Compositions and methods for the targeting of PCSK9

Abstract

Provided herein are gene repressor systems comprising fusion proteins, such as fusion proteins comprising catalytically-dead Class 2, Type V CRISPR proteins, and guide nucleic acids (gRNA) useful in t

Description

COMPOSITIONS AND METHODS FOR THE TARGETING OF PCSK9 CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and benefit of, U.S. Provisional Application Nos. 63 / 492,978, filed on March 29, 2023, and 63 / 505,888, filed on June 2, 2023, the contents of each of which are incorporated by reference herein in their entireties. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SCRB_058_02WO_SeqList_ST26.xml; Size: 141,865,746 bytes; and Date of Creation: March 26, 2024) are herein incorporated by reference in its entirety. BACKGROUND

[0003] In mammals, cholesterol is transported within lipoproteins via emulsification. The lipoprotein particles are classified based on their density: low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), high-density lipoproteins (HDL), and chylomicrons. Surface LDL receptors are internalized during cholesterol absorption. A cell with abundant cholesterol will have its LDL receptor synthesis blocked to prevent new cholesterol in LDL particles from being taken up. Conversely, LDL receptor synthesis is promoted when a cell is deficient in cholesterol. When the process is unregulated, excess LDL particles will travel in the blood without uptake by an LDL receptor. LDL particles in the blood are oxidized and taken up by macrophages, which then become engorged and form foam cells. These foam cells can become trapped in the walls of blood vessels and contribute to atherosclerotic plaque formation, which is one of the main causes of heart attacks, strokes, and other serious medical problems.

[0004] The liver protein proprotein convertase subtilisin / kexin Type 9 (PCSK9) is a secreted, globular, auto-activating serine protease that binds to the low-density lipoprotein receptor (LDL-R) during endocytosis of LDL particles, preventing recycling of the LDL-R to the cell surface and leading to reduction of LDL-cholesterol clearance. PCSK9 binds to the LDL-R (through the EGF-A domain), preventing the conformational change of the receptor- ligand complex, which redirects the LDL-R to the lysosome instead. As the receptor for low- density lipoprotein particles (LDL) typically transports thousands of fat molecules (including cholesterol) per particle within extracellular fluid, blocking or inhibiting the function ofPCSK9 to boost LDL-R-mediated clearance of LDL cholesterol can lower LDL particle concentrations. PCSK9 is expressed mainly in the liver, the intestine, the kidney, and the central nervous system, but is also highly expressed in arterial walls such as endothelium, smooth muscle cells, and macrophages, with a local effect that can regulate vascular homeostasis and atherosclerosis.

[0005] PCSK9 is a member of the proprotein convertase (PC) family and its gene is mutated in ~ 2% to 3% of individuals with familial hypercholesterolemia (FH) (Sepideh Mikaeeli, S., et al. Functional analysis of natural PCSK9 mutants in modern and archaic humans. FEBS J.2019 Aug 6. doi: 10.1111 / febs.15036). Researchers have identified several PCSK9 mutations that cause an inherited form of high cholesterol (hypercholesterolemia). These mutations change a single amino acid in the PCSK9 protein. Researchers describe the mutations responsible for hypercholesterolemia as "gain-of-function" because they appear to enhance the activity of the PCSK9 protein or give the protein a new, atypical function (Blesa, S., et al. A New PCSK9 Gene Promoter Variant Affects Gene Expression and Causes Autosomal Dominant Hypercholesterolemia. J. Clin. Endocrinol. & Metab.93:3577(2008)). The overactive PCSK9 protein substantially reduces the number of low-density lipoprotein receptors on the surface of liver cells. With fewer receptors to remove low-density lipoproteins from the blood, people with gain-of-function mutations in the PCSK9 gene have very high blood cholesterol levels. Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by increased low-density lipoprotein (LDL)-cholesterol levels, leading to high risk of premature cardiovascular disease. Approximately 10 mutations in PCSK9 have been identified as a cause of the disease in different populations. All known mutations in PCSK9 causing hypercholesterolemia produce an increase in the enzymatic activity of this protease (Bleasa, S., 2008). In addition, mutations in PCSK9 can lead to autosomal dominant familial hypobetalipoproteinemia, which can lead to hepatic steatosis, cirrhosis, and other disorders.

[0006] The advent of CRISPR / Cas systems, and the programmable nature of these minimal systems, has facilitated their use as a versatile technology for genomic manipulation and engineering. However, current methods of generating PCSK9 protective variants and loss-of- function mutants in vivo have been ineffective due to the large number of cells that need to be modified to modulate cholesterol levels. Other concerns involve off-target effects, genome instability, or oncogenic modifications that may be caused by genome editing, as well as a lack of safe delivery modalities for repressor protein systems. Additionally, in certain diseaseindications, gene silencing, or repression, is preferable to gene editing. The ability to render CRISPR nucleases such as Cas9 and CasX catalytically-inactive has been demonstrated (WO2020247882A1 and US20200087641A1, incorporated by reference herein), which makes these systems an attractive platform for the generation of fusion proteins with repressor domains capable of gene silencing. While certain repressor systems have been described, there remains a need for additional gene repressor systems that have been optimized and / or offer improvements over earlier generations of gene repressor systems, such as those based on Cas9, for utilization in a variety of therapeutic, diagnostic, and research applications. Thus, there remains a need for improved compositions and methods to regulate PCSK9. SUMMARY

[0007] The present disclosure provides systems comprising or encoding fusion proteins comprising DNA binding and linked repressor domains used in the repression and / or epigenetic modification of proprotein convertase subtilisin / kexin Type 9 (PCSK9) gene target nucleic acid sequences. In some aspects, the fusion protein comprises a DNA binding protein comprising a catalytically-dead CRISPR protein, such as a Class 2, Type V CRISPR protein, and a guide nucleic acid comprising a targeting sequence complementary to the PCSK9 gene target nucleic acid sequence. The proteins and guide nucleic acids can be modified for passive entry into target cells and are useful in a variety of methods for repression of PCSK9, which methods are also provided. The present disclosure also provides vectors and lipid nanoparticles (LNP) encoding or encapsulating the fusion proteins and guide nucleic acids components for the delivery of the systems to cells for the transcriptional repression of the PCSK9 target nucleic acid sequence. The present disclosure also provides methods for modifying a PCSK9 gene target nucleic acid sequence. The present disclosure also provides methods for treating subjects having a PCSK9-related disease. In some embodiments, the compositions and methods have utility in subjects having a metabolic disorder such as, but not limited to, familial hypercholesterolemia, familial hypobetalipoproteinemia, or elevated cholesterol levels.

[0008] In other aspects, provided herein are systems comprising PCRK9 repressor systems, or vectors comprising or encoding PCSK9 repressor systems for use in the manufacture of a medicament for the treatment of a PCSK9-related disease in a subject in need thereof.

[0009] Further features and advantages of certain embodiments of the present disclosure will become more fully apparent in the following description of embodiments and drawings thereof, and from the claims. INCORPORATION BY REFERENCE

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The contents of WO 2020 / 247882, WO 2020 / 247883, WO 2021 / 113772, WO2022 / 120095, WO 2022 / 125843, WO 2022 / 261150, WO 2022 / 261149, WO 2023 / 049872, WO 2023 / 049742and WO 2023 / 240162, which disclose CasX variants and gRNA variants, and methods of delivering same, and WO2021 / 142342 which discloses compositions and methods of modifying PCSK9 are hereby incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Understanding of the features and advantages of the present disclosure will be facilitated by reference to the following figures:

[0012] FIG.1 illustrates schematics of various configurations of repressor fusion proteins with the DNMT3A ADD domain incorporated. “D3A ADD”, “D3A CD”, and “D3L ID” denote the ADD domain of DNMT3A, the catalytic domain of DNMT3A, and the interaction domain of DNMT3L, respectively. L1-L3 are linkers. NLS is the nuclear localization signal.

[0013] FIG.2 is a bar graph showing the quantification of secreted PCSK9 levels at 6, 18, and 36 days post-transfection in Huh7 cells lipofected with mRNA encoding for CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 1. Secreted PCSK9 levels were normalized to total cell count. Naïve, untreated cells served as an experimental control.

[0014] FIG.3 is a bar plot showing the quantification of normalized secreted PCSK9 levels at 4 days post-transfection in HepG2 cells lipofected with mRNA encoding for CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 2. Secreted PCSK9 levels were normalized to total cell count. Naïve, untreated cells served as experimental controls.

[0015] FIG.4 is a bar plot showing the quantification of normalized secreted PCSK9 levels at 4 days post-transfection in Huh7 cells lipofected with mRNA encoding for CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 2. Secreted PCSK9 levels were normalized to total cell count. Naïve, untreated cells served as experimental controls.

[0016] FIG.5 is a bar plot showing the quantification of normalized secreted PCSK9 levels at 4 days post-transfection in Hep3B cells lipofected with mRNA encoding for CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 2. Secreted PCSK9 levels were normalized to total cell count. Naïve, untreated cells served as experimental controls.

[0017] FIG.6 is a bar plot showing the quantification of secreted PCSK9 levels at 4, 14, and 27 days post-transfection in Huh7 cells lipofected with mRNA encoding for CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 2. Quantification of secreted PCSK9 levels is shown as relative to the secreted levels detected in the naïve, untreated cells at the day 4 timepoint.

[0018] FIG.7 is a schematic of gRNA scaffold variant 174 (SEQ ID NO: 1744), as described in Example 3. Structural motifs are highlighted.

[0019] FIG.8 is a schematic of gRNA scaffold variant 235 (SEQ ID NO: 1745), as described in Example 3. Highlighted structural motifs are the same as in FIG.7. The differences between variant 174 and variant 235 lie in the extended stem motif and several single-nucleotide changes (indicated with asterisks). Variant 316 maintains the shorter extended stem from variant 174 but harbors the four substitutions found in scaffold 235.

[0020] FIG.9 is a schematic of gRNA scaffold variant 316 (SEQ ID NO: 1746), as described in Example 3. Highlighted structural motifs are the same as in FIG.7. Variant 316 maintains the shorter extended stem from variant 174 (FIG.7) but harbors the four substitutions found in scaffold 235 (FIG.8).

[0021] FIG.10 is a schematic illustrating versions 1-3 of chemical modifications made to gRNA scaffold variant 235, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond. For the v2 profile, the addition of three 3’ uracils (3’UUU) is annotated with “U”s in the relevant circles.

[0022] FIG.11 is a schematic illustrating versions 4-6 of chemical modifications made to gRNA scaffold variant 235, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0023] FIG.12 is a schematic illustrating versions 7-9 of chemical modifications made to gRNA scaffold variant 316, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0024] FIG.13 is a schematic illustrating versions 1-3 of chemical modifications made to gRNA scaffold variant 316, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0025] FIG.14 is a schematic illustrating versions 4-6 of chemical modifications made to gRNA scaffold variant 316, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are depicted as open circles, and 2’OMe-modified ribonucleotides are depicted as black circles. Phosphorothioate bonds are indicated with * below or beside the bond.

[0026] FIG.15 is a plot displaying a correlation between indel rate (depicted as edit fraction) at the PCSK9 locus as measured by next-generation sequencing (NGS) (x-axis) and secreted PCSK9 levels (ng / mL) detected by enzyme-linked immunosorbent assay (ELISA) (y-axis) in HepG2 cells lipofected with CasX 491 mRNA and PCSK9-targeting gRNAs containing the indicated scaffold variant and spacer combination, as described in Example 3.

[0027] FIG.16 is a plot illustrating the quantification of percent knockout of B2M in HepG2 cells co-transfected with 100 ng of CasX 491 mRNA and with the indicated doses of end-modified (v1) or unmodified (v0) B2M-targeting gRNAs with spacer 7.37, as described in Example 3. Editing level was determined by flow cytometry as the population of cells with loss of surface presentation of the HLA complex due to successful editing at the B2M locus.

[0028] FIG.17 is a plot depicting the results of an editing assay measured as indel rate detected by NGS at the human B2M locus in HepG2 cells treated with the indicated doses ofLNPs formulated with CasX 491 mRNA and the indicated B2M-targeting gRNA, as described in Example 3.

[0029] FIG.18 is a plot illustrating the quantification of percent knockout of B2M in HepG2 cells treated with the indicated doses of LNPs formulated with CasX 491 mRNA and the indicated B2M-targeting gRNA, as described in Example 3. Editing level was determined by flow cytometry as population of cells that did not have surface presentation of the HLA complex due to successful editing at the B2M locus.

[0030] FIG.19 is a plot depicting the results of an editing assay measured as indel rate detected by NGS at the mouse ROSA26 locus in Hepa1-6 cells treated with the indicated doses of LNPs formulated with CasX 676 mRNA #2 and the indicated ROSA26-targeting gRNA with either the v1 or v5 modification profile, as described in Example 3.

[0031] FIG.20 is a plot illustrating the quantification of percent editing measured as indel rate detected by NGS at the ROSA26 locus in mice treated with LNPs formulated with CasX 676 mRNA #2 and the indicated chemically-modified ROSA26-targeting gRNA, as described in Example 3.

[0032] FIG.21 is a bar graph showing the results of the editing assay measured as indel rate detected by NGS as the mouse PCSK9 locus in mice treated with LNPs formulated with CasX 676 mRNA #1 and the indicated chemically-modified PCSK9-targeting gRNA, as described in Example 3. Untreated mice served as experimental control.

[0033] FIG.22 is a diagram of the secondary structure of guide RNA scaffold 235 (SEQ ID NO: 1745), noting the regions with CpG motifs, as described in Example 6. CpG motifs in (1) the pseudoknot stem, (2) the scaffold stem, (3) the extended stem bubble, (4) the extended step, and (5) the extended stem loop are labeled on the structure.

[0034] FIG.23 is a diagram of the CpG-reducing mutations that were introduced into each of the five regions in the coding sequence of the guide RNA scaffold, as described in Example 6.

[0035] FIG.24 provides the results of an editing experiment in which AAV vectors with various CpG-reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at a multiplicity of infection (MOI) of 4e3. The bars show the mean ± the SD of two replicates per sample. “No Tx” indicates a non-transduced control, and “NT” indicates a control with a non-targeting spacer.

[0036] FIG.25 provides the results of an editing experiment in which AAV vectors with various CpG-reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at an MOI of 3e3. The bars show the mean ± the SD of two replicates per sample. “No Tx” indicates a non-transduced control.

[0037] FIG.26 provides the results of an editing experiment in which AAV vectors with various CpG-reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at an MOI of 1e3. The bars show the mean ± the SD of two replicates per sample. “No Tx” indicates a non-transduced control.

[0038] FIG.27 provides the results of an editing experiment in which AAV vectors with various CpG-reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at an MOI of MOI = 3e2. The bars show the mean ± the SD of two replicates per sample. “No Tx” indicates a non-transduced control.

[0039] FIG.28 illustrates the schematics of five configurations of long-term repressor protein (LTRP, also referred to herein as “repressor fusion proteins”) fusion proteins with repressor molecules linked to catalytically-dead CasX. D3A and D3L denote DNA methyltransferase 3 alpha (DNMT3A) and DNMT3A-like protein (DNMT3L), respectively. L1-L4 are linkers. NLS is the nuclear localization signal.

[0040] FIG.29 illustrates the schematics of the various LTRP #5 architectures, where the additional DNMT3A domains were incorporated, as described in Example 7. The additional DNMT3A domains were the ADD domain of DNMT3A (“D3A ADD”) and the PWWP domain (Pro-Trp-Trp-Pro motif) of DNMT3A (“D3A PWWP”). “D3A endo” encodes for an endogenous sequence that occurs between DNMT3A PWWP and ADD domains. “D3A CD” and “D3L ID” denote the catalytic domain of DNMT3A and the interaction domain of DNMT3L respectively. “L1-L3” are linkers. “NLS” is the nuclear localization signal. See Table 13 for LTRP sequences.

[0041] FIG.30 presents the results of a time-course experiment comparing B2M repression activities (represented as percentage of HLA-negative cells) of the indicated LTRP -ZIM3 and its variants with B2M-targeting gRNA using spacer 7.37, as described in Example 7. Data are presented as mean with standard deviation, N=3. CD = catalytic domain of DNMT3A.

[0042] FIG.31 presents the results of the same time-course experiment shown in FIG.30, but shows B2M repression activities of the indicated LTRP -ZIM3 variants with B2M- targeting gRNA using spacer 7.160, as described in Example 7. Data are presented as mean with standard deviation, N=3.

[0043] FIG.32 presents the results of the same time-course experiment shown in FIG.30, but shows B2M repression activities of the indicated LTRP -ZIM3 variants with B2M- targeting gRNA using spacer 7.165, as described in Example 7. Data are presented as mean with standard deviation, N=3.

[0044] FIG.33 presents the results of the same time-course experiment shown in FIG.30, but shows B2M repression activities of the indicated LTRP -ZIM3 variants with a non- targeting gRNA, as described in Example 7. Data are presented as mean with standard deviation, N=3.

[0045] FIG.34 is a violin plot of percent CpG methylation for CpG sites downstream of the transcription start site of the VEGFA locus for each indicated LTRP -ZIM3 variant for the three B2M-targeting gRNA and non-targeting gRNA, as described in Example 7.

[0046] FIG.35 is a scatterplot showing the relative activity (average percentage of HLA- negative cells at day 21 for spacer 7.160) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified at day 7 for spacer 7.160) for the indicated LTRP5-ZIM3 variants, as described in Example 7.

[0047] FIG.36 illustrates the schematics of the general architectures of the LTRP molecules with the ADD domain for LTRP configuration #1, #4, and #5 tested in Example 8. “D3A ADD”, “D3A CD” and “D3L ID” denote the ADD domain of DNMT3A, the catalytic domain of DNMT3A, and the interaction domain of DNMT3L respectively, as described in Example 8. “L1-L4” are linkers. “NLS” is the nuclear localization signal. See Table 11 for LTRP sequences.

[0048] FIG.37 presents the results of a time-course experiment comparing B2M repression activities (represented as percentage of HLA-negative cells) of LTRPs with the ZIM3-KRAB domain having configuration #1, #4, or #5 with or without the DNMT3A ADD domain when paired with the B2M-targeting gRNA with spacer 7.160, as described in Example 8. Data are presented as mean with standard deviation, N=3. “NT” is a gRNA with a non-targeting spacer.

[0049] FIG.38 is a plot showing the results of the same time-course experiment shown in FIG.37, but illustrates B2M repression activities for LTRP #5 with the ZNF10 or ZIM3-KRAB domain, with or without the DNMT3A ADD domain, paired with the B2M-targeting gRNA with spacer 7.160, as described in Example 8. Data are presented as mean with standard deviation, N=3. “NT” is a gRNA with a non-targeting spacer.

[0050] FIG.39 is a plot showing the results of the same time-course experiment shown in FIG.37, but illustrates B2M repression activities for LTRP5-ZIM3 with or without the DNMT3A ADD domain paired with a B2M-targeting gRNA with the indicated spacers, as described in Example 8. Data are presented as mean with standard deviation, N=3. “NT” is a gRNA with a non-targeting spacer.

[0051] FIG.40 is a plot illustrating the results of B2M repression activities on day 27 post- transfection for LTRPs with either the ZNF10 or ZIM3-KRAB domain having configuration #1 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as mean with standard deviation, N=3. “NT” is a gRNA with a non-targeting spacer.

[0052] FIG.41 is a plot illustrating the results of B2M repression activities on day 27 post- transfection for LTRPs with either the ZNF10 or ZIM3-KRAB domain having configuration #4 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as mean with standard deviation, N=3. “NT” is a gRNA with a non-targeting spacer.

[0053] FIG.42 is a plot illustrating the results of B2M repression activities on day 27 post- transfection for LTRPs with either the ZNF10 or ZIM3-KRAB domain having configuration #5 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as mean with standard deviation, N=3. “NT” is a gRNA with a non-targeting spacer.

[0054] FIG.43 is a plot illustrating the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus on day 5 post-transfection for LTRPs with either the ZNF10 or ZIM3-KRAB domain having configuration #1 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as mean percentage of CpG methylation for CpG sites near the VEGFA locus; standard error of the mean is also presented; N=3. “NT” is a gRNA with a non-targeting spacer.

[0055] FIG.44 is a plot illustrating the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus on day 5 post-transfection for LTRPs with either the ZNF10 or ZIM3-KRAB domain having configuration #4 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented asmean percentage of CpG methylation for CpG sites near the VEGFA locus; standard error of the mean is also presented; N=3. “NT” is a gRNA with a non-targeting spacer.

[0056] FIG.45 is a plot illustrating the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus on day 5 post-transfection for LTRPs with either the ZNF10 or ZIM3-KRAB domain having configuration #5 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as mean percentage of CpG methylation for CpG sites near the VEGFA locus; standard error of the mean is also presented; N=3. “NT” is a gRNA with a non-targeting spacer.

[0057] FIG.46 is a dot plot showing the relative activity (average percentage of HLA- negative cells at day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified at day 5) for the LTRP molecules with the ZIM3-KRAB domain having configurations #1, #4, and #5, for B2M-targeting gRNA with spacer 7.160, as described in Example 8.

[0058] FIG.47 is a dot plot showing the relative activity (average percentage of HLA- negative cells at day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified at day 5) for the LTRP molecules with the ZNF10-KRAB domain having configurations #1, #4, and #5, for B2M-targeting gRNA with spacer 7.160, as described in Example 8.

[0059] FIG.48 is a dot plot showing the relative activity (average percentage of HLA- negative cells at day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified at day 5) for the LTRP molecules with the ZIM3-KRAB domain having configurations #1, #4, and #5, for B2M-targeting gRNA with spacer 7.37, as described in Example 8.

[0060] FIG.49 is a dot plot showing the relative activity (average percentage of HLA- negative cells at day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified at day 5) for the LTRP molecules with the ZNF10-KRAB domain having configurations #1, #4, and #5, for B2M-targeting gRNA with spacer 7.37, as described in Example 8.

[0061] FIG.50 is a dot plot showing the relative activity (average percentage of HLA- negative cells at day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified at day 5) for the LTRP molecules with the ZIM3-KRAB domain having configurations #1, #4, and #5, for B2M-targeting gRNA with spacer 7.165, as described in Example 8.

[0062] FIG.51 is a dot plot showing the relative activity (average percentage of HLA- negative cells at day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified at day 5) for the LTRP molecules with the ZNF10-KRAB domain having configurations #1, #4, and #5, for B2M-targeting gRNA with spacer 7.165, as described in Example 8.

[0063] FIG.52 is a plot illustrating percentage of HEK293T cells, transfected with a plasmid encoding the indicated CasX or LTRP:gRNA construct, that expressed B2M six days post-treatment with the DNMT1 inhibitor 5-azadC at varying concentrations, as described in Example 9.

[0064] FIG.53 is a plot that juxtaposes the quantification of B2M repression in HEK293T cells transfected with a plasmid encoding the indicated CasX or LTRP:gRNA construct and cultured for 58 days, with the quantification of B2M reactivation upon treatment of transfected cells with 5-azadC, as described in Example 9.

[0065] FIG.54 illustrates the schematic of an LTRP5 molecule without the ADD domain of the DNMT3A, as described in Example 11. “D3A CD” and “D3L ID” denote the catalytic domain of DNMT3A and the interaction domain of DNMT3L respectively. “L1”, “L2”, “L3A”, and “L3B” are linkers. “NLS” is the nuclear localization signal. “RD1” denotes a repressor domain.

[0066] FIG.55 is a bar graph illustrating the results of a time-course experiment comparing levels of B2M repression (represented as average percentage of HLA-negative cells) in HEK293T cells transfected with the LTRP5 variant plasmids containing linker sets 1-11, as described in Example 13. Data for each timepoint (day 8, day 15, and day 45) are superimposed and presented as mean with standard deviation, N =3. A non-targeting spacer (NT) was included as experimental control.

[0067] FIG.56 is a bar graph illustrating the results of a time-course experiment comparing the level of repression of target 1 (represented as percentage of total cells with knockdown of target 1) of LTRP5 variants containing linker sets 1-11 measured in HEK293T, as described in Example 13. Data for each timepoint (day 8, day 15, and day 45) are superimposed and presented as mean with standard deviation, N =3.

[0068] FIG.57 is a bar graph illustrating the results of a time-course experiment comparing the level of repression of target 2 (represented as percentage of total cells with knockdown of target 2) of LTRP5 variants containing linker sets 1-11 measured in HEK293T, as described in Example 13. Data for each timepoint (day 8, day 15, and day 45) are superimposed andpresented as mean with standard deviation, N =3. A non-targeting spacer (NT) was included as experimental control.

[0069] FIG.58 is a bar graph illustrating the results of a time-course experiment comparing levels of B2M repression (represented as average percentage of HLA-negative cells) in HEK293T cells transfected with the LTRP5 variant plasmids containing linker sets 12-28, as described in Example 13. Data for each timepoint (day 7 and day 17) are superimposed and presented as mean with standard deviation, N =3. A non-targeting spacer (NT) was included as experimental control. A non-targeting spacer (NT) was included as experimental control.

[0070] FIG.59 is a bar graph illustrating the results of a time-course experiment comparing the level of repression of target 1 (represented as percentage of total cells with knockdown of target 1) of LTRP5 variants containing linker sets 12-28 measured in HEK293T, as described in Example 13. Data for each timepoint (day 7 and day 17) are superimposed and presented as mean with standard deviation, N =3. A non-targeting spacer (NT) was included as experimental control.

[0071] FIG.60 is a bar graph illustrating the results of a time-course experiment comparing the level of repression of target 2 (represented as percentage of total cells with knockdown of target 2) of LTRP5 variants containing linker sets 12-28 measured in HEK293T, as described in Example 13. Data for each timepoint (day 7 and day 17) are superimposed and presented as mean with standard deviation, N =3. A non-targeting spacer (NT) was included as experimental control.

[0072] FIG.61 illustrates the schematics of various configurations of LTRP molecules with the DNMT3A ADD domain. “D3A ADD”, “D3A CD”, and “D3L ID” denote the ADD domain of DNMT3A, the catalytic domain of DNMT3A, and the interaction domain of DNMT3L respectively. “L1”, “L2”, “L3A”, “L3B”, and “L4” are linkers. “NLS” is the nuclear localization signal. “RD1” denotes a repressor domain, and “RD1a” and “RD1b” represent repressor domain variants.

[0073] FIG.62 is a violin plot with each point representing mean % methylation at individual CpGs. Median methylation is indicated by dashed lines, with upper and lower quartiles indicated by dotted lines. Transcriptional start site (TSS)-proximal DNA methylation was measured from homogenized liver-extracted gDNA by amplicon enzymatic methylation sequencing (EM-seq) from N=3 mice sacrificed at days 7, 14, and 42 post- treatment, as described in example 14.

[0074] FIG.63 is a violin plot with each point representing mean % methylation at individual CpGs. Median methylation is indicated by dashed lines, with upper and lower quartiles indicated by dotted lines. Transcriptional start site (TSS)-proximal DNA methylation was measured from homogenized liver-extracted gDNA by amplicon enzymatic methylation sequencing (EM-seq) from N=3 mice sacrificed at day 7 post-treatment, as described in example 14. DETAILED DESCRIPTION

[0075] While exemplary embodiments have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the spirit of the disclosure. Thus, it is understood that various alternatives to the embodiments specifically described herein may be employed in practicing the embodiments of the disclosure.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the spirit of the disclosure and the claims. Definitions

[0077] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a host cell” includes two or more such host cells, reference to “a dCasX protein” includes one or more dCasX protein(s), reference to “a nucleic acid sequence” includes one or more nucleic acid sequences, and the like.

[0078] As used herein, the term “about” is understood by persons of ordinary skill in the art and may vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context inwhich the term “about” is used, “about” will mean up to plus or minus 10% of the particular term.

[0079] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also include any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 1-5 members refers to groups having 1, 2, 3, 4, or 5 members, and so forth.

[0080] The term “combinations thereof’ includes every possible combination of elements to which the term refers.

[0081] The term "exemplary" as used herein, refers to an example or illustration, and is not intended to imply any preference or value.

[0082] The term “CasX protein,” as used herein, refers to a family of proteins, including e.g., all naturally-occurring CasX proteins (“reference CasX”), as well as CasX proteins with sequence modifications, such as a dCasX, possessing one or more improved characteristics relative to a CasX protein from which it was derived, described more fully, herein below.

[0083] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms “polynucleotide” and “nucleic acid” encompass single- stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double- stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0084] “Hybridizable” or “complementary” are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable; it can have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity and still hybridize to the target nucleic acid. Moreover, a polynucleotide may hybridize over one or more segments such that interveningor adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a 'bulge', ‘bubble’ and the like). Thus, the skilled artisan will understand that while individual bases within a sequence may not be complementary to another sequence, the sequence as a whole is still considered to be complementary.

[0085] A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (e.g., a protein, RNA), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene may include accessory element sequences including, but not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. Coding sequences encode a gene product upon transcription or transcription and translation; the coding sequences of the disclosure may comprise fragments and need not contain a full-length open reading frame. A gene can include both the strand that is transcribed as well as the complementary strand containing the anticodons.

[0086] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0087] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0088] The term “adjacent to” with respect to polynucleotide or amino acid sequences refers to sequences that are next to, or adjoining each other in a polynucleotide or polypeptide. The skilled artisan will appreciate that two sequences can be considered to be adjacent to each other and still encompass a limited amount of intervening sequence, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or amino acids.

[0089] The term “regulatory element” is used interchangeably herein with the term “regulatory sequence,” and is intended to include promoters, enhancers, and other expression regulatory elements. It will be understood that the choice of the appropriate regulatory element will depend on the encoded component to be expressed (e.g., protein or RNA) orwhether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0090] The term “accessory element” is used interchangeably herein with the term “accessory sequence,” and is intended to include coding and non-coding sequences that enhance expression, trafficking of the nucleic acid, or the function of an mRNA or protein and include, inter alia, polyadenylation signals (poly(A) signal), enhancer elements, introns, posttranscriptional regulatory elements (PTREs), nuclear localization signals (NLS), deaminases, DNA glycosylase inhibitors, additional promoters, factors that stimulate CRISPR-mediated homology-directed repair (e.g., in cis or in trans), self-cleaving sequences, and fusion domains, for example a fusion domain fused to a CRISPR protein. It will be understood that the choice of the appropriate accessory element or elements will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0091] The term "promoter" refers to a DNA sequence that contains a transcription start site and additional sequences to facilitate polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as a TATA box, and / or B recognition element (BRE) and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter can be synthetically produced or can be derived from a known or naturally occurring promoter sequence or another promoter sequence. A promoter can be proximal or distal to the gene to be transcribed. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences to confer certain properties. A promoter of the present disclosure can include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. A promoter can be classified according to criteria relating to the pattern of expression of an associated coding or transcribable sequence or gene operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. A promoter can also be classified according to its strength. As used in the context of a promoter, “strength” refers to the rate of transcription of the gene controlled by the promoter. A “strong” promoter means the rate of transcription is high, while a “weak” promoter means the rate of transcription is relatively low.

[0092] A promoter of the disclosure can be a Polymerase II (Pol II) promoter. Polymerase II transcribes all protein coding and many non-coding genes. A representative Pol II promoterincludes a core promoter, which is a sequence of about 100 base pairs surrounding the transcription start site, and serves as a binding platform for the Pol II polymerase and associated general transcription factors. The promoter may contain one or more core promoter elements such as the TATA box, BRE, Initiator (INR), motif ten element (MTE), downstream core promoter element (DPE), downstream core element (DCE), although core promoters lacking these elements are known in the art. All Pol III promoters are envisaged as within the scope of the instant disclosure.

[0093] A promoter of the disclosure can be a Polymerase III (Pol III) promoter. Pol III transcribes DNA to synthesize small ribosomal RNAs such as the 5S rRNA, tRNAs, and other small RNAs. Representative Pol III promoters use internal control sequences (sequences within the transcribed section of the gene) to support transcription, although upstream elements such as the TATA box are also sometimes used. All Pol III promoters are envisaged as within the scope of the instant disclosure.

[0094] The term “enhancer” refers to regulatory DNA sequences that, when bound by specific proteins called transcription factors, regulate the expression of an associated gene. Enhancers may be located in the intron of the gene, or 5’ or 3’ of the coding sequence of the gene. Enhancers may be proximal to the gene (i.e., within a few tens or hundreds of base pairs (bp) of the promoter), or may be located distal to the gene (i.e., thousands of bp, hundreds of thousands of bp, or even millions of bp away from the promoter). A single gene may be regulated by more than one enhancer, all of which are envisaged as within the scope of the instant disclosure.

[0095] As used herein, a “post-transcriptional regulatory element (PTRE),” such as a hepatitis PTRE, refers to a DNA sequence that, when transcribed creates a tertiary structure capable of exhibiting post-transcriptional activity to enhance or promote expression of an associated gene operably linked thereto.

[0096] “Operably linked” means with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components; e.g., a promoter and an encoding sequence. The skilled artisan will appreciate that the two components need not be physically linked to be operably linked.

[0097] In the context of the present disclosure and with respect to a gene, “repress”, “repression”, "repressing", " transcriptional repression", “inhibition of gene expression”,“downregulation”, and “silencing” are used interchangeably herein to refer to the inhibition or blocking of transcription of a gene or a portion thereof. Accordingly, repression of a gene can result in a decrease in production of a gene product. Examples of gene repression processes which decrease transcription include, but are not limited to, those which inhibit formation of a transcription initiation complex, those which decrease transcription initiation rate, those which decrease transcription elongation rate, those which decrease processivity of transcription and those which antagonize transcriptional activation (by, for example, blocking the binding of a transcriptional activator). Gene repression can constitute, for example, prevention of activation as well as inhibition of expression below an existing level. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription; the latter can result from epigenetic modification of the gene.

[0098] “Repressor” or “repressor domain” are used interchangeably to refer to polypeptide factors that act as regulatory elements on DNA that inhibit, repress, or block transcription of DNA, resulting in repression of gene expression. In the context of the present disclosure, the linking of a repressor domain to DNA binding protein that can, when bound to the target nucleic acid, prevent transcription from a promoter or otherwise inhibit the expression of a gene. Without wishing to be bound by theory, it is thought that transcriptional repressors can function by a variety of mechanisms, including physically blocking RNA polymerase passage by steric hindrance, altering the polymerase's post-translational modification state, modifying the epigenetic state of the nascent RNA, changing the epigenetic state of the DNA through methylation, changing the epigenetic state of the DNA through histone deacetylation or modulating nucleosome remodeling, or preventing enhancer-promoter interactions, thereby leading to gene silencing or a reduction in the level of gene expression.

[0099] “Long-term repressor fusion protein” or “LTRP” is used interchangeably herein with “repressor fusion protein” and refers to a fusion protein comprising a DNA binding protein (or DNA binding domain of a protein) fused to one or more domains capable of repressing transcription of a target nucleic acid sequence. Optionally, the long-term repressor fusion proteins of the disclosure may contain additional elements, such as linkers between any of the domains of the fusion protein, nuclear localization signals, nuclear export signals, as well as additional protein domains that confer additional activities upon the long-term repressor fusion protein.

[0100] As used herein a “LTRP:gRNA system” is a system for transcriptional repression and comprises a long-term repressor fusion protein comprising a catalytically-dead CRISPRprotein and one or more linked repressor domains, and a guide nucleic acid (gRNA) that binds to the catalytically-dead CRISPR protein. For clarity, the system also includes any encoding DNA, RNA or vectors and the like that can be used to produce the long-term repressor fusion proteins and gRNA components of the system.

[0101] As used herein, a "catalytically dead DNA binding protein" refers to a protein, or domain of a protein, capable of binding to DNA but is unable to nick or cleave the DNA. As used herein a “catalytically-dead CRISPR protein” refers to a CRISPR protein that lacks endonuclease activity. The skilled artisan will appreciate that a CRISPR protein can be catalytically-dead, and still able to carry out additional protein functions, such as DNA binding. Similarly, a "catalytically-dead CasX" refers to a CasX protein that lacks endonuclease activity but is still able to carry out additional protein functions, such as DNA binding.

[0102] “Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5’ or 3’ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “enhancers” and “promoters”, above).

[0103] The term “recombinant polynucleotide” or “recombinant nucleic acid” refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative aminoacid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0104] Similarly, the term “recombinant polypeptide” or “recombinant protein” refers to a polypeptide or protein which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a protein that comprises a heterologous amino acid sequence is recombinant.

[0105] As used herein, "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, helper phospholipids, and PEG-modified lipids), as well as cholesterol. Specific components of LNP are described more fully, below. Lipid nanoparticles can be included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). The lipid nanoparticles of the disclosure can comprise a nucleic acid. Such lipid nanoparticles typically comprise neutral lipids, charged lipids, steroids and polymer conjugated lipids. The active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0106] As used herein, “lipid encapsulated” refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA, gRNA or both mRNA and gRNA), with full encapsulation, partial encapsulation, or both. The nucleic acid (e.g., mRNA) can be fully encapsulated in the lipid nanoparticle.

[0107] As used herein, “lipoprotein”, such as VLDL, LDL and HDL, refers to a group of proteins found in the serum, plasma and lymph that function in lipid transport. The chemical composition of each lipoprotein differs, for example, in that the HDL has a higher proportion of protein versus lipid, whereas the VLDL has a lower proportion of protein versus lipid.

[0108] As used herein, “atherosclerosis” means a hardening of the arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. The fattydeposits are called “atheromas” or “plaques,” which consist mainly of cholesterol and other fats, calcium and scar tissue, and damage the lining of arteries.

[0109] As used herein, “coronary heart disease (CHD)” means a narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis.

[0110] As used herein, “dyslipidemia” refers to a disorder of lipid and / or lipoprotein metabolism, including lipid and / or lipoprotein overproduction or deficiency. Dyslipidemias can be manifested by elevation of lipids such as chylomicron, cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.

[0111] As used herein, “high density lipoprotein-C” or “HDL-C” means cholesterol associated with high-density lipoprotein particles. Concentration of HDL-C in serum (or plasma) is typically quantified in mg / dL or nmol / L. “Serum HDL-C” and “plasma HDL-C” mean HDL-C in serum and plasma, respectively.

[0112] As used herein, “low density lipoprotein-cholesterol (LDL-C)” means cholesterol carried in low density lipoprotein particles. Concentration of LDL-C in serum (or plasma) is typically quantified in mg / dL or nmol / L. “Serum LDL-C” and “plasma LDL-C” mean LDL- C in the serum and plasma, respectively.

[0113] As used herein, “hypercholesterolemia” means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol and VLDL- cholesterol, as per the guidelines of the Expert Panel Report of the National Cholesterol Educational Program (NCEP) of Detection, Evaluation of Treatment of high cholesterol in adults (see, Arch. Int. Med.148: 36 (1988)).

[0114] As used herein, “hyperlipidemia” or “hyperlipemia” is a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition manifests an abnormally high concentration of fats. The lipid fractions in the circulating blood are cholesterol, low- density lipoproteins, very low density lipoproteins, chylomicrons and triglycerides. The Fredrickson classification of hyperlipidemias is based on the pattern of TG and cholesterol- rich lipoprotein particles, as measured by electrophoresis or ultracentrifugation and is commonly used to characterize primary causes of hyperlipidemias such as hypertriglyceridemia.

[0115] As used herein, “triglyceride” or “TG” means a lipid or neutral fat consisting of glycerol combined with three fatty acid molecules.

[0116] As used herein, “hypertriglyceridemia” means a condition characterized by elevated triglyceride levels. Its etiology includes primary (i.e. genetic causes) and secondary (other underlying causes such as diabetes, metabolic syndrome / insulin resistance, obesity, physical inactivity, cigarette smoking, excess alcohol and a diet very high in carbohydrates) factors or, most often, a combination of both

[0117] As used herein, “diabetes mellitus” or “diabetes” is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) resulting from insufficient levels of insulin or reduced insulin sensitivity. The characteristic symptoms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) attempting to compensate for increased urination, blurred vision due to high blood glucose effects on the eye's optics, unexplained weight loss, and lethargy.

[0118] As used herein, “diabetic dyslipidemia” or “type 2 diabetes with dyslipidemia” means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated triglycerides (TG), and elevated small, dense LDL particles.

[0119] As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a target nucleic acid with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid are made to share a physical connection; e.g., can hybridize if the sequences share sequence similarity.

[0120] “Dissociation constant”, or “Kd”, are used interchangeably and mean the affinity between a ligand “L” and a protein “P”; i.e., how tightly a ligand binds to a particular protein. It can be calculated using the formula Kd=[L] [P] / [LP], where [P], [L] and [LP] represent molar concentrations of the protein, ligand and complex, respectively.

[0121] The term "knock-down" as used herein refers to reduction in the expression of a gene or its gene product(s). As a result of a gene knock-down, the protein activity or function may be attenuated or the protein levels may be reduced or eliminated.

[0122] A polynucleotide or polypeptide has a certain percent "sequence similarity" or "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity (sometimes referred to as percent similarity, percent identity, or homology) can be determined in a number of different manners. To determine sequence similarity, sequences can be aligned using the methods and computer programs that are known in the art, including BLAST, available over the worldwide web at ncbi.nlm.nih.gov / BLAST. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0123] The terms “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence.

[0124] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, which can include another DNA segment, i.e., an expression cassette, so as to bring about the replication or expression of the other DNA segment in a cell.

[0125] The term “naturally-occurring” or “unmodified” or “wild type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.

[0126] As used herein, a “mutation” refers to an insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides as compared to a wild- type or reference amino acid sequence or to a wild-type or reference nucleotide sequence.

[0127] As used herein the term “isolated” is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[0128] A “host cell,” as used herein, denotes a eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells are used as recipients for a nucleic acid (e.g., an AAV vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (alsoreferred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an AAV vector.

[0129] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0130] As used herein, “treatment” or “treating,” are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder or disease being treated. A therapeutic benefit can also be achieved with the eradication or amelioration of one or more of the symptoms or an improvement in one or more clinical parameters associated with the underlying disease such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.

[0131] The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a drug or a biologic, alone or as a part of a composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject such as a human or an experimental animal. Such effect need not be absolute to be beneficial.

[0132] As used herein, “administering” means a method of giving a dosage of a compound (e.g., a composition of the disclosure) or a composition (e.g., a pharmaceutical composition) to a subject.

[0133] A “subject” is a mammal. Mammals include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats and other rodents.

[0134] The term “low-density lipoprotein (LDL)” refers to one of the five major groups of lipoprotein, from least dense (lower weight-volume ratio particles) to most dense (larger weight-volume ratio particles): chylomicrons, very low-density lipoproteins (VLDL), low- density lipoproteins (LDL), intermediate-density lipoproteins (IDL), and high-density lipoproteins (HDL). Lipoproteins transfer lipids (fats) around the body in the extracellular fluid thereby facilitating the transfer of fats to the cells body via receptor-mediated endocytosis. An LDL particle is about 220-275 angstroms in diameter.

[0135] “Low-density lipoprotein (LDL) receptor” refers to a receptor protein of 839 amino acids (after removal of 21-amino acid signal peptide) that mediates the endocytosis of cholesterol-rich LDL particles. It is a cell-surface receptor that recognizes the apoprotein B100 and apoE protein found in chylomicron remnants and VLDL remnants (IDL) resulting in the binding and endocytosis of LDL-cholesterol. This process occurs in all nucleated cells, but mainly in the liver which removes approximately 70% of LDL from the circulation. The human LDLR gene is described in part in the NCBI database (ncbi.nlm.nih.gov) as Reference Sequence NG_009060.1, which is incorporated by reference herein.

[0136] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. I. General Methods

[0137] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4thEd. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0138] Where a range of values is provided, it is understood that endpoints are included and that each intervening value, to the tenth of the unit of the lower limit unless the contextclearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0139] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0140] It will be appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. In other cases, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is intended that all combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. II. Systems for Epigenetic Modification and Repression of PCSK9 Genes

[0141] In a first aspect, the present disclosure provides systems comprising or encoding a long-term repressor fusion protein ("LTRP") comprising a DNA binding protein and repressor domains capable of binding a target nucleic acid sequence of a PCSK9 gene targeted for transcriptional repression, silencing, and / or epigenetic modification. The present disclosure also provides systems comprising or encoding the LTRP. In some cases, the system is designed to repress transcription of a PCSK9 gene in eukaryotic cells having a mutation.

[0142] As used herein, a "system" is used interchangeably with "composition." The disclosure also provides nucleic acids encoding the systems provided herein. Also provided herein are methods of making the systems, as well as methods of using the systems, including methods of gene repression and / or epigenetic modification and methods of treatment ofPCSK9-related diseases. For clarity, the term “system” also includes any encoding DNA, RNA or vectors and the like that can be used to produce the repressor fusion proteins and gRNA components of the system.

[0143] In some embodiments, the DNA binding proteins for use in the long-term repressor fusion proteins of the disclosure comprise zinc finger (ZF) or TALE (transcription-activator- like effector) proteins, or DNA binding domains thereof, also referred to herein as a DNA binding protein, that bind but do not cleave the target nucleic acid. The DNA binding domain of a TALE is comprised of a tandem array of 33–34 amino acid (aa)-long customizable monomers that theoretically can be assembled to recognize any genetic sequence following a one-repeat-binds-one-base-pair recognition code (Jain, S., et al. TALEN outperforms Cas9 in editing heterochromatin target sites. Nat. Commun.12:606 (2021)). The specificity of TALEs for binding DNA arises from two polymorphic amino acids, the so-called repeat variable diresidues (RVDs) located at positions 12 and 13 of a repeated unit. By re-arranging the repeats, the DNA binding specificities of TALE can be changed at will. Zinc finger proteins are transcription factors, where each finger recognizes 3-4 bases of DNA. By mixing and matching these finger modules, the ZFs can be customized for the sequence to be targeted. Exemplary ZFs that are capable of binding the PCSK9 gene are described in WO2018049009A2.

[0144] In some embodiments, the DNA binding protein for use in the long-term repressor fusion protein is a catalytically-dead Class 1 or Class 2 CRISPR protein. Catalytically-dead CRISPR proteins are also referred to in the art as “catalytically inactive” CRISPR proteins. In one embodiment, the Class 2, Type II protein is a catalytically-dead Cas9. In another embodiment, the Class 2 CRISPR protein is selected from the group consisting of a Type II, Type V, or Type VI protein. In one embodiment, the Class 2 Type V protein is selected from the group consisting of Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas14, and / or CasΦ, in each case rendered catalytically-dead by specific mutations, as described herein. The CRISPR-based systems further comprise a guide nucleic acid, for example a guide ribonucleic acid (gRNA) with a targeting sequence complementary to the target sequence of a PCSK9 gene to be bound and repressed by a complex of the fusion protein (the CRISPR protein and linked repressor domains) and the gRNA.

[0145] In some embodiments, the present disclosure provides systems comprising or encoding a long-term repressor fusion protein comprising a catalytically-dead CasX CRISPRnuclease protein and linked repressor domains and a guide ribonucleic acid (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence of a PCSK9 gene targeted for transcriptional repression, silencing, or downregulation, as well as nucleic acids encoding the long-term repressor fusion protein and / or gRNA. In some embodiments, the system comprises a long-term repressor fusion protein and a gRNA of the disclosure as a gene repressor pair ("LTRP:gRNA system") that are capable of forming a ribonucleoprotein (RNP) complex and binding a PCSK9 target nucleic acid. In other cases, the disclosure provides systems of nucleic acids encoding the long-term repressor fusion protein and the gRNA. In still other cases, the disclosure provides systems of a gRNA and an mRNA encoding the long-term repressor fusion protein for use in certain particle formulations (e.g., an LNP) described herein.

[0146] Also provided herein are methods of making long-term repressor fusion proteins and gRNA, as well as methods of using the LTRP:gRNA systems, including methods of gene repression and / or epigenetic modification of a PCSK9 gene and methods of treatment of a PCSK9-related disease or disorder. The DNA binding proteins (e.g., dCasX) and linked repressor domain(s), and the gRNA components, of the LTRP:gRNA systems and their features, as well as the delivery modalities and the methods of using the systems for the repression, down-regulation or silencing of a PCSK9 gene are described more fully, below.

[0147] In some embodiments, the disclosure provides systems specifically designed to repress or silence transcription of the PCSK9 gene. In some cases, the system is designed to repress transcription of the PCSK9 gene in eukaryotic cells having a gain of function mutation. In some cases, the system is designed to repress transcription of the wild-type PCSK9 gene in eukaryotic cells. In the alternative, the system is designed to repress transcription of a mutant allele of the PCSK9 gene in eukaryotic cells. Generally, any portion of the PCSK9 gene can be targeted using the programable systems and methods provided herein, described more fully, herein.

[0148] The PCSK9 gene encodes proprotein convertase subtilisin / kexin Type 9 (“PCSK9”), a protein that binds to the receptor for low-density lipoprotein particles (LDL) for transport of LDL into the cell. The PCSK9 gene encompasses the sequence that spans chr1:55,039,476- 55,064,853 of the human genome (GRCh38 / hg38) (the notation refers to the chromosome 1 (chr1), starting at the 55,039,476 bp to 55,064,853 bp on chromosome 1 (Homo sapiens Updated Annotation Release 109.20190905, GRCh38.p13) (NCBI). The human PCSK9 gene is described in part in the NCBI database (ncbi.nlm.nih.gov) as Reference SequenceNG_009061.1, which is incorporated by reference herein. The PCSK9 locus has 12 exons that produces an mRNA of 3636 bp encoding a 692-amino acid protein that, following its synthesis, undergoes an autocatalytic cleavage reaction that clips off the prodomain, resulting in an activated protein having 540 amino acids. The prodomain remains attached to the catalytic and resistin-like domains, likely because the prodomain serves as a chaperone and facilitates folding and secretion (Seidah, NG et al., Proc Natl Acad Sci USA 100(3):928 (2003)). The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation (Seidah NG, et al.). This protein, also called neural apoptosis regulated convertase, is a serine protease belonging to the protease K subfamily of subtilases.

[0149] The human PCSK9 gene (HGNC:20001) encodes a protein (Q8NBP7) having the sequenceIII. Catalytically-dead Proteins for Use in the Repressor Systems

[0150] In some embodiments, the DNA binding proteins for use in the long-term repressor fusion proteins of the disclosure are zinc finger (ZF) or TALE (transcription-activator-like effector) proteins that can bind, but not cleave a PCSK9 target nucleic acid.

[0151] In some embodiments, the DNA binding protein is a catalytically-dead Class 1 or Class 2 CRISPR protein. In some embodiments, the DNA binding protein is a Class 2, Type II CRISPR protein. In one embodiment, the Class 2, Type II protein is a catalytically-dead Cas9. In another embodiment, the Class 2 CRISPR protein is selected from the group consisting of a Type II, Type V, or Type VI protein. In one embodiment, the Class 2 CRISPR Type V protein is selected from the group consisting of Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, Cas12j,Cas12k, Cas14, and / or CasΦ, in each case rendered catalytically-dead by specific mutations, as described herein. In another embodiment, the Class 2 CRISPR Type V protein is a catalytically-dead CasX protein.

[0152] CasX proteins of the disclosure, including dCasX, comprise the following domains: a non-target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helical I domain, a helical II domain, an oligonucleotide binding domain (OBD), and a RuvC domain, and, in some cases, domains can be further divided into subdomains, as listed in Table 1.

[0153] In the context of the present disclosure, the CasX for use in the systems are catalytically-dead (dCasX); achieved by mutations introduced at select locations in the RuvC sequence, described below. a. Reference CasX Proteins

[0154] The disclosure provides naturally-occurring CasX proteins (referred to herein as a “reference CasX protein”), which were subsequently modified to create the engineered dCasX of the disclosure. For example, reference CasX proteins can be isolated from naturally occurring prokaryotes, such as Deltaproteobacteria, Planctomycetes, or Candidatus Sungbacteria species. A reference CasX protein (interchangeably referred to herein as a reference CasX polypeptide) is a Class 2, Type V CRISPR / Cas endonuclease belonging to the CasX (interchangeably referred to as Cas12e) family of proteins that interacts with a guide RNA to form a ribonucleoprotein (RNP) complex.

[0155] In some cases, a reference CasX protein is isolated or derived from Deltaproteobacter, and comprises a sequence of SEQ ID NO: 1.

[0156] In some cases, a reference CasX protein is isolated or derived from Planctomycetes, and comprises a sequence of SEQ ID NO: 2.

[0157] In some cases, a reference CasX protein is isolated or derived from Candidatus Sungbacteria, and comprises a sequence of SEQ ID NO: 3. b. Catalytically-dead Class 1 or Class 2 CRISPR Proteins

[0158] In the long-term repressor fusion proteins and gene repressor systems comprising same of the disclosure, the catalytically-dead Class 1 or Class 2 CRISPR protein is catalytically-dead in that it is unable to cleave DNA, but retains the ability to bind a target nucleic acid when complexed with a guide RNA (gRNA). The present disclosure provides catalytically-dead variants of Class 1 or Class 2 CRISPR proteins, wherein the catalytically- dead variants comprise multiple modifications in select domains. The present disclosure provides catalytically-dead CasX variants (interchangeably referred to herein as “dCasXvariant” or “dCasX variant protein”), wherein the catalytically-dead CasX variants comprise multiple modifications in the RuvC domain relative to the catalytically-dead versions of reference CasX proteins comprising sequences of SEQ ID NOS:1-3 (described, supra). In some embodiments, a catalytically-dead reference CasX protein comprises substitutions at residues 672, 769 and / or 935 with reference to SEQ ID NO: 1. In some embodiments, a catalytically-dead reference CasX protein comprises substitutions of D672A, E769A and / or D935A with reference to SEQ ID NO: 1. In other embodiments, a catalytically-dead reference CasX protein comprises substitutions at amino acids 659, 756 and / or 922 with reference to SEQ ID NO: 2. In some embodiments, a catalytically-dead reference CasX protein comprises D659A, E756A and / or D922A substitutions with reference to of SEQ ID NO: 2. An exemplary RuvC domain of the dCasX of the disclosure comprises amino acids 661-824 and 935-986 of SEQ ID NO: 1, or amino acids 648-812 and 922-978 of SEQ ID NO: 2, with one or more amino acid modifications relative to said RuvC cleavage domain sequence, wherein the dCasX variant exhibits one or more improved characteristics compared to the reference dCasX. In further embodiments, a catalytically-dead CasX variant protein comprises deletions of all or part of the RuvC domain of the reference CasX protein. It will be understood that the same foregoing substitutions or deletions can similarly be introduced into CasX variants known in the art, resulting in a dCasX variant (see, e.g., WO2022120095A1 and US11,560,555, incorporated by reference herein, for exemplary sequences).

[0159] In some embodiments, the long-term repressor fusion protein comprising a dCasX variant with linked repressor domains exhibits at least one improved characteristic compared to a long-term repressor fusion protein comprising the reference dCasX protein with comparable linked repressor domains. All dCasX variants that improve one or more functions or characteristics of the long-term repressor fusion protein comprising the dCasX variant protein with linked repressor domains compared to the long-term repressor fusion protein comprising the reference dCasX protein are envisaged as being within the scope of the disclosure. In some embodiments, the modification is a mutation in one or more amino acids of the reference dCasX other than those rendering the dCasX catalytically-dead. For example, dCasX variants can comprise one or more amino acid substitutions, insertions, deletions, or swapped domains, or any combinations thereof, relative to a reference dCasX protein sequence. Any amino acid can be substituted for any other amino acid in the substitutions described herein. The substitution can be a conservative substitution (e.g., a basic amino acidis substituted for another basic amino acid). The substitution can be a non-conservative substitution (e.g., a basic amino acid is substituted for an acidic amino acid or vice versa). For example, a proline in a reference dCasX protein can be substituted for any of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine or valine to generate a dCasX variant protein of the disclosure. In some embodiments, the dCasX variant exhibits an improved characteristic compared to a reference dCasX. Exemplary improved characteristics of the dCasX variant embodiments include, but are not limited to improved folding of the variant, increased binding affinity to the target nucleic acid, improved ability to utilize a greater spectrum of PAM sequences in the transcriptional repression and / or binding of target nucleic acid, improved unwinding of the target DNA, increased target strand loading, increased binding of the non-target strand of DNA, improved protein stability, increased ability to complex with gRNA, increased binding affinity to the gRNA, improved protein:gRNA (RNP) complex stability, and, with linked repressor domains and when complexed as an RNP, increased repressor activity, improved repressor specificity for the target nucleic acid, decreased off-target repression, increased percentage of a eukaryotic genome that can be efficiently repressed and / or epigenetically modified. In some embodiments, an improved characteristic of the dCasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference dCasX protein. In some embodiments, an improved characteristic of the dCasX variant is at least about 1.1 to about 10,000-fold improved, at least about 1.1 to about 1,000-fold improved, at least about 1.1 to about 500-fold improved, at least about 1.1 to about 400-fold improved, at least about 1.1 to about 300-fold improved, at least about 1.1 to about 200-fold improved, at least about 1.1 to about 100-fold improved, at least about 1.1 to about 50-fold improved, at least about 1.1 to about 40-fold improved, at least about 1.1 to about 30-fold improved, at least about 1.1 to about 20-fold improved, at least about 1.1 to about 10-fold improved, at least about 1.1 to about 9-fold improved, at least about 1.1 to about 8-fold improved, at least about 1.1 to about 7-fold improved, at least about 1.1 to about 6-fold improved, at least about 1.1 to about 5-fold improved, at least about 1.1 to about 4-fold improved, at least about 1.1 to about 3-fold improved, at least about 1.1 to about 2-fold improved, at least about 1.1 to about 1.5-fold improved, at least about 1.5 to about 3-fold improved, at least about 1.5 to about 4-fold improved, at least about 1.5 to about 5-fold improved, at least about 1.5 to about 10-fold improved, at least about 5 to about 10-fold improved, at least about 10 to about 20-foldimproved, at least 10 to about 30-fold improved, at least 10 to about 50-fold improved or at least 10 to about 100-fold improved relative to the reference dCasX protein. In some embodiments, an improved characteristic of the dCasX variant is at least about 10 to about 1000-fold improved relative to the reference dCasX protein. Additional disclosure on improved characteristics is described herein, below.

[0160] In other embodiments, the modification is a substitution of one or more domains of the reference dCasX with one or more domains from a different CasX. In some embodiments, insertion includes the insertion of a part or all of a domain from a different CasX protein. Mutations can be placed in any one or more domains of the dCasX variant, and may include, for example, deletion of part or all of one or more domains, or one or more amino acid substitutions, deletions, or insertions in any domain. The domains of dCasX proteins include the non-target strand binding (NTSB) domain, the target strand loading (TSL) domain, the helical I domain, the helical II domain, the oligonucleotide binding domain (OBD), and the RuvC DNA cleavage domain, which can further comprise subdomains, described below.

[0161] In some embodiments, the dCasX variant protein comprises between 800 and 1100 amino acids or between 900 and 1000 amino acids.

[0162] The long-term repressor fusion protein comprising dCasX variant and linked repressor domains of the disclosure have an enhanced ability to efficiently bind target nucleic acid, when complexed with a gRNA as an RNP, utilizing and binding to a PAM TC motif, including PAM sequences selected from TTC, ATC, GTC, or CTC, compared to an RNP of a fusion protein comprising the reference dCasX protein and a gRNA in a comparable assay system. In the foregoing, the PAM sequence is located at least 1 nucleotide 5’ to the non- target strand of the protospacer having identity with the targeting sequence of the gRNA.

[0163] In some embodiments, an RNP comprising the long-term repressor fusion protein comprising the dCasX variant with linked repressor domains and a gRNA of the disclosure, at a concentration of 20 pM or less, is capable of binding a double stranded DNA target with an efficiency of at least 70%, at least 80%, at least 85%, at least 90% or at least 95%. In one embodiment, an RNP of a long-term repressor fusion protein comprising a dCasX variant with linked repressor domains and a gRNA variant exhibits greater binding of a target sequence in the target nucleic acid compared to an RNP comprising a comparable long-term repressor fusion protein comprising a reference dCasX protein with linked repressor domains and a gRNA in a comparable assay system, wherein the PAM sequence of the target nucleic acid is TTC. In another embodiment, an RNP of a long-term repressor fusion proteincomprising dCasX variant with linked repressor domains and gRNA variant exhibits greater binding affinity of a target sequence in the target nucleic acid compared to an RNP comprising a comparable long-term repressor fusion protein comprising a reference dCasX protein with linked repressor domains and a gRNA in a comparable assay system, wherein the PAM sequence of the target nucleic acid is ATC. In another embodiment, an RNP of a long-term repressor fusion protein comprising a dCasX variant with linked repressor domains and gRNA variant exhibits greater binding affinity of a target sequence in the target nucleic acid compared to an RNP comprising a comparable long-term repressor fusion protein comprising a reference dCasX protein with linked repressor domains and a gRNA in a comparable assay system, wherein the PAM sequence of the target nucleic acid is CTC. In another embodiment, an RNP of a long-term repressor fusion protein comprising a dCasX variant with linked repressor domains and gRNA variant exhibits greater binding affinity of a target sequence in the target nucleic acid compared to an RNP a comparable long-term repressor fusion protein comprising a reference dCasX protein with linked repressor domains and a gRNA in a comparable assay system, wherein the PAM sequence of the target nucleic acid is GTC. In other embodiments, an RNP of a repressor fusion protein comprising the dCasX variant with linked repressor domains and a gRNA exhibits greater binding affinity for a target sequence in the target nucleic acid compared to an RNP comprising a comparable repressor fusion protein comprising the reference dCasX protein with linked repressor domains and the gRNA in a comparable assay system, wherein the PAM sequence of the target nucleic acid is GTC, TTC, ATC or CTC. In the foregoing embodiments, the increased binding affinity for the one or more PAM sequences is at least 1.5-fold greater or more compared to the binding affinity of an RNP of any one of the reference dCasX proteins (modified from SEQ ID NOS: 1-3) with linked repressor domains and the gRNA of SEQ ID NOS: 1731-1743 of Table 8 for the PAM sequences. c. dCasX Variant Proteins with Domains from Multiple Source Proteins

[0164] In certain embodiments, the disclosure provides a chimeric dCasX variant protein for use in the long-term repressor fusion proteins.

[0165] As used herein, a "chimeric dCasX" protein refers to both a catalytically-dead CasX protein containing at least two domains from different sources, as well as catalytically-dead CasX protein containing at least one domain that itself is chimeric. Accordingly, in some embodiments, a chimeric dCasX protein is one that includes at least two domains isolated or derived from different sources, such as from two different naturally occurring CasX proteins,(e.g., from two different CasX reference proteins). In other embodiments, the chimeric dCasX protein is one that contains at least one domain that is a chimeric domain, e.g. in some embodiments, part of a domain comprises a substitution from a different CasX protein (from a reference CasX protein, or another dCasX protein).

[0166] In some embodiments, the at least one chimeric domain can be any of the NTSB, TSL, helical I, helical II, OBD or RuvC domains as described herein. In the case of split or non-contiguous domains such as helical I, RuvC and OBD, a portion of the non-contiguous domain can be replaced with the corresponding portion from any other source. In some embodiments, the helical I-II domain (sometimes referred to as helical I-a) of the dCasX derived from SEQ ID NO: 2 is replaced with the corresponding helical I-II sequence from SEQ ID NO: 1, resulting in a chimeric dCasX protein.

[0167] In some embodiments, the helical I-II domain and NTSB domain of the dCasX derived from SEQ ID NO: 2 is replaced with the corresponding helical I-II and NTSB sequences from SEQ ID NO: 1, resulting in a chimeric dCasX protein.

[0168] A chimeric dCasX variant protein may comprise an NTSB, TSL, helical I-I, helical I-II, helical II, OBD-I, and OBD-II domains from a CasX protein of SEQ ID NO: 2, and a RuvC-I and / or RuvC-II domain from a CasX protein of SEQ ID NO: 1, or vice versa, in which mutations or other sequence alterations are introduced to create the catalytically-dead variant with improved properties of the variant, relative to the reference dCasX protein. As an example of the foregoing, the chimeric RuvC domain comprises amino acids 660 to 823 of SEQ ID NO: 1 and amino acids 921 to 978 of SEQ ID NO: 2. As an alternative example of the foregoing, a chimeric RuvC domain comprises amino acids 647 to 810 of SEQ ID NO: 2 and amino acids 934 to 986 of SEQ ID NO: 1. In a particular embodiment, a dCasX for use in the long-term repressor fusion protein comprises an NTSB domain and helical I-II domain from SEQ ID NO: 1 and a helical I-I domain from SEQ ID NO: 2; the latter being a chimeric domain, it being understood that the dCasX variants have additional amino acid changes at select locations (relative to the reference sequence), and the resulting chimeric dCasX proteins have improved characteristics relative to the reference dCasX proteins. Sequences of Table 2 having the NTSB domain and helical I-II domain from SEQ ID NO: 1 and a helical I- I domain from SEQ ID NO: 2 include dCasX 491 (SEQ ID NO: 4), 515 (SEQ ID NO: 6), 516 (SEQ ID NO: 7), 518-520 (SEQ ID NOS: 9-11), 522-527 (SEQ ID NOS: 12-17), 532 (SEQ ID NO: 22), 593 (SEQ ID NO: 25), 676 (SEQ ID NO: 28), with a L169K substitution in the NTSB domain), and 812 (SEQ ID NO: 29). Coordinates of CasX domains in the referenceCasX proteins of SEQ ID NO: 1 and SEQ ID NO: 2 are provided in Table 1 below. The skilled artisan will understand that the domain boundaries indicated in Table 1 below are approximate, and that protein fragments whose boundaries differ from those given in the table below by 1, 2, or 3 amino acids may have the same activity as the domains described below. Table 1: Domain coordinates in Reference CasX proteins* amino acid position

[0169] In some embodiments, a dCasX variant protein utilized in the long-term repressor fusion proteins of the disclosure comprises a sequence selected from the group consisting of SEQ ID NOS: 4-29 as set forth in Table 2, wherein the sequence comprises a RuvC domain comprising one or more mutations that inactivates the cleavage activity of the RuvC domain, In other embodiments, a dCasX variant protein utilized in the long-term repressor fusion proteins of the disclosure comprises a sequence at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or at least about 99.5% identical to a sequence of SEQ ID NOS: 4-29 as set forth in Table 2. In some embodiments, the dCasX variant protein comprises a RuvC domain comprising one or more mutations that inactivates the cleavage activity of the RuvC domain In some embodiments, the long-term repressor fusion protein comprising the dCasX retains the ability to form an RNP with a gRNA. In a particularembodiment, the dCasX variant protein utilized in the long-term repressor fusion protein of the gene repressor systems of the disclosure comprises a sequence of SEQ ID NO: 4 (dCasX 491). In another particular embodiment, the dCasX variant protein utilized in the long-term repressor fusion protein of the gene repressor systems of the disclosure comprises a sequence of SEQ ID NO: 6 (dCasX 515). In another particular embodiment, the dCasX variant protein utilized in the long-term repressor fusion protein of the gene repressor systems of the disclosure comprises a sequence of SEQ ID NO: 29 (dCasX 812). Table 2: dCasX Variant Sequencesd. Affinity for the gRNA

[0170] In some embodiments, a long-term repressor fusion protein comprising a dCasX with linked repressor domains has improved affinity for the gRNA relative to a comparable long-term repressor fusion protein comprising a reference dCasX protein with the corresponding linked repressor domains, leading to the formation of the ribonucleoprotein complex (RNP). Increased affinity of the long-term repressor fusion protein for the gRNA may, for example, result in a lower Kd for the generation of a RNP complex, which can, in some cases, result in a more stable ribonucleoprotein complex formation. In some embodiments, the Kd of a long-term repressor fusion protein for a gRNA is increased relative to a reference dCasX protein and linked repressor domains by a factor of at least about 1.1, atleast about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100. In some embodiments, the long- term repressor fusion protein comprising the dCasX variant has about 1.1 to about 10-fold increased binding affinity to the gRNA compared to the corresponding repressor fusion protein comprising the catalytically-dead variant of reference CasX protein of SEQ ID NO: 2.

[0171] In some embodiments, increased affinity of the long-term repressor fusion protein for the gRNA results in increased stability of the ribonucleoprotein complex when delivered to mammalian cells, including in vivo delivery to a subject. This increased stability can affect the function and utility of the complex in the cells of a subject, as well as result in improved pharmacokinetic properties in blood, when delivered to a subject. In some embodiments, increased affinity of the long-term repressor fusion protein, and the resulting increased stability of the ribonucleoprotein complex, allows for a lower dose of the long-term repressor fusion protein to be delivered to the subject or cells while still having the desired activity; for example in vivo or in vitro gene repression and / or epigenetic modification. The increased ability to form RNP and keep them in stable form can be assessed using in vitro assays known in the art.

[0172] In some embodiments, a higher affinity (tighter binding) of a dCasX variant protein of the long-term repressor fusion protein to a gRNA allows for a greater amount of transcriptional repression and / or epigenetic modification events when both the long-term repressor fusion protein and the gRNA remain in an RNP complex. Increased transcriptional repression events can be assessed using assays described herein.

[0173] Methods of measuring long-term repressor fusion protein binding affinity for a gRNA include in vitro methods using purified a long-term repressor fusion protein and a gRNA. The binding affinity for the long-term repressor fusion protein can be measured by fluorescence polarization if the gRNA or long-term repressor fusion protein is tagged with a fluorophore. Alternatively, or in addition, binding affinity can be measured by biolayer interferometry, electrophoretic mobility shift assays (EMSAs), or filter binding. Additional standard techniques to quantify absolute affinities of RNA binding proteins such as the dCasX proteins of the disclosure for specific gRNAs such as reference gRNAs and variantsthereof include, but are not limited to, isothermal calorimetry (ITC), and surface plasmon resonance (SPR). e. Improved Specificity for a Target Nucleic Acid Sequence

[0174] In some embodiments, a long-term repressor fusion protein comprising a dCasX variant protein with linked repressor domains has improved specificity for a target nucleic acid sequence that is complementary to the targeting sequence of the gRNA relative to a reference dCasX protein with linked repressor domains. As used herein, “specificity,” sometimes referred to as “target specificity,” refers to the degree to which a CRISPR / Cas system ribonucleoprotein complex binds off-target sequences that are similar, but not identical to the target nucleic acid sequence; e.g., an long-term repressor fusion protein containing RNP with a higher degree of specificity would exhibit reduced off-target methylation of sequences relative to an RNP of a reference dCasX with linked repressor domains. The specificity, and the reduction of potentially deleterious off-target effects, of long-term repressor fusion proteins can contribute to achieving an acceptable therapeutic index for use in mammalian subjects. Without wishing to be bound by theory, it is possible that amino acid changes in the helical I and II domains that increase the specificity of the long-term repressor fusion protein for the target nucleic acid strand and can thereby increase the specificity of the long-term repressor fusion protein for the target nucleic acid overall. In some embodiments, amino acid changes that increase specificity of long-term repressor fusion proteins for target nucleic acid may also result in decreased affinity of long-term repressor fusion proteins for DNA, but the overall benefit and safety of the composition is enhanced. f. Repressor fusion proteins with heterologous proteins

[0175] Also contemplated within the scope of the disclosure are long-term repressor fusion proteins comprising a heterologous protein fused to the long-term repressor fusion protein for use in the systems of the disclosure. This includes long-term repressor fusion proteins comprising N-terminal or C-terminal fusions to a heterologous protein or domain thereof. In some embodiments, the long-term repressor fusion protein is fused to one or more proteins or domains thereof that has a different activity of interest.

[0176] In some cases, a heterologous polypeptide (a fusion partner) for use with a long- term repressor fusion protein provides for subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence to keep the fusion protein out of the nucleus, anuclear export sequence (NES), a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like).

[0177] In some cases, a long-term repressor fusion protein includes (is fused to) a nuclear localization signal (NLS). In some cases, a long-term repressor fusion protein is fused to 2 or more, 3 or more, 4 or more, or 5 or more 6 or more, 7 or more, 8 or more NLSs. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 20 amino acids of) the N-terminus and / or the C-terminus of the long- term repressor fusion protein. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 20 amino acids of) the N- terminus of the long-term repressor fusion protein. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 20 amino acids of) the C-terminus of the long-term repressor fusion protein. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 20 amino acids of) both the N-terminus and the C-terminus of the long-term repressor fusion protein. In some cases, a single NLS is positioned at the N-terminus and a single NLS is positioned at the C-terminus of the long-term repressor fusion protein. The person of ordinary skill in the art will understand that an NLS at or near the N- or C-terminus of a protein can be within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the N- or C-terminus. In some embodiments, the NLS linked to the N-terminus of the dCasX or long-term repressor fusion protein are identical to the NLS linked to the C- terminus. In other embodiments, the NLS linked to the N-terminus of the dCasX or long-term repressor fusion protein are different to the NLS linked to the C-terminus. Representative configurations of long-term repressor fusion proteins with NLS are shown in FIG.61. In some embodiments, NLSs suitable for use with a long-term repressor fusion protein in the systems of the disclosure comprise sequences having at least about 85%, at least about 90%, or at least about 95% identity or are identical to sequences derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 30); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 31); the c-MYC NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 32) or RQRRNELKRSP (SEQ ID NO: 33). In some embodiments, the NLS and a short peptide linker linked to the N-terminus of the long-term repressor fusion protein is the sequence PKKKRKVSR (SEQ ID NO: 34). In someembodiments, the NLS and a short peptide linker linked to the N-terminus of the long-term repressor fusion protein is the sequence PKKKRKVSRVNGSGSGGG (SEQ ID NO: 3298). In some embodiments, the NLS and a short peptide linker linked to the C-terminus of the long-term repressor fusion protein is the sequence TSPKKKRKV (SEQ ID NO: 3273). In some embodiments, the NLS linked to the N-terminus of the long-term repressor fusion protein is selected from the group consisting of the N-terminal sequences as set forth in Table 3. In some embodiments, the NLS comprises a sequence selected from the group consisting of SEQ ID NOS: 30-97, 3273 and 3298. In some embodiments, the NLS comprises a sequence selected from the group consisting of SEQ ID NOS: 34-97. In some embodiments, the NLS linked to the C-terminus of the long-term repressor fusion protein is selected from the group consisting of the C-terminal sequences as set forth in Table 4. In some embodiments, NLSs suitable for use with a long-term repressor fusion protein in the systems of the disclosure include sequences having at least about 80%, at least about 90%, or at least about 95% identity or are identical to one or more sequences of Table 3 or Table 4. The person of ordinary skill in the art will appreciate that any of the NLS sequences set forth in Table 3 and Table 4 can be fused to, or proximal to either the N- or C-terminus of the long- term repressor fusion proteins described herein. Table 3: N-terminal NLS Amino Acid Sequences*Residues in bold are NLS residues, while unbolded residues are linkers. Table 4: C-terminal NLS Amino Acid Sequences

[0178] In some embodiments, the one or more NLSs are linked to the long-term repressor fusion protein or to adjacent NLS with an optional linker peptide. In some embodiments, the linker peptide is selected from the group consisting of SR, GS, GP, TS, VGS, GGS, (G)n (SEQ ID NO: 98), (GS)n (SEQ ID NO: 99), (GSGGS)n (SEQ ID NO: 100), (GGSGGS)n (SEQ ID NO: 101), (GGGS)n (SEQ ID NO: 102), GGSG (SEQ ID NO: 103), GGSGG (SEQID NO: 104), GSGSG (SEQ ID NO: 105), GSGGG (SEQ ID NO: 106), GGGSG (SEQ ID NO: 107), GSSSG (SEQ ID NO: 108), GPGP (SEQ ID NO: 109), GGP, PPP, VPPP, PPAPPA (SEQ ID NO: 110), PPPG (SEQ ID NO: 111), PPPGPPP (SEQ ID NO: 112), PPP(GGGS)n (SEQ ID NO: 113), (GGGS)nPPP (SEQ ID NO: 114), AEAAAKEAAAKEAAAKA (SEQ ID NO: 115), VPPPGGGSGGGSGGGS (SEQ ID NO: 116), TGGGPGGGAAAGSGS (SEQ ID NO: 117), GGGSGGGSGGGSPPP (SEQ ID NO: 118), TPPKTKRKVEFE (SEQ ID NO: 119), GGSGGGS (SEQ ID NO: 120), GSGSGGG (SEQ ID NO: 121), SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 122), GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSE (SEQ ID NO: 123), and GGSGGG (SEQ ID NO: 124), where n is 1 to 5.

[0179] In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of a long-term repressor fusion protein in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to a long-term repressor fusion protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly. IV. Long-term Repressor Domain Fusion Proteins

[0180] The disclosure provides systems of long-term fusion proteins comprising a DNA binding protein linked to multiple repressor domains, wherein the system is capable of binding to a PCSK9 target nucleic acid and repressing transcription of the PCSK9 gene, including by epigenetic modification of the target nucleic acid. Exemplary DNA binding proteins for use in the fusion proteins include zinc finger (ZF), TALE (transcription- activator-like effector) proteins, and catalytically-dead CRISPR proteins.

[0181] In some embodiments, the disclosure provides systems of long-term repressor fusion proteins comprising a catalytically-dead CRISPR protein, such as a dCasX, linked to multiple repressor domains that, when complexed with a guide ribonucleic acid (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence of PCSK9, wherein the system is capable of binding to a target nucleic acid of PCSK9 and repressing or silencing transcription of a PCSK9 gene. Examples of gene repression processes whichdecrease transcription include, but are not limited to, those which inhibit formation of a transcription initiation complex, those which decrease transcription initiation rate, those which decrease transcription elongation rate, those which decrease processivity of transcription and those which antagonize transcriptional activation (by, for example, blocking the binding of a transcriptional activator). Gene repression can constitute, for example, prevention of activation as well as inhibition of expression below an existing level. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription; the latter can result from epigenetic modification of the target nucleic acid.

[0182] Amongst repressor domains that have the ability to repress, or silence genes, the Krüppel-associated box (KRAB) repressor domain is amongst the most powerful in human genome systems (Alerasool, N., et al. An efficient KRAB domain for CRISPRi applications. Nat. Methods 17:1093 (2020)). KRAB-like domains are present in approximately 400 human zinc finger protein-based transcription factors that upon binding of the linked dCasX to the target nucleic acid, is capable of recruiting additional repressor domains such as, but not limited to, Trim28 (also known as Kap1 or Tif1-beta) that, in turn, assembles a protein complex with chromatin regulators such as CBX5 / HP1α and SETDB1 that induce repression of transcription of the gene, but do so in a limited, temporal fashion. Representative, non- limiting examples of KRAB domains suitable for use in the systems of the disclosure include ZIM3 (SEQ ID NO: 129) and ZNF10 (SEQ ID NO: 128). The disclosure provides additional repressor domains that are from human sources, as well as repressor domains that are from non-human sources with markedly distinct sequences (referred to herein as "RD1") that have been found to result in enhanced transcriptional repression compared to ZIM3 and ZNF10 when incorporated in long-term repressor fusion protein construct embodiments, as described herein.

[0183] In some embodiments, the disclosure provides systems in which the modification imparted by use of the LTRP:gRNA system is epigenetic, and hence the silencing of the PCSK9 gene is heritable by mechanisms other than by edited DNA replication. As used herein “epigenetic modification” means a modification to either DNA or histones associated with DNA, other than a change in the DNA sequence itself (e.g., a substitution, deletion or rearrangement), wherein the modification is either a direct modification by a component of the system or is indirect by the recruitment of one or more additional cellular components, but in which the DNA target nucleic acid sequence itself is not edited to change the sequence. For example, DNA methyltransferase 3A (DNMT3A) (or its catalytic domain) directlymodifies the DNA by methylating it, whereas KRAB recruits KAP-1 / TIF1β corepressor complexes that act as potent transcriptional repressors and can further recruit factors associated with DNA methylation and formation of repressive chromatin, such as heterochromatin protein 1 (HP1), histone deacetylases and histone methyltransferases (Ying, Y., et al. The Krüppel-associated box repressor domain induces reversible and irreversible regulation of endogenous mouse genes by mediating different chromatin states. Nucleic Acids Res.43(3): 1549 (2015)). Further, the catalytically inactive DNMT3L cofactor helps establish a heritable methylation pattern after DNA replication, together with endogenous DNMT1 of the cell. The ATRX-DNMT3-DNMT3L domain (ADD) of DNMT3A is known to have two key functions: 1) it allosterically regulates the catalytic activity of DNMT3A by serving as a methyltransferase auto-inhibitory domain, and 2) it specifically interacts with histone H3 tails that are unmethylated at lysine (K)4, leading to the preferential methylation of DNA bound to chromatin H3 tails that are unmethylated at K4 (Zhang, Y., et al. Chromatin methylation activity of Dnmt3a and Dnmt3a / 3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Research 38:4246 (2010)). In some embodiments, the inclusion of the ADD domain enhances transcriptional repression of targeted genes when incorporated into LTRP, when compared to otherwise equivalent LTRP lacking the ADD domain. In other embodiments, the inclusion of the ADD domain enhances the specificity of the transcriptional repression of targeted genes when compared to otherwise equivalent LTRP lacking the ADD domain. Supporting data for the foregoing are provided in the Examples, as well as in WO2023049742A2, incorporated by reference herein.

[0184] In some embodiments, the long-term repressor fusion protein comprises a DNA binding protein linked to a first, second, and third repressor domain, wherein each of the repressor domains are different and the fusion protein is capable of binding to the PCKS9 target nucleic acid. In some embodiments, the long-term repressor fusion protein comprises a DNA binding protein linked to a first, second, third, and fourth repressor domain, wherein each of the repressor domains are different. In any of the foregoing embodiments, the fusion protein is capable of forming an RNP with a gRNA of the system that binds to the target nucleic acid when the DNA binding domain comprises a catalytically dead CRISPR protein.

[0185] In some embodiments, the DNA binding protein comprises a TALE that can bind but not cleave the target nucleic acid. In some embodiments, the DNA binding protein comprises a zinc-finger protein modified to bind but not cleave the target nucleic acid. In some embodiments, the DNA binding protein comprises a catalytically dead CRISPR proteinthat can complex with a gRNA to form an RNP that can bind but not cleave the target nucleic acid. In some embodiments, the long-term repressor fusion protein comprises a catalytically- dead CRISPR protein sequence, a first repressor domain (hereinafter referred to as "RD1"), a DNMT3A catalytic domain (herein after referred to as "DNMT3A") from the DNMT3A protein as the second domain, and a DNMT3L interaction domain (herein after referred to as "DNMT3L") from the DNMT3L protein as the third domain. In some embodiments, the long-term repressor fusion protein comprises a catalytically-dead CRISPR protein sequence, an RD1, a DNMT3A as the second domain, a DNMT3L as the third domain, and an ATRX- DNMT3-DNMT3L domain (herein after referred to as "ADD") from the DNMT3A protein as the fourth domain. In some embodiments, the long-term repressor fusion protein comprises a dCasX and further comprises a first and a second NLS and one or more linker peptides described herein. In some embodiments, the long-term repressor fusion protein is capable of forming an RNP with a gRNA that binds to the target nucleic acid. It has been discovered that the use of the foregoing domains, when configured in select orientations relative to a dCasX in a long-term repressor fusion protein, can result in pronounced epigenetic modification of a PCSK9 target nucleic acid when complexed with a gRNA with a targeting sequence complementary to defined regions of the PCSK9 gene, and that the combination of the repressor domains work in synchrony, resulting in an additive or synergistic effect on transcriptional silencing of the targeted gene, depending on the configuration.

[0186] Representative amino acid sequences of the components utilized in the long-term repressor fusion protein constructs are provided herein.

[0187] In some embodiments, the dCasX of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 4-29, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the dCasX of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 4-29. In some embodiments, the dCasX of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 4, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto.In some embodiments, the dCasX of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 4.

[0188] In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 128-1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 128-1726. In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 128 or SEQ ID NO: 129, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 128 or SEQ ID NO: 129. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 130-1726, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 130-1726. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 130-224 or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 130-224. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 130-138 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 130-138. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 135 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 131 or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 135. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 130, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 130. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 131. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 132. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 133. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 134. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 135. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 136. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 137. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 138.

[0189] In some embodiments, the second repressor domain of the long-term repressor fusion protein is a DNMT3A, comprising a sequence of SEQ ID NO: 126, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the second repressor domain of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 126.

[0190] In some embodiments, the third repressor domain of the long-term repressor fusion protein is a DNMT3L, comprising a sequence of SEQ ID NO: 127, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0191] In some embodiments, the optional fourth repressor domain of the long-term repressor fusion protein is an ADD, comprising a sequence of SEQ ID NO: 125, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the optional fourth repressor domain of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 125. In some embodiments, the C terminal of the ADD is linked to the N-terminal of the DNMT3A. It has been discovered that the addition of the ADD to the long-term repressor fusion proteins comprising the RD1, DNMT3A, and DNMT3L greatly enhances or increases the long-term repression and / or epigenetic modification of the target nucleic acid, as well as the specificity of the repression, in comparison to long-term repressor fusion proteins lacking the ADD. Exemplary data for the improved repression and specificity of long-term repressor fusion proteins comprising the ADD are presented in the Examples, and is described in WO2023049742A2, incorporated by reference herein. In some embodiments, the fusion protein comprises one or more linker peptides selected from the group consisting of SEQ ID NOS: 98-124, 3278-3289 (exemplary sequences are shown in Tables 5 and 58), and one or more NLS comprising a sequence selected from the group consisting of SEQ ID NOS: 30-97, 32998 and 3299. Exemplary configurations of long-term repressor fusion proteins comprising the ADD are presented in FIG.61.

[0192] In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, a DNMT3A, a DNMT3L, a DNA binding protein, and an RD1. In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, an ADD, a DNMT3A, a DNMT3L, a DNA binding protein, and an RD1. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically-dead CRISPR protein. In some embodiments the long-term repressor fusion protein comprises an NLS at the N terminus, the C terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between the DNMT3A and DNMT3L, between the DNMT3L and DNA binding protein, and / or between the DNA binding protein and the RD1.

[0193] In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, a DNA binding protein, an RD1, a DNMT3A, and a DNMT3L. In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, a DNA binding protein, an RD1, and ADD, a DNMT3A, and a DNMT3L. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically-dead CRISPR protein. In some embodiments the long-term repressor fusion protein comprises an NLS at the N terminus. In some embodiments the long-term repressor fusion protein comprises an NLS between the RD1 and the DNMT3A. In some embodiments the long-term repressor fusion protein comprises an NLS at the N terminus, the C terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between an N terminal NLS and the DNA binding protein, between the DNA binding protein and the RD1, between the RD1 and the DNMT3A, or optionally, the ADD, and / or between the DNMT3A and the DNMT3A.

[0194] In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, a DNA binding protein, a DNMT3A, a DNMT3L, and an RD1. In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, a DNA binding protein, an ADD, a DNMT3A, a DNMT3L, and an RD1. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically-dead CRISPR protein. In some embodiments the long-term repressor fusion protein comprises an NLS at the N terminus, the C terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between an N terminal NLS and the DNA binding protein, between the DNA binding protein and the DNMT3A, or optionally the ADD, between the DNT3A and the DNMT3L, and / or between the DNMT3L and the RD1.

[0195] In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, an RD1, a DNMT3A, a DNMT3L, and a DNA binding protein. In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, an RD1, an ADD, a DNMT3A, a DNMT3L, and a DNA binding protein. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically-dead CRISPR protein. In some embodiments the long-term repressor fusion protein comprises an NLS at the N terminus, the C terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between the RD1 and the DNMT3A, or optionally the ADD, between the DNMT3A and the DNMT3L, between the DNMT3L and the DNA binding protein, and / or between the DNA binding protein and a C terminal NLS.

[0196] In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, a DNMT3A, a DNMT3L, an RD1, and a DNA binding protein. In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, an ADD, a DNMT3A, a DNMT3L, an RD1, and a DNA binding protein. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically-dead CRISPR protein. In some embodiments the long-term repressor fusion protein comprises an NLS at the N terminus, the C terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between the DNMT3A and the DNMT3L, between the DNTM3L and the RD1, between the RD1 and the DNA binding protein, and / or between the DNA binding protein and a C terminal NLS.

[0197] In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, a DNMT3A, a DNMT3L, an RD1, a DNA binding protein, and a second RD1. In some embodiments, the long-term repressor fusion protein comprises, from N- to C-terminus, an ADD, a DNMT3A, a DNMT3L, an RD1, a DNA binding protein. and a second RD1. In one embodiment of the foregoing, the second RD1 may be identical in sequence to the first RD1. In another embodiment of the foregoing, the second RD1 may be different in sequence to the first RD1. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically-dead CRISPR protein. In some embodiments the long-term repressor fusion protein comprises an NLS at the N terminus, the C terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between the DNMT3A and DNMT3L, between the DNMT3L and the RD1, between he RD1 and the DNA binding protein, between the DNA binding protein and the second RD1, and / or between the second RD1 and a C terminal NLS.

[0198] In some cases, the long-term repressor fusion protein also comprises one or more NLS. In some embodiments, the long-term repressor fusion protein comprises a configuration of, N-terminal to C-terminal of NLS-ADD-DNMT3A-DNMT3L-DNA binding protein-RD1- NLS, NLS-DNA binding protein-RD1-NLS-ADD-DNMT3A-DNMT3L, NLS- DNA binding protein-ADD-DNMT3A-DNMT3L- RD1-NLS), NLS-RD1-ADD-DNMT3A-DNMT3L- DNA binding protein-NLS, NLS-ADD-DNMT3A-DNMT3L-RD1-DNA binding protein- NLS, or NLS-ADD-DNMT3A-DNMT3L-RD1-DNA binding protein-RD1-NLS. In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically-dead CRISPR protein. In some embodiments, the long-term repressor fusion protein is capable of binding to and repressing or silencing transcription of the PCSK9 gene.

[0199] In some embodiments of the long-term repressor fusion protein, one or more linker peptides may be inserted between any two adjacent domains of the long-term repressor fusion protein. In some embodiments, the long-term repressor fusion protein comprises a configuration of, N-terminal to C-terminal, of NLS-ADD-DNMT3A-Linker2-DNMT3L- Linker1-Linker3A-DNA binding protein-Linker3B-RD1-NLS (configuration 1). In some embodiments, the long-term repressor fusion protein comprises a configuration of, N- terminal to C-terminal, of NLS-Linker3A-DNA binding protein-Linker3B-RD1-NLS- Linker1-ADD-DNMT3A-Linker2-DNMT3L (configuration 2). In some embodiments, the long-term repressor fusion protein comprises a configuration of, N-terminal to C-terminal, of NLS-Linker3A-DNA binding protein-Linker1-ADD-DNMT3A-Linker2-DNMT3L- Linker3B-RD1-NLS (configuration 3). In some embodiments, the long-term repressor fusion protein comprises a configuration of, N-terminal to C-terminal, of NLS-RD1-Linker3A- ADD-DNMT3A-Linker2-DNMT3L-Linker1-DNA binding protein-Linker3B-NLS (configuration 4). In some embodiments, the long-term repressor fusion protein comprises a configuration of, N-terminal to C-terminal, of NLS-ADD-DNMT3A-Linker2-DNMT3L- Linker3A-RD1-Linker1-DNA binding protein-Linker3B-NLS (configuration 5). In some embodiments, the DNA binding protein of the foregoing configurations may be a zinc finger, a TALE, or a catalytically-dead CRISPR protein. Schematics of the configurations portrayed in FIG.61. In some embodiments of the LTRP of configurations 1-5, the NLS may comprise a sequence selected from the group consisting of SEQ ID NOS: 30-97, 3298 and 3299 (Tables 3 and 4), the linker sequences may comprise sequences independently selected from the group consisting of SEQ ID NOS: 98-124, 3278-3289 (representative linkers shown in Tables 5 and 58). In some embodiments, the NLS may comprise a sequence of SEQ ID NO:30, and the linker sequences may comprise sequences independently selected from the group consisting of SEQ ID NOS: 120, and 122-124. In some embodiments, the DNA binding protein may be a dCasX sequence selected from the group consisting of SEQ ID NOS: 4-29, or a sequence having or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the second repressor domain is a DNMT3A domain comprising the sequence of SEQ ID NO: 126, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the third repressor is a DNMT3L domain comprising the sequence of SEQ ID NO: 127, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the fourth repressor is an ADD domain comprising the sequence of SEQ ID NO: 125, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the long-term repressor fusion proteins are capable of binding to and repressing or silencing the PCSK9 gene target nucleic acid.

[0200] In some embodiments, the disclosure provides a system comprising a long-term repressor fusion protein comprising two RD1 domains, a second repressor domain, a third repressor domain, and a fourth repressor domain operably linked to a DNA binding protein; e.g., a zinc finger, TALE, or catalytically-dead CRISPR protein. In some embodiments, the DNA binding protein comprises a catalytically-dead CasX comprising a sequence selected from the group consisting of SEQ ID NOS: 4-29, or sequence having at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the sequences of the two RD1 are identical. In other embodiments, the two RD1 comprise different sequences. In some embodiments, the two RD1 are located N-terminal to the DNA binding protein. In some embodiments, the two RD1 are located C- terminal to the DNA binding protein. In some embodiments, one RD1 is located N-terminal to the DNA binding protein and one RD1 is located C-terminal to the DNA binding protein.

[0201] In some embodiments, the long-term repressor fusion protein comprises two RD1. In some embodiments, the long-term repressor fusion protein comprises a configuration of, N-terminal to C-terminal, of NLS-ADD-DNMT3A-Linker2-DNMT3L-Linker3A-a RD1a- Linker1-DNA binding protein-Linker3B-RD1a-Linker 4-NLS (configuration 6a), wherein the RD1a sequences are identical (see FIG.61 as a schematic of the long-term repressor fusion protein). In some embodiments, the long-term repressor fusion protein comprises a configuration of, N-terminal to C-terminal, of NLS-ADD-DNMT3A-Linker2-DNMT3L- Linker3A-a RD1a-Linker1-DNA binding protein-Linker3B-RD1b-Linker 4-NLS (configuration 6b), wherein the RD1a and RD1b sequences are different (see FIG.61 as a schematic of the long-term repressor fusion protein). In some embodiments, the DNA binding protein may be a zinc finger, a TALE, or a catalytically-dead CRISPR protein. In some embodiments, a long-term repressor protein embodiment of the paragraph is capable of binding to and repressing or silencing the expression of the PCSK9 gene.

[0202] In some embodiments, the disclosure provides a long-term repressor fusion protein of configuration 6a, wherein the two RD1 sequences are identical. In some embodiments of the configuration 6a long-term repressor fusion protein, the DNA binding protein comprises a dCasX of the sequence of SEQ ID NO: 4, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the first and second copies of the RD1a comprise a sequence selected from the group consisting of SEQ ID NOS: 130-1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% or at least about 95% identity thereto, the second repressor domain is a DNMT3A comprising the sequence of SEQ ID NO: 126, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the third repressor is a DNMT3L comprising the sequence of SEQ ID NOS: 127, or a sequence variant having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the fourth repressor is an ADD comprising the sequence of SEQ ID NO: 125, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the NLSs comprise sequences independently selected from the group consisting of SEQ ID NOS: 30-97 (Tables 3 and 4), the L1 linker comprises a sequence of SEQ ID NO: 123, the L2 linker comprises a sequence of SEQ ID NO: 122, the L3A linker comprises a sequence of SEQ ID NO: 124, the L3B linker comprises a sequence of SEQ ID NO: 120, and the L4 linker comprises a sequence of SEQ ID NO: 3288 or SEQ ID NO: 3289. In some embodiments of the long-term repressor fusion protein of configuration 6a, the linker sequences are independently selected from the group consisting of SEQ ID NOS: 98-124, 3278-3289 (exemplary sequences shown in Tables 5 and 58). In some embodiments of the long-term repressor fusion protein of configuration 6a, wherein the two copies of the RD1 are identical, each of the RD1 comprise a sequence selected from the group consisting of SEQ ID NOS: 130, 131, and 135, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95% identity thereto. A schematic of configuration 6a is shown in FIG.61. In some embodiments, the long-term repressor fusion protein of configuration 6a is capable of forming an RNP with a gRNA of the disclosure and is capable of binding to and repressing or silencing the gene target nucleic acid.

[0203] In some embodiments of the configuration 6b long-term repressor fusion protein, wherein the two RD1 sequences are different, the DNA binding protein comprises a dCasX of the sequence of SEQ ID NO: 4, or a sequence having at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the first copy of the first repressor domain (RD1a) comprises a sequence selected from the group consisting of SEQ ID NOS: 130-1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% or at least about 95% identity thereto, the second copy of the first repressor domain (RD1b) comprises a different sequence selectedfrom the group consisting of SEQ ID NOS: 130-1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% or at least about 95% identity thereto, the second repressor domain is a DNMT3A comprising the sequence of SEQ ID NO: 126, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the third repressor is a DNMT3L comprising the sequence of SEQ ID NOS: 127, or a sequence variant having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the fourth repressor is an ADD comprising the sequence of SEQ ID NO: 125, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93% at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, the NLSs compris sequences independently selected from the group consisting of SEQ ID NOS: 30-97 of Tables 3 and 4, the L1 linker comprises a sequence of SEQ ID NO: 123, the L2 linker comprises a sequence of SEQ ID NO: 122, the L3A linker comprises a sequence of SEQ ID NO: 124, the L3B linker comprises a sequence of SEQ ID NO: 120, and the L4 linker comprises a sequence of SEQ ID NO: 3288 or SEQ ID NO: 3289. In some embodiments of the long-term repressor fusion protein of configuration 6b, the linker sequences are selected from the group consisting of SEQ ID NOS: 98-124, 3278-3289 (exemplary sequences shown in Tables 5 and 58). In some embodiments of the configuration 6b long-term repressor fusion protein, wherein the two copies of the RD1 are different, the RD1a comprises a sequence of SEQ ID NO: 130, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto, and the RD1b comprises a sequence of SEQ ID NO: 131, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto. In some embodiments of the configuration 6b long-term repressor fusion protein, wherein the two copies of the RD1 are different, the RD1a comprises a sequence of SEQ ID NO: 130, or a sequence having at least about 70%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto, and the RD1b comprises a sequence of SEQ ID NO: 135, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto. In some embodiments of the configuration 6b long-term repressor fusion protein, wherein the two copies of the RD1 are different, the RD1a comprises a sequence of SEQ ID NO: 131, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto, and the RD1b comprises a sequence of SEQ ID NO: 130, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto. In some embodiments of the configuration 6b long-term repressor fusion protein, wherein the two copies of the RD1 are different, the RD1a comprises a sequence of SEQ ID NO: 131, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90% at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto, and the RD1b comprises a sequence of SEQ ID NO: 135, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto. In some embodiments of the configuration 6b long-term repressor fusion protein, wherein the two copies of the RD1 are different, the RD1a comprises a sequence of SEQ ID NO: 135, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93% at least about 94%, or at least about 95% identity thereto, and the RD1b comprises a sequence of SEQ ID NO: 130, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto. In some embodiments of the configuration 6b long-term repressor fusion protein, wherein the two copies of the RD1 are different, the RD1a comprises a sequence of SEQ ID NO: 135, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto, and the RD1b comprises a sequence of SEQ ID NO: 131, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto. In some embodiments of the configuration 6b long-term repressor fusion protein, wherein the two copies of the RD1 are different, the RD1a and RD1b are independently selected from the group consisting of the sequences of SEQ ID NOS: 132-134 and 136-138. In some embodiments of the configuration 6b long-term repressor fusion protein, wherein the two copies of the RD1 are different, the RD1a and Rd1b are independently selected from the group consisting of the sequences of SEQ ID NOS: 130, 131 and 135. A schematic of configuration 6b is shown in FIG.61. In some embodiments, the long-term repressor fusion protein is capable of forming an RNP with a gRNA of the disclosure and is capable of binding to and repressing or silencing the gene target nucleic acid. Table 5: Exemplary linker amino acid sequences for long-term repressor fusion proteins

[0204] In some embodiments of the long-term repressor fusion protein comprising a dCasX and, optionally, an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22836- 22855, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least 99% identity thereto. Insome embodiments of the long-term repressor fusion protein comprising a dCasX and, optionally, an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22836- 22855. In some embodiments of the long-term repressor fusion protein comprising a dCasX and, optionally, an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22838 and 22847. In some embodiments of the long-term repressor fusion protein comprising a dCasX and, optionally, an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22839 and 22848. In some embodiments of the long-term repressor fusion protein comprising a dCasX and, optionally, an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22840 and 22849.

[0205] In some embodiments of the system comprising a DNA binding protein, and is configured as a configuration selected from the group consisting of configuration 1, configuration 2, configuration 3, configuration 4, configuration 5, configuration 6a, and configuration 6b, described supra, the long-term repressor fusion protein comprises a dCasX and is capable of complexing with a gRNA with a targeting sequence complementary to the PCSK9 target nucleic acid in a cell to form an RNP and, upon binding of an RNP to the PCSK9 target nucleic acid in a cell, the target nucleic acid is epigenetically-modified and transcription of the PCSK9 gene is repressed. In some embodiments, transcription of the PCSK9 gene is repressed by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least 99%. In some embodiments, transcription of the PCSK9 gene of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% or more of cells in a population of cells is repressed. In some embodiments, the transcriptional repression is assayed in an in vitro assay, including cell-based assays, when compared to untreated cells or cells treated with a comparable system comprising a non-targeting spacer. In some embodiments, the transcriptional repression is assayed in vivo, in cells obtained from a subject in which the long-term repressor fusionprotein and the gRNA with a targeting sequence complementary to the PCSK9 target nucleic acid of a gene in a cell are administered; either as a protein and gRNA, or as nucleic acids (e.g., a gRNA and an mRNA encoding the long-term repressor fusion protein), wherein the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

[0206] Most preferably, PCSK9 gene repression results in complete inhibition of gene expression, such that no gene product is detectable. However, the skilled artisan will appreciate that incomplete inhibition may still be useful and desired for various applications. In some embodiments, the repression of transcription of the PCSK9 gene by an embodiment of the system is sustained for at least about 8 hours, at least about 1 day, at least about 7 days, at least 2 weeks, at least about 3 weeks, at least about 1 month, or at least about 2 months, when assayed in an in vitro assay, including cell-based assays. In some embodiments, the repression of transcription of the PCSK9 gene by the system compositions is sustained for at least about 7 days, at least 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months in targeted cells of a subject when the composition comprising the system of the disclosure is administered as a therapeutically effective dose. In some embodiments, the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human. In some embodiments, use of an LTRP of configurations 1, 4, 5 and 6, when used in a LTRP:gRNA system of the embodiments, results in off-target methylation or off-target activity that is less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less that 0.5%, or less than 0.1% in the cells.

[0207] In some embodiments, repression of transcription of the PCSK9 gene in cells treated with an LTRP:gRNA system of the embodiments is heritable and is stable through one or more cell divisions. In some embodiments, repression of transcription is stable through 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 cell divisions, or more. V. mRNA compositions encoding long-term repressor fusion proteins

[0208] In another aspect, the disclosure relates to messenger RNA (mRNA) compositions comprising sequences of the individual components, as well as the full-length mRNA sequences of the long-term repressor domain fusion protein constructs of the disclosure. In some embodiments, the mRNA compositions have utility in the transcriptional repression and epigenetic modification of a gene, e.g. a PCSK9 gene, when used to express the long-termrepressor fusion protein. In some cases, the mRNA are designed for use in certain delivery formulations; e.g., nanoparticles such as synthetic nanoparticles or lipid nanoparticles (LNP). The disclosure also provides methods utilized to design the mRNA sequences of the mRNAs used in the compositions. In some cases, an mRNA encoding the long-term repressor fusion protein may be co-formulated in a nanoparticle (such as an LNP) with a gRNA comprising a targeting sequence complementary to a sequence of a PCSK9 gene. Upon delivery to the target cell by the LNP, the long-term repressor domain fusion protein is expressed from the mRNA and can complex with the gRNA as an RNP capable of binding the PCSK9 target nucleic acid. In other cases, the mRNA encoding the long-term repressor domain fusion protein and the gRNA may be formulated in separate nanoparticles and delivered either separately or as a mixture.

[0209] In some embodiments, the mRNA compositions of the disclosure have been modified to result in one or more improved characteristics relative to an unmodified mRNA encoding the same long-term repressor protein, and therefore may have a positive impact on the efficacy of mRNA-based delivery. Exemplary improved characteristics of the mRNA described herein include, but are not limited to improved expression upon delivery to a cell, reduced immunogenicity, increased stability, and enhanced manufacturability compared to unmodified mRNA. In some cases, the modifications to the mRNA result in an improved characteristic of at least about 1.1 to about 100,000-fold improved relative to the unmodified mRNA. In some embodiments, an improved characteristic of the modified mRNA is at least about 1.1 to about 10,000-fold improved, at least about 1.1 to about 1,000-fold improved, at least about 1.1 to about 500-fold improved, at least about 1.1 to about 400-fold improved, at least about 1.1 to about 300-fold improved, at least about 1.1 to about 200-fold improved, at least about 1.1 to about 100-fold improved, at least about 1.1 to about 50-fold improved, at least about 1.1 to about 40-fold improved, at least about 1.1 to about 30-fold improved, at least about 1.1 to about 20-fold improved, at least about 1.1 to about 10-fold improved, at least about 1.1 to about 9-fold improved, at least about 1.1 to about 8-fold improved, at least about 1.1 to about 7-fold improved, at least about 1.1 to about 6-fold improved, at least about 1.1 to about 5-fold improved, at least about 1.1 to about 4-fold improved, at least about 1.1 to about 3-fold improved, at least about 1.1 to about 2-fold improved, at least about 1.1 to about 1.5-fold improved, at least about 1.5 to about 3-fold improved, at least about 1.5 to about 4- fold improved, at least about 1.5 to about 5-fold improved, at least about 1.5 to about 10-fold improved, at least about 5 to about 10-fold improved, at least about 10 to about 20-foldimproved, at least 10 to about 30-fold improved, at least 10 to about 50-fold improved or at least 10 to about 100-fold improved relative to the unmodified mRNA. In some embodiments, an improved characteristic of the modified mRNA is at least about 10 to about 1000-fold improved relative to the unmodified mRNA.

[0210] Optimization of coding sequences and untranslated regions (UTRs) may be useful when delivering an mRNA encoding a protein of interest, as opposed to a DNA template that would be transcribed into an mRNA. DNA templates are long-lived, can replicate, and can produce many RNA transcripts over their lifetimes. For DNA templates, efficiency of transcription and pre-mRNA processing are major determinants of protein expression levels. In contrast, mRNAs generally have a much shorter half-life, on the order of hours, as they are vulnerable to degradation in the cytoplasm, and cannot produce more copies of themselves. As such, mRNA stability and translation efficiency can be key determinants of protein expression levels for mRNA-based delivery, and the specific sequences of UTRs and coding sequences that dictate mRNA stability and translation efficiency can therefore be enhanced to improve the efficacy of mRNA-based delivery. a. 5’ cap

[0211] In some embodiments of the mRNA of the disclosure, the mRNA comprises a 5’ cap linked 5’ to the 5’ UTR of the mRNA sequence of any of the embodiments described herein. In some embodiments, the 5’ cap is a 7-methylguanylate cap. In some embodiments, the 5’ cap comprises m7G(5’)ppp(5’)mAG. In other embodiments, the 5' cap comprises m7G(5′)ppp (5′(A,G(5′)ppp(5′)A or G(5′)ppp(5′)G. Exemplary caps are known in the art, and described, for example, in WO 2017 / 053297, the contents of which are incorporated by reference herein. b. 5’ untranslated region (UTR)

[0212] The 5’ UTR of an mRNA molecule can be a key determinant of both the stability of the mRNA and how efficiently it is translated into protein. Specifically, the 5’ UTR, in conjunction with the 5’ cap structure, serves as a binding site and recruitment platform for the translation pre-initiation complex as well as additional regulatory proteins that may positively or negatively affect translation. Structures within the 5’ UTR can enhance translation by recruiting initiation factors or other protein or RNA factors, reduce translation by physically blocking ribosome binding and scanning, and contribute to the stability of the mRNA by affecting both hydrolysis and nuclease digestion.

[0213] An exemplary 5’ UTR sequence for use in the mRNA of the disclosure is provided in Table 6. Table 6 lists the RNA sequence, and the RNA sequence with N1-methyl- pseudouridine substituted in place of uridine. Table 6: 5’ UTR sequences= N1-methyl-pseudouridine

[0214] In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 22787, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 22787. In some embodiments, the 5’ UTR consists of the sequence of SEQ ID NO: 22787. In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 3300, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, identity thereto. In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 3300. In some embodiments, the 5’ UTR consists of the sequence of SEQ ID NO: 3300. c. 3’ UTR

[0215] 3’ UTR sequences can have an impact on mRNA stability and translation efficiency and thus may determine both subcellular localization and tissue-specific expression. Factors influencing these properties include microRNA binding sites, AU-rich elements that recruit an array of RNA-binding proteins, Pumilio binding elements, and other binding sites for RNA-binding proteins. While many of these interactions with the 3’ UTR are known to negatively impact stability or expression, some can enhance translation. The effects of a 3’ UTR sequence can be highly cell-type specific due to differential expression of microRNAs and RNA binding proteins, which provides opportunities for engineering tissue-specific expression into a therapeutic mRNA. In some embodiments, the 3’ UTR for use in the mRNA of the disclosure is a mouse 3’ UTR. In some embodiments, the 3’ UTR is a mouse HBA gene 3’ UTR of Table 7.

[0216] Exemplary 3’ UTR sequences of the disclosure are provided in Table 7. Table 7 lists the RNA sequence, and the RNA sequence with N1-methyl-pseudouridine substituted in place of uridine. Table 7: 3’ UTR sequences= N1-methyl-pseudouridine

[0217] In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 3125, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 3125. In some embodiments, the 3’ UTR consists of the sequence of SEQ ID NO: 3125. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 3310, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 3310. In some embodiments, the 3’ UTR consists of the sequence of SEQ ID NO: 3310. d. Poly(A) sequence

[0218] Inclusion of a 3’ poly(A) tail in an mRNA sequence can contribute to the stability and translation efficiency of the mRNA. Generally, longer poly(A) tails are associated with increased mRNA stability, thereby allowing their translation and promoting high protein expression.

[0219] In some embodiments, an mRNA of the disclosure comprises a poly(A) sequence having at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides. In some embodiments, the poly(A) sequence of an mRNA of the disclosure comprises 80 adenine nucleotides. In some embodiments, the poly(A) sequence comprises the nucleic acid sequence ofembodiments, the poly(A) sequence comprises the nucleic acid sequence ofe. Sequence Modifications to the mRNA

[0220] In some embodiments, an mRNA sequence of the disclosure was modified by codon optimization of the sequence encoding the long-term repressor protein using one or more parameters to enhance expression in the target cell. Non-limiting examples of such parameters include the codon usage in human host cells (e.g., utilizing the codon adaptation index (CAI)), minimizing rare codons, utilizing codon-usage tables derived from biologics intended for use as therapeutics, utilizing an mRNA stability index, or reducing GC content. Methods of codon-optimization, and codon usage in various organisms is known in the art. See, for example, www.genscript.com / tools / codon-frequency-table.

[0221] In some embodiments, provided herein are mRNA sequences for the long-term repressor protein constructs that are codon-optimized for expression in a human cell. In other cases, various naturally-occurring or modified nucleosides may be used to produce the modified mRNA according to the present disclosure. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N-1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). In some embodiments, the mRNA comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine (“5 mC”), pseudouridine (“ψU”), and / or 2-thio-uridine (“2sU”). In a particular embodiment, one or more or all of the uridine residues of the mRNA of the disclosure are replaced with 1-methyl-pseudouridine. In someembodiments, all of the uridine residues of the mRNA of the disclosure are replaced with 1- methyl-pseudouridine. See, e.g., U.S. Pat. No.8,278,036 or WO2011012316, incorporated by reference herein, for a discussion of such residues and their incorporation into mRNA. In some embodiments, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of uridine nucleosides of the mRNA sequence are replaced with N1-methylpseudouridine.

[0222] In some embodiments, the modifications to the mRNA by codon optimization and / or substitution of modified nucleotides in the sequence results in an improved characteristic of at least about 1.1 to about 100,000-fold improved relative to the unmodified mRNA. In some embodiments, an improved characteristic of the modified mRNA is at least about 1.1 to about 10,000-fold improved, at least about 1.1 to about 1,000-fold improved, at least about 1.1 to about 500-fold improved, at least about 1.1 to about 400-fold improved, at least about 1.1 to about 300-fold improved, at least about 1.1 to about 200-fold improved, at least about 1.1 to about 100-fold improved, at least about 1.1 to about 50-fold improved, at least about 1.1 to about 40-fold improved, at least about 1.1 to about 30-fold improved, at least about 1.1 to about 20-fold improved, at least about 1.1 to about 10-fold improved, at least about 1.1 to about 9-fold improved, at least about 1.1 to about 8-fold improved, at least about 1.1 to about 7-fold improved, at least about 1.1 to about 6-fold improved, at least about 1.1 to about 5-fold improved, at least about 1.1 to about 4-fold improved, at least about 1.1 to about 3-fold improved, at least about 1.1 to about 2-fold improved, at least about 1.1 to about 1.5-fold improved, at least about 1.5 to about 3-fold improved, at least about 1.5 to about 4- fold improved, at least about 1.5 to about 5-fold improved, at least about 1.5 to about 10-fold improved, at least about 5 to about 10-fold improved, at least about 10 to about 20-fold improved, at least 10 to about 30-fold improved, at least 10 to about 50-fold improved or at least 10 to about 100-fold improved relative to the unmodified mRNA. In some embodiments, an improved characteristic of the modified mRNA is at least about 10 to about 1000-fold improved relative to the unmodified mRNA. f. LTRP mRNA Component Sequences

[0223] The present disclosure provides mRNA comprising sequences encoding the components utilized in the long-term repressor fusion proteins of the disclosure. In some embodiments, the mRNA comprises sequences encoding the DNA binding proteins, including TALE, ZF, and catalytically dead CRISPR proteins. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 3274 encoding dCasX 515 (SEQ ID NO: 6),or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 3276 encoding dCasX 812 (SEQ ID NO: 29), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 3275 encoding dCasX 491 (SEQ ID NO: 4), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 22736 encoding dCasX 676 (SEQ ID NO: 28), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0224] In some embodiments, the sequence of the mRNA encoding dCasX 491 (SEQ ID NO: 4) has pseudouridine nucleosides replacing one or more, or all uridines in the sequence (SEQ ID NO: 22729). In some embodiments, the sequence of the mRNA encoding dCasX 515 (SEQ ID NO: 6) has pseudouridine nucleosides replacing one or more, or all uridines in the sequence (SEQ ID NO: 22728). In some embodiments, the sequence of the mRNA encoding dCasX 676 (SEQ ID NO: 28) has pseudouridine nucleosides replacing one or more, or all uridines in the sequence. In some embodiments, the sequence of the mRNA encoding dCasX 812 (SEQ ID NO: 29) has pseudouridine nucleosides replacing one or more, or all uridines in the sequence (SEQ ID NOS: 22730 or 22731).

[0225] In some embodiments, the disclosure provides mRNA sequences encoding the RD1 domains. In some embodiments, the sequence of the mRNA encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3120, 22804, and 3334-6527, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto. In some embodiments, the sequence of the mRNA encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3120, 22804, and 3334-6527. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3334-3342 and 4931-4939, or a sequence having at least about 70%, at least about 80%, at least about90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3334-3342 and 4931-4939. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3334 or SEQ ID NO: 4931, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3334 or SEQ ID NO: 4931. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3335 or SEQ ID NO: 4932, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3335 or SEQ ID NO: 4932. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3336 or SEQ ID NO: 4933, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3336 or SEQ ID NO: 4933. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3337 or SEQ ID NO: 4934, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3337 or SEQ ID NO: 4934. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3338 or SEQ ID NO: 4935, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the sequenceof the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3338 or SEQ ID NO: 4935. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3339 or SEQ ID NO: 4936, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3339 or SEQ ID NO: 4936. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3340 or SEQ ID NO: 4937, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3340 or SEQ ID NO: 4937. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3341 or SEQ ID NO: 4938, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the sequence of the mRNA encoding the RD1 comprises a sequence of SEQ ID NO: 3341 or SEQ ID NO: 4938.

[0226] In some embodiments, the mRNA comprises a sequence encoding the second repressor domain. In some embodiments, the second repressor domain comprises DNMT3A. In some embodiments, the sequence of the mRNA encoding the DNMT3A comprises a sequence of SEQ ID NO: 3128 or SEQ ID NO: 3331, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the sequence of the mRNA encoding the DNMT3A comprises a sequence of SEQ ID NO: 3128 or SEQ ID NO: 3331.

[0227] In some embodiments, the mRNA comprises a sequence encoding the third repressor domain. In some embodiments, the third repressor domain comprises DNMT3L. In some embodiments, the sequence of the mRNA encoding the DNMT3L comprises a sequence SEQ ID NO: 3119 or SEQ ID NO: 3332, or a sequence having at least about 70%,at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the sequence of the mRNA encoding the DNMT3L comprises a sequence SEQ ID NO: 3119 or SEQ ID NO: 3332.

[0228] In some embodiments, the mRNA comprises a sequence encoding the fourth repressor domain. In some embodiments, the fourth repressor domain comprises the ADD. In some embodiments, the sequence of the mRNA encoding the ADD comprises a sequence of SEQ ID NO: 3296 or SEQ ID NO: 22726, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the sequence of the mRNA encoding the ADD comprises a sequence of SEQ ID NO: 3296 or SEQ ID NO: 22726.

[0229] In some embodiments, the mRNA of the disclosure comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises a GCCACCAUGG (SEQ ID NO: 22805). In some embodiments, an mRNA of the disclosure comprises a Kozak sequence and two bases upstream of the NLS (to produce the methionine and alanine upstream of the NLS). In some embodiments, the mRNA comprises the sequence GCCACCAUGGCC (SEQ ID NO: 22806) between the 5’ UTR and the sequence encoding the NLS.

[0230] In another embodiment, the mRNA comprises an NLS. In some embodiments, the sequence encoding the NLS comprises a sequence selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807, or a sequence having at least about 70%, at least about 80%, or at least about 90% identity thereto. In another embodiment, the mRNA comprises a sequence selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807. In another embodiment, for examples those embodiments wherein the long-term repressor protein comprises more than one NLS, the mRNA comprises two or more sequences independently selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807.

[0231] In some embodiments, the mRNA comprises sequences encoding the linkers of the long-term repressor fusion protein. In some embodiments, the sequence encoding the linker comprises a sequence selected from the group consisting of SEQ ID NOS: 3312-3327 and 22808-22828, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% identitythereto. In some embodiments, the sequence encoding the linker comprises a sequence selected from the group consisting of SEQ ID NOS: 3312-3327 and 22808-22828. In some embodiments, for example those embodiments where the long-term repressor fusion protein comprises more than one linker, the sequences encoding the linkers are independently selected from the group consisting of SEQ ID NOS: 3312-3327 and 22808-22828, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% identity thereto.

[0232] In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of the configurations provided herein. In some embodiments, the mRNA encodes a long-term repressor fusion protein of configuration 1, and the mRNA comprises a sequence is selected from the group consisting of SEQ ID NOS: 6529-8134, 9741-11347, 14628-16233, and 17841-19446, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence is selected from the group consisting of SEQ ID NOS: 6529-8134, 9741-11347, 14628-16233, and 17841-19446.

[0233] In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of configuration 1, and comprises a sequence encoding the RD1 comprising a sequence of SEQ ID NO: 130. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6531, 9744, 14630, or 17843, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6531, 9744, 14630, or 17843. In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of configuration 1, and comprises a sequence encoding the RD1 of SEQ ID NO: 131. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6529, 9742, 14628, or 17841, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6529, 9742, 14628, or 17841. In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein ofconfiguration 1, and the mRNA comprises a sequence encoding the RD1 of SEQ ID NO: 132. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6535, 9748, 14634, or 17847, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6535, 9748, 14634, or 17847. In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of configuration 1, and comprises a sequence encoding the RD1 of SEQ ID NO: 133. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6536, 9749, 14635, or 17848, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6536, 9749, 14635, or 17848. In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of configuration 1, and the mRNA comprises a sequence encoding the RD1 of SEQ ID NO: 134. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6534, 9747, 14633, or 17846, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6534, 9747, 14633, or 17846. In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of configuration 1, and comprises a sequence encoding the RD1 of SEQ ID NO: 135. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6530, 9743, 14629, or 17842, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6530, 9743, 14629, or 17842. In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of configuration 1, and comprises a sequence encoding the RD1 of SEQ ID NO: 136. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6537, 9750, 14636, or17849, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6537, 9750, 14636, or17849. In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of configuration 1, and comprises a sequence encoding the RD1 of SEQ ID NO: 137. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6533, 9746, 14632, or 17845, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6533, 9746, 14632, or 17845. In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of configuration 1, and comprises a sequence encoding the RD1 of SEQ ID NO: 138. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6532, 9745, 14631, or 17844, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6532, 9745, 14631, or 17844.

[0234] The disclosure provides an mRNA encoding a long-term repressor fusion protein of configuration 5. In some embodiments, the mRNA comprises a sequence is selected from the group consisting of SEQ ID NOS: 8135-9740, 11348-12953, 16234-17839, and 19447- 21052, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence is selected from the group consisting of SEQ ID NOS: 8135-9740, 11348-12953, 16234-17839, and 19447-21052.

[0235] The disclosure provides an mRNA encoding a long-term repressor fusion protein of configuration 6a, wherein the encoded RD1 are identical. In some embodiments, the mRNA comprises a sequence is selected from the group consisting of SEQ ID NOS: 12954-14553, and 21053-22652, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a sequence is selected from the group consisting of SEQ ID NOS: 12954-14553 and 21053-22652.

[0236] The disclosure provides an mRNA encoding a long-term repressor fusion protein of configuration 6b, wherein the encoded RD1 are different. In some embodiments, the mRNA comprises a e sequence is selected from the group consisting of SEQ ID NOS: 14554-14626 and 22653-22725, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA comprises a e sequence is selected from the group consisting of SEQ ID NOS: 14554-14626 and 22653-22725.

[0237] In some embodiments of the mRNA encoding a long-term repressor fusion protein of the LTRP:gRNA systems of the disclosure, upon delivery of the system to a targeted cell, the mRNA is expressed and the long-term repressor fusion protein is capable of complexing with a gRNA and binding the target DNA, resulting in repression or silencing of transcription of the PCSK9 gene in the cell. VI. Guide Nucleic Acids of the Systems

[0238] In another aspect, the disclosure relates to specifically designed guide ribonucleic acids (gRNA) comprising a scaffold and a linked targeting sequence complementary to (and are therefore able to hybridize with) a target nucleic acid sequence of a PCSK9 gene. The gRNA described herein can be used with the long-term repressor proteins, and systems comprising same, to repress transcription of the PCSK9 target nucleic acid in a eukaryotic cell. As used herein, the term “gRNA” covers naturally-occurring molecules and gRNA variants, including chimeric gRNA variants comprising domains from different gRNA. gRNAs of the disclosure comprise a scaffold and a targeting sequence complementary to a target nucleic acid of a cell linked to the 3' end of the scaffold.

[0239] In some embodiments, the systems comprising an mRNA encoding a long-term repressor fusion protein comprise a dCasX protein and one or more gRNA, upon expression of the dCasX protein in a transfected cell, form a ribonucleoprotein (RNP) complex with the gRNA, which can target and bind specific locations in the target nucleic acid sequence of the cell. The gRNA provides target specificity to the complex by including a targeting sequence (or “spacer”) having a nucleotide sequence that is complementary to a sequence of the target nucleic acid sequence while the long-term repressor fusion protein of the system provides the site-specific activity such as the binding and repression of the target sequence that is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence by virtue of its association with the gRNA.

[0240] Embodiments of gRNAs and formulations of mRNAs and gRNAs for use in the repression and / or epigenetic modification of PCSK9 target nucleic acids are described herein, below. a. Reference gRNA and gRNA variants

[0241] As used herein, a “reference gRNA" refers to a CRISPR guide ribonucleic acid comprising a wild-type sequence of a naturally-occurring gRNA. In some embodiments, a gRNA scaffold of the disclosure may be subjected to one or more mutagenesis methods, such as the mutagenesis methods described in WO2023235818A2, WO2022120095A1 and WO2020247882A1, incorporated by reference herein, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, domain swapping, or chemical modification to generate one or more gRNA variants with enhanced or varied properties relative to the gRNA scaffold that was modified. The activity of the gRNA scaffold from which a gRNA variant was derived may be used as a benchmark against which the activity of the gRNA variant is compared, thereby measuring improvements in function or other characteristics of the gRNA scaffold.

[0242] Table 8 provides the sequences of reference gRNA tracr and scaffold sequences. In some embodiments, the disclosure provides gRNA variants wherein the gRNA has a scaffold comprising a sequence having one or more nucleotide modifications relative to a reference gRNA sequence of any one of SEQ ID NOS: 1731-1743 of Table 8. Table 8: Reference gRNA tracr and scaffold sequencesb. gRNA Domains and their Function

[0243] The gRNAs of the systems of the disclosure comprise two segments: a targeting sequence and a protein-binding segment. The targeting segment of a gRNA includes a nucleotide sequence (referred to interchangeably as a spacer, a targeter, or a targeting sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within the target nucleic acid sequence (e.g., a strand of a double stranded target DNA, a target ssRNA, a target ssDNA, etc.), described more fully below. The targeting sequence of a gRNA is capable of binding to a target nucleic acid sequence, including, in the context of the present disclosure, a coding sequence, a complement of a coding sequence, a non-coding sequence, and to accessory elements. The protein-binding segment (or “activator” or “protein-binding sequence”) of the gRNA interacts with (e.g., binds to) a dCasX protein as a complex, forming an RNP (described more fully, below). As used herein, “scaffold” refers to all parts to the guide with the exception of the targeting sequence, which is comprised of several regions, described more fully, below. The properties and characteristics of CasX gRNA, both wild-type and variants, are described in WO2020247882A1, US20220220508A1, and WO2022120095A1, incorporated by reference herein.

[0244] In the case of a reference gRNA, the gRNA occurs naturally as a dual guide RNA (dgRNA), wherein the targeter and the activator portions each have a duplex-forming segment that have complementarity with one another and hybridize to one another to form a double stranded duplex (dsRNA duplex for a gRNA). The term “targeter” or “targeter RNA” is used herein to refer to a crRNA-like molecule (crRNA: "CRISPR RNA") of a CasX dual guide RNA (and therefore of a CasX single guide RNA when the “activator" and the "targeter” are linked together, e.g., by intervening nucleotides). The crRNA has a 5' region that anneals with the tracrRNA followed by the nucleotides of the targeting sequence. In the case of the gRNA for use in the systems of the disclosure, the scaffolds are designed such that the activator and targeter portions are covalently linked to one another (rather than hybridizing to one another) and comprise a single molecule, and can be referred to as a“single-molecule gRNA,” “single guide RNA”, a “single-molecule guide RNA,” a “one- molecule guide RNA”, or a “sgRNA”. The gRNA variants of the disclosure for use in the systems are all single molecule versions.

[0245] Collectively, the assembled gRNAs of the disclosure comprise distinct structured regions, or domains: the RNA triplex, the scaffold stem loop, the extended stem loop, the pseudoknot, and the targeting sequence that, in the embodiments of the disclosure is complementary to a sequence of a target nucleic acid and is located on the 3’ end of the gRNA. The RNA triplex, the scaffold stem loop, the pseudoknot and the extended stem loop, together with the unstructured triplex loop that bridges portions of the triplex, together, are referred to as the “scaffold” of the gRNA. In some cases, the scaffold stem further comprises a bubble. In other cases, the scaffold further comprises a triplex loop region. In still other cases, the scaffold further comprises a 5’ unstructured region. In some embodiments, the gRNA scaffolds of the disclosure for use in the LTRP:gRNA systems comprise a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1822), or a sequence with at least 1, 2, 3, 4 or 5 mismatches thereto.

[0246] Each of the structured domains contribute to establish the global RNA fold of the guide and retain functionality of the guide; particularly the ability to properly complex with the dCasX protein. For example, the guide scaffold stem interacts with the helical I domain of dCasX protein, while residues within the triplex, triplex loop, and pseudoknot stem interact with the OBD of the dCasX protein. Together, these interactions confer the ability of the guide to bind and form an RNP with the LTRP that retains stability, while the spacer (or targeting sequence) directs and defines the specificity of the RNP for binding a specific sequence of DNA.

[0247] Site-specific binding of a target nucleic acid sequence (e.g., genomic DNA) by the dCasX protein of the LTRP can occur at one or more locations (e.g., a sequence of a PCSK9 target nucleic acid) determined by base-pairing complementarity between the targeting sequence of the gRNA and the target nucleic acid sequence. Thus, for example, the gRNA of the disclosure have sequences complementarity to and therefore can hybridize with the target nucleic acid that is adjacent to a sequence complementary to a TC protospacer adjacent motif (PAM) motif or a PAM sequence, such as ATC, CTC, GTC, or TTC. Because the targeting sequence of a guide sequence hybridizes with a sequence of a target nucleic acid sequence, a targeting sequence can be modified by a user to hybridize with a specific target nucleic acid sequence, so long as the location of the PAM sequence is considered. In some embodiments,for design of a targeting sequence, the target nucleic acid comprises a PAM sequence located 5’ of the targeting sequence with at least a single nucleotide separating the PAM from the first nucleotide of the target nucleic acid complementary to that of the targeting sequence. This feature distinguishes the systems described herein from c Cas9 systems, andresults in the ability of the systems of the disclosure to modify different locations in the DNA sequence when compared to Cas9 systems. In some embodiments, the PAM is located on the non- targeted strand of the target region, i.e. the strand that is complementary to the target nucleic acid. In some embodiments, the targeting sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence one nucleotide from an ATC PAM sequence. In some embodiments, the targeting sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence one nucleotide from an CTC PAM sequence. In some embodiments, the targeting sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence one nucleotide from an GTC PAM sequence. In some embodiments, the targeting sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence one nucleotide from an TTC PAM sequence. By selection of the targeting sequences of the gRNA, defined regions of the target nucleic acid sequence or sequences bracketing a particular location within the target nucleic acid can be repressed using the LTRP:gRNA systems described herein. In some embodiments, the targeting sequence of the gRNA has between 15 and 22 consecutive nucleotides. In some embodiments, the targeting sequence has 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 21 consecutive nucleotides. In some embodiments, the targeting sequence consists of 20 consecutive nucleotides. In some embodiments, the targeting sequence consists of 19 consecutive nucleotides. In some embodiments, the targeting sequence consists of 18 consecutive nucleotides. In some embodiments, the targeting sequence consists of 17 consecutive nucleotides. In some embodiments, the targeting sequence consists of 16 consecutive nucleotides. In some embodiments, the targeting sequence consists of 15 consecutive nucleotides. By selection of the targeting sequences of the gRNA, defined regions of the PCSK9 target nucleic acid sequence can be repressed and / or epigenetically modified using the LTRP:gRNA systems described herein.

[0248] The gene repressor systems of the present disclosure can be designed to target any region of, or proximal to, a PCSK9 gene or region of a PCSK9 gene for which repression of transcription is sought. When the entirety of the gene is to be repressed, designing a guidewith a targeting sequence complementary to a sequence encompassing or proximal to the transcription start site (TSS) is contemplated by the disclosure. The TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. The core promoter serves as a binding platform for the transcription machinery, which comprises Pol II and its associated general transcription factors (GTFs) (Haberle, V. et al. Eukaryotic core promoters and the functional basis of transcription initiation (Nat Rev Mol Cell Biol.19(10):621 (2018)). Variability in TSS selection has been proposed to involve DNA ‘scrunching’ and ‘anti-scrunching,’ the hallmarks of which are: (i) forward and reverse movement of the RNA polymerase leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. In some embodiments, the target nucleic acid sequence bound by an RNP of the LTRP:gRNA system is within 1.5 kilobase (kb) of a transcription start site (TSS) in the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by an RNP of the system is within 20 basepairs (bp), 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bps, 1 kb, or 1.5 kb upstream of a TSS of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by an RNP of the system is within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb, or 1.5 kb downstream of a TSS of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by an RNP of the system is within 1.5 kb upstream to 1.5 downstream, 1kb upstream to 1 kb downstream, 500 bp upstream to 500 bp downstream, or 300 bp upstream to 300 bp downstream, or 100 bp upstream to 100 bp downstream of a TSS of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by an RNP of the system is within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb, or 1.5 kb of an enhancer of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by an RNP of the system of the disclosure is within 1 kb 3' to a 5' untranslated region of the PCSK9 gene. In other embodiments, the target nucleic acid sequence bound by an RNP of the system is within the open reading frame of the PCSK9 gene, inclusive of introns (if any). In some embodiments, the targeting sequence of a gRNA of the system of the disclosure is complementary to a sequence of an exon of the PCSK9 gene. In a particular embodiment, the targeting sequence of a gRNA of the system of the disclosure is complementary to a sequence of exon 1 of the PCSK9 gene. In other embodiments, the targeting sequence of a gRNA of the system of the disclosure is complementary to a sequence of an intron of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system of the disclosure is complementary to asequence of an intron-exon junction of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system of the disclosure is complementary to a sequence of a regulatory element of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system of the disclosure is complementary to a sequence of an intergenic region of the PCSK9 gene. In other embodiments, the targeting sequence of a gRNA of the system of the disclosure is complementary to a sequence of a junction of the exon, an intron, and / or a regulatory element of the PCSK9 gene. In those cases where the targeting sequence is complementary to a sequence of a regulatory element, such regulatory elements include, but are not limited to promoter regions, enhancer regions, intergenic regions, 5' untranslated regions (5' UTR), 3' untranslated regions (3' UTR), conserved elements, and regions comprising cis-regulatory elements. The promoter region is intended to encompass nucleotides within 5 kb of the initiation point of the encoding sequence or, in the case of gene enhancer elements or conserved elements, can be thousands of bp, hundreds of thousands of bp, or even millions of bp away from the encoding sequence of the PCSK9 gene. In the foregoing, the targets are those in which the encoding PCSK9 gene of the target is intended to be repressed such that the PCSK9 gene product is not expressed or is expressed at a lower level in a cell. In some embodiments, upon binding of the RNP of the system of the disclosure to the binding location of the target nucleic acid, the system is capable of repressing transcription of the PCSK9 gene 5’ to the binding location of the RNP. In other embodiments, upon binding of the RNP of the system to the binding location of the target nucleic acid, the system is capable of repressing transcription of the PCSK9 gene 3’ to the binding location of the RNP.

[0249] In some embodiments, the target nucleic acid comprises a PAM sequence located 5’ of the targeting sequence with at least a single nucleotide separating the PAM from the first nucleotide of the targeting sequence. In some embodiments, the PAM is located on the non- targeted strand of the target region, i.e. the strand that is complementary to the target nucleic acid. , and are shown below as Table 9, representing targeting sequences for PCSK9 target nucleic acid for linkage to the gRNA scaffolds of the disclosure; e.g., gRNA 174 (SEQ ID NO: 1744), 235 (SEQ ID NO: 1745), 316 (SEQ ID NO: 1746), or chemically-modified versions thereof. In some embodiments, the PAM sequence is TTC.

[0250] In some embodiments, the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1824-2544, 2672, 2675, 2694, and 2714, as set forth in Table 9. In some embodiments, the targeting sequence of the gRNA comprisesa sequence selected from the group consisting of SEQ ID NOS: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments, the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1834, 1849, 1853, 1855-1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875. In some embodiments, the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1855, 1867 and 1869. In a particular embodiment, the targeting sequence of the gRNA consists of a sequence of SEQ ID NO: 1855. In another particular embodiment, the targeting sequence of the gRNA consists of a sequence of SEQ ID NO: 1867. In another particular embodiment, the targeting sequence of the gRNA consists of a sequence of SEQ ID NO: 1869. In some embodiments, the foregoing targeting sequences are linked to the gRNA scaffolds of gRNA 174 (SEQ ID NO: 1744), 235 (SEQ ID NO: 1745), or 316 (SEQ ID NO: 1746), or chemically-modified versions thereof. Table 9: Targeting Sequences Specific to PCSK9 using long-term repressor fusion proteinsc. gRNA Modifications

[0251] In another aspect, the disclosure relates to gRNAs (sometimes referred to as gRNA variants herein), which comprise modifications relative to a reference gRNA from which the gRNAs were derived. The gRNAs can be used in the long-term gene repressor fusion proteinsystems described herein. In some embodiments, a gRNA variant for use in the systems of the disclosure comprises one or more nucleotide substitutions, insertions, deletions, or swapped or replaced domains relative to a gRNA sequence of the disclosure that improve a characteristic. Exemplary regions for modifications and swapped regions or domains include the RNA triplex, the pseudoknot, the scaffold stem loop, and the extended stem loop. In some embodiments, the gRNA variant of the disclosure comprises at least a first swapped region from a different gRNA, resulting in a chimeric gRNA. A representative example of such a chimeric gRNA is guide 316 (SEQ ID NO: 1746), in which the extended stem loop of gRNA scaffold 235 (SEQ ID NO: 1745) is replaced with the extended stem loop of gRNA scaffold 174 (SEQ ID NO: 1744), wherein the resulting 316 variant retains the ability to form an RNP with a long-term repressor fusion protein and exhibits an improved characteristic compared to the parent 235, when assessed in an in vitro or in vivo assay under comparable conditions.

[0252] All gRNAs that have one or more improved functions, characteristics, or add one or more new functions when the gRNA scaffold variant is compared to a gRNA scaffold from which it was derived, while retaining the functional properties of being able to complex with the long-term repressor fusion protein and guide the ribonucleoprotein holo RNP complex to the PCSK9 target nucleic acid are envisaged as within the scope of the disclosure. In some embodiments, the gRNA has an improved characteristic selected from the group consisting of increased pseudoknot stem stability, increased triplex region stability, increased scaffold stem stability, extended stem stability, reduced off-target folding intermediates, increased binding affinity to a long-term repressor fusion protein, and increased repression activity when complexed with a long-term repressor fusion protein, or any combination thereof. In some cases, the improved characteristic is assessed in an in vitro assay, including the assays of the Examples. In other cases, the improved characteristic is assessed in vivo.

[0253] Table 10 provides exemplary gRNA variant scaffold sequences of the disclosure that are utilized as gRNA scaffolds or for the generation of the gRNAs for use in the LTRP:gRNA systems of the disclosure. In some embodiments, the gRNA variant scaffold for use in the systems comprises a sequence selected from the group consisting of SEQ ID NOS: 1744-1746, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto, wherein the gRNA variant retains the ability to form an RNP with a long-term repressor fusion protein of the disclosure. In some embodiments, the gRNA variant scaffold for use inthe systems comprises a sequence selected from the group consisting of SEQ ID NOS: 1744- 1746. In some embodiments, the gRNA variant scaffold for use in the systems comprises SEQ ID NO: 1744. In some embodiments, the gRNA variant scaffold for use in the systems comprises SEQ ID NO: 1745. In some embodiments, the gRNA variant scaffold for use in the systems comprises SEQ ID NO: 1746. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gRNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gRNA sequence embodiments described herein. Similarly, any RNA sequence disclosed herein can be encoded by a DNA in which the uracil bases are substituted by thymine. In some embodiments, the disclosure provides gRNA variants of Table 10 that are chemically-modified, described below. In some embodiments, the gRNA comprises a scaffold comprising a sequence of SEQ ID NOS: 1744- 1746, and is chemically modified. Table 10: gRNA Scaffold Sequences

[0254] Additional gRNA variants contemplated for use in the systems of the disclosure are selected from the group consisting of SEQ ID NOS: 1747-1821. In some embodiments, the gRNA comprises a scaffold comprising a sequence of SEQ ID NOS: 1747-1821, and is chemically modified.

[0255] Guide scaffolds can be made by several methods, including recombinantly or by solid-phase RNA synthesis. However, the length of the scaffold can affect the manufacturability when using solid-phase RNA synthesis, with longer lengths resulting in increased manufacturing costs, decreased purity and yield, and higher rates of synthesis failure. For use in particle formulations, such as lipid nanoparticle (LNP) formulations, solid- phase RNA synthesis of the scaffold is preferred to generate the quantities needed for commercial development. While previous experiments had identified gRNA scaffold 235 (SEQ ID NO: 1745) as having enhanced properties relative to gRNA scaffold 174 (SEQ ID NO: 1744), its increased length (in nucleotides) rendered its use for LNP formulations less desirable due to synthetic manufacturing constraints. Accordingly, alternative sequences weresought. In some embodiments, the disclosure provides gRNA variant scaffolds having improved manufacturability compared to the gRNA scaffold from which it was derived. In some embodiments, the disclosure provides a gRNA wherein the gRNA scaffold and linked targeting sequence has a sequence that is less than about 115 nucleotides, less than about 110 nucleotides, or less than about 100 nucleotides. In a particular embodiment, the 316 gRNA scaffold (SEQ ID NO: 1746) has a shorter sequence compared to the 235 scaffold from which it was derived. The 316 gRNA scaffold was designed wherein the scaffold 235 sequence was modified by a domain swap in which the extended stem loop of scaffold 174 replaced the extended stem loop of the 235 scaffold, resulting in the chimeric gRNA scaffold 316, having the sequencehaving 89 nucleotides, compared with the 99 nucleotides of gRNA scaffold 235. The resulting 316 scaffold had the further advantage in that the extended stem loop does not contain CpG motifs; an enhanced property conferring reduced potential to elicit an immune response. In some embodiments, the shorter sequence length of the 316 scaffold confers the improvements of a higher fidelity in the ability to create the guide synthetically with the correct and complete sequence, as well as an enhanced ability to be successfully incorporated into an LNP. In some embodiments, the disclosure provides gRNA 316 variants that are chemically- modified, described below. d. Chemically-modified gRNAs

[0256] In some embodiments, the gRNAs have one or more chemical modifications. In some embodiments, the chemical modification is the addition of a 2’O-methyl group to one or more nucleotides of the sequence. In some embodiments, the chemical modification is substitution of a phosphorothioate bond between two or more nucleotides of the sequence.. In some embodiments, the chemical modification is a substitution of phosphorothioate bonds between two or more nucleotides on each terminal end of the gRNA. In some embodiments, the gRNA comprises a substitution of phosphorothioate bonds between two or more nucleotides located 1, 2, 3 or 4 nucleotides the from the 5’ terminal end, the 3’ terminal, or both terminal ends of the gRNA. In some embodiments, the gRNA comprises an addition of a 2’O-methyl group to one or more nucleotides of the gRNA. In some embodiments, one or more nucleotides located 1, 2, 3, or 4 nucleotides from the 5’ terminal end, the 3’ terminal, or both terminal ends of the gRNA are modified by an addition of a 2’O-methyl group. Insome embodiments, the first 1, 2, or 3 nucleotides of the 5’ end of the scaffold (i.e., A, C, and U in the case of gRNA 174, 235, and 316) are modified by the addition of a 2’O-methyl group and each of the modified nucleotides is linked to the adjoining nucleotide by a phosphorothioate bond. Similarly, the last 1, 2, or 3 nucleotides of the 3’ end of the targeting sequence linked to the 3’ end of the scaffold are similarly modified. In some embodiments, the disclosure provides gRNA with chemical modifications selected from the group consisting of the sequences of SEQ ID NOS: 2948-2956, 2958-2966, and 2968-2976, as set forth in Table 22, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In any of the foregoing, the 20 nucleotides on the 3' end of the sequence are replaced with a targeting sequence complementary to a PCSK9 target nucleic acid sequence, optionally the targeting sequence is selected from the group consisting of SEQ ID NOS: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714, but includes the chemical modifications on the 3' end of the gRNA. In some embodiments, the 20 nucleotides on the 3' end of the sequence are replaced with a targeting sequence complementary to a PCSK9 target nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1834, 1849, 1853, 1855-1858,1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875, but includes the chemical modifications on the 3' end of the gRNA. In some embodiments, the 20 nucleotides on the 3' end of the sequence are replaced with a targeting sequence complementary to a PCSK9 target nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1855, 1867 and 1869, but includes the chemical modifications on the 3' end of the gRNA. In some embodiments, the gRNA with chemical modifications comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2948-2956, 2958-2966, and 2968-2976 with the 20 nucleotides on the 3' end replaced with a targeting sequence selected from the group consisting of SEQ ID NOS: 1824- 1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714 but having the chemical modifications on the 3' end. In some embodiments, the chemically modified gRNA comprises the sequence of SEQ ID NO: 2968 with the 20 nucleotides on the 3' end replaced with a targeting sequence selected from the group consisting of SEQ ID NOS: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714 but having the chemical modifications on the 3' end. In some embodiments, the chemically modified gRNA comprises the sequence of SEQ ID NO: 2968 with the 20 nucleotides on the 3' end replaced with a targeting sequence selected from the group consisting of SEQ ID NOS: 1855, 1867 and 1869, but having thechemical modifications on the 3' end. In some embodiments, the modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 22780- 22803. In some embodiments, the modified gRNA a sequence selected from the group consisting of SEQ ID NOS: 22788-22790. A schematic of the structure of gRNA variants 174, 235, and 316 are shown in FIGS.7-9, respectively, and chemically modified versions of gRNA variants 235 and 316 are shown in FIGS.10-14. In some embodiments, the gRNA with chemical modifications exhibit improved stability compared to a gRNA without chemical modifications. e. Complex Formation with Repressor Fusion Protein

[0257] Upon delivery or expression of the components of the system in a target cell, the gRNA variant is capable of complexing with a long-term repressor fusion protein as an RNP, and binding to the target nucleic acid of the PCSK9 gene. In some embodiments, a gRNA variant has an improved ability to form an RNP complex with a long-term repressor fusion protein when compared to a reference gRNA. Improving ribonucleoprotein complex formation may, in some embodiments, improve the efficiency with which functional RNPs are assembled. In some embodiments, greater than 90%, greater than 93%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% of RNPs comprising a gRNA variant and its targeting sequence and a long-term repressor fusion protein of the disclosure are competent for gene repression of a target nucleic acid. VII. Polynucleotides and Vectors

[0258] The disclosure provides polynucleotides encoding the long-term repressor fusion protein and / or gRNA that have utility in the repression and epigenetic modification of the PCSK9 gene. The disclosure provides polynucleotides encoding mRNA encoding the long- term repressor fusion protein, e.g. DNA polynucleotides encoding the corresponding mRNA.

[0259] A long-term repressor fusion protein or an mRNA encoding the long-term repressor fusion protein of the disclosure may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids or nucleotides (as applicable) may be substituted with unnatural amino acids or nucleotides. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. A gRNA can also be produced synthetically; for example, by use of a T7 RNA polymerase system known in the art.

[0260] The long-term repressor fusion protein and / or the gRNA may also be prepared by recombinantly producing a polynucleotide sequence coding for the long-term repressor fusion protein or gRNA of any of the embodiments described herein using standard recombinant techniques known in the art and incorporating the encoding gene into an expression vector appropriate for a host cell. For production of the encoded long-term repressor fusion protein and / or gRNA of any of the embodiments described herein, the methods include transforming an appropriate host cell with an expression vector comprising the encoding polynucleotide, and culturing the host cell under conditions causing or permitting the resulting long-term repressor fusion protein or gRNA of any of the embodiments described herein to be expressed or transcribed in the transformed host cell, which are recovered by methods described herein or by standard purification methods known in the art or as described in the Examples. Standard recombinant techniques in molecular biology are used to make the polynucleotides and expression vectors of the present disclosure.

[0261] A long-term repressor fusion protein and / or a gRNA of the disclosure may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise 50% or more by weight of the desired product, more usually 75% or more by weight, preferably 95% or more by weight, and for therapeutic purposes, usually 99.5% or more by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein. Thus, in some cases, a long-term repressor fusion protein or gRNA of the present disclosure is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants or other macromolecules, etc.).

[0262] Additionally, the disclosure provides vectors comprising polynucleotides encoding the long-term repressor fusion proteins and the gRNAs described herein. In some cases, the vectors are utilized for the expression and recovery of the CasX and gRNA components of the LTRP:gRNA system, when the system is delivered as a repressor fusion protein and gRNA, or an RNP. In other cases, the vectors are utilized for the delivery of the encoding polynucleotides to target cells for the repression and / or epigenetic modification of the target nucleic acid, as described more fully, below. In some embodiments, sequences encoding the long-term repressor fusion protein and a gRNA are templated on the same vector. In someembodiments, sequences encoding the long-term repressor fusion protein and a gRNA are templated on different vectors. Suitable vectors are described, for example, in WO2023235818A2, WO2022120095A1 and WO2020247882A1, incorporated by reference herein. As described in WO2023235818A2, WO2022120095A1 and WO2020247882A1, depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

[0263] The present disclosure provides polynucleotide sequences encoding the long-term repressor fusion protein of any of the embodiments described herein. In some embodiments, the disclosure provides a DNA sequence encoding a long-term repressor fusion protein for use in a vector. In some embodiments, the disclosure provides an mRNA sequence encoding a long-term repressor fusion protein of any of the embodiments described herein for use in a particle system for delivery to a cell. In some embodiments, the disclosure provides an mRNA sequence encoding a long-term repressor fusion protein of any of the embodiments described herein for use in an LNP particle formulation for delivery to a cell. In a particular embodiment, the disclosure provides a gRNA and an mRNA sequence encoding a long-term repressor fusion protein of any of the embodiments described herein for use in an LNP particle formulation for delivery to a cell. In some embodiments, the disclosure provides an isolated polynucleotide sequence encoding a gRNA variant of any of the embodiments described herein, with a linked targeting sequence complementary to a PCSK9 target nucleic acid sequence.

[0264] In some embodiments, the disclosure relates to methods to produce polynucleotide sequences encoding the long-term repressor fusion protein or the gRNA of any of the embodiments described herein, including variants thereof, as well as methods to express the proteins or RNA transcribed by the polynucleotide sequences. In general, the methods include producing a polynucleotide sequence coding for the long-term repressor fusion protein or the gRNA of any of the embodiments described herein and incorporating the encoding gene into an expression vector. In some embodiments, the vector is designed for transduction of cells for repression and / or epigenetic modification of the PCSK9 target nucleic acid. Such vectors can include a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex virus (HSV) vector, a plasmid, a minicircle, a nanoplasmid, a DNA vector, and an RNA vector. In other embodiments, the expression vector is designed for production of a long-term repressorfusion protein, mRNA encoding the long-term repressor fusion protein, or gRNA in either a cell-free system or in a host cell. For production of the encoded long-term repressor fusion protein or the gRNA of any of the embodiments described herein in a host cell, the methods include transforming an appropriate host cell with an expression vector comprising the encoding polynucleotide, and culturing the host cell under conditions causing or permitting the resulting long-term repressor fusion protein or the gRNA of any of the embodiments described herein to be expressed or transcribed in the transformed host cell, thereby producing the long-term repressor fusion protein or the gRNA, which are recovered by methods described herein (e.g., in the Examples, below) or by standard purification methods known in the art. Standard recombinant techniques in molecular biology are used to make the polynucleotides and expression vectors of the present disclosure.

[0265] In accordance with the disclosure, nucleic acid sequences that encode the long-term repressor fusion protein or the gRNA of any of the embodiments described herein are used to generate recombinant DNA molecules that direct the expression in appropriate host cells. Several cloning strategies are suitable for performing the present disclosure, many of which are used to generate a construct that comprises a gene coding for a long-term repressor fusion protein or gRNA of the present disclosure, or its complement. In some embodiments, the cloning strategy is used to create a gene that encodes a construct that comprises nucleotides encoding the long-term repressor fusion protein or the gRNA. In some embodiments, the gene, for example as part of a vector, is used to transform a host cell for expression of the gene, e.g. expression of the long-term repressor fusion protein or gRNA.

[0266] In one approach, a construct is first prepared containing the DNA sequence encoding a long-term repressor fusion protein or a gRNA. Exemplary methods for the preparation of such constructs are described in the Examples. The construct is then used to create an expression vector suitable for transforming a host cell, such as a prokaryotic or eukaryotic host cell for the expression and recovery of the protein construct, in the case of the long-term repressor fusion protein, or the gRNA. Where desired, the host cell is an E. coli. In other embodiments, the host cell is a eukaryotic cell. The eukaryotic host cell can be selected from Baby Hamster Kidney fibroblast (BHK) cells, human embryonic kidney 293 (HEK293), human embryonic kidney 293T (HEK293T), NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, NIH3T3 cells, CV-1 (simian) in Origin with SV40 genetic material (COS), HeLa, Chinese hamster ovary (CHO), yeast cells, or other eukaryotic cells known in the art suitable for the production ofrecombinant products. Exemplary methods for the creation of expression vectors, the transformation of host cells and the expression and recovery of the long-term repressor fusion protein or the gRNA are described in the Examples.

[0267] The gene encoding the long-term repressor fusion protein or the gRNA construct can be made in one or more steps, either fully synthetically or by synthesis combined with enzymatic processes, such as restriction enzyme-mediated cloning, PCR and overlap extension, including methods more fully described in the Examples. The methods disclosed herein can be used, for example, to ligate sequences of polynucleotides encoding the various components into a gene of a desired sequence. Genes encoding polypeptide compositions are assembled from oligonucleotides using standard techniques of gene synthesis.

[0268] In some embodiments, the nucleotide sequence encoding a long-term repressor fusion protein is codon optimized. This type of optimization can entail a mutation of an encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell of the long-term repressor fusion protein was a human cell, a human codon-optimized long-term repressor fusion protein-encoding nucleotide sequence could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized long-term repressor fusion protein-encoding nucleotide sequence could be generated. The gene design can be performed using algorithms that optimize codon usage and amino acid composition appropriate for the host cell utilized in the production of the long-term repressor fusion protein or the gRNA. In one method of the disclosure, a library of polynucleotides encoding the components of the constructs is created and then assembled, as described above. The resulting genes are then assembled and the resulting genes used to transform a host cell and produce and recover the long-term repressor fusion protein or the gRNA compositions for evaluation of its properties or for use in the modification of the PCSK9 target nucleic acid, as described herein.

[0269] In some embodiments, a nucleotide sequence encoding a gRNA is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. In some embodiments, a nucleotide sequence encoding a long-term repressor fusion protein is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. Insome cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in eukaryotic cells, e.g., hepatocytes or a liver sinusoidal endothelial cell.

[0270] Non-limiting examples of Pol II promoters operably linked to the polynucleotide encoding the long-term repressor fusion protein of the disclosure include, but are not limited to EF-1alpha, EF-1alpha core promoter, Jens Tornoe (JeT), promoters from cytomegalovirus (CMV), CMV immediate early (CMVIE), CMV enhancer, herpes simplex virus (HSV) thymidine kinase, early and late simian virus 40 (SV40), the SV40 enhancer, long terminal repeats (LTRs) from retrovirus, mouse metallothionein-I, adenovirus major late promoter (Ad MLP), CMV promoter full-length promoter, the minimal CMV promoter, the chicken β-actin promoter (CBA), CBA hybrid (CBh), chicken β-actin promoter with cytomegalovirus enhancer (CB7), chicken beta-Actin promoter and rabbit beta-Globin splice acceptor site fusion (CAG), the rous sarcoma virus (RSV) promoter, the HIV-Ltr promoter, the hPGK promoter, the HSV TK promoter, a 7SK promoter, the Mini-TK promoter, the human synapsin I (SYN) promoter which confers neuron-specific expression, beta-actin promoter, super core promoter 1 (SCP1), the Mecp2 promoter for selective expression in neurons, the minimal IL-2 promoter, the Rous sarcoma virus enhancer / promoter (single), the spleen focus- forming virus long terminal repeat (LTR) promoter, the TBG promoter, promoter from the human thyroxine-binding globulin gene (Liver specific), the PGK promoter, the human ubiquitin C promoter (UBC), the UCOE promoter (Promoter of HNRPA2B1-CBX3), the synthetic CAG promoter, the Histone H2 promoter, the Histone H3 promoter, the U1a1 small nuclear RNA promoter (226 nt), the U1a1 small nuclear RNA promoter (226 nt), the U1b2 small nuclear RNA promoter (246 nt) 26, the GUSB promoter, the CBh promoter, rhodopsin (Rho) promoter, silencing-prone spleen focus forming virus (SFFV) promoter, a human H1 promoter (H1), a POL1 promoter, the TTR minimal enhancer / promoter, the b-kinesin promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, the human eukaryotic initiation factor 4A (EIF4A1) promoter, the ROSA26 promoter, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, tRNA promoters, and truncated versions and sequence variants of the foregoing. In a particular embodiment, the Pol II promoter is EF-1alpha, wherein the promoter enhances transfection efficiency, thetransgene transcription or expression of the CRISPR nuclease, the proportion of expression- positive clones and the copy number of the episomal vector in long-term culture.

[0271] Non-limiting examples of Pol III promoters operably linked to the polynucleotide encoding the gRNA variants of the disclosure include, but are not limited to U6, mini U6, U6 truncated promoters,7SK, and H1 variants, BiH1 (Bidrectional H1 promoter), BiU6, Bi7SK, BiH1 (Bidirectional U6, 7SK, and H1 promoters), gorilla U6, rhesus U6, human 7SK, human H1 promoters, and truncated versions and sequence variants thereof. In the foregoing embodiment, the pol III promoter enhances the transcription of the gRNA. In a particular embodiment, the Pol III promoter is U6, wherein the promoter enhances expression of the gRNA.. Experimental details and data for the use of such promoters are provided in the Examples.

[0272] Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, as it related to controlling expression. The expression vector may also contain a ribosome binding site for translation initiation, and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag, fluorescent protein, etc.) that can be fused to the long-term repressor fusion protein, thus resulting in a chimeric protein that are used for purification or detection.

[0273] Recombinant expression vectors of the disclosure can also comprise elements that facilitate robust expression of the proteins and the gRNAs of the disclosure. For example, recombinant expression vectors can include one or more of a polyadenylation signal (poly(A)), an intronic sequence or a post-transcriptional regulatory element such as a woodchuck hepatitis post-transcriptional regulatory element (WPRE). Exemplary poly(A) sequences include hGH poly(A) signal (short), HSV TK poly(A) signal, synthetic polyadenylation signals, a split poly(A) sequence with a SphI restriction site between two 60 A stretches (SEQ ID NO: 3307), SV40 poly(A) signal, β-globin poly(A) signal and the like. A person of ordinary skill in the art will be able to select suitable elements to include in the recombinant expression vectors described herein.

[0274] The polynucleotides encoding the long-term repressor fusion protein or the gRNA sequences can be individually cloned into an expression vector. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, as it relates to controlling expression, e.g., for repressing expression and / or epigenetic modification of the PCSK9 gene. The expression vector may also contain a ribosome binding site for translationinitiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression.

[0275] The nucleic acid sequence is inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. Such techniques are well known in the art and well described in the scientific and patent literature. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage that may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. Once introduced into a suitable host cell, expression of the long-term repressor fusion protein or gRNA can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of the long-term repressor fusion protein can be detected and / or quantified by conventional hybridization assays (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Pat. No. 5,695,937), and array-based technologies (see e.g., U.S. Pat. Nos.5,405,783, 5,412,087 and 5,445,934), using probes complementary to any region of the polynucleotide.

[0276] In some embodiments, a vector is created for the transcription of the long-term repressor fusion protein, and expression and recovery of the resulting encoding mRNA. In some embodiments, the mRNA is generated by in vitro transcription (IVT) using a PCR product or linearized plasmid DNA template and a T7 RNA polymerase, wherein the plasmid contains a T7 promoter. If using a PCR product, DNA sequences encoding candidate mRNAs will be cloned into a plasmid containing a T7 promoter, wherein the plasmid DNA template will be linearized and then used to perform IVT reactions for expression of the mRNA. Exemplary methods for the generation of such vectors and the production and recovery of the mRNA are provided in the Examples, below.VIII. Particles for Delivery of LTRP:gRNA systems

[0277] In another aspect, the present disclosure provides particle compositions for delivery of the LTRP:gRNA systems to cells or to subjects for the transcriptional repression or silencing of the PCSK9 gene. Particles envisaged as within the scope of the instant disclosure include, but are not limited to, nanoparticles such as synthetic nanoparticles, polymeric nanoparticles, lipid nanoparticles, viral particles and virus-like particles. Particles of the disclosure may encapsulate payloads such as gRNA variants, as described herein, optionally in combination with mRNA encoding the long-term repressor fusion proteins of any of the embodiments described herein. Alternatively, or in addition, particles of the disclosure may encapsulate payloads of gRNA variants and long-term repressor fusion proteins, for example when associated as a ribonucleoprotein (RNP) complex. In some embodiments, the particles are synthetic nanoparticles that encapsulate payloads of gRNA variants and mRNA encoding long-term repressor fusion protein of any of the embodiments described herein. In some embodiments, the synthetic nanoparticles comprise biodegradable polymeric nanoparticles (PNP). In some embodiments, materials for the creation of biodegradable polymeric nanoparticles (PNP) include polylactide, poly (lactic-co-glycolic acid) (PLGA), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), and poly(isohexyl cyanoacrylate), polyglutamic acid (PGA), poly (ɛ-caprolactone) (PCL), cyclodextrin, and natural polymers for instance chitosan, albumin, gelatin, and alginate, which are the most utilized polymers for the synthesis of PNP (Production and clinical development of nanoparticles for gene delivery. Molecular Therapy-Methods & Clinical Development 3:16023; doi:10.1038 (2016)). In other embodiments, the particles are lipid nanoparticles that encapsulate a gRNA variant and an mRNA encoding the long-term repressor fusion protein of any of the embodiments described herein, described more fully, below. In other embodiments, the particles are lipid nanoparticles (LNPs) that separately encapsulate a gRNA variant and a mRNA encoding long-term repressor fusion protein of any of the embodiments described herein in different particles, that are the co-formulated as a mixture for administration, described more fully, below. In other embodiments, the particles are lipid nanoparticles that separately encapsulate a gRNA variant and a mRNA encoding long-term repressor fusion protein of any of the embodiments, and the two types of particles are administered separately.a. Lipid Nanoparticles (LNP)

[0278] In another aspect, the present disclosure provides lipid nanoparticles (LNP) for delivery of the LTRP:gRNA systems of the disclosure to cells or to subjects for the transcriptional repression of the PCSK9 gene. In some embodiments, the LNPs of the disclosure are tissue- or organ-specific (e.g., the liver), have excellent biocompatibility, and can deliver the systems with high efficiency, and thus can be usefully used for the transcriptional repression of the PCSK9 gene.

[0279] The disclosure further provides LNP compositions and pharmaceutical compositions comprising a plurality of the LNP described herein.

[0280] In their native forms, nucleic acid polymers are generally unstable in biological fluids and cannot penetrate into the cytoplasm of target cells, thus requiring delivery systems. Lipid nanoparticles (LNP) have proven useful for both the protection and delivery of nucleic acids to tissues and cells. Furthermore, the use of mRNA in LNPs to encode the long-term repressor fusion protein eliminates the possibility of undesirable genome integration, as compared to DNA vectors. Moreover, mRNA efficiently transfects both mitotic and non- mitotic cells, as it does not require entry into the nucleus since it exerts its function in the cytoplasmic compartment. LNPs as a delivery platform thus offer the additional advantage of being able to co-formulate both the mRNA encoding the long-term repressor fusion protein and a gRNA into single LNP particles.

[0281] Accordingly, in various embodiments, the disclosure encompasses lipid nanoparticles and compositions that may be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells, both in vitro and in vivo. In certain embodiments, the disclosure encompasses methods of treating or preventing diseases or disorders in a subject in need thereof by contacting the subject with a lipid nanoparticle that encapsulates or is associated with a suitable therapeutic agent complexed through various physical, chemical or electrostatic interactions between one or more of the lipid components used in the compositions to make LNPs. In some embodiments, the suitable therapeutic agent comprises a LTRP:gRNA system as described herein.

[0282] In certain embodiments, the lipid nanoparticles are useful for the delivery of nucleic acids, including, e.g., the mRNA encoding the long-term repressor fusion proteins and the gRNA variants of the disclosure. In some embodiments, an LNP comprises an mRNA comprising a sequence encoding a long-term repressor fusion protein selected from the groupconsisting of SEQ ID NOS: 6528-14626 (unmodified mRNA), and 14627-22725 (mRNA modified with N1-methyl-pseudouridine), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, an LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 6528-8134, 9741-11347, 14628-16233, and 17840-19446 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, an LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 6529-6537, 9742-9750, 14628, 14636, and 17841-17849, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, an LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 6529-6537, 9742-9750, 14628, 14636, and 17841-17849. In some embodiments, an LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 6531, 9744, 14630, and 17843, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, an LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 6531, 9744, 14630, and 17843. In some embodiments, an LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 6529, 9742, 14628, and 17841, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, an LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 6529, 9742, 14628, and 17841. In some embodiments, an LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 6530, 9743,14629, and 17842, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, an LNP comprises an mRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 6530, 9743, 14629, and 17842. In some embodiments, an LNP comprises a gRNA comprising a scaffold sequence selected from the group consisting of SEQ ID NOS: 1744-1746, 2948- 2956, 2958-2966, and 2968-2974, and a linked targeting sequence complementary to a sequence of the PCSK9 gene (with, in the case of SEQ ID NOS: 2948-2956, 2958-2966, and 2968-2974, the 20 non-targeting nucleotides on the 3' end replaced with a targeting sequence complementary to a sequence of the PCSK9 gene but retaining the chemical modifications on the 3' end). In some embodiments, an LNP comprises a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 2278- 22803. In some embodiments, an LNP comprises a gRNA comprising a sequence selected from the group consisting of SEQ ID NOS: 2278-22790. In some embodiments, the present disclosure provides LNP in which the gRNA and mRNA encoding the long-term repressor fusion protein are incorporated into single LNP particles. In other embodiments, the present disclosure provides LNP in which the gRNA and mRNA encoding the long-term repressor fusion protein are incorporated into separate populations of LNPs, which can be formulated together in varying ratios for administration.

[0283] The lipid nanoparticles and lipid nanoparticle compositions of the disclosure may be used to repress expression of a desired protein both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more ionizable lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA encoding the long-term repressor fusion protein). In some embodiments, the lipid nanoparticles and compositions may be used to repress the expression of a target gene both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with one or more nucleic acids of the LTRP:gRNA systems of the disclosure. The lipid nanoparticles and compositions of embodiments of the disclosure may also be used for co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination, such as may be useful to provide an effectrequiring colocalization of different nucleic acids (e.g., mRNA encoding for a suitable gene repressing factor or enzyme and gRNA for targeting of the gene).

[0284] In some embodiments, LNPs and LNP compositions described herein include at least one cationic lipid, at least one conjugated lipid, at least one steroid or derivative thereof, at least one additional lipid, or any combination thereof. Alternatively, the lipid compositions of the disclosure can include an ionizable lipid, such as an ionizable cationic lipid, a helper lipid (usually a phospholipid), cholesterol, and a polyethylene glycol-lipid conjugate (PEG- lipid) to improve the colloidal stability in biological environments by, for example, reducing a specific absorption of plasma proteins and forming a hydration layer over the nanoparticles. Such lipid compositions can be formulated at typical mole ratios of 50:10:37-39:1.5-2.5 or 20-50:8-65:25-40:1-2.5, with variations made to adjust individual properties.

[0285] The LNPs and LNP compositions of the present disclosure are configured to protect and deliver an encapsulated payload of the systems of the disclosure to tissues and cells, both in vitro and in vivo. Various embodiments of the LNPs and LNP compositions of the present disclosure are described in further detail herein. Cationic lipid

[0286] In some embodiments, the LNPs and LNP compositions of the present disclosure include at least one cationic lipid. The term “cationic lipid,” refers to a lipid species that has a net positive charge. In some embodiments, the cationic lipid is an ionizable cationic lipid that has a net positive charge at a selected pH, such as physiological pH. In some embodiments, the ionizable cationic lipid has a pKa less than 7 such that the LNPs and LNP compositions achieve efficient encapsulation of the payload at a relatively low pH. In some embodiments, the cationic lipid has a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. In some embodiments, the cationic lipid may be protonated at a pH below the pKa of the cationic lipid, and it may be substantially neutral at a pH over the pKa. The LNPs and LNP compositions may be safely delivered to a target organ (for example, the liver, lung, heart, spleen, as well as to tumors) and / or cell(hepatocyte, LSEC, cardiac cell, cancer cell, etc.) in vivo, and after endocytosis, exhibit a positive charge to release the encapsulated payload through electrostatic interaction with an anionic protein of the endosome membrane.

[0287] Early formulations of LNP utilizing permanently cationic lipids resulted in LNPs with positive surface charge that proved toxic in vivo, plus were rapidly cleared by phagocytic cells. By changing to ionizable cationic lipids bearing tertiary amines, especially those with pKa < 7, results in LNP achieving efficient encapsulation of nucleic acid polymersat low pH by interacting electrostatically with the negative charges of the phosphate backbone of mRNA, that also result in largely neutral systems at physiological pH values, thus alleviating problems associated with permanently-charged cationic lipids.

[0288] As used herein, “ionizable lipid” means an amine-containing lipid which can be easily protonated, and, for example, it may be a lipid of which charge state changes depending on the surrounding pH. The ionizable lipid may be protonated (positively charged) at a pH below the pKa of a cationic lipid, and it may be substantially neutral at a pH over the pKa. In one example, the LNP may comprise a protonated ionizable lipid and / or an ionizable lipid showing neutrality. In some embodiments, the LNP has a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. The pKa of the LNP can influence in vivo stability and release of the nucleic acid payload of the LNP in the target cell or organ. In some embodiments, the LNP having the foregoing pKa ranges may be safely delivered to a target organ (for example, the liver, lung, heart, spleen, as well as to tumors) and / or target cell (hepatocyte, LSEC, cardiac cell, cancer cell, etc.) in vivo, and after endocytosis, exhibit a positive charge to release the encapsulated payload through electrostatic interaction with an anionic protein of the endosome membrane.

[0289] The ionizable lipid is an ionizable compound having characteristics similar to lipids generally, and through electrostatic interaction with a nucleic acid (for example, an mRNA of the disclosure), may play a role of encapsulating the nucleic acid payloads within the LNP with high efficiency.

[0290] According to the type of the amine and the tail group comprised in the ionizable lipid, (i) the nucleic acid encapsulation efficiency, (ii) PDI (polydispersity index) and / or (iii) the nucleic acid delivery efficiency to tissue and / or cells constituting an organ (for example, hepatocytes or liver sinusoidal endothelial cells in the liver) of the LNP may be different. In certain embodiments, the ionizable lipid is an ionizable cationic lipid, and comprises from about 46 mol % to about 66 mol % of the total lipid present in the particle.

[0291] The LNP comprising an ionizable lipid comprising an amine may have one or more kinds of the following characteristics: (1) the ability to encapsulate a nucleic acid with high efficiency; (2) uniform size of prepared particles (or having a low PDI value); and / or (3) excellent nucleic acid delivery efficiency to organs such as liver, lung, heart, spleen, bone marrow, as well as to tumors, and / or cells constituting such organs (for example, hepatocytes, LSEC, cardiac cells, cancer cells, etc.).

[0292] In particular embodiments, the cationic lipid form plays an important role both in nucleic acid encapsulation through electrostatic interactions and intracellular release by disrupting endosomal membranes. The nucleic acid payloads are encapsulated within the LNP by the ionic interactions they form with the positively charged cationic lipid. Non- limiting examples of cationic lipid components utilized in the LNP of the disclosure are selected from DLin-MC3-DMA (heptatriaconta-6,9,28,31-tetraen-19-yl4- (dimethylamino)butanoate), DLin- KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)- [1,3]-dioxolane), and TNT (1,3,5-triazinane-2,4,6-trione) and TT (N1,N3,N5-tris(2- aminoethyl)benzene-1,3,5-tricarboxamide). Non-limiting examples of helper lipids utilized in the LNP of the disclosure are selected from DSPC (1,2-distearoyl-sn-glycero-3- phosphocholine), POPC (2-Oleoyl-1- palmitoyl-sn-glycero-3-phosphocholine) and DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), 1,2-dioleoyl-sn-glycero-3-phospho-(1'- rac-glycerol) DOPG, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2- dilauroyl-sn-glycero-3-phosphocholine (DLPC), sphingolipid, and ceramide. Cholesterol and PEG-DMG ((R)-2,3- bis(octadecyloxy)propyl-1-(methoxy polyethylene glycol 2000) carbamate), PEG-DSG (1,2-Distearoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000), or DSPE-PEG2k (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]), are components utilized in the LNP of the disclosure for the stability, circulation, and size of the LNP.

[0293] In some embodiments, the cationic lipid in the LNP of the disclosure comprises a tertiary amine. In some embodiments, the tertiary amine includes alkyl chains connected to N of the tertiary amine with ether linkages. In some embodiments, the alkyl chains comprise C12-C30 alkyl chains having 0 to 3 double bonds. In some embodiments, the alkyl chains comprise C16-C22 alkyl chains. In some embodiments, the alkyl chains comprise C18 alkyl chains. A number of cationic lipids and related analogs have been described in U.S. Patent Publication Nos.20060083780, 20060240554, 20110117125, 20190336608, 20190381180 and 20200121809; U.S. Pat. Nos.5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; 5,785,992; 9,738,593; 10,106,490; 10,166,298; 10,221,127; and 11,219,634; and PCT Publication No. WO 96 / 10390, the disclosures of which are herein incorporated by reference in their entirety.

[0294] In some embodiments, the cationic lipid in the LNP of the disclosure may comprise, for example, one or more ionizable cationic lipids wherein the ionizable cationic lipid is a dialkyl lipid. In other embodiments, the ionizable cationic lipid is a trialkyl lipid.

[0295] In some embodiments, the cationic lipid in the LNP of the disclosure is selected from 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]- dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4- DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2- dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3- dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3- dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S- DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2- dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2- distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3- (N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3- dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1- propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12- octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3- dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), and any combination of the forgoing.

[0296] In some embodiments, the cationic lipid in the LNP of the disclosure is selected from heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin- KC2-DMA), (1,3,5- triazinane-2,4,6-trione) (TNT), N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT), and any combination of the forgoing.

[0297] In some embodiments, the N / P ratio (nitrogen from the cationic / ionizable lipid and phosphate from the nucleic acid) in the LNP of the disclosure is in the range of is about 3:1 to 7:1, or about 4:1 to 6:1, or is 3:1, or is 4:1, or is 5:1, or is 6:1, or is 7:1. Conjugated Lipid

[0298] In some embodiments, the LNPs and LNP compositions of the present disclosure include at least one conjugated lipid. In some embodiments, the conjugated lipid may be selected from a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, a cationic-polymer-lipid conjugate (CPL), and any combination of the foregoing. In some cases, conjugated lipids can inhibit aggregation of the LNPs of the disclosure.

[0299] In some embodiments, the conjugated lipid of the LNP of the disclosure comprises a pegylated lipid. The terms “polyethyleneglycol (PEG)-lipid conjugate,” “pegylated lipid” "lipid-PEG conjugate", "lipid-PEG", "PEG-lipid", "PEG-lipid", or "lipid-PEG" are used interchangeably herein and refer to a lipid attached to a polyethylene glycol (PEG) polymer which is a hydrophilic polymer. The pegylated lipid contributes to the stability of the LNPs and LNP compositions and reduces aggregation of the LNPs.

[0300] As the PEG-lipid can form the surface lipid, the size of the LNP can be readily varied by varying the proportion of surface (PEG) lipid to the core (ionizable cationic) lipids. In some embodiments, the PEG-lipid of the LNP of the disclosure can be varied from ~1 to 5 mol% to modify particle properties such as size, stability, and circulation time.

[0301] The lipid-PEG conjugate contributes to the particle stability in serum of the nanoparticle within the LNP, and plays a role of preventing aggregation between nanoparticles. In addition, the lipid-PEG conjugate may protect nucleic acids, such as mRNAs encoding the long-term repressor fusion proteins of the disclosure, or gRNAs of the disclosure, from degrading enzymes during in vivo delivery of the nucleic acids and enhance the stability of the nucleic acids in vivo and increase the half-life of the delivered nucleic acids encapsulated in the nanoparticle. Examples of PEG-lipid conjugates include, but are not limited to, PEG-DAG conjugates, PEG-DAA conjugates, and mixtures thereof. In certain embodiments, the PEG-lipid conjugate is selected from the group consisting of a PEG- diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, a PEG-phospholipid conjugate, a PEG-ceramide (PEG-Cer) conjugate, and a mixture thereof.

[0302] In some embodiments, the pegylated lipid of the LNP of the disclosure is selected from a PEG-ceramide, a PEG-diacylglycerol, a PEG-dialkyloxypropyl, a PEG- dialkoxypropylcarbamate, a PEG-phosphatidylethanoloamine, a PEG-phospholipid, a PEG- succinate diacylglycerol, and any combination of the foregoing.

[0303] In some embodiments, the pegylated lipid of the LNP of the disclosure is a PEG- dialkyloxypropyl. In some embodiments, the pegylated lipid is selected from PEG- didecyloxypropyl (C10), PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), and any combination of the foregoing.

[0304] In other embodiments, the lipid-PEG conjugate of the LNP of the disclosure may be PEG bound to phospholipid such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide (PEG-CER, ceramide-PEG conjugate, ceramide-PEG, cholesterol or PEG conjugated to derivative thereof, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE(DSPE-PEG), and a mixture thereof, and for example, may be C16- PEG2000 ceramide (N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}), DMG-PEG 2000, 14:0 PEG2000 PE.

[0305] In some embodiments, the pegylated lipid of the LNP of the disclosure is selected from 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, 4-O-(2′,3′- di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, 2,3- di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, and any combination of the foregoing.

[0306] In some embodiments, the pegylated lipid of the LNP of the disclosure is selected from mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG), 1-[8′-(1,2- dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-w-methyl- poly(ethylene glycol) (2 KPEG-DMG), and any combination of the foregoing.

[0307] In some embodiments, the PEG is directly attached to the lipid of the pegylated lipid. In other embodiments, the PEG is attached to the lipid of the pegylated lipid by a linker moiety selected from an ester-free linker moiety or an ester-containing linker moiety. Non- limiting examples of the ester-free linker moiety include amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (- O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide and combinations thereof. For example, the linker may contain a carbamate linkermoiety and an amido linker moiety. Non-limiting examples of the ester-containing linker moiety include carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0308] The PEG moiety of the pegylated lipid of the LNP of the disclosure described herein may have an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain embodiments, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons, about 1,000 daltons to about 4,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, or about 1750 daltons to about 2,000 daltons.

[0309] In some embodiments, the conjugated lipid (e.g., pegylated lipid) comprises from about 1 mol % to about 60 mol %, from about 2 mol % to about 50 mol %, from about 5 mol % to about 40 mol %, or from about 5 mol % to about 20 mol % of the total lipid present in the LNPs and / or LNP compositions. In certain embodiments, the conjugated lipid comprises from about 0.5 mol % to about 3 mol % of the total lipid present in the particle.

[0310] In additional embodiments, the conjugated lipid (e.g., pegylated lipid) of the LNP of the disclosure comprises at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol %, or an intermediate range of any of the foregoing, of the total lipid present in the LNPs and / or LNP compositions.

[0311] For the lipid in the lipid-PEG conjugate of the LNP of the disclosure, any lipid capable of binding to polyethyleneglycol may be used without limitation, and the phospholipid and / or cholesterol which are other elements of the LNP may be also used. In some embodiments, the lipid in the lipid-PEG conjugate may be ceramide, dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), or cholesterol, but not limited thereto.

[0312] In the lipid-PEG conjugate of the LNP of the disclosure, the PEG may be directly conjugated to the lipid or linked to the lipid via a linker moiety. Any linker moiety suitable for binding PEG to the lipid may be used, and for example, includes an ester-free linker moiety and an ester-containing linker moiety. The ester-free linker moiety includes amido (- C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (- NHC(O)CH2CH2C(O)NH-), ether, disulfide and combinations thereof (for example, a linker containing both a carbamate linker moiety and an amido linker moiety), but not limitedthereto. The ester-containing linker moiety includes for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof, but not limited thereto. Steroids

[0313] In some embodiments, the LNPs and LNP compositions of the present disclosure include at least one steroid or derivative thereof. In some embodiments, the steroid comprises cholesterol. In some embodiments, the LNPs and LNP compositions comprise a cholesterol derivative selected from cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, and any combination of the foregoing.

[0314] In some embodiments, the steroid (e.g., cholesterol) of the LNP of the disclosure comprises from about 1 mol % to about 60 mol %, from about 2 mol % to about 50 mol %, from about 5 mol % to about 40 mol %, or from about 5 mol % to about 20 mol % of the total lipid present in the LNPs and / or LNP compositions. In other embodiments, the steroid (e.g., cholesterol) of the LNP of the disclosure comprises at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol %, or an intermediate range of any of the foregoing, of the total lipid present in the LNPs and / or LNP compositions. Additional Lipid

[0315] In some embodiments, the LNPs and LNP compositions of the present disclosure include at least one additional lipid. In some embodiments, the additional lipid is non-cationic lipid selected from an anionic lipid, a neutral lipid, or both. In some embodiments, the additional lipid comprises at least one phospholipid. In some embodiments, the phospholipid is selected from an anionic phospholipid, a neutral phospholipid, or both. The phospholipid of the elements of the LNPs and LNP compositions can play a role in covering and protecting a core of the LNP formed by interaction of the cationic lipid and nucleic acid in the LNP, and may facilitate cell membrane permeation and endosomal escape during intracellular delivery of the nucleic acid by binding to the phospholipid bilayer of a target cell. A phospholipid which can promote fusion of the LNP to a cell may include without limitation, any of the phospholipids selected from the group described below.

[0316] In some embodiments, the LNPs and LNP compositions comprise at least one phospholipid selected from, but not limited to, dipalmitoyl-phosphatidylcholine (DPPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylethanolamine (DOPE), dioleoyl-phosphatidylcholine (DOPC), dioleoyl-phosphatidylglycerol (DOPG),palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl- phosphatidylethanolamine (DPPE), dipalmitoyl-phosphatidylglycerol (DPPG), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), phosphatidylethanolamine (PE), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L- serine], and any combination of the foregoing. In one example, the LNP comprising DOPE may be effective in mRNA delivery (excellent drug delivery efficacy).

[0317] In some embodiments, the additional lipid (e.g., phospholipid) of the LNP of the disclosure comprises from about 1 mol % to about 60 mol %, from about 2 mol % to about 50 mol %, from about 5 mol % to about 40 mol %, or from about 5 mol % to about 20 mol % of the total lipid present in the LNPs and / or LNP compositions. In other embodiments, the additional lipid (e.g., phospholipid) of the LNP of the disclosure comprises at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol %, or an intermediate range of any of the foregoing, of the total lipid present in the LNPs and / or LNP compositions.

[0318] It will be appreciated that the total lipid present in the LNPs and / or LNP compositions comprises the combination of the cationic lipid or ionizable cationic lipid, the conjugated lipid, (e.g., pegylated lipid), the steroid (e.g., cholesterol), and the additional lipid (e.g., phospholipid).

[0319] The LNPs and / or LNP compositions may be prepared by dissolving the total lipids (or a portion thereof) in an organic solvent (e.g., ethanol) followed by mixing through a micromixer with the payload (e.g., nucleic acids of the systems) dissolved in an acidic buffer (e.g., pH 4). At this pH the cationic lipid is positively charged and interacts with the negatively-charged nucleic acid polymers. The resulting nanostructures containing the nucleic acids are then converted to neutral LNPs when dialyzed against a neutral buffer which may then be followed by removal of the organic solvent (e.g., ethanol) and exchange the LNPs into physiologically relevant buffer. The LNPs and / or LNP compositions thus formed have a distinct electron-dense nanostructured core where the cationic lipids are organized into inverted micelles around the encapsulated payload, as opposed to traditionalbilayer liposomal structures. In another embodiment, the LNP may form a bleb-like structure with nucleic acids in aqueous pockets along the non-electron dense lipid core. b. Lipid nanoparticle properties

[0320] In some embodiments, the LNPs and / or LNP compositions comprise from about 50 mol % to about 85 mol % of the cationic lipid or ionizable cationic lipid, from about 0.5 mol % to about 10 mol % of the conjugated lipid, (e.g., pegylated lipid), from about 0.5 mol % to about 10 mol % of the steroid (e.g., cholesterol) and from about 5 mol % to about 50 mol % of the additional lipid (e.g., phospholipid). In some embodiments, the LNPs and / or LNP compositions comprise from about 50 mol % to about 85 mol % of the cationic lipid or ionizable cationic lipid, from about 0.5 mol % to about 5 mol % of the conjugated lipid, (e.g., pegylated lipid), from about 0.5 mol % to about 5 mol % of the steroid (e.g., cholesterol) and from about 5 mol % to about 20 mol % of the additional lipid (e.g., phospholipid).

[0321] In some embodiments, the LNPs and / or LNP compositions of the disclosure comprise cationic lipid : additional lipid (e.g., phospholipid) : steroid (e.g., cholesterol) : conjugated lipid, (e.g., pegylated lipid) at a molar ratio of 20 to 50:10 to 30:30 to 60:0.5 to 5, at a molar ratio of 25 to 45:10 to 25:40 to 50:0.5 to 3, at a molar ratio of 25 to 45:10 to 20:40 to 55:0.5 to 3, or at a molar ratio of 25 to 45:10 to 20:40 to 55:1.0 to 1.5.

[0322] In some embodiments, the LNPs and / or LNP compositions of the disclosure have a total lipid : payload ratio (mass / mass) of from about 1 to about 100. In some embodiments, the total lipid : payload ratio is about 1 to about 50, from about 2 to about 25, from about 3 to about 20, from about 4 to about 15, or from about 5 to about 10. In some embodiments, the total lipid : payload ratio is about 5 to about 15, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or an intermediate range of any of the foregoing.

[0323] In certain embodiments, the LNPs of the disclosure comprise a total lipid: nucleic acid mass ratio of from about 5:1 to about 15:1. In some embodiments, the weight ratio of the cationic lipid and nucleic acid comprised in the LNP may be 1 to 20:1, 1 to 15:1, 1 to 10:1, 5 to 20:1, 5 to 15:1, 5 to 10:1, 7.5 to 20:1, 7.5 to 15:1, or 7.5 to 10:1.

[0324] In some embodiments, the LNP of the disclosure may comprise the cationic lipid of 20 to 50 parts by weight, the phospholipid of 10 to 30 parts by weight, cholesterol of 20 to 60 parts by weight (or 20 to 60 parts by weight), and lipid-PEG conjugate of 0.1 to 10 parts by weight (or 0.25 to 10 parts by weight, 0.5 to 5 parts by weight). Alternatively, the LNP may comprise the cationic lipid of 20 to 50 % by weight, phospholipid of 10 to 30 % by weight, cholesterol of 20 to 60 % by weight (or 30 to 60 % by weight), and lipid-PEG conjugate of0.1 to 10 % by weight (or 0.25 to 10 % by weight, 0.5 to 5 % by weight) based on the total nanoparticle weight. As a further alternative, the LNP may comprise the cationic lipid of 25 to 50 % by weight, phospholipid of 10 to 20 % by weight, cholesterol of 35 to 55 % by weight, and lipid-PEG conjugate of 0.1 to 10 % by weight (or 0.25 to 10 % by weight, 0.5 to 5 % by weight), based on the total nanoparticle weight.

[0325] In some embodiments, the LNPs of the present disclosure have a mean diameter of from about 20 to 200 nm, 20 to 180 nm, 20 to 170 nm, 20 to 150 nm, 20 to 120 nm, 20 to 100 nm, 20 to 90 nm, 30 to 200 nm, 30 to 180 nm, 30 to 170 nm, 30 to 150 nm, 30 to 120 nm, 30 to 100 nm, 30 to 90 nm, 40 to 200 nm, 40 to 180 nm, 40 to 170 nm, 40 to 150 nm, 40 to 120 nm, 40 to 100 nm, 40 to 90 nm, 40 to 80 nm, 40 to 70 nm, 50 to 200 nm, 50 to 180 nm, 50 to 170 nm, 50 to 150 nm, 50 to 120 nm, 50 to 100 nm, 50 to 90 nm, 60 to 200 nm, 60 to 180 nm, 60 to 170 nm, 60 to 150 nm, 60 to 120 nm, 60 to 100 nm, 60 to 90 nm, 70 to 200 nm, 70 to 180 nm, 70 to 170 nm, 70 to 150 nm, 70 to 120 nm, 70 to 100 nm, 70 to 90 nm, 80 to 200 nm, 80 to 180 nm, 80 to 170 nm, 80 to 150 nm, 80 to 120 nm, 80 to 100 nm, 80 to 90 nm, 90 to 200 nm, 90 to 180 nm, 90 to 170 nm, 90 to 150 nm, 90 to 120 nm, or 90 to 100 nm, or an intermediate range of any of the foregoing.

[0326] In some embodiments, the LNPs and / or LNP compositions of the disclosure have a positive charge at acidic pH and may encapsulate the payload (e.g., therapeutic agent, such as the LTRP:gRNA systems, or polynucleotides encoding same) through electrostatic charges produced by negative charges of the payload (e.g., therapeutic agent). The term “encapsulation,” refers to the mixture of lipids surrounding and embedding the payload (e.g., therapeutic agent) at physiological conditions, forming the LNPs. The term “encapsulation efficiency,” as used herein is the amount of payload (e.g., therapeutic agent) encapsulated by the LNPs divided by the total amount of payload (e.g., therapeutic agent) used to load the payload (e.g., therapeutic agent) into the LNPs. The encapsulation efficiency of the LNPs and / or LNP compositions may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 94% or more, or 95% or more. In other embodiments, the encapsulation efficiency of the LNPs and / or LNP compositions is about 80% to 99%, about 85% to 98%, about 88% to 95%, about 90% to 95%, or the payload (e.g., nucleic acids of the systems) may be fully encapsulated within the lipid portion of the LNPs compositions, and thereby protected from enzymatic degradation. In some embodiments, the payload (e.g., therapeutic agent) is not substantially degraded after exposure of the LNPs and / or LNP compositions to a nuclease at 37° C. for at least about 20, 30, 45, or 60 minutesor at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In some embodiments, the payload (e.g., nucleic acids of the systems) is complexed with the lipid portion of the LNPs and / or LNP compositions. The LNPs and / or LNP compositions of the present disclosure are non-toxic to mammals such as humans.

[0327] The term “fully encapsulated” indicates that the payload (e.g., the nucleic acids of the system) in the LNPs and / or LNP compositions is not significantly degraded after exposure to conditions that degrade free DNA, RNA, or protein. In a fully encapsulated system, less than about 25%, more preferably less than about 10%, and most preferably less than about 5% of the payload (e.g., nucleic acids of the system) in the LNPs and / or LNP compositions is degraded by conditions that would degrade 100% of a non-encapsulated payload. “Fully encapsulated” also indicates that the LNPs and / or LNP compositions are serum-stable, and do not decompose into their component parts upon in vivo administration.

[0328] In some embodiments, the amount of the LNPs and / or LNP compositions having the payload (e.g., therapeutic agent), encapsulated therein is from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, %, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or an intermediate range of any of the foregoing.

[0329] In some embodiments, the amount of the payload (e.g., the nucleic acids), encapsulated within the LNPs and / or LNP compositions is from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, %, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at leastabout 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or an intermediate range of any of the foregoing.

[0330] In some embodiments, the nucleic acids of the disclosure, such as the mRNA encoding the long-term repressor fusion protein, and / or the gRNA, may be provided in a solution to be mixed with a lipid solution such that the nucleic acids may be encapsulated in the lipid nanoparticles. A suitable nucleic acid solution may be any aqueous solution containing the nucleic acid to be encapsulated at various concentrations. For example, a suitable nucleic acid solution may contain the nucleic acid (or nucleic acids) at a concentration of or greater than about 0.01 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.25 mg / ml, 1.5 mg / ml, 1.75 mg / ml, or 2.0 mg / ml. In some embodiments, the nucleic acid comprises an mRNA encoding an long-term repressor fusion protein, and a suitable mRNA solution may contain the mRNA at a concentration ranging from about 0.01-2.0 mg / ml, 0.01-1.5 mg / ml, 0.01-1.25 mg / ml, 0.01-1.0 mg / ml, 0.01-0.9 mg / ml, 0.01-0.8 mg / ml, 0.01-0.7 mg / ml, 0.01-0.6 mg / ml, 0.01-0.5 mg / ml, 0.01-0.4 mg / ml, 0.01-0.3 mg / ml, 0.01-0.2 mg / ml, 0.01-0.1 mg / ml, 0.05-1.0 mg / ml, 0.05-0.9 mg / ml, 0.05-0.8 mg / ml, 0.05-0.7 mg / ml, 0.05-0.6 mg / ml, 0.05-0.5 mg / ml, 0.05-0.4 mg / ml, 0.05-0.3 mg / ml, 0.05-0.2 mg / ml, 0.05-0.1 mg / ml, 0.1-1.0 mg / ml, 0.2-0.9 mg / ml, 0.3- 0.8 mg / ml, 0.4-0.7 mg / ml, or 0.5-0.6 mg / ml. In some embodiments, a suitable mRNA solution may contain an mRNA at a concentration up to about 5.0 mg / ml, 4.0 mg / ml, 3.0 mg / ml, 2.0 mg / ml, 1.0 mg / ml, 0.9 mg / ml, 0.8 mg / ml, 0.7 mg / ml, 0.6 mg / ml, 0.5 mg / ml, 0.4 mg / ml, 0.3 mg / ml, 0.2 mg / ml, 0.1 mg / ml, 0.05 mg / ml, 0.04 mg / ml, 0.03 mg / ml, 0.02 mg / ml, 0.01 mg / ml, or 0.05 mg / ml. In some embodiments, a suitable gRNA solution may contain an gRNA at a concentration up to about 5.0 mg / ml, 4.0 mg / ml, 3.0 mg / ml, 2.0 mg / ml, 1.0 mg / ml, 0.9 mg / ml, 0.8 mg / ml, 0.7 mg / ml, 0.6 mg / ml, 0.5 mg / ml, 0.4 mg / ml, 0.3 mg / ml, 0.2 mg / ml, 0.1 mg / ml, 0.05 mg / ml, 0.04 mg / ml, 0.03 mg / ml, 0.02 mg / ml, 0.01 mg / ml, or 0.05 mg / ml.

[0331] In some embodiments, the LNP may have an average diameter of 20nm to 200nm, 20 to 180nm, 20nm to 170nm, 20nm to 150nm, 20nm to 120nm, 20nm to 100nm, 20nm to 90nm, 30nm to 200nm, 30 to 180nm, 30nm to 170nm, 30nm to 150nm, 30nm to 120nm, 30nm to 100nm, 30nm to 90nm, 40nm to 200nm, 40 to 180nm, 40nm to 170nm, 40nm to 150nm, 40nm to 120nm, 40nm to 100nm, 40nm to 90nm, 40nm to 80nm, 40nm to 70nm, 50nm to 200nm, 50 to 180nm, 50nm to 170nm, 50nm to 150nm, 50nm to 120nm, 50nm to100nm, 50nm to 90nm, 60nm to 200nm, 60 to 180nm, 60nm to 170nm, 60nm to 150nm, 60nm to 120nm, 60nm to 100nm, 60nm to 90nm, 70nm to 200nm, 70 to 180nm, 70nm to 170nm, 70nm to 150nm, 70nm to 120nm, 70nm to 100nm, 70nm to 90nm, 80nm to 200nm, 80 to 180nm, 80nm to 170nm, 80nm to 150nm, 80nm to 120nm, 80nm to 100nm, 80nm to 90nm, 90nm to 200nm, 90 to 180nm, 90nm to 170nm, 90nm to 150nm, 90nm to 120nm, or 90nm to 100nm for easy introduction into liver tissue, hepatocytes and / or LSEC (liver sinusoidal endothelial cells). The LNP may be sized for easy introduction into organs or tissues, including but not limited to liver, lung, heart, spleen, as well as to tumors. When the size of the LNP is smaller than the above range, it can be difficult to maintain stability as the surface area of the LNP is excessively increased, and thus delivery to the target tissue and / or drug effect may be reduced. The LNP may specifically target liver tissue. Without wishing to be bound by theory, it is thought that one mechanism by which LNP may be used to deliver therapeutic agents is through the imitation of the metabolic behaviors of natural lipoproteins, and so LNP may be usefully delivered to a subject through the lipid metabolism processes carried out by the liver. During the delivery of therapeutic agents to hepatocytes or and / or LSEC (liver sinusoidal endothelial cells), the diameter of the fenestrae leading from the sinusoidal lumen to the hepatocytes and LSEC is about 140 nm in mammals and about 100 nm in humans, so the LNP composition for therapeutic agent delivery having LNPs with a diameter in the above ranges may have excellent delivery efficiency to hepatocytes and LSEC when compared to LNP having the diameter outside the above range.

[0332] According to one example, the LNPs of the LNP composition may comprise the cationic lipid : phospholipid : cholesterol : lipid-PEG conjugate in the range described above or at a molar ratio of 20 to 50:10 to 30:30 to 60:0.5 to 5, at a molar ratio of 25 to 45:10 to 25:40 to 50:0.5 to 3, at a molar ratio of 25 to 45:10 to 20:40 to 55:0.5 to 3, or at a molar ratio of 25 to 45:10 to 20:40 to 55:1.0 to 1.5. The LNP comprising components at a molar ratio in the above range may have excellent delivery efficiency of therapeutic agents specific to cells of target organs.

[0333] In certain aspects, the LNP exhibit a positive charge under the acidic pH condition by showing a pKa of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7, and may encapsulate a nucleic acid with high efficiency by easily forming a complex with a nucleic acid through electrostatic interaction with a therapeutic agent such as a nucleic acid showing a negative charge. In such cases, the LNP may be usefully used as a composition for intracellular or in vivo delivery of a therapeutic agent (for example, nucleic acid).

[0334] Herein, "encapsulate" or "encapsulation" refers to incorporation of a therapeutic agent efficient delivery, i.e., by surrounding it by the particle surface and / or embedding it within the particle interior. The encapsulation efficiency means the content of the therapeutic agent encapsulated in the LNP relative the total therapeutic agent content used for preparation of the LNP.

[0335] The encapsulation of the nucleic acids of the composition in the LNP may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 94% or more, or 95% or more of LNP in the composition encapsulate nucleic acids. In some embodiments, the encapsulation of the nucleic acids of the composition in the LNP is such that between 80% to 99%, between 80% to 97%, between 80% to 95%, between 85% to 95%, between 87% to 95%, between 90% to 95%, between 91% or more to 95% or less, 91% or more to 94% or less, over 91% to 95% or less, 92% to 99%, between 92% to 97%, or between 92% to 95% of the LNP in the composition encapsulate nucleic acids. In some embodiments, the mRNA encoding the long-term repressor fusion protein and a gRNA of any of the embodiments of the disclosure are fully encapsulated in the LNP.

[0336] The target organs to which a nucleic acid is delivered by the LNP include, but are not limited to the liver, lung, heart, spleen, as well as to tumors. The LNP according to one example is liver tissue-specific and has excellent biocompatibility and can deliver the nucleic acids of a composition with high efficiency, and thus it can be usefully used in related technical fields such as lipid nanoparticle-mediated gene therapy. In a particular embodiment, the target cell to which the nucleic acids are delivered by the LNP according to one example may be a hepatocyte and / or LSEC in vivo. In other embodiments, the disclosure provides LNP formulated for delivery of the nucleic acids of the embodiments to cells ex vivo.

[0337] The disclosure provides a pharmaceutical composition comprising a plurality of LNPs comprising nucleic acids, such as mRNA encoding a long-term repressor fusion protein and / or a gRNA variant described herein, and a pharmaceutically acceptable carrier.

[0338] In certain embodiments, the LNP comprising the nucleic acid(s) has an electron dense core.

[0339] The disclosure provides LNP comprising one or more nucleic acids comprising: (a) an mRNA encoding the long-term repressor fusion protein, and / or a gRNA variant described herein; (b) one or more cationic lipids or ionizable cationic lipids or salts thereof comprising from about 50 mol % to about 85 mol % of the total lipid present in the LNP; (c) one or more non-cationic lipids comprising from about 13 mol % to about 49.5 mol % of the total lipidpresent in the LNP; and (d) one or more conjugated lipids that inhibit aggregation of LNPs comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle. In another embodiment, the disclosure provides LNP comprising one or more nucleic acids comprising: (a) an mRNA encoding the long-term repressor fusion protein, and / or a gRNA variant described herein; (b) one or more cationic lipids or ionizable cationic lipids or salts thereof comprising from about 22 mol % to about 85 mol % of the total lipid present in the LNP; (c) one or more non-cationic / phospholipids comprising from about 10 mol % to about 70 mol % of the total lipid present in the LNP; (d) 15 mol % to about 50 mol % sterol, and (d) 1 mol % to about 5 mol % lipid-PEG or lipid-PEG-peptide in the particle. In certain embodiments the long-term repressor fusion protein mRNA and gRNA may be present in the same nucleic acid-lipid particle, or they may be present in different nucleic acid-lipid particles.

[0340] The disclosure provides LNP comprising one or more nucleic acids comprising: (a) an mRNA encoding the long-term repressor fusion protein described herein; (b) a cationic lipid or a salt thereof comprising from about 52 mol % to about 62 mol % of the total lipid present in the LNP; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 36 mol % to about 47 mol % of the total lipid present in the LNP; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the LNP. In particular embodiments, the formulation is a four-component system comprising about 1.4 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 57.1 mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 7.1 mol % DPPC (or DSPC), and about 34.3 mol % cholesterol (or derivative thereof). In some embodiments, the LNP comprises the mRNA encoding the CasX described herein and a gRNA.

[0341] In other embodiments, the LNP comprising one or more nucleic acids comprises: (a) an mRNA encoding the long-term repressor fusion protein and / or a gRNA of any of the embodiments described herein; (b) a cationic lipid or a salt thereof comprising from about 46.5 mol % to about 66.5 mol % of the total lipid present in the LNP; (c) cholesterol or a derivative thereof comprising from about 31.5 mol % to about 42.5 mol % of the total lipid present in the LNP; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the LNP. In particular embodiments, the formulation is a three-component system which is phospholipid-free and comprises about 1.5 mol % PEG- lipid conjugate (e.g., PEG2...

Claims

CLAIMS What is claimed is 1. A system for transcriptional repression of a proprotein convertase subtilisin / kexin Type 9 (PCSK9) gene, the system comprising: (a) a guide ribonucleic acid (gRNA) comprising a targeting sequence complementary to a PCSK9 gene target nucleic acid sequence, and (b) an mRNA encoding a long-term repressor fusion protein (LTRP), wherein the LTRP comprises: a. a catalytically-dead CasX (dCasX); b. a DNA methyltransferase (DNMT) 3A catalytic domain (DNMT3A); c. a DNMT3 like interaction domain (DNMT3L); and d. a first repressor domain (RD1), wherein the LTRP is capable of forming a ribonucleoprotein (RNP) with the gRNA.

2. The system of claim 1, wherein the LTRP comprises, from N- to C- terminus: a. the DNMT3A; b. the DNMT3L; c. the dCasX; and d. the RD1.

3. The system of claim 1, wherein the LTRP comprises, from N- to C- terminus: a. the DNMT3A; b. the DNMT3L; c. the RD1; and d. the dCasX.

4. The system of claim 3, wherein the LTRP further comprises an additional RD1 C- terminal to the dCasX.

5. The system of claim 4, wherein the RD1 sequences are identical.

6. The system of claim 4, wherein the RD1 sequences are different.

7. The system of any one of claims 1-6, wherein the LTRP comprises a DNA DNMT3A ATRX-DNMT3-DNMT3L domain (ADD) linked to the N-terminus of the DNMT3A.

8. The system of any one of claims 1-7, wherein the LTRP comprises one or more linker peptides.

9. The system of claim 8, wherein at least one of the one or more linker peptides comprises a sequence independently selected from the group consisting of SEQ ID NOS: 98- 124, and 3278-3289.

10. The system of any one of claims 1-9, wherein the LTRP comprises one or more nuclear localization signals (NLS).

11. The system of claim 10, wherein at least one of the one or more NLS comprises a sequence selected from the group consisting of SEQ ID NOS: 30-97.

12. The system of claim 10 or claim 11, wherein at least one of the one or more NLS is a simian virus 40 (SV40) NLS.

13. The system of claim 12, wherein the SV40 NLS comprises the sequence of SEQ ID NO:

30.

14. The system of claim 10 or claim 11, wherein at least one of the one or more NLS is a c-MYC NLS.

15. The system of claim 14, wherein the c-MYC NLS comprises the sequence of SEQ ID NO:

32.

16. The system of any one of claims 1-2, or 8-15, wherein the LTRP comprises, from N- to C-terminus, a. a first NLS; b. the DNMT3A; c. a first linker peptide; d. the DNMT3L; e. a second linker peptide; f. a third linker peptide; g. the dCasX;h. a fourth linker peptide; i. the RD1, and j. a second NLS.

17. The system of claim 16, wherein the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 22836, and SEQ ID NOS: 22838-22846.

18. The system of any one of claims 7-15, wherein the LTRP comprises, from N- to C- terminus, a. a first NLS; b. the ADD; c. the DNMT3A; d. a first linker peptide; e. the DNMT3L; f. a second linker peptide; g. a third linker peptide; h. the dCasX; i. a fourth linker peptide; j. the RD1, and k. a second NLS.

19. The system of claim 18, wherein the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 22837 and SEQ ID NOS: 22847-22855.

20. The system of any one of claims 1, 3, or 8-15, wherein the LTRP comprises, from N- to C-terminus, a. a first NLS; b. the DNMT3A; c. a first linker peptide; d. the DNMT3L; e. a third linker peptide; f. the RD1; g. a second linker peptide; h. the dCasX; i. a fourth linker peptide, andj. a second NLS.

21. The system of any one of claims 7-15, wherein the LTRP comprises, from N- to C- terminus, a. a first NLS; b. the ADD; c. the DNMT3A; d. a first linker peptide; e. the DNMT3L; f. a second linker peptide; g. the RD1 h. a third linker peptide; i. the dCasX; j. a fourth linker peptide, and k. a second NLS.

22. The system of any one of claims 7-15, wherein the LTRP comprises, from N- to C- terminus, a. a first NLS; b. the ADD; c. the DNMT3A; d. a first linker peptide; e. the DNMT3L; f. a second linker peptide; g. the RD1 h. a third linker peptide; i. the dCasX; j. a fourth linker peptide; k. an additional RD1; l. a fifth linker, and m. a second NLS.

23. The system of claim 22, wherein the RD1 and the additional RD1 sequences are identical.

24. The system of claim 22, wherein the RD1 and the additional RD1 sequences are different.

25. The system of any one of claims 1-24, wherein the dCasX comprises a sequence selected from the group consisting of SEQ ID NOS: 4-29, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

26. The system of claim 25, wherein the dCasX comprises a sequence of SEQ ID NO: 4, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

27. The system of claim 26, wherein the dCasX consists of a sequence of SEQ ID NO:

4.

28. The system of any one of claims 1-27, wherein the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 128-1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

29. The system of any one of claims 1-27, wherein the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 130-138, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

30. The system of any one of claims 1-27, wherein the RD1 comprises a sequence of SEQ ID NO: 130, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

31. The system of any one of claims 1-27, wherein the RD1 comprises a sequence of SEQ ID NO: 131, or sequence having at least about 70%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

32. The system of any one of claims 1-27, wherein the RD1 comprises a sequence of SEQ ID NO: 132, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

33. The system of any one of claims 1-27, wherein the RD1 comprises a sequence of SEQ ID NO: 133, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

34. The system of any one of claims 1-27, wherein the RD1 comprises a sequence of SEQ ID NO: 134, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

35. The system of any one of claims 1-27, wherein the RD1 comprises the sequence of SEQ ID NO: 135, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

36. The system of any one of claims 1-27, wherein the RD1 comprises a sequence of SEQ ID NO: 136, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

37. The system of any one of claims 1-27, wherein the RD1 comprises a sequence of SEQ ID NO: 137, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

38. The system of any one of claims 1-27, wherein the RD1 comprises a sequence of SEQ ID NO: 138, or sequence having at least about 70%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

39. The system of any one of claims 7-15, 18, 19 or 19-38, wherein the ADD comprises a sequence of SEQ ID NO: 125, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

40. The system of any one of claims 1-39, wherein the DNMT3A comprises a sequence of SEQ ID NO: 126, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

41. The system of any one of claims 1-40, wherein the DNMT3L comprises a sequence of SEQ ID NO: 127, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

42. The system of any one of claims 1-41, wherein the sequence encoding the dCasX comprises a sequence selected from the group consisting of SEQ ID NOS: 3122, and 22727, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

43. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3334-6527, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

44. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3334-3342, and 4931-4939, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

45. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3335 and SEQ ID NO: 4932, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

46. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3339 and SEQ ID NO: 4936, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

47. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3334 and SEQ ID NO: 4931, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

48. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3336 and SEQ ID NO: 4933, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

49. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3337 and SEQ ID NO: 4934, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

50. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3338 and SEQ ID NO: 4935, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

51. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3340 and SEQ ID NO: 4937, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

52. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3341 and SEQ ID NO: 4938, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

53. The system of any one of claims 1-41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of SEQ ID NO: 3342 and SEQ ID NO: 4939, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

54. The system of any one of claims 1-53, wherein the sequence encoding the DNMT3A comprises a sequence selected from the group consisting of SEQ ID NO: 3128 and SEQ ID NO: 3331, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

55. The system of any one of claims 1-54, wherein the sequence encoding the DNMT3L comprises a sequence selected from the group consisting of SEQ ID NO: 3119 and SEQ ID NO: 3332, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

56. The system of any one of claims 7-15, 18, 19, or 21-55, wherein the sequence encoding the ADD comprises a sequence selected from the group consisting of SEQ ID NOS: 3127, 3296, and 22726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

57. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 6529-8134, and 14628-16233, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

58. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 6529-6537, and 14628-14636, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

59. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6529 and SEQ ID NO: 14628, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

60. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6530 and SEQ ID NO: 14629, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

61. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6531 and SEQ ID NO: 14630, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

62. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6532 and SEQ ID NO: 14631, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

63. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6533 and SEQ ID NO: 14632, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

64. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6535 and SEQ ID NO: 14632, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

65. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6534 and SEQ ID NO: 14633, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

66. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6536 and SEQ ID NO: 14635, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

67. The system of any one of claims 1, 2, 8-17 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6537 and SEQ ID NO: 4636, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

68. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 9742-11347, and 17840-19446, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

69. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 9742-9750, and 17841-17849, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

70. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9742 and SEQ ID NO: 17841, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

71. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9743 and SEQ ID NO: 17842, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

72. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9744 and SEQ ID NO: 17843, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

73. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9745 and SEQ ID NO: 17844, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

74. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9746 and SEQ ID NO: 17845, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

75. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9747 and SEQ ID NO: 17846, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

76. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9748 and SEQ ID NO: 17847, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

77. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9749 and SEQ ID NO: 17848, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

78. The system of any one of claims 1, 2, 7-15, 18, 19 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from SEQ ID NO: 9750 and SEQ ID NO: 17849, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

79. The system of any one of claims 1, 3, 8-15, 20 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 8135- 9740 and 16234-17839, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

80. The system of any one of claims 1, 3, 8-15, 20 or 25-55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 8135- 8143 and 16234-16242, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

81. The system of any one of claims 1, 3, 7-15, 21 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 11348- 12953 and 19447-21052, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

82. The system of any one of claims 1, 3, 7-15, 21 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 11348- 11356 and 19447-19455, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

83. The system of any one of claims 1, 3-5, 7-15, 22, 23 or 25-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 12954-14553, and 21053-22652, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

84. The system of any one of claims 1, 3-4, 6-15, 22, or 24-56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 14554-14626, and 22653-22725, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

85. The system of any one of claims 1-84, wherein the mRNA is codon-optimized.

86. The system of claim 85, wherein the mRNA is codon-optimized for expression in a human cell.

87. The system of any one of claims 1-86, wherein the mRNA is chemically modified, optionally wherein one or more or all of the uridine residues of the mRNA are replaced with 1-methyl-pseudouridine.

88. The system of any one of claims 1-87, wherein the mRNA comprises a 5’ untranslated region (UTR), a 3’ UTR, a poly(A) sequence, and / or a 5’cap.

89. The system of claim 88, wherein the 5' UTR comprises a sequence of SEQ ID NO:3300.

90. The system of claim 88 or claim 89, wherein the 3' UTR comprises a sequence of SEQ ID NO: 3310.

91. The system of any one of claims 88-90, wherein the poly(A) comprises a sequence of SEQ ID NO: 3057.

92. The system of any one of claims 88-91, wherein the 5' cap is linked 5’ to the mRNA.

93. The system of any one of claims 87-92, wherein the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 14628-16233, 16234-17839, 17841- 19446, 19447-21052, 21053-22652, and 22653-22725, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

94. The system of any one of claims 87-92, wherein the mRNA comprises a sequence of SEQ ID NOS: 14628-14635, or 14636, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

95. The system of any one of claims 87-92, wherein the mRNA comprises a sequence of SEQ ID NO: 14628, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

96. The system of any one of claims 87-92, wherein the mRNA comprises a sequence of SEQ ID NO: 14629, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

97. The system of any one of claims 87-92, wherein the mRNA comprises a sequence of SEQ ID NO: 14630, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

98. The system of any one of claims 87-92, wherein the mRNA comprises a sequence of SEQ ID NO: 14631 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

99. The system of any one of claims 87-92, wherein the mRNA comprises a sequence SEQ ID NO: 14632, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

100. The system of any one of claims 87-92, wherein the mRNA comprises a sequence of SEQ ID NO: 14633 or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

101. The system of any one of claims 87-92, wherein the mRNA comprises a sequence of SEQ ID NO: 14634, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

102. The system of any one of claims 87-92, wherein the mRNA comprises a sequence of SEQ ID NO: 14635, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

103. The system of any one of claims 87-92, wherein the mRNA comprises a sequence of SEQ ID NO: 14636, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

104. The system of any one of claims 1-103, wherein the PCSK9 gene target nucleic acid sequence is within 1.5 kb of a transcription start site (TSS) in the gene.

105. The system of any one of claims 1-103, wherein the PCSK9 gene target nucleic acid sequence is within 500 bps upstream to 500 bps downstream of a TSS of the gene.

106. The system of any one of claims 1-103, wherein the PCSK9 gene target nucleic acid sequence is within 300 bps upstream to 300 bps downstream or is within 100 bps upstream to 100 bps downstream of a TSS of the gene.

107. The system of any one of claims 1-103, wherein the PCSK9 gene target nucleic acid sequence is within 100 bps upstream to 100 bps downstream of a TSS of the gene.

108. The system of any one of claims 1-107, wherein the PCSK9 gene target nucleic acid sequence is within 1 kb of an enhancer of the gene.

109. The system any one of claims 1-107, wherein the PCSK9 gene target nucleic acid sequence is within the 3’ untranslated region of the PCSK9 gene.

110. The system of any one of claims 1-109, wherein the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1824-1881, 1883- 1897, 1899-1934, 1937-1991, 1993-1999, 2001-2026, 2029-2057, 2059-2106, 2108-2114, 2116-2122, 2124-2139, 2141-2209, 2211-2212, 2214-2233, 2235-2252, 2254-2269, 2271- 2281, 2283-2284, 2286-2320, 2322-2334, 2236-2346, 2348-2362, 2364-2375, 2377-2395, 2397-2428, 2430-2438, 2440-2463, 2465-2509, 2511-2544, 2672, 2675, 2694 and 2714.

111. The system of any one of claims 1-109, wherein the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714.

112. The system of any one of claims 1-109, wherein the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1834, 1849, 1853, 1855-1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875.

113. The system of any one of claims 1-109, wherein the targeting sequence of the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 1855, 1867, and 1869.

114. The system of any one of claims 110-113, wherein the targeting sequence of the gRNA comprises a sequence wherein 1, 2, 3, 4, or 5 nucleotide(s) are removed from the 3’ end of the sequence.

115. The system of any one of claims 1-114, wherein the gRNA is a single-molecule gRNA (sgRNA).

116. The system of any one of claims 1-115, wherein the gRNA comprises a scaffold stem loop comprising the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1822), or a sequence having 1, 2, 3, 4, or 5 mismatches thereto.

117. The system of any one of claims 1-116, wherein the gRNA comprises a scaffold comprising a sequence selected from the group consisting of SEQ ID NOS: 1744-1746, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto.

118. The system of any one of claims 1-116, wherein the gRNA comprises a scaffold comprising a sequence of SEQ ID NOS: 1746, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto.

119. The system of any one of claims 1-118, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 3074-3081, 3145-3147, 3150-3153, and 3158-3176, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto.

120. The system of any one of claims 1-119, wherein the gRNA is chemically modified.

121. The system of claim 120, wherein the chemical modification to the gRNA comprises an addition of a 2’O-methyl group to one or more nucleotides of the gRNA.

122. The system of claim 121, wherein one or more nucleotides located 1, 2, 3, or 4 nucleotides from the 5’ terminal end, the 3’ terminal, or both terminal ends of the gRNA are modified by an addition of a 2’O-methyl group.

123. The system of any one of claims 120-122, wherein the chemical modification to the gRNA comprises a substitution of a phosphorothioate bond between two or more nucleotides of the gRNA.

124. The system of claim 123, wherein the chemical modification comprises a substitution of phosphorothioate bonds between two or more nucleotides located 1, 2, 3 or 4 nucleotides the from the 5’ terminal end, the 3’ terminal, or both terminal ends of the gRNA.

125. The system of any one of claims 104-124, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 3074-3081, 3147, 3151, 3153, 3159- 3164, 3166-3171, 3173-3176 and 22788- 22803, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto.

126. The system of any one of claims 104-124, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 22788-22803.

127. The system of any one of claims 104-124, wherein the gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 22788-22790.

128. A lipid nanoparticle comprising the system of any one of claims 1-127.

129. A lipid nanoparticle (LNP) comprising the mRNA of any one of claims 87-103 and the gRNA of any one of claims 120-127.

130. The LNP of claim 128 or 129, wherein the LNP comprises one or more components selected from the group consisting of ionizable lipids, one or more helper phospholipids, one or more polyethylene glycol (PEG)-modified lipids, and cholesterol or a derivative thereof.

131. The LNP of any one of claims 128-130, wherein the LNP comprises an ionizable lipid, a helper phospholipid, a polyethylene glycol (PEG)-modified lipid, and cholesterol or a derivative thereof.

132. The LNP of any one of claims 128-131, comprising a cationic lipid comprising a pKa of 5 to 8.

133. A pharmaceutical composition comprising: a. the system of any one of claims 1-127; or b. the LNP of any one of claims 128-132, and one or more pharmaceutically suitable excipients.

134. A pharmaceutical composition comprising a plurality of the lipid nanoparticles of any one of claims 128-132 and a pharmaceutically acceptable carrier or diluent.

135. The pharmaceutical composition of claim 134, wherein an average diameter of lipid nanoparticles in the plurality is between about 20 nm and about 200 nm.

136. The pharmaceutical composition of claim 134 or claim 135, wherein the pharmaceutical composition is formulated for a route of administration selected from the group consisting of intravenous, intraarterial, intraportal vein injection, intraperitoneal, intramuscular, intracerebroventricular, intracisternal, intrathecal, intracranial, intralumbar, intraocular, subcutaneous, and oral routes.

137. A method of repressing transcription of a PCSK9 gene in a population of cells, the method comprising introducing into cells of the population: a. the system of any one of claims 1-127; b. the LNP of any one of claims 128-132; c. the pharmaceutical composition of any one of claims 133-136; or d. combinations of two or more of (a)-(c), thereby repressing transcription of the PCSK9 gene in the population of cells.

138. The method of claim 137, wherein transcription of the PCSK9 gene is repressed in at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more of the cells of the population.

139. The method of claim 137 or claim 138, wherein transcription of the PCSK9 gene in the cells of the population is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in comparison to an untreated cell.

140. The method of any one of claims 137-139, wherein the cells of the population are eukaryotic cells.

141. The method of claim 140, wherein the eukaryotic cells are selected from the group consisting of rodent cells, mouse cells, rat cells, and non-human primate cells.

142. The method of claim 140, wherein the eukaryotic cells are human cells.

143. The method of any one of claims 140-142, wherein the eukaryotic cells are selected from the group consisting of cells of the liver, cells of the intestine, cells of the kidney, cells of the central nervous system, smooth muscle cells, macrophages, and cells of arterial walls.

144. The method of any one of claims 137-143, wherein the repression of the PCSK9 gene of the population of cells occurs in vitro or ex vivo.

145. The method of any one of claims 137-143, wherein the repression of the PCSK9 gene of the population of cells occurs in vivo in a subject.

146. The method of claim 145, wherein the subject is selected from the group consisting of a rodent, a mouse, a rat, and a non-human primate.

147. The method of claim 145, wherein the subject is a human.

148. The method of any one of claims 137-147, wherein the repression of transcription is stable through one or more cell divisions.

149. The method of any one of claims 137-148, wherein the repression of transcription is heritable.

150. The method of any one of claims 137-149, wherein the repression is reversible.

151. The method of claim 150, wherein the repression is reversible by use of an inhibitor of DNMT.

152. The method of claim 151, wherein the inhibitor of DNMT is a cytidine analog.

153. The method of claim 151 or claim 152, wherein the inhibitor of DNMT is selected from the group consisting of azacytidine, decitabine, clofarabine, and zebularine.

154. A method of treating a PCSK9-related disease in a subject in need thereof, comprising administering a therapeutically effective dose of: a. the system of any one of claims 1-127; b. the LNP of any one of claims 128-132; or c. the pharmaceutical composition of any one of claims 133-136, thereby treating the PCSK9-related disease.

155. The method of claim 154, wherein the subject is treated with a therapeutically effective dose of the LNP.

156. The method of claim 154 or claim 155, wherein the LNP is administered by a route of administration selected from the group consisting of intravenous, intraarterial, intraportal vein injection, intraperitoneal, intramuscular, intracerebroventricular, intracisternal, intrathecal, intracranial, intralumbar, intraocular, subcutaneous, and oral routes.

157. The method of any one of claims 154-156, wherein the PCSK9-related disease is selected from the group consisting of autosomal dominant hypercholesterolemia (ADH), hypercholesterolemia, elevated total cholesterol levels, hyperlipidemia, elevated low-density lipoprotein (LDL) levels, elevated LDL- cholesterol levels, reduced high-density lipoprotein levels, liver steatosis, coronary heart disease, ischemia, stroke, peripheral vascular disease, thrombosis, type 2 diabetes, high elevated blood pressure, atherosclerosis, obesity, aortic stenosis, elevated PCSK9 levels, or a combination thereof.

158. The method of any one of claims 154-157, wherein the method results in improvement in at least one clinically-relevant endpoint selected from the group consisting of change from baseline in LDL-cholesterol, decrease in plaque atheroma volume, reduction in in coronary plaque, reduction in atherosclerotic cardiovascular disease (ASCVD), cardiovascular death, nonfatal myocardial infarction, ischemic stroke, nonfatal stroke, coronary revascularization, unstable angina, and visual acuity.

159. The method of any one of claims 154-158, wherein the method results in improvement in at least two clinically-relevant endpoints selected from the group consisting of change from baseline in LDL-cholesterol, decrease in plaque atheroma volume, reduction in in coronary plaque, reduction in atherosclerotic cardiovascular disease (ASCVD), cardiovascular death, nonfatal myocardial infarction, ischemic stroke, nonfatal stroke, coronary revascularization, unstable angina and visual acuity.

160. A kit comprising the system of any one of claims 1-127, the LNP of any one of claims 128-132, the pharmaceutical composition of any one of claims 133-136, or combinations thereof, and a suitable container.

161. The kit of claim 160, comprising a buffer, an excipient, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, instructionsfor use, or any combination of the foregoing.

162. A composition comprising: a. the system of any one of claims 1-127; b. the LNP of any one of claims 128-132; or c. the pharmaceutical composition of any one of claims 133-136.

163. The composition of claim 162, for use in the manufacture of a medicament for the treatment of a PCSK9-related disease in a subject in need thereof.

164. The composition of claim 162, for use in the treatment of a PCSK9-related disease in a subject in need thereof.

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