose, PS is the phsophorothioate linkage and RN is N unmodified ribonucleotides. In some embodiments, the first half-site of the first guide polynucleotide and the second half-site of the second guide polynucleotide are capable of hybridizing to a target sequence selected from the group consisting of an enhancer sequence, a promoter sequence, an exon sequence, and an intron sequence. In some embodiments, the first half-site of the first guide polynucleotide and the second half-site of the second guide polynucleotide are capable of hybridizing to a sequence in a gene selected from Table 3, e.g., a sequence selected from Table 4 or Table 5 [insert SEQ ID NOs:]. In some embodiments, the composition further comprises a lipid nanoparticle (LNP) encapsulating the nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex, the first guide polynucleotide and the second guide polynucleotide. In some embodiments, the lipid nanoparticle comprises a lipid phase comprising an ionizable cationic lipid at about 46-50%, cholesterol at about 38-43%, a phospholipid at about 9-10%, and a polyethylene glycol (PEG) derivative at about 1-2%. In some embodiments, the lipid phase comprises 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l-aminium (ALC-0315), cholesterol, l,2-Distearoyl-sn-glycero-3-PC (1,2-DSPC), and Methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (ALC-0159), e.g., ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about 1-2%. In some embodiments, the lipid phase comprises 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM102), cholesterol, 1,2-DSPC, and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), e.g., SM102 at about 50%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and DMG-PEG2000 at about 1-2%. In some embodiments, the lipid phase comprises 4-(dimethylamino)-butanoic acid, ( 10Z, 13Z)- 1 -(9Z, 12Z)-9, 12-octadecadien- 1 -yl- 10, 13 -nonadecadien-l-yl ester (MC3), cholesterol, 1,2-DSPC, and DMG-PEG2000, e.g., MC3 at about 50%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and DMG-PEG2000 at about 1-2%. In some embodiments, the LNP are characterized by encapsulation efficiency of 70-100%, poly dispersity index of 0-0.25, and diameter of 65-100 nanometers.PATENT Docket No.: CB 1062.30

[0009] In one embodiment, the invention is a therapeutic composition for modifying a sequence of a target nucleic acid in a somatic cell in a living organism the composition comprising a lipid nanoparticle (LNP) including a lipid phase comprising ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about 1-2%, and the LNP contains a therapeutically effective amount of (i) a nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex; (ii) a first guide polynucleotide comprising a first half-site capable of hybridizing to a first target site within the target nucleic acid, and (iii) a second guide polynucleotide comprising a second half-site capable of hybridizing to a second target site within the target nucleic acid, wherein the first and the second target sites in the nucleic acid are separated by an inter-nicking distance, and wherein the nCas3 is capable of nicking the first and the second target site. In some embodiments, the therapeutically effective amount is between 0.25 mg / kg and 2.5 mg / kg of total nucleic acid. In some embodiments, the composition comprises one or more of excipient, antimicrobial agent, an antioxidant, a surfactant, and a freezing agent. In some embodiments, the therapeutically effective amount is capable of achieving the rate of sequence modification at the first target site, or the second target site or in the inter-nicking distance between the first target site and the second target site of at least 65%.

[0010] In one embodiment, the invention is a therapeutic composition for modifying a sequence of a target nucleic acid in a somatic cell in a living organism comprising the composition described above.

[0011] In one embodiment, the invention is a method of treating a disease or a condition in a human patient comprising a step of systemic administration to a human patient having the disease or the condition of the composition described above. In some embodiments, at least one of the first half-site and the second half-site is in a gene is selected from Table 3. In some embodiments, the gene is TTR, and the level of the TTR protein in the patient’s plasma is reduced and the method further comprises assessing the level of the TTR protein in the patient’s plasma. In some embodiments, the gene is PCSK9 gene, and the level of the PCSK9 protein or the level of LDL cholesterol in the patient’s plasma is reduced, and the method further comprises assessing the level of the PCSK9 protein or the level of LDL cholesterol in the patient’s plasma. In some embodiments, the gene sANGPTL3, and the level of the ANGPTL3PATENT Docket No.: CB 1062.30 protein the level of triglycerides in the patient’s plasma is reduced, and the method further comprises assessing the level of the ANGPTL3 protein the level of triglycerides in the patient’s plasma. In some embodiments, the method further comprises monitoring the patient for excessive immune response and for change in the function of the liver by assessing the amount of liver-secreted enzymes.

[0012] In one embodiment, the invention is a method of making the therapeutic composition of claim 44, the method comprising combining the lipid phase comprising ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about 1-2%, and the therapeutically effective amount of (i) a nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex; (ii) a first guide polynucleotide comprising a first half-site capable of hybridizing to a first target site within the target nucleic acid, and (iii) a second guide polynucleotide comprising a second half-site capable of hybridizing to a second target site within the target nucleic acid, wherein the first and the second target sites in the nucleic acid are separated by an inter-nicking distance, and wherein the nCas3 is capable of nicking the first and the second target site. In some embodiments, the method further comprises adding one or more of excipient, antimicrobial agent, an antioxidant, a surfactant, and a freezing agent.

[0013] In one embodiment, the invention is a method of treating a disease or condition comprising administering to a patient having the disease or condition a nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex, a first guide polynucleotide comprising a first half-site capable of hybridizing to a first target site within the target nucleic acid, and a second guide polynucleotide comprising a second half-site capable of hybridizing to a second target site within the target nucleic acid, wherein the first and the second target sites in the nucleic acid are separated by an inter-nicking distance, and nicking the first and the second target site with the nCas3. In some embodiments, the nCas3 is the Pseudomonas sp. S-6-2 nCas3 selected from the group consisting of D448A nCas3, D448R nCas3, D448C nCas3, D448N nCas3, D448Q nCas3, D448G nCas3, D448K nCas3, D448M nCas3, D448S nCas3, D448T nCas3, and D448V nCas3. In some embodiments, the nucleic acid coding for the nCas3 Cascade effector complex comprises an mRNA coding for a nCas3 protein, a Cas7 protein, a Cas5 protein, a Cas8 protein, a Casll protein, and a Cas6 protein. In some embodiments, the mRNA comprises a 5’-cap having a formula selected from the group consisting of N7-(4-PATENT Docket No.: CB 1062.30 chlorophenoxyethyl)-m3'-OG(5')ppp(5')G, N7-(4-bromophenoxyethyl)-m3'-OG(5')ppp(5')G, N7mG(5’)ppp(5’)G, N7mG(3’OMe)(5’)ppp(5')m6A(2’OMe)pG, and N7mG(3 ’ OMe)(5 ’ )ppp(5 ’ )m6G(2 ’ OMe)pG, N7mG(3 ’ OMe)(5 ’ )ppp(5 ')m6 A(2 ’ OMe)pG or N7mG(5’)ppp(5’)G, or the structure 3’-G(5’)PPP-5' wherein the G is a modified guanosine selected from the group consisting of N7-(4-chlorophenoxyethyl)- guanosine, N7-(4-chloro-phenoxy ethyl)- guanosine, N7-(4-chlorophenoxyethyl)-m3'-O-guanosine, and N7-(4-bromophenoxyethyl)-m3'-O-guanosine. In some embodiments, the mRNA comprises codon optimization for optimizing mRNA expression in mammalian cells, at least one uridine modification selected from the group consisting of 5-methoxyuridine, 5-methyluridine, 5-carboxymethytl ester uridine, 2-thiouridine and pseudouridine and derivatives thereof, and at least one cytidine modification 2-methoxy cytidine or its derivatives. In some embodiments, the mRNA is a single polycistronic mRNA encoding the nCas3 protein, the Cas7 protein, the Cas5 protein, the Cas8 protein, the Casll protein, and the Cas6 protein. In some embodiments, the mRNA consists of a group of mRNA molecules comprising an mRNA encoding the nCas3 protein, an mRNA encoding the Cas7 protein, an mRNA encoding the Cas5 protein, an mRNA encoding the Cas8 protein, an mRNA encoding the Casl 1 protein, and an mRNA encoding the Cas6 protein, e.g., the mRNA encoding the nCas3 protein comprises SEQ ID NO: 6, the mRNA encoding the Cas7 protein comprises SEQ ID NO: 3, the mRNA encoding the Cas5 protein comprises SEQ ID NO: 1, the mRNA encoding the Cas8 protein comprises SEQ ID NO: 4, the mRNA encoding the Casll protein comprises SEQ ID NO: 5, and the mRNA encoding the Cas6 protein comprises SEQ ID NO: 2. In some embodiments, the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for a nuclear localization signal (NLS) selected from the group consisting of SV40 large T-antigen, nucleoplasmin, 53BP1, VACM-1 / CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrpl, c-Myc, Mouse c-able IV, Mata2 and MINIYO NLS. In some embodiments, the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for a tag selected from the group consisting of a FLAG-tag, a HA tag, a FC tag, a GFP tag, a HIS tag, a MYC tag. In some embodiments, the nucleic acid encoding the nCas3 Cascade effector complex comprises a nucleic acid coding for an effector selected from the group consisting of a nuclease, a phosphatase, a transcription factor, a histone acetyltransferase, a histone deacetylase, a kinase,PATENT Docket No.: CB 1062.30 a HUH endonuclease. In some embodiments, the first guide polynucleotide and the second guide polynucleotide comprise the structure 5’-2’OMe-PS-2’OMe-PS-2’OMe-R.N-2’OMe-PS-2’0Me-PS-2’0Me-3’ wherein 2’OMe is the 2’-O-methyl ribose, PS is the phsophorothioate linkage and RN is N unmodified ribonucleotides. In some embodiments, the first half-site of the first guide polynucleotide and the second half-site of the second guide polynucleotide hybridize to a sequence in a gene selected from Table 3. In some embodiments, the first half-site of the first guide polynucleotide and the second half-site of the second guide polynucleotide are capable of hybridizing to a sequence selected from Table 4 or Table 5 (SEQ ID NOs: insert). In some embodiments, the nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex, the first guide polynucleotide and the second guide polynucleotide are administered encapsulated in a lipid nanoparticle (LNP).BRIEF DESCRIPTION OF THE FIGURES

[0014] FIGURE 1 (prior art) illustrates the Class I Type I (Cascade) operon from E. coli K-12 and the assembled Class I Type I (Cascade) effector complex.

[0015] FIGURE 2 illustrates the double nicking step performed by two Cascade effector complexes comprising nCas3 (nickase).

[0016] FIGURE 3 illustrates Cascade gDNA encoding the Cascade gRNA and the transcribed Cascade gRNA.

[0017] FIGURE 4 illustrates Cascade target selection in the murine Psck9 gene.

[0018] FIGURE 5 illustrates Cascade target selection in the murine Ttr gene.

[0019] FIGURE 6 depicts the results of editing of the Psck9 locus and the Ttr locus in the mouse hepatic cell line H2.35 with nCas3 Cascade effector complexes delivered via LNPs (lipofection).

[0020] FIGURE 7 depicts the results of editing of the Psck9 locus and the Ttr locus in the mouse hepatic cell line H2.35 with nCas3 Cascade effector complexes delivered via electroporation (nucleofection).

[0021] FIGURE 8 depicts genomic deletions in the Ttr locus following cleavage by nCas3 Cascade effector complexes determined by next-generation nucleic acid sequencing (NGS).PATENT Docket No.: CB 1062.30

[0022] FIGURE 9 illustrates chemical modifications of both ends of the Cascade gRNA.

[0023] FIGURE 10 depicts the results of editing of the Ttr locus in the mouse hepatic cell line H2.35 by nCas3 Cascade effector complexes comprising chemically modified and unmodified gRNA.

[0024] FIGURE 11 depicts the results of editing of the Pcsk9 and the Ttr targets in the mouse hepatic cell line H2.35 by nCas3 Cascade effector complexes with varying amounts of gRNA and using LNPs to deliver the nCas3 -Cascade mRNAs and gRNAs.

[0025] FIGURE 12 depicts genomic deletions in the Ttr locus following cleavage by nCas3 Cascade effector complexes determined by nucleic acid sequencing (NGS), where the complexes were delivered to cells via LNP.

[0026] FIGURE 13 depicts the design of the mRNAs for the protein components of the Cascade effector complex. The length of each coding sequence is shown in nucleotide bases. Figure discloses SEQ ID NOs: 327, 327, 327, 327, 327, and 327, respectively, in order of appearance.

[0027] FIGURE 14 depicts the results of editing of Pcsk9 and Ttr in primary mouse hepatocytes with nCas3 Cascade effector complexes comprising different designs of proteincoding mRNA (LNP delivery).

[0028] FIGURE 15 depicts the results of in vivo editing of Pcsk9 and Ttr with nCas3 Cascade effector complexes comprising unmodified mRNA and either unmodified gRNA or unmodified gDNA.

[0029] FIGURE 16 depicts genomic deletions in the Pcsk9 and Ttr loci following cleavage with nCas3 Cascade effector complexes in vivo determined by next-generation nucleic acid sequencing (NGS).

[0030] FIGURE 17 depicts the results of editing of the Pcsk9 and Ttr targets by nCas3 Cascade effector complexes in vivo with different designs of mRNA encoding the components of the Cascade effector complex.

[0031] FIGURE 18 depicts the results of editing of the Ttr locus by nCas3 Cascade effector complexes in vivo combining optimized mRNA with different designs of gRNA.PATENT Docket No.: CB 1062.30

[0032] FIGURE 19 depicts the results of serum TTR measurement in animals with the edited Ttr gene.

[0033] FIGURE 20 demonstrates tolerability of the LNP composition comprising nucleic acids encoding nCas3 Cascade effector complexes as assessed by measuring changes in the body weight of the treated animals.

[0034] FIGURE 21 demonstrates tolerability of the LNP composition comprising nucleic acids encoding nCas3 Cascade effector complexes as assessed by measuring serum levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) following LNP administration.

[0035] FIGURE 22 depicts the results of a dose escalation study testing increasing amounts of total nucleic acid.

[0036] FIGURE 23 depicts a gene knock-in in human induced pluripotent stem cells (iPSCs) mediated by nCas3 Cascade effector complexes.

[0037] FIGURE 24 depicts validation of the gene knock-in into the CBLB locus in human iPSCs.

[0038] FIGURE 25 depicts the results of the gene knock-in in human iPSCs with nCas3 Cascade effector complexes comprising different designs and amounts of protein-coding mRNA.

[0039] FIGURE 26 depicts results of the gene knock-in in human iPSCs with nCas3 Cascade effector complexes comprising different amounts of gRNA.

[0040] FIGURE 27 depicts the results of in vitro genome editing with FLAG-tagged components of the Cascade effector complex.

[0041] FIGURE 28 shows durability of plasma TTR depletion after one year of a single gene editing event (unmodified and end-modified guides, see FIGURE 9).

[0042] FIGURE 29 depicts an exemplary workflow for isolating and transfecting primary mouse hepatocytes (PMH) from transgenic mice.

[0043] FIGURE 30 shows the results of editing the human APOC3 gene in a human cell line HEK293T.

[0044] FIGURE 31 shows the results of editing the human APOC3 gene in PMH of transgenic mice carrying the human APOC3 gene.PATENT Docket No.: CB 1062.30

[0045] FIGURE 32 shows the results of editing of targets in the human SERPINA1 gene in PMH of transgenic mice carrying the human SERPINA1 gene and in the human cell lineHEK293T.

[0046] FIGURE 33 shows the results of editing the human LPA gene in HEK293T.

[0047] FIGURE 34 shows the results of editing of targets in the mouse Ttr gene in mice using chemically modified gRNA.

[0048] FIGURE 35 shows the results of editing in the mouse Ttr and Pcsk9 genes in PMH using gRNA with truncated repeats.DETAILED DESCRIPTION OF THE INVENTION

[0049] Definitions

[0050] The following definitions aid in understanding this disclosure.

[0051] The terms “Type I CRISPR-Cas nucleoprotein (NP) complex,” “Type I Cascade NP complex,” “Cascade NP complex,” and “Type I NP complex,” are used interchangeably to refer to a complex comprising Cascade proteins in a complex with a guide polynucleotide (crRNA or modifications thereof including CRISPR RNA-DNA hybrids, chRDNA). A typical Cascade NP complex comprises six Cas7 proteins, a Cas5 protein, a Cas8 protein, two Casl 1 proteins, and a Cas6 protein. The complex is capable of binding a target nucleic acid sequence. After association with a Cas3 protein (or mCas3 protein or nCas3 protein) and crRNA, the Cascade NP complex is capable of cleaving the target nucleic acid. Unless otherwise indicated, the full set of proteins listed above is present in the complex. The term “nCas3 Cascade NP complex” refers to the Cascade NP complex associated with nCas3 (“nickase”) instead of the wild-type Cas3. For brevity, the “nCas3” is sometimes omitted from the term “nCas3 Cascade effector complex” when it is clear from the context that the Cascade effector complex comprises the nCas3 nickase.

[0052] As used herein, the term “mCas3 protein” refers to a Cas3 protein comprising one or more mutations relative to its corresponding wild-type Cas3 protein. As used herein, the term “nCas3 protein” refers to a type of mCas3 protein that retains nicking activity but not the wild-type DNA shredding activity. The nCas3 protein is sometimes referred to as a nickase.

[0053] As used herein, the terms “guide” and “guide polynucleotide” as used herein refer to one or more polynucleotides that form a nucleoprotein complex with a Cas protein,PATENT Docket No.: CB 1062.30 wherein the nucleoprotein complex preferentially binds a nucleic acid target sequence in a polynucleotide (relative to a polynucleotide that does not comprise the nucleic acid target sequence). Such guides can comprise ribonucleotide bases (e.g., RNA), deoxyribonucleotide bases (e.g., DNA), combinations of ribonucleotide bases and deoxyribonucleotide bases (e.g., RNA / DNA), nucleotide analogs, modified nucleotides, and the like, as well as synthetic, naturally occurring, and non-naturally occurring modified backbone residues or linkages. Many such guides are known, such as but not limited to, single-guide RNA (including miniature and truncated single-guide RNAs), crRNA, dual-guide RNAs, including but not limited to, crRNA / tracrRNA molecules, and the like, the use of which depends on the particular Cas protein. The term “gRNA” is used to refer to guide RNA with or chemical modifications. The term “gDNA” is used to refer to DNA encoding gRNA.

[0054] As used herein, a “CRISPR polynucleotide” is a polynucleotide sequence comprising at least a portion of a CRISPR guide molecule. In some embodiments, the CRISPR polynucleotide includes a targeting region and / or an activating region. CRISPR polynucleotides include gRNA and gDNA.

[0055] With reference to a guide molecule, a “spacer,” “spacer sequence,” “spacer element,” or “targeting region,” as used herein refers to a polynucleotide sequence that is capable of specifically (i.e., to the exclusion of other sequences) hybridizing to a target nucleic acid sequence. The targeting region determines the location of the sequence-specific binding and nucleolytic cleavage of the target DNA by the CRISPR endonuclease. For convenience, the targeting region is often about 20-30 bases in length. In the NATNA, the “targeting region” is a region that is capable of hybridizing to a sequence in a target nucleic acid.

[0056] The terms “target” and “genomic target” refer to the sequences of the target cell (e.g., human sequences) that are to be targeted by a sequence-guided endonuclease. For CRISPR endonucleases, the target comprises a sequence capable of hybridizing the targeting region of the NATNA as well as the protospacer adjacent motif (PAM). It is well known that the formation of a stable hybrid between two nucleic acid strands depends on several parameters including the length of complementary regions, the degree of complementarity (less than 100% complementarity may be sufficient), any chemical modifications of the nucleic acid strands, and temperature and ionic strength of the reaction mixture (see e.g., Lewin, B., Genes IV,PATENT Docket No.: CB 1062.30 Oxford University Press, 1990, Chapter 23, The extraordinary power of DNA technology). One of skill in the art is capable of designing a nucleotide sequence of a desired length capable of specifically hybridizing to another nucleic acid sequence under a set of reaction conditions. However, it is not possible to reliably predict which of the targets will be more efficiently cleaved by a CRISPR endonuclease paired with a NATNA capable of hybridizing to the target.

[0057] As used herein, the term “capable of hybridizing” refers to a property of complementary nucleic acid strands to form a stable duplex. One of skill in the art would recognize that perfect complementarity over the entire length of the strands is not required for a stable duplex to form. For example, a certain number of mismatches between opposite nucleotides can be tolerated in a stable duplex. Furthermore, not all nucleotides in the duplex need to form Watson-Crick pairs in order for the duplex to remain stable. For example, some nucleotides may comprise non-canonical bases and even abasic (apurinic or apyrimidinic) sites. The number of tolerable mismatches depends on the length of the region of complementarity between the two nucleic acid strands. For example, a stable duplex formed of a longer region of complementarity will tolerate more mismatches than a duplex formed of a shorter region of complementarity. Furthermore, the degree of complementarity required to form a stable duplex varies depending on reaction conditions. For example, a stable duplex present in a high ionic strength solution will tolerate more mismatches than a duplex present in a low ionic strength solution.

[0058] With reference to a guide molecule, the term “activating region” refers to a portion of a polynucleotide capable of associating, or binding with, a CRISPR endonuclease polypeptide, such as for example, a Casl2a polypeptide.

[0059] As used herein, the terms “nucleotide analog,” “non-canonical nucleotide,” and “chemically-modified nucleotide” refer to a compound having structural similarity to a canonical purine or pyrimidine nucleotide occurring in DNA or RNA. The nucleotide analog may contain a modified sugar and / or a modified nucleobase, as compared to a purine or pyrimidine base occurring naturally in DNA or RNA. In some embodiments, the nucleotide analog is inosine or deoxyinosine, such as 2’ -deoxyinosine. In other embodiments, the nucleotide analog is a 2’-deoxyribonucleotide (in an RNA molecule), or a ribonucleotide (in a DNA molecule). In some embodiments, the nucleotide analog includes a modified base (suchPATENT Docket No.: CB 1062.30 as, for example, xanthine, uridine, oxanine (oxanosine), 7-methlguanosine, dihydrouridine, 5-methylcytidine, C3 spacer, 5-hydroxybutynl-2’ -deoxyuridine, 5-nitroindole, 5-methyl isodeoxycytosine, iso deoxy guanosine, other 0-1 purine analogs, N-6-hydroxylaminopurine, nebularine, 7-deaza hypoxanthine, and other 7-deazapurines, a fluoroinosine or a chloroinosine, such as 2-chloroinosine, 6-chloroinosine, 8-chloroinosine, 2-fluoroinosine, 6-fluoroinosine, or 8-fluoroinosine. A nucleotide analog or modified nucleotide may comprise a modified sugar moiety or a modified phosphodiester linkage, e.g. 2’-O-methyl, 2’-O-methoxyethyl, 2’-aza, protein-nucleic acid (PNA), linked nucleic acid (LNA), xeno nucleic acids (XNA), phosphoro-thioate and the like.

[0060] As used herein, the term “CRISPR hybrid RNA / DNA guide” (chRDNA) refers to a polynucleotide guide molecule comprising a targeting region and an activating region, wherein one or both of the targeting region and the activating region comprises one or more deoxyribonucleotides in addition to the ribonucleotides.

[0061] As used herein, the terms “engineered,” “genetically engineered,” “genetically modified,” “recombinant,” “modified,” “non-naturally occurring,” and “non-native” indicate intentional human manipulation of the genome of an organism or a cell. The terms encompass methods of genomic modification that include genomic editing, as defined herein, as well as techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like.

[0062] As used herein, the terms “protospacer adjacent motif’ or “PAM” as used herein refers to double-stranded nucleic acid sequences comprising a CRISPR endonuclease recognition sequence, wherein amino acids of endonuclease protein directly interact with the recognition sequence (e.g., Casl2a protein interacts with the PAM 5’-TTTN-3’ or the PAM 5’-TTTV-3’). PAM sequences are on the non-target strand and can be 5’ or 3’ of a target complement sequence (e.g., in CRISPR-Casl2a systems the PAM 5’-TTTN-3’ or the PAM 5’-TTTV-3’ sequence is on the non-target strand and is 5’ of the target-complement sequence).

[0063] As used herein, the terms “nuclear localization sequence” or “nuclear localization signal” (both abbreviated NLS) refer to a polypeptide sequence within a protein that preferentially increases the subcellular localization of a protein to the nucleus of a cell.PATENT Docket No.: CB 1062.30 NLS sequences are typically positively changed stretches of amino acids located at the terminus of a protein (N-terminus or C-terminus) or internally within the protein sequence. A protein may comprise more than one NLS, a protein (or a combination thereof, i.e., one or more NLS at the N-terminus and one or more NLS at the C-terminus). NLS sequences can be covalently linked to a prokaryotic protein to enable trafficking of the engineered protein to the nucleus of a eukaryotic cell. NLS sequences can be engineered or derived from existing proteins sequences.

[0064] As used herein, the terms “target,” “target sequence,” “nucleic acid target sequence,” “target nucleic acid sequence,” and “on-target sequence” are used interchangeably herein to refer to a nucleic acid sequence that is wholly, or in part, complementary to a nucleic acid target binding sequence of a CRISPR guide polynucleotide (e.g., the targeting region). Typically, the nucleic acid target binding sequence is selected to be 100% complementary to a nucleic acid target sequence to which binding of a CRISPR nucleoprotein complex is being directed; however, to attenuate binding to a nucleic acid target sequence, lower percent complementarity can be used. In connection with the nCas3 (nickase) Cascade effector complex, the target comprises the first half-site capable of hybridizing to the first CRISPR (Cascade) guide polynucleotide and the second half-site capable of hybridizing to the second CRISPR (Cascade) guide polynucleotide. The sequence between the two half-sites is the “internicking distance.”

[0065] As used herein, the terms “donor polynucleotide,” “donor oligonucleotide,” “donor template,” refer to a nucleic acid at least a portion of which is to be incorporated into the genome of a cell. Donor polynucleotides (or portions thereof) may be incorporated into the genome by a variety of mechanisms including homology directed repair (HDR), nonhom ologous end joining (NHEJ), template-based copying by a DNA polymerase or a reverse transcriptase or integration utilizing viral integrases ortransposases. Donor polynucleotides can comprise homology arms flanking the insertion sequence (e.g., DSBs in the DNA) to facilitate HDR

[0066] As used herein, the term “homology-directed repair” (HDR) refers to the biochemical pathway of DNA repair that takes place in cells, for example, during repair of a DSB in DNA. HDR requires nucleotide sequence homology and uses a donor polynucleotidePATENT Docket No.: CB 1062.30 to repair the sequence wherein the DSB (e.g., within a target DNA sequence) occurred. For example, a donor polynucleotide can be used for repair of the break in the target DNA sequence, wherein the repair results in the transfer of genetic information e.g., polynucleotide sequences) from the donor polynucleotide at the site or in close proximity of the break in the DNA. Accordingly, new genetic information (e.g., polynucleotide sequences) may be inserted or copied at a target DNA sequence.

[0067] As used herein, the term “genomic region” refers to a segment of a chromosome in the genome of a host cell that is present on either side of the nucleic acid target sequence site or, alternatively, also includes a portion of the nucleic acid target sequence site. The homology arms of the donor polynucleotide have sufficient homology to undergo homologous recombination with the corresponding genomic regions.

[0068] As used herein, the term “non-homologous end joining” (NHEJ) refers to the biochemical pathway of repairing a DSB in DNA by direct ligation of one terminus of the break to the other terminus of the break without a requirement for a donor polynucleotide. NHEJ is a DNA repair pathway available to cells to repair DNA without the use of a repair template. NHEJ in the absence of a donor polynucleotide often results in nucleotides being randomly inserted or deleted at the site of the DSB. NHEJ may use a donor polynucleotide to repair the sequence wherein the DSB occurred (e.g., within a target DNA sequence).

[0069] As used herein, the term “microhomology-mediated end joining” (MMEJ) refers to the biochemical pathway for repairing a DSB in DNA. MMEJ involves deletions flanking a DSB and alignment of microhomologous sequences internal to the break site before joining. MMEJ is genetically defined and requires the activity of, for example, CtIP, Poly(ADP -Ribose) Polymerase 1 (PARP1), DNA polymerase theta (Pol 0), DNA Ligase 1 (Lig 1), or DNA Ligase 3 (Lig 3). Additional genetic components are known in the art. See, e.g., Sfeir et al. (Trends in Biochemical Sciences, 2015, 40:701-714).

[0070] As used herein, the term “DNA repair” encompasses any biochemical process whereby cellular machinery repairs damage to a DNA molecule contained in the cell. The damage repaired can include single-strand breaks or double-strand breaks (DSBs). At least three mechanisms exist to repair DSBs: HDR, NHEJ, and MMEJ. “DNA repair” is also used herein to refer to DNA repair resulting from human manipulation, wherein a target locus is modified,PATENT Docket No.: CB 1062.30 e.g., by inserting, deleting, or substituting nucleotides, all of which represent forms of genome editing.

[0071] The term “genome editing” refers to altering a nucleotide sequence within the genome of an organism, e.g., a cell, including a cell of a multicellular organism. The term “gene editing” is used to refer to a type of genome editing that occurs within a gene with the intention of eliminating or altering the function of the gene in the cell.

[0072] As used herein, the terms “regulatory sequences,” “regulatory elements,” and “control elements” are interchangeable and refer to polynucleotide sequences that are upstream (5’ non-coding sequences), within, or downstream (3’ non-translated sequences) of a polynucleotide target to be expressed. Regulatory sequences influence, for example, the timing of transcription, amount or level of transcription, RNA processing or stability, and / or translation of the related structural nucleotide sequence. Regulatory sequences may include activator binding sequences, enhancers, introns, poly adenylation recognition sequences, promoters, transcription start sites, repressor binding sequences, stem-loop structures, translational initiation sequences, internal ribosome entry sites (IRES), translation leader sequences, transcription termination sequences (e.g., polyadenylation signals and poly-U sequences), translation termination sequences, primer binding sites, and the like.

[0073] As used herein, the term “modulate” refers to a change in the quantity, degree or amount of a function. For example, a CRISPR complex, as disclosed herein, may modulate the activity of a gene sequence by binding to a nucleic acid target sequence. Depending on the action occurring after binding, the CRISPR complex can transiently or permanently induce, enhance, suppress, or inhibit, transcription of a gene, e.g., by cleaving the sequence which is then and imperfectly repaired by cellular DNA repair thereby disrupting the gene sequence. Thus, “modulation” of gene expression includes both gene activation and gene repression, including complete repression of gene transcription.

[0074] As used herein, the term "Significant Mutant Fraction” (SMF) refers to a parameter and a method of calculating gene editing at a particular site in the genome in a population of cells using next-generation sequencing data obtained from a test cell population and a control cell population. SMF is calculated by first determining the "significant" mutations in the matched test and control samples. A mutation is only considered as "significant" if (1) itPATENT Docket No.: CB 1062.30 falls within ±3bp of the predicted cut-site coordinates, and (2) its frequencies in the test and control samples differ significantly as determined by a chi-squared test with p-value threshold of 10'4with Bonferroni multiple-comparison correction. The total frequency of "significant" mutations is calculated for the test and control samples and the control frequency is subtracted from the test frequency to produce the final Significant Mutation Fraction statistic.

[0075] As used herein, the term “lipid nanoparticle” (LNP) refers to a water-in-oil droplet typically between 60 nm and 100 nm in size. One of skill in the art will appreciate that the size of a stable LNP depends on the composition and ionic strength of aqueous solutions within and without the LNP.

[0076] As used herein, the term “encapsulation” refers to successful enclosure of nucleic acids within LNPs. Encapsulation may be expressed as fraction or nucleic acid present in the composition that is enclosed in LNP. For example, 95% encapsulation means that only 5% of nucleic acid is present outside of the LNPs. The non-encapsulated nucleic acid can be reacted with detection reagents or nucleases and thereby quantified. For example, RIBOGREEN® is a fluorescent dye that binds single-stranded nucleic acids including oligonucleotides and can be used to measure encapsulation.

[0077] As used herein, the terms “LNP composition” and “nCas3 Cascade LNP composition” refer to an aqueous solution comprising LNPs encapsulating the components of the nCas3 Cascade system. Unless otherwise indicated, the LNPs in the LNP composition comprise all of the components of the nCas3 Cascade system necessary to produce editing of the targeted locus in the subject organism.

[0078] As used herein, the terms “subject,” “individual,” or “patient” refer to humans and other animals such as laboratory animals. In some embodiments, a cell is derived from a subject. In some embodiments, the subject is a non-human subject.

[0079] As used herein, the terms “effective amount” or “therapeutically effective amount” of a composition or agent refer to a sufficient amount of the composition or agent to provide the desired response. Such responses will depend on the particular disease in question. For example, in a patient being treated for hypercholesteremia using the therapy disclosed herein, a desired response may include reduction in blood LDL levels, or blood triglyceride levels, or reduction or elimination or cardiovascular (CV) adverse events. Preferably, thePATENT Docket No.: CB 1062.30 effective amount also prevents or avoids one or more harmful side effects. The exact effective amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the type of somatic cell being subjected to treatment.

[0080] As used herein, the terms “treatment” or “treating” a particular disease includes preventing, reversing or ameliorating symptoms of the disease. For example, for a patient being treated for hypercholesteremia, treatment may include reducing blood LDL levels, or blood triglyceride levels, or reducing or eliminating any cardiovascular (CV) adverse events.

[0081] As used herein, the terms “pharmaceutically acceptable carrier” and “excipient” refer to aqueous solvents (e.g., water, aqueous solutions of alcohols, saline solutions, sodium chloride, Ringer's solution, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters), as well as dispersion media, coatings, surfactants, gels, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, stabilizers, binders, disintegration agents, lubricants, sweetening agents, flavoring agents, and dyes. The concentration and pH of the various components in a pharmaceutical composition are adjusted according to well-known parameters for each component.

[0082] The present disclosure relies on the ordinary skill in the art as it pertains conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant polynucleotides, as taught, for example, by the following standard publication: Sambrook, Joseph. Molecular Cloning: a Laboratory Manual. 2001, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press; E.A. Greenfield, Antibodies: A Laboratory Manual, 2014, Second edition, Cold Spring Harbor Laboratory Press; R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 2016, 7th Edition, Wiley-Blackwell; J.M. Walker, Methods in Molecular Biology (Series), Humana Press; and Innis, J., Gelfand, D., et al., PCR Protocols: a Guide to Methods and Applications, 1989, Academic Press.

[0083] Then first two approved genome editing therapies involved ex vivo modification of patient’s cells. LYFGENIA®, a treatment for sickle cell disease works by adding a functional P-globin gene to patients’ own hematopoietic stem cells (HSCs). Durable production of adultPATENT Docket No.: CB 1062.30 hemoglobin with anti-sickling properties (HbAT87Q) is possible following reinfusion and successful engraftment of the edited HSCs. Similarly, CASGEVY® involves ex vivo genome editing of patient’s HSCs with CRISPR / Cas9 at the erythroid-specific enhancer region of the BCL11A gene. Reduced BCL11A expression in reinfused and engrafted HSCc leads to increase in fetal hemoglobin (HbF) production in edited HSCs and their progeny.

[0084] In addition to the successful ex vivo gene editing therapies described above, some in vivo gene therapies are currently in development or in clinical trials.

[0085] Hereditary amyloidogenic transthyretin amyloidosis is caused by instability and proteolysis of the mutant transthyretin protein resulting in deposition of amyloid and fatal damage of the peripheral nervous system. One of the approaches to treatment has been gene silencing with small interfering RNA (siRNAs) or anti-sense oligonucleotides (ASO). See Adams, D., et al., (2018) Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis, NEJM 379:11. While showing promise, RNAi and ASO therapies must be frequently readministered due to the short half-life of RNA molecules. A program using CRISPR / Cas9 to disrupt the TTR gene is ongoing, see Gillmore, J., et al., (2021) CRISPR Cas9 in vivo gene editing for transthyretin amyloidosis, NEJM 385:493. (Phase 1 study completed in 2022).

[0086] One of the major risk factors for cardiovascular disease is abnormal elevation of serum triglycerides and serum cholesterol. One of the factors affecting serum cholesterol levels is the protein PSCK9 that binds to LDL receptors and promotes their internalization and lysosomal degradation thus leaving more LDL in circulation. Depletion of PSCK9 with monoclonal antibodies leads to increased numbers of LDL receptors on the surface of hepatocytes and lowering of serum cholesterol (LDL) levels. Anti-PSCK9 antibody drugs such as REPATHA® and PRALUENT® require monthly or bi-weekly injections in order to maintain the cholesterol-lowering effect. A PSCK9 gene-based therapy is yet to be developed.

[0087] Triglycerides in serum are present in triglyceride-rich lipoproteins chylomicrons and VLDL. For tissue use triglycerides are liberated from these lipoproteins by lipoprotein lipase (LPL). ANGPTL-family proteins are negative regulators of LPL. Loss-of-function mutations in ANGPTL3 are associated with reduced blood triglycerides in experimental animals. An antisense drug blocking ANGPTL3 expression is in clinical trials and hasPATENT Docket No.: CB 1062.30 demonstrated the ability to lower blood triglycerides by 36-47%. Gaudet, D., et al., (2020) Vupanorsen, an N-acetyl galactosamine-conjugated antisense drug to ANGPTL3 mRNA, lowers triglycerides and atherogenic lipoprotein in patients with diabetes, hepatic steatosis, and hypertriglyceridaemia, Eur. Heart J. 41(40):3936. As with PSCK9 inhibitors, weekly or bimonthly injections are required to maintain the physiological effect. An ANGPTL3 gene-based therapy is yet to be developed.

[0088] The instant disclosure describes methods and compositions for successful onetime gene editing of somatic cells in vivo using engineered CRISPR systems with desired long-lasting physiological effect attributable to gene editing.

[0089] The methods disclosed herein utilize a CRISPR endonuclease, i.e., the endonuclease associated with the CRISPR system and encoded by a CRISPR locus. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus is found many prokaryotic genomes and provides resistance to invasion of foreign nucleic acids. Structure, nomenclature, and classification of CRISPR loci are reviewed in Makarova et al., Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology. 2011 lune; 9(6): 467-477.

[0090] Briefly, a typical CRISPR locus includes a number of short repeats regularly interspaced with spacers. The CRISPR locus also includes coding sequences for CRISPR-associated (Cas) genes. A spacer-repeat sequence unit encodes a crisprRNA (crRNA). In vivo, a mature crRNAs is processed from a polycistronic transcript referred to as pre-crRNA or pre-crRNA array. The repeats in the pre-crRNA array are recognized by Cas-encoded proteins that bind to and cleave the repeats liberating mature crRNAs. CRISPR systems perform cleavage of a target nucleic acid wherein Cas proteins and crRNA form a CRISPR ribonucleoproteins (crRNP). The crRNA molecule guides the crRNP to the target nucleic acid (e.g., a foreign nucleic acid invading a bacterial cell) and the Cas nuclease proteins cleave the target nucleic acid.

[0091] Class 1, Type I CRISPR systems include means for processing the pre-crRNA array that include a multi-protein complex called CASCADE (CRISPR-associated complex for antiviral defense) comprised of subunits CasA, B, C, D and E. The CASCADE-crRNA complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA.PATENT Docket No.: CB 1062.30 The bound nucleoprotein complex recruits the Cas3 helicase / nuclease to facilitate cleavage of target nucleic acid.

[0092] Class 2, Type II CRISPR systems include a trans-activating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to a crRNA repeat in the pre-crRNA array and recruits endogenous RNaselll to cleave the pre-crRNA array. The tracrRNA / crRNA complex can associate with a nuclease, e.g., Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. Hybridization of the crRNA to the target nucleic acid activates the Cas9 nuclease, for target nucleic acid cleavage.

[0093] Class 1, Type III CRISPR systems include the RAMP superfamily of endoribonucleases (e.g., Cas6) that cleave the pre-crRNA array with the help of one or more CRISPR polymerase-like proteins.

[0094] Class 2, Type V CRISPR systems comprise a different set of Cas-like genes, including Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl4, Casl2g, Casl2h, Casl2i, Casl2j, and Casl2k proteins which are distant homologues of Cas genes in Type I-III CRISPR systems.

[0095] CRISPR nucleases do not cleave a fixed sequence but instead are guided by a nucleic acid guide to a target sequence. In addition to the target sequence hybridizing to the nucleic acid guide, the CRISPR endonucleases recognize a sequence termed protospacer adjacent motif (PAM). The CRISPR Class 1 (including CASCADE) endonuclease recognize a PAM sequence selected from 5’-AAG-3’, 5’-AGG-3’, 5’-ATG-3’, 5’-GAG-3’, 5’-CAG-3’, 5’-GTG-3’, 5’-TAA-3’, 5’-TGG-3’, 5 ’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5 ’-ATA-3’, 5 ’-TAG-3’, and 5’-TTG-3’. The CRISPR Class 2 endonucleases (including Casl2a) recognize a PAM consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3’, and 5'-NNNACA-3', 5’-TTN-3’, 5’-TTTN-3’ and 5’-TTTV-3’.

[0096] The CRISPR guide nucleic acid comprises a targeting region capable of binding the target nucleic acid and an activating region capable of binding the CRISPR endonuclease. In some embodiments, the guide nucleic acid is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, a guide nucleic acid can comprise, in the order of 5' to 3', a spacer extension, a spacer, a minimum CRISPR repeat, a single guide connector, a minimum tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some instances, a nucleicPATENT Docket No.: CB 1062.30 acid-targeting nucleic acid can comprise, a tracrRNA extension, a 3' tracrRNA sequence, a minimum tracrRNA, a single guide connector, a minimum CRISPR repeat, a spacer, and a spacer extension in any order. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. Other chRDNA may be a single guide D(R)NA for use with a Type II CRISPR system comprising a targeting region, and an activating region composed of and a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. For example, the targeting region may comprise DNA or a mixture of DNA and RNA, and an activating region may comprise RNA or a mixture of DNA and RNA.

[0097] In some embodiments, the single guide nucleic acid comprises a spacer sequence located 5' of a first duplex which comprises a region of hybridization between a minimum CRISPR repeat and minimum tracrRNA sequence. The first duplex can be interrupted by a bulge. The bulge facilitates recruitment of the endonuclease to the guide nucleic acid. The bulge can be followed by a first stem comprising a linker connecting the minimum CRISPR repeat and the minimum tracrRNA sequence. The last paired nucleotide at the 3' end of the first duplex can be connected to a second linker connecting the first duplex to a mid-tracrRNA. The mid-tracrRNA can comprise one or more additional hairpins.

[0098] In some embodiments, the guide nucleic acid can comprise a dual guide nucleic acid structure. The double guide nucleic acid comprises a spacer extension, a spacer, a minimum CRISPR repeat, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and a tracrRNA extension. The dual guide nucleic acid does not include the single guide connector. Instead, the minimum CRISPR repeat sequence comprises a 3' CRISPR repeat sequence and the minimum tracrRNA sequence comprises a 5' tracrRNA sequence and the dual guide nucleic acids can hybridize via the minimum CRISPR repeat and the minimum tracrRNA sequence.

[0099] In some embodiments, gene editing with CRISPR endonucleases involves disruption of a gene sequence. In some embodiments, disruption of a gene sequence reduces or eliminates transcription of the mRNA from the gene (e.g., by disrupting a promoter or enhancer region). In some embodiments, disruption of a gene sequence reduces or eliminates the amount of the functional protein encoded by the gene (e.g., by disrupting an mRNA splicing site orPATENT Docket No.: CB 1062.30 disrupting the coding sequence with missense or nonsense mutations). Gene disruption with CRISPR endonucleases involves cleavage of the target sequence and subsequent imperfect repair by cellular DNA repair pathways.

[0100] Eukaryotic cells, e.g., mammalian cells possess an innate diversity of DNA repair pathways. The DNA repair pathway involved in repairing double strand breaks (DSB) (such as the ones introduced by the CRISPR-Cas nucleases) includes highly accurate homologous recombination (HR) as well as less accurate pathways of non-homologous end joining (NHEJ) and micro-homology -mediated end joining (MMEJ). NHEJ and MMEJ generate a variety of small insertions and deletions at the target site see Xue and Greene, (2021) DNA repair pathway choices in CRISPR-Cas9 mediated genome editing, Trends Genet.37:639.). These error-prone pathways are capable of producing the desired gene disruption through frameshift, nonsense, or missense mutations resulting in reduction or elimination of protein expression.

[0101] Unfortunately, it has been reported that programmable endonucleases such as CRISPR bring about undesirable consequences including large genomic deletions, chromosomal translocations, chromotripsis and other chromosomal abnormalities. A recent review article states “These editing outcomes, while rare, pose safety risks that could negatively impact certain clinical uses of nucleases. These drawbacks of nuclease editing, combined with the fact that nuclease-initiated HDR is inefficient in most therapeutically relevant cell types, have motivated the development of alternative strategies for more precise gene editing [such as primer editing and base editing].” Raguram, A., et al., (2022) Therapeutic in vivo delivery of gene editing agents, Cell 185 :P2806. The instant disclosure overcomes these alleged drawbacks of wild-type CRISPR endonucleases by presenting engineered CRISPR compositions for accurate and efficient method of in vivo gene editing. In the animal models tested so far, the gene editing is followed by a successful therapeutic outcome.

[0102] In some embodiments, the invention comprises methods of modifying a genome of a somatic cell with the Type I CRISPR complex. The Type I CRISPR complex (CRISPR-Associated Complex for Anti-viral Defense or “Cascade”) comprises multiple proteins and a guide RNA, all transcribed from the Type I CRISPR operon. The proteins include a Cas5 subunit protein, a Cas6 subunit protein, a large Cas8 subunit protein capable of enveloping thePATENT Docket No.: CB 1062.30 5’ end of the guide RNA, several Cas7 subunit proteins capable of interacting with the guide RNA backbone, and a Casl 1 subunit protein. (Van der Dost, J., et al., (2014) Unraveling the structural and mechanistic basis of CRISP R-C as systems, Nature Rev Microbiol. 12:479-492. The gene order in the E. coli K12 Cascade operon is cas3, cas8, casll, cas7, cas5, cas6, casl, and cas2. Koonin, E.V., (2017) supra and FIGURE 1.

[0103] In some embodiments, the nucleic acids (DNA) encoding the proteins of the Cascade effector complex are selected from Table 1.

[0104] Table 1. Protein components of the Cascade effector complex

[0105] The Type I CRISPR (Cascade) complex further comprises the Cas3 effector protein. The Cas3 protein has helicase and nuclease domains responsible for unwinding and processive cleavage (“shredding”) of the target DNA (Sinkunas, T., et al., (2011) Cas3 is a single stranded DNA nuclease and A TP-dependent helicase in the CRlSPRlCas immune system, EMBO J. 30:1335.) To date, seven subtypes of the Type I system have been identified: I-A, I-B, I-C, I-D, I-E, and I-F (including variants I-Fl, I-F2 and I-F3), see Koonin, E. et al., (2023) Discovery of diverse CRISPR-Cas systems and expansion of the genome engineering toolbox, Biochemistry, 62:3465. The Type I cas genes include cas 7, cas5, cas8, casll (cse2), cas5, cas3, cas2, cas4, casl, and cas6. The Cascade complex comprises 11 proteins and one guide RNA and has a molecular weight of approximately 400 kDa. (FIGURE 1). Examples of organisms having Type I systems include: I-A, Archaeoglobus fulgidus; I-B, Clostridium kluyveri; I-C, Bacillus halodurans; I-D, Cyanothece sp. 8802; I-E, Escherichia coli K12 (E. coll K12); I-F, Yersinia pseudo-tuberculosis and Shewanella putrefaciens CN-32 (Koonin, supra).PATENT Docket No.: CB 1062.30

[0106] The DNA encoding the crRNA is transcribed into a precursor crRNA and processed by the Cascade RNA endonuclease into a mature crRNA. A mature crRNA is capable of interacting with the Cascade protein complex to form a Cascade nucleoprotein complex, and is further capable of binding a complementary DNA sequence in the genome of a cell. In some embodiments, the crRNA comprises a minimal CRISPR array. The minimal CRISPR array (the 3 ’-portion of the wild-type operon shown in FIGURE 1) comprises a series of structures where two repeat sequences flank a spacer sequence. RNA processing by the Cascade endonuclease Cas6 generates a series of crRNAs each having repeat sequences on both the 5’ and 3’ ends flanking the guide sequence.

[0107] CRISPR Type I (Cascade) effector complex cleaves the target nucleic acid adjacent to a protospacer adjacent motif (PAM). The Cascade PAM consists of a sequence selected from 5’-AAG-3’, 5’-AGG-3’, 5’-ATG-3’, 5 ’-GAG-3’, 5 ’-C AG-3’, 5’-GTG-3’, 5’-TAA-3’, 5’-TGG-3’, 5’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5’-ATA-3’, 5’-TAG-3’, and 5’-TTG-3’.

[0108] In some embodiments, the invention includes an engineered Type I CRISPR-Cas (Cascade) effector complex. In some embodiments, the engineering comprises replacing the wild-type Cas3 protein in complex with a mutant Cas3 protein (mCas3 protein), wherein the mCas3 protein is capable of site-specific cleavage of a strand of genomic DNA but is devoid of the DNA shredding activity of the wild-type Cas3 protein. In some embodiments, the mCas3 protein is from an organism selected from the group consisting of E. coli, Pseudomonas sp. S-6-2 (also known as Halopseudomonas phragmitis and Pseudomonas phragmitis and having the NCBI Taxonomy ID: 1931241), Thermobifida fusca, Saccharomonospora viridis, Thermomonospora curvata, Streptomyces avermitihs. Streptomyces bottropensis, Thermus thermophilus, Vibrio cholera, Salmonella enterica, Geothermobacter sp. EPR-M, Methanocella arvoryzae MRE50, and Streptococcus thermophilus (strain ND07).

[0109] In some embodiments, the mCas3 protein is an A coli mCas3 protein comprising one or more mutations selected from the group consisting of G317A, S318A, G319A, K320N, T321N, Q297E, D452E, D452N, E453N, R662A, R665Q, T346A, Q347N, G375A, K412G, T423A, D425H, Q426T, H601A, A602V, R603Q, R609S, T635A, Q636A, and Q640H. InPATENT Docket No.: CB 1062.30 come embodiments, the mutation is selected from the group consisting of K320N and D452N and D452E.

[0110] In some embodiments, the mCas3 protein is a Pseudomonas sp. S-6-2 mCas3 protein. In some embodiments, the mutant Cas3 protein (mCas3) retains only a nicking (single strand cleavage) activity. The nicking mCas3 protein is sometimes referred to as “nickase” or “nCas3.”

[0111] In some embodiment, e Pseudomonas sp. S-6-2 mCas3 protein comprises one or more mutations in the ATP hydrolyzing DEXD / H domains. In some embodiments, the mutation is in the Motif I (Walker A), Motif II (Walker B), or Motif III (Walker C) of the DEXD / H domain. In some embodiments, the Pseudomonas sp. S-6-2 mCas3 protein comprises one or more mutations in the Motif II of the DEXD / H domains. In some embodiments, the mCas3 protein comprises a substitution mutation at the first aspartic acid (“D”) in the DEXD / H domain and the mutation is selected from the group consisting of the following amino acid substitutions: alanine (D448A), arginine (D448R), cysteine (D448C), asparagine (D448N), glutamine (D448Q), glycine (D448G), lysine (D448K), methionine (D448M), serine (D448S), threonine (D448T), and valine (D448V).

[0112] In some embodiments, nCas3 is encoded by SEQ ID NO: 6.

[0113] In some embodiments, at least one protein in the Cascade effector complex comprises a nuclear localization signal (NLS). The NLS can be appended to the N-terminus or the C-terminus of a protein. In some embodiments, the NLS is connected via a linker. The NLS sequence may be engineered or derived from another protein. In some embodiments, the NLS sequence is derived from a protein selected from the group consisting of SV40 large T-antigen, Nucleoplasmin, 53BP1, VACM-1 / CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrpl, c-Myc, Mouse c-able IV, Mata2 and MINIYO.

[0114] In some embodiments, a tag or an effector is added to a protein component of the Cascade complex for delivery to the target site in the genome.

[0115] In some embodiments, the tag is used for the detection of the Cascade protein or Cascade protein complex, or Cascade effector complex in a sample or a cell. Detection includes detecting the localization of the Cascade protein (or complex) to a cell, cellular organelle, orPATENT Docket No.: CB 1062.30 export of the protein from an organelle. In some embodiments, the tag is used for purification of the Cascade protein or Cascade protein complex, or Cascade effector complex.

[0116] In some embodiments, the tag is selected from a nuclear localization signal, a nuclear export signal, a mitochondrial localization signal, a cell penetrating peptide, a solubility tag, a FLAG tag, a HA tag, a FC tag, a GFP tag, a HIS tag, a MYC tag.

[0117] In some embodiments, the effector performs one or more chemical reactions selected from deamination of DNA, ligation of DNA, polymerization of DNA, cleavage of DNA or RNA, reverse transcription of RNA, phosphorylation or dephosphorylation of DNA, RNA, or protein, modification (e. ., acetylation or deacetylation) of a histone protein, and activation or repression of DNA transcription. In some embodiments, the effector is selected from a deaminase, a ligase, a polymerase, a transposase, a reverse transcriptase, a nuclease, a phosphatase, a transcription factor, a histone acetyltransferase, a histone deacetylase, a kinase, a HUH endonuclease, and a methylase.

[0118] In some embodiments, the FLAG-tag is the sequence (DYKDHDG)n(SEQ ID NO: 7), wherein n is between 1 and 10, c.g., n=3. In some embodiments, the FLAG-tag is the sequence DYKDHDGDYKDHDIDYKDDDDK, SEQ ID NO: 8. In some embodiments, the FLAG-tag is conjugated to the N-terminus of at least one protein of the Cascade effector complex. In some embodiments, the FLAG-tag is conjugated to the C-terminus of at least one protein of the Cascade effector complex.

[0119] In some embodiments, the editing efficiency is not diminished or is not substantially diminished by the presence of the tag or the effector. The FLAG-tag experiment (Example 18, FIGURE 27) demonstrates the feasibility of this approach within the functional nCas3 Cascade effector complex.

[0120] In some embodiments, for genome modification in a cell, the Cascade proteins are introduced into the cell in the form of individual proteins. In some embodiments, the Cascade proteins are introduced into the cell in the form of a pre-assembled Cascade protein complex. In some embodiments, the Cascade proteins are introduced into the cell in the form of mRNA encoding the proteins. In some embodiments, an individual expression construct for each protein is introduced into the cell. In some embodiments, a polycistronic mRNA encoding two or more, or all six of the Cascade proteins is introduced into the cell.PATENT Docket No.: CB 1062.30

[0121] In some embodiments, the mRNA encoding the one or more proteins of the Cascade effector complex are of prokaryotic origin (e.g., E. coli or Pseudomonas). In some embodiments, the mRNA is modified for optimal function in a eukaryotic cell within a eukaryotic organism.

[0122] In some embodiments, the mRNA is codon-optimized for translation in eukaryotic cells. Codon optimization is the process of altering the nucleic acid sequence without changing the polypeptide sequence encoded thereby in order to utilize the most prevalent tRNAs present in an organism and increase the efficiency of translation. Codon optimization may be performed manually or with the help of any of the codon optimization software such as GeneArt (ThermoFisher Scientific), GenSmart (Genscript), and Codon Optimization Tool (Integrated DNA Technologies).

[0123] In one embodiment, the present invention relates to engineered polynucleotides encoding Cascade components that are engineered by modifying naturally occurring Cascade polynucleotides.

[0124] In some embodiments, the mRNA encoding the endonuclease has undergone uridine depletion, z.e., a sequence design process whereby synonymous mRNA codons with no uridines or fewer uridines are substituted for uridine-containing codons. Uridine depletion has been shown to reduce immunogenicity of the mRNA in humans and protect the mRNA from intracellular RNases.

[0125] In some embodiments, the mRNA comprises one or more chemical modifications. In some embodiments, the chemical modifications comprise modified nucleobases or non-canonical nucleobases (i.e., bases other than adenosine, cytosine, guanosine and uridine). In some embodiments, one or more or all of uridines in the nucleic acid are substituted with less immunogenic uridine derivatives in order to further reduce immunogenicity of the nucleic acid. In some embodiments, the uridine derivative is selected from 5-methoxyuridine, 5-methyluridine, 5-carboxymethytl ester uridine, 2-thiouridine and pseudouridine and their various derivatives for which methods of making and methods of use in nucleic acids are disclosed e.g., in the U.S. Patent Nos. 9,428,535 and 9,751,925 and Morais P., el al., (2021) The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines, Front Cell Dev Biol. V. 9 article 789427.PATENT Docket No.: CB 1062.30

[0126] In some embodiments, the combination of uridine depletion and the use of uridine derivatives results in less immunogenic nucleic acid suitable for in vivo administration as disclosed in Vaidyanathan, S., el al., (2018). Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification. Molecular Therapy - Nucleic Acids, 12:530.

[0127] In some embodiments, the mRNA encoding the proteins of the Cascade effector complex is selected from SEQ ID NOs: 1-6.

[0128] In some embodiments, the nucleic acid is an mRNA containing a 5’-cap. In some embodiments, the cap comprises the traditional terminal guanine or “inverted G” structure 3’G(5’)PPP-5’. In some embodiments, the 5’-cap comprises one or more chemical modifications selected from modifications to the guanosine base, the ribose sugar moiety or to the phosphate moiety. In some embodiments, the cap comprises one of more modifications selected from N7-methyl guanosine, 2’-O-methyl ribose, a-thiophosphate, a-methyl phosphate, boranophosphate and selenophosphate.

[0129] In some embodiments, the cap is an engineered mRNA cap comprising modifications of the naturally-occurring mRNA cap consisting of two guanines arranged in the anti-reverse configuration, e.g., G(5’)ppp(5')G. In some embodiments, the engineered cap is a mononucleotide cap. In some embodiments, the engineered cap is a dinucleotide cap. In some embodiments, the engineered cap comprises modifications of the nucleotide base, ribose and the phosphate group.

[0130] In some embodiments, the modified nucleotide base is guanosine (G) and the modified G is selected from N7-(4-chlorophenoxyethyl-G, N7-(4-bromophenoxyethyl)-G, N7-methyl G (N7mG), N7-methyl(3’O-methyl)-G (N7mG(3’OMe) and N7-methyl(2’O-methyl)-G (N7mG(2’OMe).

[0131] In some embodiments, the modified nucleotide base is adenosine (A) and the modified A is selected from 6-methyl A (m6A), 6-methyl(3’O-methyl)-A (m6A(3’OMe) and (2’O-methyl)-A (A(2’OMe).

[0132] In some embodiments, the cap comprises an N-7-methylated guanosine N7mG(5’)ppp(5’)G cap, anti-reverse cap analog (ARCA), catalog no. N-7003, TriLink Biotechnologies, San Diego, Cal. In some embodiments, the cap comprisesPATENT Docket No.: CB 1062.30 N7mG(3’OMe)(5’)ppp(5’)m6A(2’OMe)pG (e.g., CLEANCAP® Reagent M6, catalog no. N-7453, TriLink Biotechnologies.)

[0133] In some embodiments, the cap is added post-transcriptionally, using enzymes such as the recombinant vaccinia virus capping enzyme and the recombinant 2'-O-methyltransferase enzyme.

[0134] In some embodiments, the mRNA further comprises a poly-A tail, i.e., a structure of about 100-250 adenine ribonucleotides at the 3 ’-end of the mRNA.

[0135] In some embodiments, the mRNA comprises untranslated regions (UTRs). In some embodiments, the mRNA comprises a 5’-UTR, or a 3’-UTR or both the 5’-UTR and the 3 ’-UTR.

[0136] In some embodiments, the mRNA comprises a UTR from a mammalian mRNA. UTR can be selected based on its known effect on stability and expression of the mRNA. In some embodiments, the UTR is from the organism undergoing in vivo gene therapy, e.g., human. In some embodiments, the UTR is from a human gene selected from alpha globin (Hba), beta-globin (Hbb), actin, glyceraldehyde 3 -phosphate dehydrogenase (Gapdh), growth hormone (Ghl), or another gene with high level of expression.

[0137] In some embodiments, the mRNA comprises a Kozak sequence that plays a role in translation initiation. In some embodiments, the Kozak sequence includes the AUG start codon of the mRNA. In some embodiment, the Kozak sequence is placed between the 5’-UTR and the AUG start codon of the mRNA.

[0138] In some embodiments, for genome modification in a cell, the Cascade guide nucleic acids (gRNAs) are introduced into the cell. In some embodiments, the Cascade guide nucleic acids are introduced into the cell in the form of DNA, i.e., a DNA sequence encoding the gRNA and a suitable promoter, e.g., a U6 promoter (“gDNA” in FIGURE 3). In some embodiments, the Cascade guide nucleic acids are introduced into the cell in the form of RNA.

[0139] In some embodiments, the invention comprises a Cascade CRISPR array engineered for the dual-nicking system described herein. FIGURE 3 illustrates the Cascade gRNAs containing two separate spacers referred as “Half-site 1” and “Half-site 2.” In some embodiments, a single gDNA contains both half-sites, each site flanked by CRISPR repeats and under the control of a promoter (e.g., hU6 as illustrated in FIGURE 3). In some embodiments,PATENT Docket No.: CB 1062.30 two separate gRNAs are used, each gRNA comprising one half-site flanked by CRISPR repeats and under the control of a promoter. In some embodiments, each half site of the gRNA contains a 32-bp spacer capable of hybridizing to a target nucleic acid. The spacer is flanked by two CRISPR repeats of 29 bp. In some embodiments, the minimal CRISPR arrays shown FIGURE 3 is further truncated without compromising their function.

[0140] In some embodiments, the gRNA comprises one or more chemical modifications selected from nitrogenous base modifications and ribophosphate backbone modifications. In some embodiments, the gRNA comprises 2’O-methyl (2’0-Me) modifications of one or more bases.

[0141] In some embodiments, the 2’0-Me bases are present in one or both 5’- and d’terminal nucleotides of each half-site (FIGURE 9). In some embodiments, the crRNA comprises phosphorothioate (PS) modification of the phosphate groups. In some embodiments, the PS modifications are present in one or both 5’- and 3’-terminal nucleotides of each half-site (FIGURE 9). In some embodiments, each half-site is modified on both ends with 2’0-Me— PS--2’0-Me-PS-2’0-Me and the gRNA has the overall structure 2’0-Me— PS— 2’0-Me— PS- 2’0-Me— RN— 2’0-Me— PS— 2’0-Me— PS— 2’0-Me where R is the remaining N ribonucleotides (less the three 5 ’-terminal nucleotides and the three 3 ’-terminal nucleotides) present in the gRNA (FIGURE 9).

[0142] In some embodiments, the gRNA comprises a sequence capable of hybridizing to a target sequence selected from the sequences listed in Table 4 and Table 5.

[0143] In some embodiments, the gRNA comprises a sequence selected from Table 2.

[0144] Table 2. gRNA sequencesPATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30

[0145] In some embodiments, the delivery of CRISPR components (e.g., mRNA encoding the CRISPR endonuclease and the guide) is achieved by packaging the components into a compartment. The compartment comprising the CRISPR components can be administered in vivo intravenously, intrathecally or into a desired organ. In some embodiments, the compartment is a non-biological compartment selected from nanospheres, liposomes, nanoparticles, microparticles, nanocapsules, vesicles, polyethylene glycol particles, hydrogels, and micelles. In some embodiments, the compartment is a lipid nanoparticle (LNP).

[0146] In some embodiments, the LNP used herein has a diameter of between about 100 nm and about 1 pm, preferably <100 nm. In some embodiments, the LNP includes one or more cationic lipids. The cationic lipids can be selected such that, when combined, the measured value of the pKaof the combination is no less than 6.1 and no greater than 6.7, e.g., between 6.2 and 6.6; or between 6.3 and 6.5. The cationic lipids can have a head group, one or more hydrophobic tails, and a linker between the head group and the one or more tails. The head group can include an amine which is a site of positive charge. The amine can be a primary, secondary, or tertiary amine, or a quaternary amine. The one or more hydrophobic tails can include two hydrophobic chains, which may be the same or different. The tails can be aliphatic chains, fatty acid chains or other hydrophobic chains. The linker can include, for example, a glyceride linker, an acyclic glyceride analog linker, or a cyclic linker. The linker can include functional groups such as an ether, an ester, a phosphate, a phosphonate, a phosphorothioate, a sulfonate, a disulfide, an acetal, a ketal, an imine, a hydrazone, or an oxime. Cationic lipids include one or more amine group(s) which bear the positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral depending on pH. The ionization of the cationic lipid affects the surface charge of a lipid nanoparticle (LNP) and can influence plasma protein absorption, blood clearance, tissue distribution and the ability to fuse with cellular membranes.

[0147] Methods of making and using lipid nanoparticles for in vivo delivery of nucleic acids are disclosed e.g., in the U.S. Patent Nos. 9,415,109, 9,533,047, and 11,420,931. Briefly, a typical LNP lipid phase comprises an ionizable cationic lipid, cholesterol, a phospholipid, and a polyethylene glycol (PEG) derivative. In some embodiments, the LNP lipid phase comprises an ionizable cationic lipid at about 46-50%, cholesterol at about 38-43%, aPATENT Docket No.: CB 1062.30 phospholipid at about 9-10%, and a PEG derivative at about 1-2%. Polyethylene glycol (PEG) is included in the lipid phase of the LNP as it has an effect of reducing aggregation of LNPs. PEG and PEG derivatives useful for in vivo delivery of nucleic acids are disclosed e.g., in U.S. Patent Application Publication No. US20220047518.

[0148] In some embodiments, the method comprises a step of forming LNPs enclosing (encapsulating) nucleic acids. To form LNPs enclosing nucleic acids, the lipids are diluted in ethanol to a desired concentration, e.g., 25-50 nM and mixed at predetermined molar ratios. The nucleic acid is diluted in a suitable aqueous buffer (e.g., a buffer maintaining stability of the nucleic acid such as sodium citrate buffer) to a desired concentration, e.g., 0.1-10 mg / mL. Formation of lipid nanoparticles can be achieved my mixing the lipid / ethanol solution and the aqueous nucleic acid containing solution at various ratios. The mixing can be effected manually or with the help of pumps or syringes. Ethanol and buffers may be removed via dialysis and additional sterilization steps can be performed according to methods known in the art.

[0149] In some embodiments, the lipid phase of the LNP comprises an ionizable cationic lipid at about 46-50%, cholesterol at about 38-43%, a phospholipid at about 9-10%, and a polyethylene glycol (PEG) derivative at about 1-2%.

[0150] In some embodiments, the lipid phase of the LNP comprises 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l-aminium (ALC-0315), cholesterol, l,2-Distearoyl-sn-glycero-3-PC (1,2-DSPC), and Methoxypoly ethyleneglycol oxy(2000)-N,N-ditetradecylacetamide (ALC-0159).

[0151] In some embodiments, the lipid phase of the LNP comprises 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM102), cholesterol, 1,2-DSPC, and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

[0152] In some embodiments, the lipid phase of the LNP comprises 4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester (MC3), cholesterol, 1,2-DSPC, and DMG-PEG2000.

[0153] In some embodiments, the surface of LNPs is further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) or coupled to targeting molecules (antibodies specific for cell-surface receptors or natural ligands of cell surface receptors) toPATENT Docket No.: CB 1062.30 direct the nanoparticle to the appropriate cell type and increase the likelihood of cellular uptake as described e.g.. in Jian et al., (2012) Cationic core shell liponanopar ticles for ocular gene delivery, Biomaterials 33(30): 7621-30).

[0154] In some embodiments, prior to administration to a patient, LNPs are evaluated to verify that the physical properties are in a suitable range for in vivo administration. In some embodiments, LNPs are evaluated for encapsulation efficiency using a nucleic-acid binding dye that quantifies the amount of non-encapsulated nucleic acid. In some embodiments, the LNPs are used for in vivo administration if encapsulation efficiency is in the range of 70-100%. In some embodiments, LNPs are evaluated for diameter. In some embodiments, the LNPs are used for patient administration if the diameter is in the range of 65-100 nm. In some embodiments, LNPs are evaluated for uniformity. In some embodiments, the LNPs are used for in vivo administration if the poly dispersity index (PDI) is at or about 0.25.

[0155] In some embodiments, the method described herein comprises selective cleavage of a target nucleic acid sequence in a cellular genome by a nCas3 Cascade effector complex.

[0156] In some embodiments, the spacer sequence is capable of hybridizing to a target site in a human gene. In some embodiments, the target site is in a gene selected from Table 3. In some embodiments, the target site is in a gene selected from Pcsk9, Ttr, Angptl3, CBLB, TRAC and CISH. One of skill in the art can design a Cascade gRNA spacer capable of hybridizing to any target nucleic acid in a gene of interest in a genome of a cell relying on the knowledge of Watson-Crick complementarity, the knowledge of stability of partially complementary nucleic acid hybrids, and further, using the guidance provided herein and e.g., in U.S. Patent 11,939,604 and U.S. Patent Application Pub. No. 20210102183 related to the Cascade systems.

[0157] In the context of the nCas3 Cascade effector complex cleavage, the target site comprises two target sequences, each recognized by each of the gRNA half-sites. The distance between the cleavage sites in each of the two target sequences is the “inter-nicking distance” of the target site.

[0158] In some embodiments, the target selection process comprises scanning the sequence of the gene for the presence of two copies of the protospacer adjacent motif (PAM)PATENT Docket No.: CB 1062.30 recognized by the Cascade complex. The two copies of the PAM must be on opposite strands of the target site for the desired paired nicking to occur (FIGURE 2). In some emboidiments, the Cascade PAM is selected from the sequences 5’-AAG-3’, 5’-AGG-3’, 5’-ATG-3’, 5’-GAG-3’, 5’-CAG-3’, 5’-GTG-3’, 5’-TAA-3’, 5’-TGG-3’, 5’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5’-ATA-3’, 5 ’-TAG-3’, and 5’-TTG-3.’ In the exemplary embodiments shown in FIGURE 4 and FIGURE 5, the sequences of the mouse genes Pcsk9 and Ttr were scanned for the presence of the 5’-AAG-3’ two PAMs.

[0159] In some embodiments, the target sites are selected based on the distance between the two PAMs in the site which determines the inter-nicking distance. In some embodiments, the desired range of the inter-nicking distance is 2-30 nucleotides. In some embodiments, the desired range of the inter-nicking distance is 3-17 nucleotides.

[0160] In some embodiments, where gene disruption is desired, it is preferred to target the first exon of the gene or the first two or three exons of the gene to ensure the earliest possible interruption of transcription or translation of the gene. In some embodiments, at least one of the first and the second target sites is selected to fall into first two or three exons of the gene. In some embodiments, the first and the second target sites are selected to fall into the desired range of the inter-nicking distance.

[0161] In some embodiments, the target gene is selected from Table 3.

[0162] Table 3. Diseases and conditions and genes that can be targeted in vivo.PATENT Docket No.: CB 1062.30

[0163] In some embodiments, the target gene is selected from human genes HSD17B 13, DGAT2, PNPLA3, HNF1, HNF4, SERPINA1, TTR, LPA, ANGPTL3, PCSK9, AGT, APO A, APOB, APOC3, TM6SF2, HMGCR, TERT-hTR, BMPR2, ALAS1, GSDla, HAO1, LDHA, XDH, SERPINC1, F7, F8, F9, F10, F12, F13, IDS, C5, C3, CFB, ALDH2, KLKB1, F12, SERPING1, USH2A, OT, HFE, IDUA, CEP290, RHO and an HBV gene. In somePATENT Docket No.: CB 1062.30 embodiments, the human target sequence is selected from Table 4 (SEQ ID NOs: 31-279). In some embodiments, the guide polynucleotide is selected from SEQ ID NOs 336-347.

[0164] Table 4. Target sequences for human therapeutics.PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30

[0165] In some embodiments, for development of the method of the invention in animal models, the mouse target sequence is selected from Table 5.

[0166] Table 5. Target sequences for use in animal modelsPATENT Docket No.: CB 1062.30PATENT Docket No.: CB 1062.30

[0167] In some embodiments, genome editing by Cascade effector systems produces a genome modification resulting in reduced expression or elimination of expression of one or more genes listed in Table 3 in the modified cell and this brings about alleviation of the symptoms of the disease or condition. In some embodiments, the gene expression is aberrant (i.e., is or becomes abnormally high) and reducing or eliminating the excessive gene transcript results is in alleviation of the symptoms of the disease or condition. In some embodiment, the gene contains a mutation and expression of the mutant protein results in the symptoms of the disease so that eliminating expression of the mutant protein results is in alleviation of the symptoms of the disease or condition.

[0168] In some embodiments, genome editing by Cascade effector systems allows insertion of a nucleic acid sequence that results in expression of one or more genes listed in Table 3 in the modified cell and this brings about alleviation of the symptoms of the disease or condition. In some embodiment, the gene contains a mutation and expression of the gene is either abolished or produced a non-functional protein. In some embodiments, the genePATENT Docket No.: CB 1062.30 expression is or becomes aberrant (i.e., abnormally low or absent) and inserting a functional copy of the gene results in restoring gene expression and alleviation of the symptoms of the disease or condition.

[0169] In some embodiments, the target nucleic acid comprises a gene that is expressed (or aberrantly expressed) in the liver, e.g., in hepatocytes or sinusoidal endothelial cells of the liver. In some embodiments the gene is expressed in hematopoietic cells throughout the body. In such embodiments, the LNP comprising the CRISPR system is administered systemically, i.e., intravenously.

[0170] In some embodiments, the target nucleic acid comprises a gene that is expressed (or aberrantly expressed) in the cells of the eye. In some embodiments, the LNPs comprising the Cascade effector system are delivered into the eye (intraocular delivery). I some embodiments, the delivery is intravitreal. In some embodiments, the delivery is directly to the retina to reach the retinal pigment epithelium. In some embodiments, the delivery is intrathecal or intraperitoneal.

[0171] In some embodiments, the invention comprises a method of modifying a target nucleic acid with the Cascade effector complex described herein. In some embodiments, the target nucleic acid is present in the genome of a cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a somatic cell or a pluripotent stem cell (z.e., a stem cell lacking the totipotent potential). In some embodiments, the eukaryotic cell is a germline cell of a plant, a fungal organism or a non-human animal. In some embodiments, the eukaryotic cell is a germline human cell within the boundaries set forth by local laws pertaining to human genome editing. In some embodiments, the eukaryotic somatic cell is present in vitro or ex vivo. In some embodiments, the eukaryotic somatic cell is present in vivo.

[0172] In some embodiments, the invention is a method of staggered cleavage of a target nucleic acid. In some embodiments, the method utilizes a first Cascade effector complex comprising an nCas3 (nickase) and the first guide polynucleotide comprising a first half-site capable of hybridizing to a first target site a second Cascade effector complex comprising an nCas3 (nickase) and the second guide polynucleotide comprising a second half-site capable of hybridizing to a second target site, wherein the first and a second Cascade effector complexesPATENT Docket No.: CB 1062.30 are capable for binding the target nucleic acid at a distance from each other referred to as the inter-nicking distance, and the region between the first and the second target sites is referred to as the inter-nicking region. In some embodiments, the inter-nicking distance is 1-150 bp. The first and a second Cascade effector complexes are capable of binding to and nicking (via the action of nCas3) the first and the second target sites in the target nucleic acid thereby accomplishing paired nicking, FIGURE 2. In some embodiments, the paired nicking facilitates genome modification in or near the inter-nicking region. In some embodiments, the genome modification comprises a deletion of at least a portion of the inter-nicking region. In some embodiments, the genome modification further comprises a deletion of a sequence adjacent to the inter-nicking region.

[0173] Without intending to be bound by a particular theory, the inventors hypothesize that one or more of DNA repair pathways selected from HDR, NHEJ and MMEJ take place in the cell to produce modifications of the inter-nicking region following paired nicking introduced by the two Cascade effector complexes. In the presence of a repair template (e.g., an exogenous donor nucleic acid introduced into the cell and having homology arms capable of hybridizing to the target nucleic acid) the HDR pathway may predominate. In the absence of a repair template (e.g., any homologous nucleic acid), the NHEJ pathway or the MMEJ pathways may predominate.

[0174] In some embodiments, the genome modification comprises a deletion centering in or near the inter-nicking region and comprising between about 10 and about 350 base pairs (FIGURE 8, FIGURE 12)

[0175] In some embodiments, the method comprises a genome modification including insertion of a donor nucleic acid into the inter-nicking region. If a donor nucleic acid comprises a gene expression cassette, the insertion of such a donor and subsequent expression of the gene are referred to as “gene knock-in.” In some embodiments, the donor nucleic acid is delivered by lipofection (in LNPs) or by nucleofection (electroporation) along with the components of the Cascade effector complex.

[0176] In some embodiments, the invention is a therapeutic composition comprising the LNP described above (“the LNP composition”) suitable for administration to humans. In some embodiments, the LNP composition comprises a therapeutically effective amount of the nCas3PATENT Docket No.: CB 1062.30 Cascade effector system components. As previously detailed in this disclosure, the nCas3 Cascade proteins may be introduced into the cell in the form of individual proteins, or a preassembled Cascade protein complex, or a pre-assembled Cascade effector complex (proteins and gRNA). In some embodiments, the nCas3 Cascade effector complex is introduced into the cell in the form of mRNA encoding the Cascade proteins nCas3, Cas5, Cas6, Cas7, Cas8 and Casll, and the gRNA. In some embodiments, the therapeutically effective amount comprises between 0.25 mg / kg and 2.5 mg / kg of total nucleic acid (including the mRNA and the gRNA).

[0177] In some embodiments, the therapeutic composition comprises one or more guide nucleic acids selected from Table 2 (SEQ ID NOs: [insert]) and the mRNAs for each of nCas3, Cas5, Cas6, Cas7, Cas8, and Casl 1 selected from Table 1 (SEQ ID NOs: 1-6).

[0178] In some embodiments, the amount of total nucleic acid has been shown to achieve sufficient genome editing to produce the desired physiological response. In some embodiments, the sufficient genome editing is editing in fewer than 100% of the cells of a target organ. In some embodiments, the sufficient genome editing is editing in at least 65% of the cells of a target organ.

[0179] In some embodiments, the LNP composition undergoes validation studies and safety studies prior to being administered to a human patient. In some embodiments, validation is in vitro validation. In some embodiments, validation is in vivo validation in experimental animals. In some embodiments, validation is a combination of in vitro validation and in vivo validation in experimental animals.

[0180] In some embodiments, prior to administration to a patient, the LNP composition comprising the Cascade effector system targeting a particular gene is tested in vitro to assess genome editing properties. Preferably, the system is tested on the cell type that is to be edited in vivo, e.g., hepatocyte cell line or primary hepatocytes for liver editing, or retinal cell lines (retinal pigment epithelium cell lines) for editing cells of the retina.

[0181] In some embodiments, for in vitro testing gene of the editing capabilities of the Cascade effector system, the components (e g., individual proteins, or a pre-assembled Cascade protein complex, or a pre-assembled Cascade effector nucleoprotein complex, or a combination of mRNA encoding the Cascade proteins and the gRNA) are transfected into the appropriate cell type by nucleofection, e.g., using the NUCLEOFECTOR™ 96-well Shuttle SystemPATENT Docket No.: CB 1062.30 (Lonza, Allendale, N.J.). In some embodiments, after nucleofection, the cells are allowed to incubate, e.g., for 48 hours prior to assessing gene editing.

[0182] In some embodiments, to assess genome editing, genomic DNA from cells is isolated and the site of desired editing is assessed by DNA sequencing. In some embodiments, DNA sequencing is next-generation sequencing (NGS).

[0183] In some embodiments, the sequencing step utilizes an adaptor added at least one end of a nucleic acid or nucleic acid strand. The adaptor can be double-stranded or partially double-stranded and comprises a double-stranded portion that can be ligated to the double stranded nucleic acid to be sequenced. Adaptors of various shapes and functions are known in the art, see e.g., U.S. Patent Nos. 8,822,150 (Y-shaped adaptor); 8,455,193 (stem-loop / hairpin adaptor); and 11,085,084 (various shapes of partially double-stranded adaptors). In some embodiments, the function of an adaptor is to introduce certain useful elements into a nucleic acid, such as barcodes, amplification primer binding sites, sequencing primer binding sites, enzyme recognition sites, and ligation-enabling sites. In some embodiments, the adaptor molecules are in vitro synthesized artificial sequences. In other embodiments, the adaptor molecules are in vitro synthesized naturally occurring sequences. In yet other embodiments, the adaptor molecules are isolated naturally occurring molecules or isolated non-naturally occurring molecules.

[0184] Adaptor ligation can be performed according to methods widely known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, 4thEd. Cold Spring Harbor Lab Press (2012). A suitable ligase enzyme catalyzes the formation of phosphodi ester linkages between the strands of two nucleic acids strand, e.g., a single-strand DNA ligase such as CIRCLIGASE™ ssDNA ligase (Epicentre Biotechnologies, Madison, Wise., or Lucigen, Middleton, Wise.), or a double-strand DNA ligase selected from T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, or E. coli DNA ligase. In some embodiments, the ligation step is preceded by addition of the 5' phosphate e.g., with a polynucleotide kinase such as T4 polynucleotide kinase. In some embodiments, the ligation step is preceded by addition of the 3’-dA (“dA-tailing”) e.g., with a DNA polymerase capable of template-independent addition of a nucleotide such as Taq DNA polymerase.PATENT Docket No.: CB 1062.30

[0185] In some embodiments, the sequencing step utilizes barcodes. Analyzing individual nucleic acid molecules by massively parallel sequencing typically requires a separate level of barcoding for sample identification and for error correction.

[0186] The use of unique molecular barcodes is described e.g., in U.S. Patent Nos.7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368. A unique molecular identifying barcode (abbreviated UMI or UID) is added to each molecule to be sequenced to mark the molecule and its progeny (e.g., amplicons generated by PCR). In some embodiments, a UMI is present in the 5 ’-portion of an amplification primer. In some embodiments, a UMI is present in an adaptor ligated to the nucleic acid.

[0187] A UMI has multiple uses including counting the number of original target molecules in the sample and error correction (Newman, A., et al., (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nature Medicine doi:10.1038 / nm.3519). Briefly, the entire progeny of a single target molecule is marked with the same UMI barcode and thus forms a barcoded family. A variation in the sequence not shared by all (or the majority) of the members of the barcoded family is discarded as an artefact. UMI barcodes can also be used for positional deduplication and target quantification, as the entire family represents a single molecule in the original sample (Newman, A., et al., (2016) Integrated digital error suppression for improved detection of circulating tumor DNA, Nature Biotechnology 34:547).

[0188] A sample identifying barcode is used for multiplex sequencing. A multiplex sample ID barcode (abbreviated MID or SID) is used to identify the source of the nucleic acid where two or more samples are mixed prior to the sequencing step (e.g., application to a flowcell of a sequencing instrument).

[0189] In some embodiments, the nucleic acid molecule to be sequenced includes a UMI and an MID. In some embodiments, a single barcode is used as both UMI and MID. In some embodiments, a barcode is composed of several parts. For example, the unique identifying information is comprised of a barcode sequence and a nucleic acid end sequence. In some embodiments, a barcode is comprised of several subcodes as described in the U.S. Patent Application Pub. No. 20230081899 "Modular Nucleic Acid Adaptors."PATENT Docket No.: CB 1062.30

[0190] In some embodiments, each barcode comprises a predefined sequence. In other embodiments, the barcode comprises a random sequence. The barcodes are about 4-20 bases long, so that between 96 and 384 different adaptors, each with a different pair of identical barcodes can be added to a human genomic sample. In some embodiments, the number of UMIs in the reaction can be in excess of the number of molecules to be labelled. A person of ordinary skill in the relevant art would recognize that the number of barcodes depends on the complexity of the sample (z.e., expected number of unique target molecules) and would be able to design a suitable number of barcodes of suitable lengths for each sequencing run.

[0191] In some embodiments, the method includes sequencing the nucleic acid adapted by the methods described herein. Any of a number of sequencing technologies or sequencing assays can be utilized. The term "Next Generation Sequencing (NGS)" as used herein refers to sequencing methods that allow for massively parallel sequencing of single molecules or clonally amplified single molecules.

[0192] Non-limiting examples of sequence assays that are suitable for use with the methods disclosed herein include nanopore sequencing (U.S. Pat. Publ. Nos. 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0119259 and 2015 / 0337366), Sanger sequencing, capillary array sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman etal., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., Nature Biotech., 16:381-384 (1998)), sequencing by hybridization (Drmanac et al., Nature Biotech., 16:54-58 (1998), and NGS methods, including but not limited to sequencing by synthesis (e.g., HISEQ™, MISEQ™, or Genome Analyzer, each available from Illumina), sequencing by ligation (e.g., SOLID™, Life Technologies), ion semiconductor sequencing (e.g., ION TORRENT™, Life Technologies), and SMRT® sequencing (e.g., Pacific Biosciences).

[0193] Commercially available sequencing technologies include sequencing-by-hybridization platforms from Affymetrix Inc. (Sunnyvale, Calif.), sequencing-by-synthesis platforms from Illumina / Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.). Other sequencing technologies include, but are not limited to, the ION TORRENT™ technologyPATENT Docket No.: CB 1062.30 (ThermoFisher Scientific, Waltham, Mass.), Single Molecule Real-Time (SMRT®) sequencing (Pacific Biosciences, Menlo Park, Calif.) and Oxford Nanopore Technologies (Oxford, UK).

[0194] In some embodiments, the sequencing step involves sequence aligning and determining a consensus sequence. In some embodiments, a consensus sequence is determined from a plurality of sequences all having an identical UMI. The sequenced having an identical UMI are presumed to derive from the same original molecule through amplification. In other embodiments, UMI is used to eliminate artifacts, z.e., variations existing in the progeny of a single molecule resulting from PCR errors or sequencing errors.

[0195] In some embodiments, the number or representation of each sequence in a sample can be quantified by quantifying relative numbers of sequences with each UMI among the population having the same multiplex sample ID (MID). A person skilled in the relevant art will be able to determine the number of sequence reads per UMI (“sequence depth”) necessary to determine a consensus sequence with a desired degree of confidence. In some embodiments, the desired depth is 5-50 reads per UMI.

[0196] In some embodiments, the computational script was designed to executes the following tasks: align reads to the mouse genome (e.g., the latest build, currently mml 0) using any suitable software; compare aligned reads to the expected wild type genomic locus sequence; discard reads not aligning to any part of the wild type locus; tally reads matching the wild type sequence; categorize reads with indels (insertion or deletion of bases) by indel type and tally; and determine the proportion (e.g., percentage) of mutant (edited) reads by dividing the tally of indel reads by the sum of wild type reads and indel reads.

[0197] In some embodiments, the invention comprises an amplification step preceding the sequencing step. The amplification step can involve linear or exponential amplification, e.g., PCR. Amplification may be isothermal or involve thermocycling. In some embodiments, the amplification is exponential and involves PCR or any of its variations including real-time PCR, digital droplet PCR (ddPCR), emulsion PCR and the like. In some embodiments, a universal amplification primer is used, i.e., a primer that hybridizes to a universal primer binding site present in the adaptor ligated to all nucleic acids in the sample. The number of amplification cycles where universal primers are used can be low, but also can be 10, 20 or as high as 30 or more cycles, depending on the amount of amplification product needed for thePATENT Docket No.: CB 1062.30 subsequent steps. Because amplification with universal primers has reduced sequence bias, the number of amplification cycles need not be limited out of concern for amplification bias.

[0198] In some embodiments, successful editing is assessed by amplification. In some embodiments, the amplification primers are designed to flank the editing site and only yield amplification products in case of a successful edit or yield different size amplification products with and without the edits (FIGURE 24).

[0199] In some embodiments, the LNP composition comprising the Cascade effector system is used for patient administration if editing efficiency is at least 65%. The inventors discovered that 100% editing efficiency in the liver is not required to achieve the desired physiological effect. As is seen from FIGURE 18 and FIGURE 19, 65% editing results in nearly complete elimination of Ttr gene expression.

[0200] One of skill in the relevant art would be able to experimentally determine the minimal editing efficiency required to achieve physiological effect for each target gene in each target organ. The therapeutically effective amount of the LNP composition would include the sufficient amount of Cascade effector system components to produce at least the minimal editing efficiency.

[0201] In some embodiments, prior to patient administration, the LNP composition comprising the CRISPR system is assessed for causing chromosomal translocations. It has been reported that genome editing involving double-strand breaks (e.g., editing with CRISPR endonucleases) occasionally results in balanced chromosomal translocations between two cleavage sites located on different chromosomes. A detection assay can be designed for the predicted most likely translocation e.g., between a target cleavage site and the predicted most likely off-target cleavage site. One example of such a detection assay is disclosed in the International Application Pub. No. WO2025024676 In vitro validation methods for CD19-targeting cell therapies, filed on July 26, 2023. Briefly, a series of amplification primers can be designed adjacent to each known or predicted cleavage site involved in the translocation to be detected. In some embodiments, the LNP composition comprising the CRISPR system is administered to a patient if no translocations are detected during the testing or if the rate of translocations falls below a predetermined safety threshold.PATENT Docket No.: CB 1062.30

[0202] In some embodiments, prior to patient administration, the LNP composition comprising the CRISPR system is assessed for causing off-target editing.

[0203] In some embodiments, potential sites for off-target editing in a given genome are found empirically, e.g., by performing whole-genome sequencing of edited cells and identifying and rating any genome changes not present in unedited genomes. In some embodiments, potential off-target sites are located using algorithms developed for that purpose, e.g., DeWierdt, P., el al. (2021) Optimization of AsCasl2a for combinatorial genetic screens in human cells, Nat. Biotech. 39(1):94), SITE-Seq, Cas-OFFinder, CRISPRme, and GUIDE-Seq. In some embodiments, the most likely off-target sites are selected for testing, e.g., off-target sites with no more than 4 mismatches with the target site, or no more than 6 mismatches with the target site.

[0204] In some embodiments, amplification primers are designed to amplify and sequence each of the selected potential off-target sites in edited cells to assess genome editing. In some embodiments, the editing rates are determined as Significant Mutant Fraction (SMF) calculated by first determining the "significant" mutations in the matched test and control samples. A mutation (edit) is only considered "significant" if (1) it falls within ±3bp of the predicted cut-site coordinates, and (2) its frequencies in the test and control samples differ significantly as determined by a chi-squared test with p-value threshold of 10’4with Bonferroni multiple-comparison correction. The total frequency of "significant" mutations (edits) is calculated for the test and control samples and the control frequency is subtracted from the test frequency to produce the final Significant Mutation Fraction statistic. In some embodiments, subtraction of the control mutant rate results in a negative number (mutation frequency less than in the control sample). In some embodiments, the negative numbers are recorded as zero.

[0205] In some embodiments, the Cascade effector system is used for patient administration if off-target editing is no greater than 0.02% across all off-sites tested.

[0206] With respect to the precision of genomic editing, the inventors devised methods and compositions for in vivo gene editing that substantially improve upon the state of the art. Gillmore etal., report that in the case of in vivo TTR gene editing with CRISPR-Cas9, up to 7% of off-target editing has occurred. (Gillmore, J. etal., (2021) CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis, NEJM 385:493, appendix, Table SI).PATENT Docket No.: CB 1062.30

[0207] In some embodiments, the LNP composition comprising the Cascade effector system is administered to a patient if no off-target editing is detected or if the rate of off-target editing falls below a predetermined safety threshold.

[0208] In some embodiments, prior to administration to a patient, the LNP composition comprising the Cascade effector system is tested in vivo in experimental animals to extra-organ editing (e.g., extrahepatic editing in case of targeting the liver). In some embodiments, the LNP composition comprising the Cascade effector system is used for patient administration if extraorgan editing is no greater than 0.5% across all (or relevant) extra organs tested.

[0209] In some embodiments, prior to administration to a patient, the LNP composition comprising the CRISPR system is tested in vivo for tolerability in experimental animals. In some embodiments, the tolerability is assessed by measuring immune response following the treatment, e.g., 6 hours following the treatment. In some embodiments, immune response is assessed by measuring serum levels of cytokines selected from IL-1, IL-2, lL-2-receptor-a, IL-6, IL-8, IL-10, JFNy, TNFa, MCP-1 and GM-CSF. In some embodiments cytokine levels are compared before and after the treatment. In some embodiments, cytokine levels are remeasured, e.g,, after 1 or 2 days. In some embodiments, cytokine levels are measured by ELISA.

[0210] In some embodiments, the LNP composition comprising the CRISPR system is used for patient administration if no excessive cytokine levels are observed in experimental animals after 6 hours and cytokine levels return to baseline after one or two days.

[0211] In some embodiments, in cases of liver editing, the tolerability of the LNP composition is assessed by measuring the amount of liver enzymes. In some embodiments, liver enzymes are aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In some embodiments, the amount of liver enzymes is assessed in vivo in experimental animals. In some embodiments, the LNP composition comprising the Cascade effector system is used for patient administration if no significant change in the amount of liver enzymes (e.g., AST and ALT) is observed.

[0212] In some embodiments, the composition also includes one or more pharmaceutically acceptable excipients. Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water,PATENT Docket No.: CB 1062.30 alcohols, polyols, glycerin, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; di saccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0213] In some embodiments, the composition further comprises an antimicrobial agent for preventing or deterring microbial growth. In some embodiments, the antimicrobial agent is selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

[0214] In some embodiments, the composition further comprises an antioxidant added to prevent the deterioration of the lymphocytes. In some embodiments, the antioxidant is selected from ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0215] In some embodiments, the composition further comprises a surfactant. In some embodiments, the surfactant is selected from polysorbates, sorbitan esters, lipids, such as phospholipids (lecithin and other phosphatidylcholines), phosphatidylethanolamines, fatty acids and fatty esters; steroids, such as cholesterol.

[0216] In some embodiments, the composition further comprises a freezing agent such as 3% to 12% dimethylsulfoxide (DMSO) or 1% to 5% human albumin.

[0217] In some embodiments, the invention is a method of treating a patient by administering to the patient in need thereof an amount of the LNP composition comprising 0.25 mg / kg and 2.5 mg / kg of total nucleic acid (including the mRNA encoding the Cascade proteinsPATENT Docket No.: CB 1062.30 and the gRNA) and a suitable excipient and optionally, also one or more of a suitable antimicrobial agent, an antioxidant, a surfactant, and a freezing agent.

[0218] In some embodiments, the method of treating a patient further involved steps of monitoring the patient for safety and effectiveness of the treatment.

[0219] In some embodiments, the patient is monitored for tolerability of the treatment with LNPs containing the Cascade effector system components described herein. In some embodiments, the tolerability is assessed by measuring immune response following the treatment, e.g., 6 hours following the treatment. In some embodiments, immune response is assessed by measuring serum levels of one or more cytokines selected from IL-1, IL-2, IL-2-receptor-a, IL-6, IL-8, IL-10, IFNy, TNFa, MCP-1 and GM-CSF. In some embodiments cytokine levels are compared before and after the treatment. In some embodiments, cytokine levels are remeasured, e.g., after 1 or 2 days. In some embodiments, cytokine levels are measured by ELISA.

[0220] In some embodiments, in cases of liver editing, the tolerability is assessed by measuring the amount of liver enzymes. In some embodiments, liver enzymes are aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In some embodiments, the levels of AST and ALT are measured 1, 2, 3, 4, 5, 6, 7 10 or more days after LNP administration. In some embodiments, AST and ALT levels are measured by ELISA.

[0221] In some embodiments, the patient is periodically assessed for durability of response to treatment with LNPs containing the Cascade effector system components described herein. In some embodiments, the patient is assessed for the presence of the protein whose gene has been edited or for physiological response to the editing of the gene. In some embodiments, e.g., when the gene is TTR, PSCK9 or ANGPTL3, the patient is assessed for serum level of the respective protein. In some embodiments, e.g., when the gene is PSCK9, the patient is assessed for serum cholesterol level, e.g., LDL cholesterol level or total cholesterol level. In some embodiments, e.g., when the gene is ANGPTL3, the patient is assessed for serum triglyceride level, e.g., VLDL or chylomicron level or total triglyceride level. In some embodiments, the patient is assessed monthly or bi-weekly.

[0222] EXAMPLES

[0223] Example 1. nCas3 Cascade effector complexes targeting mouse Pcsk9 and TtrPATENT Docket No.: CB 1062.30

[0224] This example describes design of nucleic acids encoding the protein components of the Cascade effector complex and design of the guide RNA (gRNA) targeting mouse Psck9 and Ttr genes.

[0225] A. Nucleic acids encoding Cascade proteins

[0226] Taking the Cascade effector complex from Pseudomonas sp. S-6-2, each of the protein-coding mRNA of the nCas3 Cascade effector complex was codon-optimized for translation in mouse cells and further optimized by uridine depletion and substitution of uridine for N1 -methyl pseudouridine.

[0227] B. Nucleic acids encoding Cascade guide RNA

[0228] The nucleic acids encoding Cascade guide RNAs were designed based on the wild-type minimal CRISPR array encoding the Cascade gRNA shown in FIGURE 1. The Cascade gRNAs were engineered for the dual-nicking system and contained two separate spacers referred as “Half-site 1” and “Half-site 2,” FIGURE 3. Two separate gRNAs were used each containing a 32-bp spacer targeting the desired locus flanked by two CRISPR repeats of 29 bp. (FIGURE 3).

[0229] To design the targeting region of the gRNA, mouse genes Pcsk9 and Ttr were scanned for the presence of one of the Cascade PAMs “AAG.” Targets were prioritized based on the shortest inter-nicking distance between two half sites targeting Exons 1, 2 or 3 of the selected loci. FIGURE 4 and FIGURE 5 illustrate the location of the targets in the murine Psck9 and Ttr genes respectively.

[0230] Example 2. Initial attempt at in vitro genome editing with nCas3 effector complexes

[0231] This example describes editing of t\&Psck9 locus and the Ttr locus in the murine hepatic cell line H2.35 (ATCC CRL-1995) with nCas3 Cascade effector complexes delivered either via electroporation (nucleofection) or via LNPs (lipofection).

[0232] A. RNA

[0233] The mRNAs encoding the nCas3 Cascade effector complex proteins were synthesized by TriLink Biotechnologies (San Diego, Cal.). The gRNAs were synthesized by Integrated DNA Technologies (San Diego, Cal.). The mRNA:gRNA mixture atPATENT Docket No.: CB 1062.30 ratio (nCas3:Cas5:Cas6: Cas7:Cas8:Casll: gRNA(Half-sitel):gRNA(Half-site2) was prepared.

[0234] B. LNP preparation

[0235] The lipid mixture “ALC0315” was prepared in ethanol as shown in Table 6.

[0236] Table 6. Lipid components of the LNP

[0237] 1 mL of the lipid mixture was loaded into a syringe for the encapsulation step.

[0238] C. LNP formation (encapsulation)

[0239] For LNP formation (encapsulation), a syringe with 3 mL of the RNA mixture and a syringe with 1 mL of the lipid mixture was inserted into the NANOASSEMBLR® IGNITE™ (Precision Nanosystems, Vancouver, BC). The NANOASSEMBLR® IGNITE™ was pre-loaded with a NANOASSEMBLR® IGNITE™ Cartridges (NxGen) to form water-in-oil droplets using rapid mixing. Encapsulation efficiency was assessed using the QUANT-IT™ RiboGreen Assay Kit (ThermoFisher Scientific). LNP size and poly dispersity was measured with an UNCLE instrument (Unchained Labs, Pleasanton, Cal.) using Dynamic Light Scattering (DLS).

[0240] D. LNP delivery (lipofection)

[0241] Cells were plated for 24 hours prior to LNP delivery. Human ApoE4 Recombinant Protein lyophilized (PeproTech, ThermoFisher Scientific, Cranbury, N.J.) was resuspended in PBS for a stock concentration of lOOug / ml. Resuspended ApoE4 was added to cultured cells at a final concentration of lug / ml. After addition of ApoE4 to the media, LNPs encapsulating 50-500ng of Casl2a mRNA and gRNA were also added to the cultured cells.

[0242] E. Electroporation (nucleofection)

[0243] Nucleofection was performed essentially as follows. The RNA mixture described in Example 2(A) was transfected into H2.35 cells using the NUCLEOFECTOR™PATENT Docket No.: CB 1062.30 96-well Shuttle System (Lonza, Allendale, N.J.). The RNA mixture was dispensed in a 2.5 pl final volume into individual wells of a 96-well plate. The adherent H2.35 cells were washed with 10 mL of calcium and magnesium-free phosphate-buffered saline (PBS) and then PBS aspirated, followed by the addition of 5 mL of ACCUTASE™ (Innovative Cell Technologies, Inc., San Diego, Cal.) and incubated for 5-10 minutes at room temperature. Flask was rocked side to side and tapped against the palm of the hand to detach cells. H2.35 cell suspension was pelleted by centrifugation for 5 minutes at 200 x g, washed with calcium and PBS, and the cell pellet was resuspended in 10 ml of calcium and magnesium-free PBS. The cells were counted using the COUNTESS® II Automated Cell Counter (Life Technologies; Grand Island, N.Y.).

[0244] 107cells were transferred to a 15 ml conical tube and pelleted. The PBS was aspirated, and the cells resuspended in CTS Xenon Electroporation Buffer (ThermoFisher Scientific, Wilmington, Del.) solution to a density of 105- 2.5 x 105cells / ml per sample. 18 pl of the cell suspension was then added to each well containing 2.5 pl of the RNA mixture, and the entire volume from each well was transferred to a well of a 96-well NUCLEOCUVETTE™ Plate (Lonza). The plate was loaded onto the NUCLEOFECTOR™ 96-well Shuttle and cells nucleofected using the DS-137 NUCLEOFECTOR™ program (Lonza). Post-nucleofection, 77.5 pl of DMEM (4g / L glucose) medium supplemented with lOOnM dexamethasone and 4% fetal bovine serum was added to each well, and the entire volume of transfected cell suspension was transferred to a 96-well cell culture plate containing 100 pl pre-warmed DMEM complete medium. The plate was transferred to a tissue culture incubator and maintained at 33°C in 10% CO2 for 48 hours before downstream analysis.

[0245] F. Assessing genome editing by NGS

[0246] NGS was performed essentially as follows. Genomic DNA (gDNA) was isolated from the H2.35 cells 48 hours after contact with Cascade effector complex using the QUICKEXTRACT™ DNA extraction solution (Epicentre, Madison, Wise.) The isolated gDNA was diluted with 50 pL sterile water and samples were stored at -80°C.

[0247] Using the isolated gDNA, a first PCR was performed using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, Mass.) at lx concentration, primers designed to amplify the region around the Cascade targets were used at 0.5 pM each, and 3.75 pL of gDNA was used in a final volume of 10 pL. Amplification was conducted by an initialPATENT Docket No.: CB 1062.30 cycle at 98°C for 1 minute, 35 cycles of 10s at 98°C, and 20 seconds at 60°C, 30 seconds at 72°C; and a final extension at 72°C for 2 minutes. The PCR reactions were diluted 1:100 in water.

[0248] PCR with barcoded primers was performed using a reaction mix comprising Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) at lx concentration, primers at 0.5 pM each, and 1 pL of 1:100 diluted first PCR in a final volume of 10 pL. The reaction mixtures were amplified as follows: 98°C for 1 minute; followed by 12 cycles of 10s at 98°C, 20s at 60°C, and 30s at 72°C; with a final extension reaction at 72°C for 2 minutes.

[0249] The PCR reactions were pooled and transferred into a single microfuge tube for SPRIselect (Beckman Coulter, Pasadena, Cal.) bead-based cleanup of amplicons for sequencing.

[0250] The purified amplicons were quantified using the NANODROP™ 2000 System (ThermoFisher Scientific, Wilmington, Del.) and library quality analyzed using the Fragment Analyzer™ System and the DNF-910 dsDNA Reagent Kit (Advanced Analytical Technologies, Ames, Iowa).

[0251] The pooled amplicons were normalized to a 4 nM concentration as calculated from the NANODROP™ 2000 System values and the average size of the amplicons. The library was analyzed on a MiSeq Sequencer with MiSeq Reagent Kit v2 (Illumina, San Diego, Cal.) for 300 cycles with two 151-cycle paired-end runs and two 8-cycle index reads.

[0252] The identities of products in the sequencing data were determined based on the index barcode sequences adapted onto the amplicons in the barcoding PCR. A computational script was used to process the MiSeq data that executes, for example, the following tasks: a. Reads were aligned to the mouse genome (build mm 10) using Bowtie software; b. Aligned reads were compared to the expected wild type genomic locus sequence, and reads not aligning to any part of the wild type locus discarded;c. Reads matching wild type sequence were tallied;d. Reads with indels (insertion or deletion of bases) were categorized by indel type and tallied; ande. Total indel reads were divided by the sum of wild type reads and indel reads to give percent-mutated reads.PATENT Docket No.: CB 1062.30

[0253] Through the identification of indel sequences at regions targeted by the Casl2a-guide nucleoprotein complexes, the resulting genome editing efficiency was determined.

[0254] FIGURE 6 depicts results of editing of the Psck9 locus and the Ttr loci with Cascade delivered via lipofection (LNP delivery) and FIGURE 7 depicts results of editing of the same loci with Cascade delivered via nucleofection (electroporation).

[0255] FIGURE 8 depicts the pattern of genomic deletions in the Ttr locus with nucleofection (electroporation) delivery and FIGURE 12 depicts the pattern of genomic deletions in the Ttr locus with lipofection (LNP) delivery. Targets Pcsk9-tgtl3 and Ttr-tgt5 were selected for further study.

[0256] Example 3. Optimizing in vitro genome editing with nCas3 Cascade effector complexes.

[0257] This example describes improvements upon the genome editing methods of Example 2 including improvements of gRNA and protein-coding mRNA.

[0258] A. Chemical modifications and titrations of gRNA

[0259] The chemical modifications of gRNA are shown in FIGURE 9. The 3 nucleotides on the 5’ and 3’ end of each half-site were chemically modified adding a 2'-O-Methyl and a phosphorothioate group in the 3 last nucleotides as indicated.

[0260] Genome editing was performed using the nCas3 Cascade effector complex containing an unmodified gRNA, end-modified gRNAs and a gDNA from Example 1(B). The editing of each locus was assessed essentially as described in Example 2. Results are shown in FIGURE 10

[0261] B. Optimizing the amount of gRNA

[0262] Cascade mRNA (Example 1(A)) and gDNA (Example 1(B)) targeting Pcsk9-tgtl3 or Ttr-tgt5 were encapsulated using NANOASSEMBLR® SPARK™ nanoparticle formulation system (Cytiva, Danaher Corp.). Genome editing in the H2.35 cells was assessed by PCR and next-generation sequencing essentially as described in Example 2. Results are shown in FIGURE 11 (% edits) and FIGURE 12 (pattern of genomic deletions at the locus Ttr-tgt5).

[0263] C. Optimizing the mRNA encoding Cascade proteinsPATENT Docket No.: CB 1062.30

[0264] Two mRNA structures were used to edit the targets Pcsk9-tgtl3 or Ttr-tgt5 in primary hepatocytes. The first mRNA design “In vitro” had the structure optimized for in vitro applications and containing a 5’-cap Anti-Reverse Cap Analog (ARCA) (Catalog # N-7003, TriLink Biotechnologies, San Diego, Cal.), enzymatic A-tailing and no modified nucleotides. The mRNA was manufactured by Aldevron (Fargo, N.D.). The second mRNA design “In vivo” is shown in FIGURE 13 and contains a 5’ cap m6AG (3’OMe) sold under the trademark CLEANCAP®-Reagent M6, catalog # N-7453 by TriLink Biotechnologies, UTRs, and a 120-bppoly-Atail (SEQ ID NO: 327). The protein coding sequence in the mRNA was mouse codon-optimized (codon adaptation index, CAI), uridine depleted and fully substituted with Nl-methyl-pseudouridine. The mRNA was manufactured by TriLink Biotechnologies.

[0265] Unmodified gRNAs were used in this experiment to assess editing efficiencies with both mRNA structures. Primary hepatocytes where isolated from Balb / c mice using a perfusion method. Cascade delivery via LNP was performed essentially as described in Example 2. Results are shown in FIGURE 14 with references to “In vitro” mRNA design and “In vivo” mRNA design.

[0266] Example 4. In vivo genome editing with Cascade effector complexes.

[0267] This example describes in vivo genome editing using the mRNA “Aldevron” (Example 3(C)) and either gRNA or gDNA for the Cascade effector complex (Example 1) encapsulated in LNPs as described in Example 2.

[0268] Seven weeks-old BALB / c mice were injected intravenously with a 350-400 uL volume containing the mRNA and either gRNA (Example 1(B)) or gDNA (Example 1(B)) at 2 mg of nucleic acid / kg of body weight (2 mg / kg). TTR protein was deleted in serum by ELISA on days zero and 16 post-LNP injection using a commercial ELISA kit (Cat. No. OKIA00111, Aviva Systems Biology, San Diego, CaL). Briefly, 5ul of plasma were diluted to a final dilution of 1 / 20,000 following manufacturer’s recommendations. Results are shown in FIGURE 15 (% editing) and FIGURE 16 (genomic deletions detected by NGS).

[0269] Example 5. Optimizing in vivo genome editingwith Cascade effector complexes.

[0270] A. Modifications of mRNA

[0271] In this example, the mRNA designs “In vivo” and “In vitro” from Example 3(C) were compared in the in vivo study designed essentially as in Example 4, except the amount ofPATENT Docket No.: CB 1062.30 nucleic acid was increased from 2 mg / kg to 2.5 mg / kg and the mice were sacrificed after 6 days. Results of editing Ttr-tgt5 and Pcsk9-tgtl3 are shown in FIGURE 17.

[0272] B. Modifications of guide RNA

[0273] In this example, three gRNA structures were assessed and compared to gDNA. “Ultramer” is a gRNA described in Example 1(B) without any chemical modifications. “AltR” is a gRNA using an ALT-R™ end-modification from Integrated DNA Technologies (Coralville, la). “End-mod” is a gRNAs that has chemical modifications as illustrated in FIGURE 9

[0274] C. In vivo gene editing

[0275] The in vivo study was designed as described in Example 4, except the mice were sacrificed after 14 days. FIGURE 18 depicts results of editing of Ttr-tgt5. Serum levels of the TTR protein were assessed essentially as described in Example 4 and the results are shown in FIGURE 19. Tolerability of the LNP composition was assessed by monitoring body weight of the mice (FIGURE 20) and by measuring serum levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) 24 hours post-LNP injection (FIGURE 21)

[0276] Example 6. In vivo dose escalation of the Cascade effector complex

[0277] In this example, the LNP delivery of Cascade complex was performed in accordance with Example 5 with the end-modified gRNA targeting Ttr and mRNAs generated by TriLink Biotechnologies. The doses of total nucleic acid administered to the animals was varied 10-fold between 0.25 mg / kg and 2.5 mg / kg (0.25, 0.5, 1, 2, and 2.5 mg / kg). Results (% edits) are show in FIGURE 22.

[0278] Example 7. Gene knock-in (KI) in human cells with nCas3 Cascade effector complexes

[0279] Human iPSCs were nucleofected with mRNAs encoding the nCas3 Cascade effector complex proteins (FIGURE 13) generated by TriLink Biotechnologies. gDNAs were designed as described in Example 1(B) targeting the human loci CISH, CBLB and TRAC. A donor DNA plasmid was co-transfected at the same time. The donor constructs contained a 500bp 5’-and 3’-homology arms for the CBLB locus and a EFla promoter driving the expression of eGFP. Results are shown in FIGURE 23. For CBLB, targeted knock-in of thePATENT Docket No.: CB 1062.30 eGFP insert was validated using targeted PCR with primer pairs P1 / P2 and P3 / P4 (Table 7) flanking each integration site (FIGURE 24). The reaction conditions were:

[0280] Table 7. Amplification primers (artificial sequences)

[0281] The identify of the PCR products was confirmed by NGS.

[0282] Example 8. Optimizing gene knock-in (KI) in human cells with nCas3 Cascade effector complexes.

[0283] In this example, the gene knock-in experiment was optimized by varying the design and the amount of protein-coding mRNA and further optimized by varying the amounts of guide RNA (gRNA).

[0284] The mRNA designs “In vitro” and “In vivo” described in Example 3(C) were compared in the human iPSC study designed essentially as in Example 7, except for each mRNA, four doses were tested: 0.75 pg, 0.5 pg, 0.25 pg, and 0.125 pg for each mRNA component of the Cascade effector complex. Results are shown in FIGURE 25.

[0285] The gRNA design described in Example 3(A) were compared in the human iPSC study designed essentially as in Example 7, except for total mRNA, the dose was fixed at 0.5 pg, while the dose of gRNA was either 0.5 pg or 0.8 pg. Results are shown in FIGURE 26.

[0286] Example 9. In vitro genome editing with tagged Cascade effector complexes

[0287] In this experiment, we designed several DNA constructs encoding the proteins nCas3 or Cas5 tagged with 3 FLAG-tag (DYKDHDGDYKDHDIDYKDDDDK, SEQ ID NO: 8) at either the N-terminus or C-terminus using molecular cloning techniques. Constructs werePATENT Docket No.: CB 1062.30 inserted into suitable expression vectors with EFl -alpha promoter for expression in mammalian cells essentially as described in Example 17. Cells were transfected with the plasmids with no FLAG-tag, N-terminus-3xFLAG or C-terminus-3xFLAG on either nCas3 or Cas5. Editing of the loci TTR, CISH or CBLB was evaluated to assess potential disruption of Cascade protein function. Results are shown in FIGURE 27.

[0288] Example 9. Durable Ttr suppression for at least 12 months

[0289] In this experiment, the mice with Ttr gene disruption (Example 5, FIGURE 19) were monitored for 12 months. Every two months serum TTR level was assessed by ELISA using the commercial kit described in Example 4. Results are shown in FIGURE 28.

[0290] Example 10. Primary hepatocytes from transgenic mice carrying human liveractive genes.

[0291] Transgenic mice carrying human genes were obtained from The Jackson Laboratory.

[0292] The human APOC3 transgenic mice B6;CBA-Tg(APOC3)3707Bres / J, strain # 006907 are described in Reaven G.M., et al., (1994) Hypertriglyceridemic mice transgenic for the human apolipoprotein C-1I1 gene are neither insulin resistant nor hyperinsulinemic, J Lipid Res 35(5): 820-4. According to the description provided by the supplier, to produce the strain, transgenic construct containing the human apolipoprotein C-III gene (including 2.5 kb 5' and 1.1 kb 3' flanking sequences) was injected into the male pronucleus of fertilized eggs (both from (C57BL / 6J x CBA / J)F1 mice). The resulting animals were bred to (C57BL / 6J x CBA / J)F1 to establish a founder line. Given the high expression of the transgene in the mice, multiple copies are likely.

[0293] The human SERPINA1 transgenic mice C57BL / 6J-Tg(SERPINAl*E366K)lMlb / J, strain # 037670 are described in Lu Y., et al., (2022) The unfolded protein response to PI*Z alpha-1 antitrypsin in human hepatocellular and murine models, Hepatol Commun 6(9):2354-2367. The PI*Z mice express a mutant human SERPINA 1 gene which carries the mutation E366K. According to the description provided by the supplier, to produce the strain, the linearized transgenic construct containing the entire mutant human E366K SERPINA 1 gene under its endogenous promoters (both hepatocyte and macrophage), plus 5 kB of the 5' and 3 kb of the 3' flanking genomic DNAPATENT Docket No.: CB 1062.30 sequences was microinjected into fertilized C57BL / 6J oocytes. Founder 1 carrying 5-6 copies of the transgene was maintained on the C57BL / 6J background.

[0294] Primary hepatocytes from transgenic mice (PMH) (FIGURE 29) were isolated with a Liver Perfusion Kit (Miltenyi Biotec, 130-128-030) following the manufacturer’s protocol. The isolated cells were plated in 96-well plate at 4xl04cells / well and transfected within 2-4 hours after plating using the Cascade mRNAs (FIGURE 13) and unmodified crRNA as described in the next example.

[0295] Example 11. Editing APOC3 and SERPINAI in a human cell line and PMH.

[0296] In this experiment, the nCas3 Cascade editing was applied to cells of a human cell line (HEK293T, kidney, ATCC® CRL-3216) and to the primary mouse hepatocytes (PMH) of transgenic mice carrying the human APOC3 gene or the human SERPINAI gene (Example 10). APOC3 or SERPINAI were targeted with Cascade mRNA and unmodified crRNA delivered using LIPOFECTAMINE™ MESSENGERMAX™ transfection reagent (Invitrogen, ThermoFisher Scientific, Carlsbad, Cal.) per manufacturer’s instructions. The targeting regions of gRNAs for each APOC3 and SERPINAI target (half-spacers) are listed in Table 4. Genome editing was assessed by NGS as described in Example 2(F). Results are shown FIGURE 30 and FIGURE 31 (APOC3) and FIGURE 32 (SERPINAI).

[0297] Example 12. Editing LPA in a human cell line

[0298] In this experiment, the nCas3 Cascade targeted the LPA gene in the HEK293T cell line essentially as described in Example 11. The targeting regions of gRNAs for each LPA target (half-spacers) are listed in Table 4. Genome editing was assessed by NGS as described in Example 2(F). Results are shown in FIGURE 33.

[0299] Example 13. Modified gRNA used in vivo

[0300] In this example, the nCas3 Cascade with chemically modified gRNA that was previously used in an in vitro editing experiment (Example 3(A), FIGURE 9) was used for in vivo gene editing of target 5 in the Ttr gene (TTR-tg5, Table 4) in Balb / c mice. The nCas3 Cascade effector complex (Example 1) with SEQ ID NOs: 350 and 351 was codelivered with gRNAs via LNPs as described in Example 2. Results are shown in FIGURE 34.

[0301] Example 14. Truncated gRNAPATENT Docket No.: CB 1062.30

[0302] In this example, the gRNA (FIGURE 3) was modified to truncate the 29-bp repeats situated around each spacer. The 29-bp repeats were shortened to 21 bp or to 7 bp. Editing Pcsk9 target 13 (tgt 13 guides SEQ ID NOs: 334 and 335) and Ttr targets 5 and 25 (tgt5 guides having spacers SEQ ID NOs: 318 and 316 truncated to 7 and 21 bp respectively, tgt25: guides SEQ ID NOs: 330 and 331) in PMH of Balb / c mice (Example 3) with all other reagents and procedure steps described in Example 10. Results are shown in FIGURE 35.

[0303] While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus, the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

Claims

PATENT Docket No.: CB 1062.30 CLAIMS:

1. A composition for nucleic acid modification comprising:(i) a nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex, (ii) a first guide polynucleotide comprising a first half-site capable of hybridizing to a first target site within the target nucleic acid, and(iii) a second guide polynucleotide comprising a second half-site capable of hybridizing to a second target site within the target nucleic acid, wherein the first and the second target sites in the nucleic acid are separated by an inter-nicking distance, and wherein the nCas3 is capable of nicking the first and the second target site.

2. The composition of claim 1, wherein the nCas3 is the Pseudomonas sp. S-6-2 nCas3.

3. The composition of claim 2, wherein the nCas3 is selected from the group consisting of D448A nCas3, D448R nCas3, D448C nCas3, D448N nCas3, D448Q nCas3, D448G nCas3, D448K nCas3, D448M nCas3, D448S nCas3, D448T nCas3, and D448V nCas3.

4. The composition of claim 1 , wherein the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for an nCas3 protein, a Cas7 protein, a Cas5 protein, a Cas8 protein, a Cast 1 protein, and a Cas6 protein.

5. The composition of claim 1, wherein the nucleic acid encoding the nCas3 Cascade effector complex in an mRNA having the SEQ ID NOs.: 1-6.

6. The composition of claim 5, wherein the mRNA comprises codon optimization for optimizing mRNA expression in mammalian cells.

7. The composition of claim 5, wherein the mRNA comprises modifications minimizing immunogenicity in mammalian recipients of the mRNA.

8. The composition of claim 7, wherein the modification minimizing immunogenicity is a uridine modification or a cytidine modification.

9. The composition of claim 8, wherein the uridine modification is selected from the group consisting of 5-methoxyuridine, 5-methyluridine, 5 -carboxy methytl ester uridine, 2-thiouridine and pseudouridine and derivatives thereof.PATENT Docket No.: CB 1062.30 10. The composition of claim 8, wherein the cytidine modification is 2 -methoxy cytidine or its derivatives.

11. The composition of claim 5, wherein the mRNA comprises a 5’-cap having a formula selected from the group consisting of N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G, N7-(4-bromophenoxyethyl)-m3'-OG(5')ppp(5')G, N7mG(5’)ppp(5’)G,N7mG(3 ’ 0Me)(5 ’ )ppp(5 ')m6 A(2’ OMe)pG, and N7mG(3 ’ 0Me)(5 ’ )ppp(5 ’ )m6G(2 ’ OMe)pG, 12. The composition of claim 5, wherein the mRNA comprises a 5’-cap and the cap comprises the structure 3’-G(5’)PPP-5' wherein the G is a modified guanosine selected from the group consisting of N7-(4-chlorophenoxyethyl)- guanosine, N7-(4-chloro-phenoxyethyl)-guanosine, N7-(4-chlorophenoxyethyl)-m3'-O-guanosine, and N7-(4-bromophenoxyethyl)-m3'-O-guanosine.

13. The composition of claim 5, wherein the mRNA comprises a 5’-cap and the cap consists of the structure N7mG(3’OMe)(5’)ppp(5')m6A(2’OMe)pG or the structure N7mG(5 ’)ppp(5 ’)G.

14. The composition of claim 5, wherein the mRNA is a single polycistronic mRNA encoding the nCas3 protein, the Cas7 protein, the Cas5 protein, the Cas8 protein, the Casl 1 protein, and the Cas6 protein.

15. The composition of claim 5, wherein the mRNA consists of a group of mRNA molecules comprising an mRNA encoding the nCas3 protein, an mRNA encoding the Cas7 protein, an mRNA encoding the Cas5 protein, an mRNA encoding the Cas8 protein, an mRNA encoding the Casl 1 protein, and an mRNA encoding the Cas6 protein.

16. The composition of claim 15, wherein the mRNA encoding the nCas3 protein comprises SEQ ID NO: 6.

17. The composition of claim 15, wherein the mRNA encoding the Cas7 protein comprises SEQ ID NO: 3.

18. The composition of claim 15, wherein the mRNA encoding the Cas5 protein comprises SEQ ID NO: 1.

19. The composition of claim 15, wherein the mRNA encoding the Cas8 protein comprises SEQ ID NO: 4.PATENT Docket No.: CB 1062.30 20. The composition of claim 15, wherein the mRNA encoding the Casl 1 protein comprises SEQ ID NO: 5.

21. The composition of claim 15, wherein the mRNA encoding the Cas6 protein comprises SEQ ID NO: 2.

22. The composition of claim 1, wherein the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for a nuclear localization signal (NLS).

23. The composition of claim 22, wherein the NLS is selected from the group consisting of SV40 large T-antigen, nucleoplasmin, 53BP1, VACM-1 / CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrpl, c-Myc, Mouse c-able IV, Mata2 and MINIYO NLS.

24. The composition of claim 1, wherein the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for a tag selected from the group consisting of a FLAG-tag, a HA tag, a FC tag, a GFP tag, a HIS tag, a MYC tag.

25. The composition of claim 1, wherein the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for an effector selected from the group consisting of a nuclease, a phosphatase, a transcription factor, a histone acetyltransferase, a histone deacetylase, a kinase, a HUH endonuclease, and a methylase.

26. The composition of claim 1, wherein the first guide polynucleotide and the second guide polynucleotide are RNA comprising a chemical modification of one or more nucleotides at the termini of the RNA.

27. The composition of claim 26, wherein the chemical modification comprises 2’0-methyl ribose.

28. The composition of claim 26, wherein the chemical modification comprises a phsophorothioate linkage.

29. The composition of claim 26, wherein the first guide polynucleotide and the second guide polynucleotides comprise the structure 5’-2’OMe-PS-2’OMe-PS-2’OMe-RN-2’OMe-PS-2’OMe-PS-2’OMe-3’ wherein 2’0Me is the 2’-O-methyl ribose, PS is the phsophorothioate linkage and RN is N unmodified ribonucleotides.

30. The composition of claim 26, wherein the first guide polynucleotide and the second guide polynucleotide comprise a sequence selected from the group consisting of SEQ ID NOs: 336-347.PATENT Docket No.: CB 1062.30 31. The composition of claim 1, wherein the first half-site of the first guide polynucleotide and the second half-site of the second guide polynucleotide are capable of hybridizing to a sequence in a gene selected from Table 3.

32. The composition of claim 1, wherein the first half-site of the first guide polynucleotide and the second half-site of the second guide polynucleotide are capable of hybridizing to a sequence selected from Table 4 (SEQ ID NOs: 31-279).

33. The composition of claim 1, further comprising a lipid nanoparticle (LNP) encapsulating the nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex, the first guide polynucleotide and the second guide polynucleotide.

34. The composition of claim 33, wherein the lipid nanoparticle comprises a lipid phase comprising an ionizable cationic lipid at about 46-50%, cholesterol at about 38-43%, a phospholipid at about 9-10%, and a polyethylene glycol (PEG) derivative at about 1-2%.

35. The composition of claim 34, wherein the lipid phase comprises 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l-aminium (ALC-0315), cholesterol, l,2-Distearoyl-sn-glycero-3-PC (1,2-DSPC), and Methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (ALC-0159).

36. The composition of claim 34, wherein the lipid phase comprises ALC-0315 at about 46%o, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about l-2%>.

31. The composition of claim 34, wherein the lipid phase comprises 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM102), cholesterol, 1,2-DSPC, and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

38. The composition of claim 34, wherein the lipid phase comprises SM102 at about 50%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and DMG-PEG2000 at about 1-2%.

39. The composition of claim 34, wherein the lipid phase comprises 4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester (MC3), cholesterol, 1,2-DSPC, and DMG-PEG2000.

40. The composition of claim 34, wherein the lipid phase comprises MC3 at about 50%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and DMG-PEG2000 at about 1-2%.PATENT Docket No.: CB 1062.30 41. The composition of claim 34, wherein the LNP are characterized by encapsulation efficiency of 70-100%.

42. The composition of claim 33, wherein the LNP are characterized by poly dispersity index of 0-0.25.

43. The composition of claim 33, wherein the LNP are characterized by diameter of 65- 100 nanometers.

44. A therapeutic composition for modifying a sequence of a target nucleic acid in a somatic cell in a living organism the composition comprising a lipid nanoparticle (LNP) including a lipid phase comprising ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about l-2%>, and the LNP contains a therapeutically effective amount of (i) a nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex; (ii) a first guide polynucleotide comprising a first half-site capable of hybridizing to a first target site within the target nucleic acid, and (iii) a second guide polynucleotide comprising a second half-site capable of hybridizing to a second target site within the target nucleic acid, wherein the first and the second target sites in the nucleic acid are separated by an inter-nicking distance, and wherein the nCas3 is capable of nicking the first and the second target site.

45. The therapeutic composition of claim 44, wherein the therapeutically effective amount is between 0.25 mg / kg and 2.5 mg / kg of total nucleic acid.

46. The therapeutic composition of claim 44, further comprising one or more of excipient, antimicrobial agent, an antioxidant, a surfactant, and a freezing agent.

47. The therapeutic composition of claim 44, wherein the therapeutically effective amount is capable of achieving the rate of sequence modification at the first target site, or the second target site or in the inter-nicking distance between the first target site and the second target site of at least 65%.

48. A therapeutic composition for modifying a sequence of a target nucleic acid in a somatic cell in a living organism comprising the composition of any of the claims 1-43.

49. A method of treating a disease or a condition in a human patient comprising a step of systemic administration to a human patient having the disease or the condition of the composition of any of the claims 1-48.PATENT Docket No.: CB 1062.30 50. The method of claim 49, wherein at least one of the first half-site and the second halfsite is in a gene is selected from Table 3.

51. The method of claim 50, wherein the gene is TTR, and the level of the TTR protein in the patient’s plasma is reduced.

52. The method of claim 51, wherein the gene is TTR, and the method further comprises assessing the level of the TTR protein in the patient’s plasma.

53. The method of claim 50, wherein the gene is PCSK9 gene, and the level of the PCSK9 protein or the level of LDL cholesterol in the patient’s plasma is reduced.

54. The method of claim 53, wherein the gene is PCSK9 gene, and the method further comprises assessing the level of the PCSK9 protein or the level of LDL cholesterol in the patient’s plasma.

55. The method of claim 50, wherein the gene is ANGPTL3, and the level of the ANGPTL3 protein the level of triglycerides in the patient’s plasma is reduced.

56. The method of claim 55, wherein the gene is ANGPTL3, and the method further comprises assessing the level of the ANGPTL3 protein the level of triglycerides in the patient’s plasma.

57. The method of claim 49, further comprising monitoring the patient for excessive immune response.

58. The method of claim 49, further comprising monitoring the patient for change in the function of the liver by assessing the amount of liver-secreted enzymes.

59. A method of making the therapeutic composition of claim 44, the method comprising combining the lipid phase comprising ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about 1-2%, and the therapeutically effective amount of (i) a nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex; (ii) a first guide polynucleotide comprising a first half-site capable of hybridizing to a first target site within the target nucleic acid, and (iii) a second guide polynucleotide comprising a second half-site capable of hybridizing to a second target site within the target nucleic acid, wherein the first and the second target sites in the nucleic acid are separated by an inter-nicking distance, and wherein the nCas3 is capable of nicking the first and the second target site.PATENT Docket No.: CB 1062.30 60. The method of claim 59, further comprising adding one or more of excipient, antimicrobial agent, an antioxidant, a surfactant, and a freezing agent.

61. A method of treating a disease or condition comprising administering to a patient having the disease or condition(i) a nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex, (ii) a first guide polynucleotide comprising a first half-site capable of hybridizing to a first target site within the target nucleic acid, and(iii) a second guide polynucleotide comprising a second half-site capable of hybridizing to a second target site within the target nucleic acid, wherein the first and the second target sites in the nucleic acid are separated by an inter-nicking distance, andnicking the first and the second target site with the nCas3.

62. The method of claim 61, wherein the nCas3 is the Pseudomonas sp. S-6-2 nCas3 selected from the group consisting of D448A nCas3, D448R nCas3, D448C nCas3, D448N nCas3, D448Q nCas3, D448G nCas3, D448K nCas3, D448M nCas3, D448S nCas3, D448T nCas3, and D448V nCas3.

63. The method of claim 61, wherein the nucleic acid coding for the nCas3 Cascade effector complex comprises an mRNA coding for a nCas3 protein, a Cas7 protein, a Cas5 protein, a Cas8 protein, a Casl 1 protein, and a Cas6 protein.

64. The method of claim 63, wherein the mRNA comprises a 5 ’-cap having a formula selected from the group consisting of N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G, N7-(4-bromophenoxy ethyl)-m3 '-OG(5 ')ppp(5 ')G, N7mG(5 ’ )ppp(5 ’ )G,N7mG(3 ’ 0Me)(5 ’ )ppp(5 ')m6 A(2’ OMe)pG, and N7mG(3 ’ 0Me)(5 ’ )ppp(5 ’ )m6G(2 ’ OMe)pG, N7mG(3 ’ OMe)(5 ’ )ppp(5 ')m6A(2’ OMe)pG or N7mG(5 ’ )ppp(5 ’ )G.

65. The method of claim 63, wherein the mRNA comprises a 5’-cap comprising the structure 3’-G(5’)PPP-5' wherein the G is a modified guanosine selected from the group consisting of N7-(4-chlorophenoxy ethyl)- guanosine, N7-(4-chloro-phenoxyethyl)-guanosine, N7-(4-chlorophenoxyethyl)-m3'-O-guanosine, and N7-(4-bromophenoxyethyl)-m3'-O-guanosine.PATENT Docket No.: CB 1062.30 66. The method of claim 63, wherein the mRNA comprises codon optimization for optimizing mRNA expression in mammalian cells, at least one uridine modification selected from the group consisting of 5-methoxyuridine, 5-methyluridine, 5 -carboxy methytl ester uridine, 2-thiouridine and pseudouridine and derivatives thereof, and at least one cytidine modification 2-meth oxy cytidine or its derivatives.

67. The method of claim 63, wherein the mRNA is a single polycistronic mRNA encoding the nCas3 protein, the Cas7 protein, the Cas5 protein, the Cas8 protein, the Casl 1 protein, and the Cas6 protein.

68. The method of claim 63, wherein the mRNA consists of a group of mRNA molecules comprising an mRNA encoding the nCas3 protein, an mRNA encoding the Cas7 protein, an mRNA encoding the Cas5 protein, an mRNA encoding the Cas8 protein, an mRNA encoding the Casl 1 protein, and an mRNA encoding the Cas6 protein.

69. The method of claim 68, wherein the mRNA encoding the nCas3 protein comprises SEQ ID NO: 6, the mRNA encoding the Cas7 protein comprises SEQ ID NO: 3, the mRNA encoding the Cas5 protein comprises SEQ ID NO: 1, the mRNA encoding the Cas8 protein comprises SEQ ID NO: 4, the mRNA encoding the Casl 1 protein comprises SEQ ID NO: 5, and the mRNA encoding the Cas6 protein comprises SEQ ID NO: 2.

70. The method of claim 61, wherein the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for a nuclear localization signal (NLS) selected from the group consisting of SV40 large T-antigen, nucleoplasmin, 53BP1, VACM-1 / CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrpl, c-Myc, Mouse c-able IV, Mata2 and MINIYO NLS.

71. The method of claim 61, wherein the nucleic acid coding for the nCas3 Cascade effector complex comprises a nucleic acid coding for a tag selected from the group consisting of a FLAG-tag, a HA tag, a FC tag, a GFP tag, a HIS tag, a MYC tag.

72. The method of claim 61, wherein the nucleic acid encoding the nCas3 Cascade effector complex comprises a nucleic acid coding for an effector selected from the group consisting of a nuclease, a phosphatase, a transcription factor, a histone acetyltransferase, a histone deacetylase, a kinase, a HUH endonuclease.PATENT Docket No.: CB 1062.30 73. The method of claim 61, wherein the first guide polynucleotide and the second guide polynucleotide comprise the structure 5’-2’OMe-PS-2’OMe-PS-2’OMe-RN-2’OMe-PS-2’0Me-PS-2’0Me-3’ wherein 2’0Me is the 2’-O-methyl ribose, PS is the phsophorothioate linkage and RN is N unmodified ribonucleotides.

74. The method of claim 61, wherein the first half-site of the first guide polynucleotide and the second half-site of the second guide polynucleotide hybridize to a sequence in a gene selected from Table 3.

75. The composition of claim 61, wherein the first half-site of the first guide polynucleotide and the second half-site of the second guide polynucleotide are capable of hybridizing to a sequence selected from Table 4 or Table 5 (SEQ ID NOs: insert).

76. The method of claim 61, wherein the nucleic acid coding for a nickase Cas3 (nCas3) Cascade effector complex, the first guide polynucleotide and the second guide polynucleotide are administered encapsulated in a lipid nanoparticle (LNP).

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