Compositions and methods for targeting pcsk9

Abstract

This article provides a gene repressor system comprising fusion proteins, such as fusion proteins containing two types of CRISPR proteins that catalyze cell death, and guide nucleic acids (gRNAs) that can be used to repress the preprotein convertase subtilisin / kexin type 9 (PCSK9) gene. Methods for preparing such systems and for using such systems to repress PCSK9 transcription are also provided.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to U.S. Provisional Application No. 63 / 492,978, filed March 29, 2023, and U.S. Provisional Application No. 63 / 505,888, filed June 2, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Reference to the electronic sequence list

[0004] The contents of the electronic sequence list (SCRB_058_02WO_SeqList_ST26.xml; size: 141,865,746 bytes; and creation date: March 26, 2024) are incorporated herein by reference in their entirety. Background Technology

[0005] In mammals, cholesterol is transported in lipoproteins via emulsification. Lipoprotein particles are classified according to their density: low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), high-density lipoprotein (HDL), and chylomicrons. Surface LDL receptors are internalized during cholesterol uptake. Cholesterol-rich cells prevent the synthesis of their LDL receptors to prevent the uptake of new cholesterol from LDL particles. Conversely, when cells lack cholesterol, LDL receptor synthesis is promoted. When this process is uncontrolled, excess LDL particles circulate in the bloodstream without being absorbed by LDL receptors. LDL particles in the blood are oxidized and absorbed by macrophages, then become congested and form foam cells. These foam cells can become trapped in the blood vessel walls, leading to the formation of atherosclerotic plaques, a major cause of heart attacks, strokes, and other serious medical problems.

[0006] Hepatic proprotein convertase subtilisin / kexin type 9 (PCSK9) is a secreted, globular, self-activating serine protease that binds to the low-density lipoprotein receptor (LDL-R) during the endocytosis of LDL particles, preventing LDL-R from recirculating to the cell surface and leading to a decrease in LDL-cholesterol clearance. PCSK9 binds to LDL-R (via the EGF-A domain), preventing conformational changes in the receptor-ligand complex, thereby redirecting LDL-R to lysosomes. Since LDL receptors typically transport thousands of fat molecules (including cholesterol) per particle in the extracellular fluid, blocking or inhibiting PCSK9 function to promote LDL-R-mediated LDL-cholesterol clearance can reduce LDL particle concentration. PCSK9 is primarily expressed in the liver, intestine, kidney, and central nervous system, but is also highly expressed in arterial walls, such as endothelial cells, smooth muscle cells, and macrophages, exhibiting local effects in regulating vascular homeostasis and atherosclerosis.

[0007] PCSK9 is a member of the proprotein convertase (PC) family, and its gene is mutated in approximately 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. *Journal of the Federation of European Biochemical Societies (FEBS J)*. August 6, 2019. doi:10.1111 / febs.15036). Researchers have identified several PCSK9 mutations that lead to genetically inherited forms of high cholesterol (hypercholesterolemia). These mutations alter individual amino acids in the PCSK9 protein. Researchers describe mutations leading to hypercholesterolemia as “gain-of-function” because they appear to enhance the activity of the PCSK9 protein or confer a new atypical function to the protein (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)). Overactive PCSK9 protein significantly reduces the number of low-density lipoprotein receptors on the surface of hepatocytes. People with gain-of-function mutations in the PCSK9 gene have very high blood cholesterol levels due to fewer receptors removing low-density lipoprotein from the blood. Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by elevated low-density lipoprotein (LDL) cholesterol levels, leading to a high risk of premature cardiovascular disease. In different populations, approximately 10 mutations in PCSK9 have been identified as contributing factors to this disease. All known mutations in PCSK9 that cause hypercholesterolemia increase the activity of this protease (Bleasa, S., 2008). Additionally, PCSK9 mutations may contribute to autosomal dominant familial hypobeta-lipoproteinemia, leading to hepatic steatosis, cirrhosis, and other diseases.

[0008] The advent of CRISPR / Cas systems and the programmable nature of these minimal systems has facilitated their use as a general-purpose technology for genome manipulation and engineering. However, current methods for generating protective variants and loss-of-function mutants of PCSK9 in vivo are ineffective because they require modification of large numbers of cells to regulate cholesterol levels. Other issues involve off-target effects, genome instability, or potentially oncogenic modifications due to genome editing, as well as the lack of safe delivery methods for repressor protein systems. Furthermore, in certain disease indications, gene silencing or repression is superior to gene editing. The ability to render CRISPR nucleases such as Cas9 and CasX catalytically inactive has been demonstrated (WO2020247882A1 and US20200087641A1, incorporated herein by reference), making these systems an attractive platform for generating fusion proteins with repressor domains capable of gene silencing. While some repressor systems have been described, other gene repressor systems that have been optimized and / or provide improvements over earlier gene repressor systems, such as Cas9-based systems, are still needed for a variety of therapeutic, diagnostic, and research applications. Therefore, improved compositions and methods are still needed to regulate PCSK9. Summary of the Invention

[0009] This disclosure provides a system comprising or encoding a fusion protein comprising a DNA-binding domain and a linked repressor domain for repression and / or epigenetic modification of the target nucleic acid sequence of the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene. In some aspects, the fusion protein comprises a DNA-binding protein including a death-catalyzing CRISPR protein such as class 2 type V CRISPR proteins and a guide nucleic acid including a targeting sequence complementary to the PCSK9 gene target nucleic acid sequence. The protein and guide nucleic acid can be modified for passive entry into target cells and can be used in various methods for PCSK9 repression, and these methods are also provided. This disclosure also provides carriers and lipid nanoparticles (LNPs) encoding or encapsulating the fusion protein and guide nucleic acid components for delivering the system to cells for transcriptional repression of the PCSK9 target nucleic acid sequence. This disclosure also provides methods for modifying the PCSK9 gene target nucleic acid sequence. This disclosure also provides methods for treating subjects with PCSK9-related diseases. In some embodiments, the compositions and methods may be used in subjects suffering from metabolic disorders such as, but not limited to, familial hypercholesterolemia, familial hypobeta-lipoproteinemia, or elevated cholesterol levels.

[0010] In other respects, this article provides systems comprising a PCRK9 repressor system, or vectors comprising or encoding a PCSK9 repressor system, for the preparation of medicaments for the treatment of PCSK9-related diseases in subjects of need.

[0011] Further features and advantages of certain embodiments of this disclosure will become clearer from the following description of the embodiments and their accompanying drawings, as well as from the claims.

[0012] By incorporating references

[0013] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated as incorporated by reference. The contents of WO 2020 / 247882, WO 2020 / 247883, WO2021 / 113772, WO2022 / 120095, WO 2022 / 125843, WO 2022 / 261150, WO 2022 / 261149, WO2023 / 049872, WO 2023 / 049742, and WO 2023 / 240162, which disclose CasX variants and gRNA variants and methods for delivering said variants, and WO2021 / 142342, which discloses compositions and methods for modifying PCSK9, are hereby incorporated in their entirety by reference. Attached Figure Description

[0014] A better understanding of the features and advantages of this disclosure will be facilitated by referring to the accompanying drawings:

[0015] Figure 1 This diagram illustrates various conformations of the repressor fusion protein integrating the DNMT3A ADD domain. "D3A ADD", "D3A CD", and "D3L ID" represent 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.

[0016] Figure 2 This is a bar graph showing the quantification of secreted PCSK9 levels in Huh7 cells lipid-transfected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated target gRNA at 6, 18, and 36 days post-transfection, as described in Example 1. Secreted PCSK9 levels were normalized relative to the total cell count. Untreated, pristine cells were used as experimental controls.

[0017] Figure 3This is a bar graph showing the quantification of secreted PCSK9 levels in HepG2 cells lipid-transfected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated target gRNA, as described in Example 2, 4 days post-transfection. Secreted PCSK9 levels were normalized relative to the total cell count. Untreated, pristine cells were used as experimental controls.

[0018] Figure 4 This is a bar graph showing the quantification of secreted PCSK9 levels in Huh7 cells transfected with lipid-transfected mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated target gRNA, as described in Example 2, 4 days post-transfection. Secreted PCSK9 levels were normalized relative to the total cell count. Untreated, pristine cells were used as experimental controls.

[0019] Figure 5 This is a bar graph showing the quantification of secreted PCSK9 levels in Hep3B cells lipid-transfected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated target gRNA, as described in Example 2, 4 days post-transfection. Secreted PCSK9 levels were normalized relative to the total cell count. Untreated, pristine cells were used as experimental controls.

[0020] Figure 6 This is a bar graph showing the quantification of secreted PCSK9 levels in Huh7 cells lipid-transfected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated target gRNA, at days 4, 14, and 27 post-transfection, as described in Example 2. The quantification of secreted PCSK9 levels is shown relative to the secretion levels detected in untreated cells at day 4.

[0021] Figure 7 This is a schematic diagram of gRNA scaffold variant 174 (SEQ ID NO: 1744), as described in Example 3. The structural motif is highlighted.

[0022] Figure 8 This is a schematic diagram of gRNA scaffold variant 235 (SEQ ID NO: 1745), as described in Example 3. The highlighted structural motifs are related to... Figure 7 Same. The difference between variant 174 and variant 235 lies in the elongated stem motif and several single nucleotide variations (indicated by asterisks). Variant 316 maintains the shorter, elongated stem from variant 174 but includes the four substitutions found in scaffold 235.

[0023] Figure 9 This is a schematic diagram of gRNA scaffold variant 316 (SEQ ID NO: 1746), as described in Example 3. The highlighted structural motifs are... Figure 7 Same. Variant 316 maintains the same characteristics as variant 174. Figure 7 The shorter, elongated stem, but carried on a support 235 ( Figure 8 Four substitutions were found in ).

[0024] Figure 10 This is a schematic diagram illustrating versions 1-3 of the 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 ribonucleotides modified with 2'OMe are depicted as black circles. Phosphothioester bonds are indicated with * below or next to the bond. For v2 spectra, three 3' uracils (3'UUU) are added in the relevant circles with a “U” annotation.

[0025] Figure 11 This is a schematic diagram illustrating versions 4-6 of the 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. Phosphothioester bonds are indicated with an asterisk (*) below or next to the bonds.

[0026] Figure 12 This is a schematic diagram illustrating versions 7-9 of the 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. Phosphothioester bonds are indicated with an asterisk (*) below or next to the bonds.

[0027] Figure 13 This is a schematic diagram illustrating versions 1-3 of the 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. Phosphothioester bonds are indicated with an asterisk (*) below or next to the bonds.

[0028] Figure 14 This is a schematic diagram illustrating versions 4-6 of the 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. Phosphothioester bonds are indicated with an asterisk (*) below or next to the bonds.

[0029] Figure 15The graph shows the correlation between the insertion / deletion rate at the PCSK9 locus (plotted as edit fraction) as measured by next-generation sequencing (NGS) (x-axis) and the secreted PCSK9 level (ng / mL) in HepG2 cells transfected with CasX 491 mRNA and gRNA targeting PCSK9 containing the indicated scaffold variant and spacer combination as detected by enzyme-linked immunosorbent assay (ELISA), as described in Example 3.

[0030] Figure 16 This is a graph illustrating the quantitative percentage of B2M knockout in HepG2 cells co-transfected with 100 ng CasX 491 mRNA and a specified dose of terminally modified (v1) or unmodified (v0) B2M-targeting gRNA with a spacer of 7.37, as described in Example 3. The editing level was determined by flow cytometry as the cell population that lost surface presentation of the HLA complex due to successful editing at the B2M locus.

[0031] Figure 17 This is a graph depicting the results of an editing assay measuring the insertion / deletion rate at the human B2M locus by NGS in HepG2 cells treated with LNPs formulated with the indicated dose of CasX 491 mRNA and the indicated B2M-targeting gRNA, as described in Example 3.

[0032] Figure 18 This is a graph quantifying the percentage of B2M knockout in HepG2 cells treated with LNPs formulated with the indicated dose of CasX 491 mRNA and the indicated B2M-targeting gRNA, as described in Example 3. The editing level was determined by flow cytometry as a cell population lacking surface presentation of the HLA complex due to successful editing at the B2M locus.

[0033] Figure 19 This is a graph depicting the results of an editing assay measuring the insertion / deletion rate at the mouse ROSA26 locus by NGS in Hepa1-6 cells treated with LNPs of LNPs modulated with CasX 676 mRNA number 2 and the indicated gRNA targeting ROSA26 with v1 or v5 modification profiles, as described in Example 3.

[0034] Figure 20 This is a graph showing the percentage of editing as measured by NGS as the insertion / deletion rate at the ROSA26 locus in mice treated with LNPs modulated using CasX 676 mRNA number 2 and the indicated chemically modified gRNA targeting ROSA26, as described in Example 3.

[0035] Figure 21 This is a bar graph showing the results of an editing assay measuring the insertion / deletion rate at the PCSK9 locus in mice treated with LNPs modulated using CasX 676 mRNA number 1 and the indicated chemically modified gRNA targeting PCSK9, as described in Example 3. Untreated mice were used as experimental controls.

[0036] Figure 22 This is a diagram of the secondary structure of guide RNA scaffold 235 (SEQ ID NO: 1745), indicating the regions with CpG motifs, as described in Example 6. The CpG motifs in (1) pseudo-stalk, (2) scaffold stalk, (3) elongated stalk vesicle, (4) elongated step, and (5) elongated stalk loop are structurally labeled.

[0037] Figure 23 This is a diagram of CpG reduction mutations in each of the five regions introduced into the coding sequence of the guide RNA scaffold, as described in Example 6.

[0038] Figure 24 Results of the editing experiments are provided, in which the B2M locus in induced neurons was edited using AAV vectors with guide RNA scaffolds of various CpG reduction or CpG depletion, as described in Example 6. The AAV vectors were administered at a multiplicity of infection (MOI) of 4e3. The bars show the mean ± SD of two replicates for each sample. “No Tx” indicates an untransduced control, and “NT” indicates a control with a non-targeted spacer.

[0039] Figure 25 Results of the editing experiments are provided, in which the B2M locus in induced neurons was edited using AAV vectors with guide RNA scaffolds of various CpG reduction or CpG depletion, as described in Example 6. The AAV vectors were administered at an MOI of 3e3. Bars show the mean ± SD of two replicates for each sample. “No Tx” indicates the untransduced control.

[0040] Figure 26 Results of the editing experiments are provided, in which the B2M locus in induced neurons was edited using AAV vectors with guide RNA scaffolds of various CpG reduction or CpG depletion, as described in Example 6. The AAV vectors were administered at an MOI of 1e3. Bars show the mean ± SD of two replicates for each sample. “No Tx” indicates the untransduced control.

[0041] Figure 27Results of the editing experiments are provided, in which the B2M locus in induced neurons was edited using AAV vectors with guide RNA scaffolds of various CpG reduction or CpG depletion, as described in Example 6. The AAV vectors were administered at an MOI of 3e2. Bars show the mean ± SD of two copies for each sample. “No Tx” indicates the untransduced control.

[0042] Figure 28 A schematic diagram of five conformations of the long-term repressor protein (LTRP, also referred to as the "repressor fusion protein" in this paper) fusion protein is shown, in which the repressor molecule is linked to the CasX that catalyzes death. D3A and D3L represent DNA methyltransferase 3α (DNMT3A) and DNMT3A-like protein (DNMT3L), respectively. L1-L4 are linkers. NLS is the nuclear localization signal.

[0043] Figure 29 Schematic diagrams of various LTRP number 5 architectures are shown, incorporating additional DNMT3A domains as described in Example 7. These additional DNMT3A domains are the ADD domain of DNMT3A (“D3A ADD”) and the PWWP domain of DNMT3A (Pro-Trp-Trp-Pro motif) (“D3A PWWP”). “D3A endogenous” encodes the endogenous sequence occurring between the DNMT3A PWWP and ADD domains. “D3A CD” and “D3L ID” represent the catalytic domain of DNMT3A and the interacting domain of DNMT3L, respectively. “L1-L3” are linkers. “NLS” is the nuclear localization signal. LTRP sequences are shown in Table 13.

[0044] Figure 30 Results of time-course experiments comparing the B2M repressive activity (expressed as the percentage of HLA-negative cells) of the indicated LTRP-ZIM3 and its variant using spacer 7.37 with a B2M-targeting gRNA are presented as described in Example 7. Data are presented as mean with standard deviation N = 3. The catalytic domain of CD = DNMT3A is also shown.

[0045] Figure 31 Presented Figure 30 Results of the same time-course experiment are shown, but B2M repression activity of the LTRP-ZIM3 variant with a B2M-targeting gRNA using spacer 7.160 is presented as described in Example 7. Data are presented as mean with a standard deviation of N = 3.

[0046] Figure 32 Presented Figure 30Results of the same time-course experiments are shown, but B2M repression activity of the LTRP-ZIM3 variant with a B2M-targeting gRNA using spacer 7.165 is presented as described in Example 7. Data are presented as mean with a standard deviation of N = 3.

[0047] Figure 33 Presented Figure 30 Results of the same time-course experiments are shown, but B2M repressive activity of the indicated LTRP-ZIM3 variant with non-targeting gRNA is presented as described in Example 7. Data are presented as mean with a standard deviation of N = 3.

[0048] Figure 34 This is a violin plot of the percentage of CpG methylation at the CpG site downstream of the transcription start site at the VEGFA locus for each of the indicated LTRP-ZIM3 variants for the three B2M-targeting and non-targeting gRNAs, as described in Example 7.

[0049] Figure 35 The scatter plot shows the relative activity (mean percentage of HLA-negative cells for spacer 7.160 on day 21) for the indicated LTRP5-ZIM3 variant relative to specificity (percentage of off-target CpG methylation at the VEGFA locus quantified for spacer 7.160 on day 7), as described in Example 7.

[0050] Figure 36 A schematic diagram of the general architecture of an LTRP molecule having ADD domains numbered 1, 4, and 5 in the LTRP configurations tested in Example 8 is shown. “D3A ADD,” “D3A CD,” and “D3L ID” represent 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. The LTRP sequence is shown in Table 11.

[0051] Figure 37 Results of time-course experiments comparing the B2M repression activity (expressed as the percentage of HLA-negative cells) of LTRPs with or without the ZIM3-KRAB domain (configuration number 1, 4, or 5) when paired with a B2M-targeting gRNA having spacer 7.160 are presented, as described in Example 8. Data are presented as mean with a standard deviation of N = 3. “NT” refers to gRNAs with a non-targeting spacer.

[0052] Figure 38 It shows Figure 37The figure shows the results of the same time-course experiment, but illustrates the B2M repression activity of LTRP number 5 with or without the ZNF10 or ZIM3-KRAB domain, paired with a B2M-targeting gRNA having a spacer 7.160, as described in Example 8. Data are presented as mean with a standard deviation N = 3. “NT” is a gRNA with a non-targeting spacer.

[0053] Figure 39 It shows Figure 37 The figure shows the results of the same time-course experiment, but illustrates B2M repression activity for LTRP5-ZIM3 with or without the indicated spacer and a DNMT3A ADD domain paired with the B2M-targeting gRNA, as described in Example 8. Data are presented as mean with a standard deviation of N = 3. “NT” represents a gRNA with a non-targeting spacer.

[0054] Figure 40 This is a graph showing the results of B2M repression activity on day 27 post-transfection for LTRPs with or without the ZNF10 or ZIM3-KRAB domain (configuration number 1) for the DNMT3A ADD domain used for the indicated gRNA, as described in Example 8. Data are presented as mean with a standard deviation of N = 3. “NT” is a gRNA with a non-targeting spacer.

[0055] Figure 41 This is a graph showing the results of B2M repression activity on day 27 post-transfection for LTRPs with or without the ZNF10 or ZIM3-KRAB domain (configuration number 4) for the indicated gRNA, with or without the DNMT3A ADD domain. Data are presented as mean with a standard deviation of N = 3. “NT” is a gRNA with a non-targeting spacer.

[0056] Figure 42 This is a graph showing the results of B2M repression activity of LTRPs with or without the ZNF10 or ZIM3-KRAB domain (configuration number 5) for the indicated gRNA, on day 27 post-transfection, as described in Example 8. Data are presented as mean with a standard deviation of N = 3. “NT” is a gRNA with a non-targeting spacer.

[0057] Figure 43This is a graph showing the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus on day 5 post-transfection for LTRPs with or without the ZNF10 or ZIM3-KRAB domain of configuration number 1 (DNMT3A ADD domain for the indicated gRNA), as described in Example 8. Data are presented as the mean percentage of CpG methylation at CpG sites near the VEGFA locus; the standard error of the mean is also presented; N = 3. “NT” is a gRNA with an off-target spacer.

[0058] Figure 44 This is a graph showing the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus on day 5 post-transfection for LTRPs with or without the ZNF10 or ZIM3-KRAB domain (configuration number 4) for the indicated gRNA, with or without the DNMT3A ADD domain. Data are presented as the mean percentage of CpG methylation at CpG sites near the VEGFA locus; the standard error of the mean is also presented; N = 3. "NT" represents gRNAs with non-target spacers.

[0059] Figure 45 This is a graph showing the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus on day 5 post-transfection for LTRPs with or without the ZNF10 or ZIM3-KRAB domain (configuration number 5) for the indicated gRNA, with or without the DNMT3A ADD domain. Data are presented as the mean percentage of CpG methylation at CpG sites near the VEGFA locus; the standard error of the mean is also presented; N = 3. "NT" represents a gRNA with an off-target spacer.

[0060] Figure 46 This is a dot plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with ZIM3-KRAB domains numbered 1, 4, and 5 for gRNAs targeting B2M with spacer 7.160, as described in Example 8.

[0061] Figure 47 This is a dot plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with ZNF10-KRAB domains numbered 1, 4, and 5 for gRNAs targeting B2M with spacer 7.160, as described in Example 8.

[0062] Figure 48 This is a dot plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with ZIM3-KRAB domains numbered 1, 4, and 5 for gRNAs targeting B2M with spacer 7.37, as described in Example 8.

[0063] Figure 49 This is a dot plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with ZNF10-KRAB domains numbered 1, 4, and 5 for gRNAs targeting B2M with spacer 7.37, as described in Example 8.

[0064] Figure 50 This is a dot plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with ZIM3-KRAB domains numbered 1, 4, and 5 for gRNAs targeting B2M with spacer 7.165, as described in Example 8.

[0065] Figure 51 This is a dot plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with ZNF10-KRAB domains numbered 1, 4, and 5 for gRNAs targeting B2M with spacer 7.165, as described in Example 8.

[0066] Figure 52 This is a graph showing the percentage of HEK293T cells transfected with plasmids encoding the CasX or LTRP:gRNA constructs indicated by the formula, which express B2M six days after treatment with different concentrations of the DNMT1 inhibitor 5-azadC, as described in Example 9.

[0067] Figure 53 This is a graph juxtaposed with the quantification of B2M repression in HEK293T cells transfected with plasmids encoding the indicated CasX or LTRP:gRNA constructs and cultured for 58 days, and the quantification of B2M reactivation when transfected cells were treated with 5-azadC, as described in Example 9.

[0068] Figure 54 A schematic diagram of the LTRP5 molecule without the ADD domain of DNMT3A is shown, as described in Example 11. “D3A CD” and “D3L ID” represent the catalytic domain of DNMT3A and the interacting domain of DNMT3L, respectively. “L1”, “L2”, “L3A”, and “L3B” are linkers. “NLS” is the nuclear localization signal. “RD1” represents the repressor domain.

[0069] Figure 55 This is a bar graph showing the results of a time-course experiment comparing B2M repression levels (expressed as the mean percentage of HLA-negative cells) in HEK293T cells transfected with plasmids containing adapter groups 1-11, as described in Example 13. Data from each time point (day 8, day 15, and day 45) are stacked and expressed as a mean with a standard deviation of N = 3. Untargeted spacers (NTs) are included as experimental controls.

[0070] Figure 56 This is a bar graph showing the results of a time-course experiment comparing the repression level of LTRP5 variants containing adapter groups 1-11 (expressed as the percentage of total cells with target 1 knocked down) measured in HEK293T, as described in Example 13. Data from each time point (day 8, day 15, and day 45) are stacked and expressed as a mean with a standard deviation N = 3.

[0071] Figure 57 This is a bar graph showing the results of a time-course experiment comparing the repression levels of target 2 (expressed as the percentage of total cells with target 2 knocked down) of LTRP5 variants containing adapter groups 1–11, as measured in HEK293T, as described in Example 13. Data from each time point (day 8, day 15, and day 45) are stacked and expressed as a mean with a standard deviation N = 3. Non-target spacers (NTs) are included as experimental controls.

[0072] Figure 58 This is a bar graph showing the results of a time-course experiment comparing B2M repression levels (expressed as the mean percentage of HLA-negative cells) in HEK293T cells transfected with plasmids containing adapter group 12–28, as described in Example 13. Data from each time point (day 7 and day 17) are stacked and expressed as a mean with a standard deviation of N = 3. Non-targeting spacers (NTs) are included as experimental controls.

[0073] Figure 59This is a bar graph showing the results of a time-course experiment comparing the repression levels of LTRP5 variants containing adapter group 12–28 (expressed as the percentage of total cells with target 1 knocked down) measured in HEK293T, as described in Example 13. Data from each time point (day 7 and day 17) are stacked and expressed as a mean with a standard deviation of N = 3. Non-target spacers (NTs) are included as experimental controls.

[0074] Figure 60 This is a bar graph showing the results of a time-course experiment comparing the repression levels of LTRP5 variants containing adapter group 12–28 (expressed as the percentage of total cells with target 2 knocked down) measured in HEK293T, as described in Example 13. Data from each time point (day 7 and day 17) are stacked and expressed as a mean with a standard deviation of N = 3. Non-target spacers (NTs) are included as experimental controls.

[0075] Figure 61 A schematic diagram of various configurations of the LTRP molecule with the DNMT3A ADD domain is shown. “D3A ADD,” “D3A CD,” and “D3L ID” represent 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” represents the repressor domain, and “RD1a” and “RD1b” represent repressor domain variants.

[0076] Figure 62 This is a violin plot, where each dot represents the average methylation % at each CpG. Median methylation is indicated by a dashed line, and the upper and lower quartiles are indicated by dotted lines. Transcription start site (TSS)-proximal DNA methylation was measured from gDNA extracted from homogenized livers of N=3 mice sacrificed at days 7, 14, and 42 post-treatment by amplicon enzymatic methylation sequencing (EM-seq), as described in Example 14.

[0077] Figure 63 This is a violin plot, where each dot represents the average methylation % at each CpG. Median methylation is indicated by a dashed line, and the upper and lower quartiles are indicated by dotted lines. Transcription start site (TSS)-proximal DNA methylation was measured from gDNA extracted from homogenized livers of N=3 mice sacrificed on day 7 post-treatment by amplicon enzymatic methylation sequencing (EM-seq), as described in Example 14. Detailed Implementation

[0078] Although 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. Many variations, modifications, and substitutions will now be apparent to those skilled in the art without departing from the spirit of this disclosure. It should be understood that different alternatives to the embodiments described herein can be used to practice the embodiments of this disclosure.

[0079] 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 pertains. While methods and materials similar to or equivalent to those described herein may be used to practice or test the embodiments herein, suitable methods and materials are described below. In case of conflict, the present patent specification, including its definitions, shall prevail. Furthermore, materials, methods, and examples are merely illustrative and not intended to be limiting. Many variations, alterations, and substitutions will now be apparent to those skilled in the art without departing from the spirit of this disclosure and the claims.

[0080] definition

[0081] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates 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 proteins, reference to “a nucleic acid sequence” includes one or more nucleic acid sequences, and so on.

[0082] As used herein, the term "about" will be understood by those skilled in the art and may vary to some extent depending on the context in which it is used. Where there is a use of a term that is not readily apparent to those skilled in the art, the term "about" will, taking into account the context in which it is used, mean at most ±10% of the particular term.

[0083] As those skilled in the art will understand, for any and all purposes, all scopes disclosed herein also include any and all possible subscopes and combinations thereof. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1-3 members refers to a group having 1, 2, or 3 members. Similarly, a group having 1-5 members refers to a group having 1, 2, 3, 4, or 5 members, etc.

[0084] The term "combinations thereof" includes every possible combination of the elements referred to by the term.

[0085] As used herein, the term “exemplary” refers to an instance or illustration and is not intended to imply any preference or value.

[0086] As used herein, the term "CasX protein" refers to a family of proteins, including, for example, all naturally occurring CasX proteins ("reference CasX"), as well as sequence-modified CasX proteins, such as dCasX, which have one or more modified properties relative to their derived CasX proteins, described more fully below.

[0087] The terms “polynucleotide” and “nucleic acid”, used interchangeably in this document, refer to polymeric forms of nucleotides of any length, namely ribonucleotides or deoxyribonucleotides. Therefore, the 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 polymers containing purine and pyrimidine bases or other natural, chemically modified, or biochemically modified, non-natural or derived nucleotide bases.

[0088] The terms "hybridizable" and "complementary" are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) contains a nucleotide sequence that enables it to non-covalently bind to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid) under appropriate temperature and solution ionic strength conditions in vitro and / or in vivo, forming Watson-Crick base pairs and / or G / U base pairs, "annealing," or "hybridizing." It should be understood that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid for specific hybridization; 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 with the target nucleic acid. Furthermore, polynucleotides can hybridize on one or more segments such that intermediate or adjacent segments do not participate in the hybridization event (e.g., loop structures or hairpin structures, 'bulges,' 'bubbles,' etc.). Therefore, those skilled in the art will understand that although individual bases within a sequence may not be complementary to another sequence, the sequence as a whole is still considered complementary.

[0089] For the purposes of this disclosure, "gene" includes the DNA region encoding a gene product (e.g., protein, RNA) and all DNA regions regulating the production of the gene product, regardless of whether such regulatory sequences are adjacent to the coding and / or transcribed sequences. Therefore, a gene may include accessory element sequences, including but not limited to: promoter sequences, terminators, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions. The coding sequence encodes the gene product during transcription or transcription and translation; the coding sequences of this disclosure may comprise fragments and do not necessarily contain a full-length open reading frame. A gene may include both the transcribed strand and the complementary strand containing anticodons.

[0090] The term "downstream" refers to the nucleotide sequence located at the 3' end of a reference nucleotide sequence. In some embodiments, the downstream nucleotide sequence refers to a sequence following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0091] The term "upstream" refers to the nucleotide sequence located at the 5' end of a reference nucleotide sequence. In some embodiments, the upstream nucleotide sequence involves a sequence located 5' to the side of a coding region or transcription start site. For example, most promoters are located upstream of the transcription start site.

[0092] The term "adjacent to" in relation to polynucleotide or amino acid sequences refers to sequences that are adjacent or neighboring to each other in a polynucleotide or polypeptide. Those skilled in the art will understand that two sequences can be considered to be adjacent to each other and still encompass a limited number of intermediate sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or amino acids.

[0093] The term “regulatory element” is used interchangeably with the term “regulatory sequence” in this document and is intended to include promoters, enhancers, and other expression regulatory elements. It should be understood that the selection of an appropriate regulatory element will depend on whether the encoded component (e.g., protein or RNA) to be expressed or whether the nucleic acid contains multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0094] The term “auxiliary element” is used interchangeably with the term “auxiliary sequence” herein and is intended to include coding and non-coding sequences that enhance expression, nucleic acid transport, or the function of mRNA or protein, and includes, in particular, poly(A) signals, enhancer elements, introns, post-transcriptional regulatory elements (PTREs), nuclear localization signals (NLS), deaminases, DNA glycosyltransferase inhibitors, additional promoters, factors that stimulate CRISPR-mediated homology-directed repair (e.g., in cis or trans), self-cleaving sequences, and fusion domains, such as fusion domains fused to CRISPR proteins. It should be understood that the selection of one or more appropriate auxiliary elements will depend on whether the encoded component (e.g., protein or RNA) or nucleic acid to be expressed contains multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0095] The term "promoter" refers to a DNA sequence containing a transcription start site and additional sequences that promote polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as TATA boxes and / or B recognition elements (BREs) and assist or promote the transcription and expression of associated transcribed polynucleotide sequences and / or genes (or transgenes). Promoters can be synthetically generated or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can be located proximal or distal to the gene to be transcribed. Promoters can also include chimeric promoters, which comprise combinations of two or more heterologous sequences to confer certain properties. Promoters disclosed herein may include variants of promoter sequences that are compositionally similar to but not identical to one or more other promoter sequences known or provided herein. Promoters can be classified according to criteria associated with the expression pattern of the associated coding or transcribed sequence or the gene operatively linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. Promoters can also be classified according to their strength. As used in the context of promoters, "strength" refers to the transcription rate of the gene controlled by the promoter. A "strong" promoter means a high transcription rate, while a "weak" promoter means a relatively low transcription rate.

[0096] The promoter disclosed herein may be a polymerase II (Pol II) promoter. Polymerase II transcribes all protein-coding and many non-coding genes. A representative Pol II promoter includes a core promoter, which is a sequence of approximately 100 base pairs surrounding the transcription start site and serves as a binding platform for Pol II polymerase and associated universal transcription factors. The promoter may contain one or more core promoter elements, such as a TATA box, BRE, initiator (INR), decamosome element (MTE), downstream core promoter element (DPE), and downstream core element (DCE), although core promoters lacking these elements are known in the art. All Pol III promoters are contemplated within the scope of this disclosure.

[0097] The promoter disclosed herein may be a polymerase III (Pol III) promoter. Pol III transcribes DNA to synthesize small ribosomal RNAs, such as 5S rRNA, tRNA, and other small RNAs. Representative Pol III promoters use internal control sequences (sequences within the transcribed portion of a gene) to support transcription, although upstream elements such as TATA boxes are sometimes used as well. All Pol III promoters are contemplated within the scope of this disclosure.

[0098] The term "enhancer" refers to a regulatory DNA sequence that, when bound by a specific protein called a transcription factor, regulates the expression of a related gene. Enhancers can be located within introns of a gene or at the 5' or 3' end of the gene's coding sequence. Enhancers can be located proximal to the gene (i.e., within tens or hundreds of base pairs (bp) of the promoter) or distal to the gene (i.e., thousands, hundreds of thousands, or even millions of bps away from the promoter). A single gene can be regulated by more than one enhancer, all of which are envisioned within the scope of this disclosure.

[0099] As used in this article, "posttranscriptional regulatory elements (PTREs)," such as hepatitis PTREs, refer to tertiary DNA sequences that are produced during transcription and can exhibit posttranscriptional activity to enhance or promote the expression of related genes that are operatively linked to them.

[0100] "Operably connected" means juxtaposing two or more components (such as sequence elements) such that both components are arranged to function properly, and allowing for the possibility that at least one of the components can mediate functionality over at least one of the other components (e.g., promoters and coding sequences). Those skilled in the art will understand that a physical connection is not required for operably connected components.

[0101] In the context of this disclosure and relating to genes, the terms “repress,” “repression,” “repressing,” “transcriptional repression,” “inhibition of gene expression,” “downregulation,” and “silencing” are used interchangeably herein to refer to the repression or blockage of transcription of a gene or a portion thereof. Thus, gene repression can reduce the production of gene products. Examples of gene repression processes that reduce transcription include, but are not limited to: gene repression processes that inhibit the formation of the transcription initiation complex, gene repression processes that reduce the rate of transcription initiation, gene repression processes that reduce the rate of transcriptional elongation, gene repression processes that reduce the sustained synthetic capacity of transcription, and gene repression processes that antagonize transcriptional activation (e.g., by blocking the binding of transcriptional activators). For example, gene repression can constitute prevention of activation and suppression of expression below existing levels. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription; the latter may be caused by epigenetic modifications of the gene.

[0102] The terms "repressor" or "repressor domain" are used interchangeably to refer to polypeptide factors that act as regulatory elements on DNA, inhibiting, repressing, or blocking DNA transcription, thereby repressing gene expression. In the context of this disclosure, the repressor domain, when linked to a DNA-binding protein, can prevent promoter transcription or otherwise suppress gene expression when it binds to a target nucleic acid. It is not intended to be theoretically constrained, but rather that transcriptional repressors may function through a variety of mechanisms, including physically blocking RNA polymerase channels through steric hindrance, altering the post-translational modification state of polymerase, modifying the epigenetic state of nascent RNA, altering the epigenetic state of DNA through methylation, altering the epigenetic state of DNA through histone deacetylation or regulation of nucleosome remodeling, or preventing enhancer-promoter interactions, thereby silencing genes or reducing gene expression levels.

[0103] The terms "long-term repressor fusion protein" or "LTRP" are used interchangeably with "repressor fusion protein" herein, and refer to a fusion protein comprising a DNA-binding protein (or a protein's DNA-binding domain) fused to one or more domains capable of repressing transcription of a target nucleic acid sequence. Optionally, the long-term repressor fusion protein of this disclosure may contain additional elements, such as linkers between any domains of the fusion protein, nuclear localization signals, nuclear export signals, and additional protein domains that confer additional activity to the long-term repressor fusion protein.

[0104] As used herein, the “LTRP:gRNA system” is a system for transcriptional repression and comprises a long-term repressor fusion protein including a CRISPR protein that catalyzes death and one or more linked repressor domains, and a guide nucleic acid (gRNA) that binds to the CRISPR protein that catalyzes death. For clarity, the system also includes any encoding DNA, RNA, or vector, etc., that can be used to generate the long-term repressor fusion protein and gRNA component of the system.

[0105] As used herein, "catalytically dead DNA-binding protein" refers to a protein or protein domain that can bind to DNA but cannot cleave or cut DNA. As used herein, "catalytically dead CRISPR protein" refers to a CRISPR protein lacking endonuclease activity. Those skilled in the art will understand that CRISPR proteins can be catalytically dead and can still perform other protein functions, such as DNA binding. Similarly, "catalytically dead CasX" refers to a CasX protein lacking endonuclease activity but still capable of performing other protein functions, such as DNA binding.

[0106] As used herein, “recombination” means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps, producing a construct with a structural coding or non-coding sequence that differs from the endogenous nucleic acid found in natural systems. Typically, the DNA sequence 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 capable of being expressed by a recombinant transcription unit contained in a cellular or cell-free transcription and translation system. Such sequences can be provided in the form of open reading frames (ORFs) unaffected by internal untranslated sequences or introns typically present in eukaryotic genes. Genomic DNA containing the relevant sequences can also be used to form recombinant genes or transcription units. Untranslated DNA sequences may appear at the 5' or 3' of the ORF, where such sequences do not interfere with the operation or expression of the coding region and can indeed regulate the production of the desired product through various mechanisms (see “enhancers” and “promoters” above).

[0107] The term "recombinant polynucleotide" or "recombinant nucleic acid" refers to non-naturally occurring nucleic acids, for example, created by artificially combining segments of two otherwise separated sequences. This artificial combination is typically achieved through chemical synthesis or by manipulating the separated segments of the nucleic acid, for example, through genetic engineering techniques. This is often done by replacing codons with redundant codons encoding the same or conserved amino acids, while typically introducing or removing sequence recognition sites. Alternatively, the artificial combination is performed to join nucleic acid segments having the desired function together to generate the desired functional combination. This artificial combination is typically achieved through chemical synthesis or by manipulating the separated segments of the nucleic acid, for example, through genetic engineering techniques.

[0108] Similarly, the terms "recombinant polypeptide" or "recombinant protein" refer to polypeptides or proteins that are not naturally occurring, for example, created by artificially combining segments of two otherwise separated amino acid sequences. Thus, for example, proteins containing heterologous amino acid sequences are recombinant.

[0109] As used herein, “lipid nanoparticle” or “LNP” refers to a particle comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, cofactor phospholipids, and PEG-modified lipids) and cholesterol, with at least one dimension on the nanometer scale (e.g., 1-1,000 nm). Specific components of LNPs are described more fully below. Lipid nanoparticles may be included in formulations that can be used to deliver active agents or therapeutic agents, such as nucleic acids (e.g., mRNA), to target sites of interest (e.g., cells, tissues, organs, tumors, etc.). The lipid nanoparticles of this disclosure may contain nucleic acids. Such lipid nanoparticles typically comprise neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Active agents or therapeutic agents, such as nucleic acids, may be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space encapsulated by some or all of the lipid portion of the lipid nanoparticle, thereby protecting them from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cells (e.g., adverse immune responses).

[0110] As used herein, “lipid encapsulation” refers to lipid nanoparticles that provide an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA, gRNA, or both), with complete, partial, or both encapsulation. Nucleic acids (e.g., mRNA) can be completely encapsulated in lipid nanoparticles.

[0111] As used herein, “lipoproteins,” such as VLDL, LDL, and HDL, refer to a group of proteins found in serum, plasma, and lymph that play a role in lipid transport. Each lipoprotein has a different chemical composition; for example, HDL has a higher protein-to-lipid ratio, while VLDL has a lower protein-to-lipid ratio.

[0112] As used herein, “atherosclerosis” refers to arteriosclerosis affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. Fatty deposits, known as “atherosclerotic plaques” or “atherosclerotic plaques,” are primarily composed of cholesterol and other fats, calcium, and scar tissue, and they damage the arterial intima.

[0113] As used in this article, "coronary heart disease (CHD)" refers to the narrowing of the small blood vessels that supply blood and oxygen to the heart, which is usually a result of atherosclerosis.

[0114] As used in this article, "dyslipidemia" refers to disorders of lipid and / or lipoprotein metabolism, including excessive or insufficient production of lipids and / or lipoproteins. Dyslipidemia can manifest as elevated levels of lipids such as chylomicrons, cholesterol, and triglycerides, as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.

[0115] As used in this article, "high-density lipoprotein-C" or "HDL-C" refers to cholesterol associated with high-density lipoprotein particles. The concentration of HDL-C in serum (or plasma) is usually quantified in mg / dL or nmol / L. "Serum HDL-C" and "Plasma HDL-C" refer to HDL-C in serum and plasma, respectively.

[0116] As used in this article, "low-density lipoprotein cholesterol (LDL-C)" refers to cholesterol carried by low-density lipoprotein particles. The concentration of LDL-C in serum (or plasma) is usually quantified in mg / dL or nmol / L. "Serum LDL-C" and "Plasma LDL-C" refer to LDL-C in serum and plasma, respectively.

[0117] As used in this article, “hypercholesterolemia” refers to a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol, and VLDL-cholesterol, according to the expert panel report guidelines of the National Cholesterol Education Program (NCEP) on the detection and evaluation of treatment of hypercholesterolemia in adults (see Arch. Int. Med. 148: 36(1988)).

[0118] As used in this article, "hyperlipidemia" or "hyperlipidemia" is a condition characterized by elevated serum or circulating (plasma) lipids. This condition manifests as abnormally high concentrations of fats. The lipid components in circulating blood are cholesterol, low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), chylomicrons, and triglycerides. The Fredrickson classification of hyperlipidemia is based on patterns of triglycerides (TG) and cholesterol-rich lipoprotein particles, as measured by electrophoresis or ultracentrifugation, and is commonly used to characterize the primary causes of hyperlipidemia, such as hypertriglyceridemia.

[0119] As used in this article, "triglycerides" or "TG" refers to lipids or neutral fats composed of glycerol and three fatty acid molecules.

[0120] As used in this article, "hypertriglyceridemia" refers to a condition characterized by elevated triglyceride levels. Its causes include primary (i.e., genetic factors) and secondary (other underlying causes such as diabetes, metabolic syndrome / insulin resistance, obesity, lack of exercise, smoking, excessive alcohol consumption, and a high-carbohydrate diet) factors, or often a combination of both.

[0121] As used in this article, “diabetes mellitus” or “diabetes” is a syndrome characterized by metabolic disorder and abnormally high blood sugar (hyperglycemia), caused by insufficient insulin levels or decreased insulin sensitivity. Characteristic symptoms include excessive urination (polyuria) due to high blood sugar levels, excessive thirst and increased fluid intake in an attempt to compensate for increased urination (polydipsia), blurred vision due to the optical effects of high blood sugar on the eyes, unexplained weight loss, and somnolence.

[0122] As used in this article, "diabetic dyslipidemia" or "type 2 diabetes with dyslipidemia" refers to a condition characterized by type 2 diabetes, low HDL-C, high triglycerides (TG), and high levels of small, dense LDL particles.

[0123] As used herein, the term "contact" refers to 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 share a physical connection; for example, hybridization is possible if the sequences share sequence similarity.

[0124] "Dissociation constant" or "K" d "Can be used interchangeably and refers to the affinity between the ligand "L" and the protein "P"; that is, the degree to which the ligand binds tightly to a specific protein. Formula K can be used. d=[L] [P] / [LP] is calculated, where [P], [L] and [LP] represent the molar concentrations of the protein, ligand and complex, respectively.

[0125] As used herein, the term "knockdown" refers to a reduction in the expression of a gene or one or more of its gene products. As a result of gene knockdown, protein activity or function may be reduced or eliminated.

[0126] A polynucleotide or polypeptide has a certain percentage of “sequence similarity” or “sequence identity” with another polynucleotide or polypeptide, meaning that when aligned, the percentages of bases or amino acids are the same and in the same relative positions. Sequence similarity (sometimes referred to as similarity percentage, identity percentage, or homology) can be determined in a variety of different ways. To determine sequence similarity, sequences can be aligned using methods and computer programs known in the art, including BLAST (available at ncbi.nlm.nih.gov / BLAST). The percentage of complementarity between specific segments of nucleic acid sequences within a nucleic acid can be determined using any convenient method. Exemplary methods include the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (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, GeneticsComputer Group, University Research Park, Madison Wisconsin), for example, using the default settings that employ the algorithm of Smith and Waterman (Advances in Appl. Math., 1981, 2, 482-489).

[0127] The terms “peptide” and “protein” are used interchangeably herein and refer to a polymeric form of amino acids of any length, including encoded and non-encoded amino acids, chemically modified or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences.

[0128] A "vector" or "expression vector" is a replicon, such as a plasmid, bacteriophage, virus, or kinase, which may include another DNA segment, i.e., an expression cassette, to induce the replication or expression of another DNA segment in the cell.

[0129] As used herein, the terms “naturally occurring,” “unmodified,” or “wild-type” used for nucleic acids, peptides, cells, or organisms refer to nucleic acids, peptides, cells, or organisms found in nature.

[0130] As used herein, “mutation” means the insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides compared to the wild-type or reference amino acid sequence or the wild-type or reference nucleotide sequence.

[0131] As used herein, the term "isolated" is intended to describe a polynucleotide, polypeptide, or cell in an environment different from the environment in which the polynucleotide, polypeptide, or cell naturally exists. Isolated genetically modified host cells can exist within a mixed population of genetically modified host cells.

[0132] As used herein, “host cell” refers to a eukaryotic, prokaryotic, or multicellular cell (e.g., a cell line) cultured as a single-celled entity, said eukaryotic or prokaryotic cell being used as a recipient of nucleic acids (e.g., an AAV vector), and includes the progeny of the original cell that has been genetically modified with nucleic acids. It should be understood that the progeny of a single cell may not necessarily be identical to the original parent in morphology, genome, or total DNA complement due to natural, accidental, or intentional mutations. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which a heterologous nucleic acid, such as an AAV vector, has been introduced.

[0133] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. For example, a group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains consists of serine and threonine; a group of amino acids with amide-containing side chains consists of asparagine and glutamine; a group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains consists of lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conserved amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0134] As used herein, the terms "treatment" or "treating" are used interchangeably and refer to a method of achieving a beneficial or desired outcome, including but not limited to therapeutic and / or preventative benefits. A therapeutic benefit means the eradication or improvement of the underlying condition or disease being treated. A therapeutic benefit can also be achieved by eradicating or improving one or more symptoms or improving one or more clinical parameters associated with the underlying disease, resulting in an observed improvement in the subject, although the subject may still have the underlying condition.

[0135] As used herein, the terms "therapeutic effective amount" and "therapeutic effective dose" refer to the amount of a drug or biological agent, alone or as part of a composition, which, when administered in a single or repeated dose to a subject such as a human or laboratory animal, is capable of having any detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition. Such effects are not necessarily absolutely beneficial.

[0136] As used herein, “administration” means the method of giving a subject a dose of a compound (e.g., a composition of this disclosure) or a composition (e.g., a pharmaceutical composition).

[0137] "Subjects" are mammals. Mammals include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats, and other rodents.

[0138] The term "low-density lipoprotein (LDL)" refers to one of the five major groups of lipoproteins, ranging from the lowest density (particles with a lower weight-to-volume ratio) to the highest density (particles with a larger weight-to-volume ratio): chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL). Lipoproteins transport lipids (fats) around the body in the extracellular fluid, thereby facilitating the transfer of fats into the cell body via receptor-mediated endocytosis. LDL particles have a diameter of approximately 220–275 angstroms.

[0139] The "low-density lipoprotein (LDL) receptor" is an 839-amino acid receptor protein (after removing the 21-amino acid signal peptide) that mediates the endocytosis of cholesterol-rich LDL particles. It is a cell surface receptor that recognizes the apoE proteins B100 and apoE, found in chylomicron remnants and VLDL remnants (IDL), leading to LDL-cholesterol binding and endocytosis. This process occurs in all nucleated cells, but primarily in the liver, which removes approximately 70% of LDL from circulation. The human LDLR gene is partially described as reference sequence NG_009060.1 in the NCBI database (ncbi.nlm.nih.gov), which is incorporated herein by reference.

[0140] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated as incorporated by reference.

[0141] I. General Methods

[0142] Unless otherwise indicated, the practice of this invention employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, techniques found in standard textbooks such as *Molecular Cloning: A Laboratory Manual*, 3rd edition (Sambrook et al., Harbor Laboratory Press, 2001); *Short Protocols in Molecular Biology*, 4th edition (edited by Ausubel et al., John Wiley & Sons, 1999); *Protein Methods* (Bollag et al., John Wiley & Sons, 1996); *Nonviral Vectors for Gene Therapy* (edited by Wagner et al., Academic Press, 1999); and *Viral Vectors* (edited by Kaplift and Loewy). The publications of the aforementioned standard textbooks are incorporated herein by reference: Academic Press, 1995; *Immunology Methods Manual* (edited by I. Lefkovits, Academic Press, 1997); and *Cell and Tissue Culture: Laboratory Procedures in Biotechnology* (Doyle and Griffiths, John Willie & Son Publishing, 1998).

[0143] Where a range of values ​​is provided, it should be understood that the range includes the endpoints and encompasses every intermediate value between the upper and lower limits of the range (to one-tenth of the unit of the lower limit, unless otherwise explicitly stated) and any other stated values ​​or intermediate values ​​within the range. The upper and lower limits of these smaller ranges may be independently included in and also encompassed by any explicitly excluded limits within the stated range. Where the stated range includes one or both of the included limits, it also includes ranges excluding one or both of these included limits.

[0144] 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 pertains. All publications referenced herein are incorporated by way of citation to disclose and describe the methods and / or materials in connection with the cited publications.

[0145] It should be understood that, for clarity, certain features of this disclosure described in the context of individual embodiments may also be provided in combination in a single embodiment. In other instances, for brevity, various features of this disclosure described in the context of a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments contemplated in relation to this disclosure are specifically covered by this disclosure and disclosed herein, as if each and every combination were individually and explicitly disclosed herein. Furthermore, all sub-combinations of various embodiments and their elements are also specifically covered by this disclosure and disclosed herein, as if each and every such sub-combination were individually and explicitly disclosed herein.

[0146] II. Systems for epigenetic modification and repression of the PCSK9 gene

[0147] In a first aspect, this disclosure provides a system comprising or encoding a long-term repressor fusion protein (“LTRP”), said LTRP comprising a DNA-binding protein and a repressor domain capable of binding to a target nucleic acid sequence of the PCSK9 gene that targets transcriptional repression, silencing, and / or epigenetic modifications. This disclosure also provides a system comprising or encoding an LTRP. In some cases, said system is designed to repress transcription of the PCSK9 gene in eukaryotic cells with mutations.

[0148] As used herein, the terms "system" and "composition" are used interchangeably. This disclosure also provides nucleic acids encoding the systems provided herein. Methods for preparing said systems and for using said systems, including methods for gene repression and / or epigenetic modification and methods for treating PCSK9-related diseases, are also provided herein. For clarity, the term "system" also includes any encoding DNA, RNA, or vectors, etc., that can be used to generate the repressor fusion protein and gRNA component of said systems.

[0149] In some embodiments, the DNA-binding protein used for the long-term repressor fusion protein of this disclosure comprises a zinc finger (ZF) or TALE (transcription activator-like effector) protein or its DNA-binding domain, also referred to herein as a DNA-binding protein, which binds to the target nucleic acid but does not cleave it. The DNA-binding domain of TALE comprises a tandem array of customizable monomers 33-34 amino acids (aa) long, which can theoretically be assembled to recognize any genetic sequence that follows a repeat sequence binding a one-base-pair recognition code (Jain, S. et al., TALEN outperforms Cas9 in editing heterochromatin targetsites. *Nature Communications* 12:606 (2021)). TALE's specificity for binding DNA stems from two polymorphic amino acids, namely the so-called repeat variable double residue (RVD) located at positions 12 and 13 of the repeat unit. The DNA-binding specificity of TALE can be arbitrarily altered by rearranging the repeat sequence. Zinc finger proteins are transcription factors, with each finger recognizing 3-4 DNA bases. By mixing and matching these finger modules, ZFs can be customized to target specific sequences. An exemplary ZF capable of binding to the PCSK9 gene is described in WO2018049009A2.

[0150] In some embodiments, the DNA-binding protein used for the long-term repressor fusion protein is a catalytically dead CRISPR protein of type 1 or type 2. Catalytically dead CRISPR proteins are also referred to in the art as “catalytically inactivated” CRISPR proteins. In one embodiment, the type 2 protein is the catalytically dead Cas9. In another embodiment, the type 2 CRISPR protein is selected from the group consisting of type II, type V, or type VI proteins. In one embodiment, the type 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 catalytically dead due to a specific mutation, as described herein. CRISPR-based systems further include guide nucleic acids, such as guide ribonucleic acid (gRNA), which have a target sequence complementary to the target sequence of the PCSK9 gene, so as to be bound and repressed by a complex of the fusion protein (CRISPR protein and linked repressor domain) and gRNA.

[0151] In some embodiments, this disclosure provides a system comprising or encoding a long-term repressor fusion protein comprising a CasX CRISPR nuclease protein that catalyzes death and a linked repressor domain, a guide ribonucleic acid (gRNA) comprising a target sequence complementary to a target nucleic acid sequence of a PCSK9 gene that targets transcriptional repression, silencing, or downregulation, and nucleic acid encoding the long-term repressor fusion protein and / or gRNA. In some embodiments, the system comprises the long-term repressor fusion protein and the gRNA of this disclosure as a gene repressor pair (“LTRP:gRNA system”), the gene repressor pair being capable of forming a ribonucleoprotein (RNP) complex and binding to the PCSK9 target nucleic acid. In other embodiments, this disclosure provides a system encoding nucleic acid of the long-term repressor fusion protein and gRNA. Still in other embodiments, this disclosure provides a system of gRNA for certain particulate modulators (e.g., LNPs) described herein and mRNA encoding the long-term repressor fusion protein.

[0152] This article also provides methods for preparing long-term repressor fusion proteins and gRNAs, as well as methods for using the LTRP:gRNA system, including methods for gene repression and / or epigenetic modification of the PCSK9 gene and methods for treating PCSK9-related diseases or conditions. The DNA-binding proteins (e.g., dCasX) of the LTRP:gRNA system, the linked repressor domains, the gRNA components and their characteristics, delivery methods, and methods for using said system to repress, downregulate, or silence the PCSK9 gene are described in more detail below.

[0153] In some embodiments, this 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 with acquired functional mutations. In some cases, the system is designed to repress transcription of the wild-type PCSK9 gene in eukaryotic cells. Alternatively, the system is designed to repress transcription of a mutant allele of the PCSK9 gene in eukaryotic cells. Generally, any part of the PCSK9 gene can be targeted using the programmable systems and methods provided herein, which are described in more detail.

[0154] The PCSK9 gene encodes the proprotein convertase subtilisin / kexin type 9 (“PCSK9”), a protein that binds to receptors on low-density lipoprotein particles (LDL) for transporting LDL into cells. The PCSK9 gene covers the sequence chr1:55,039,476–55,064,853 across the human genome (GRCh38 / hg38) (symbol refers to chromosome 1 (chr1), starting at 55,039,476 bp to 55,064,853 bp on chromosome 1 (Homo sapiens Update Annotation Release 109.20190905, GRCh38.p13) (NCBI). The human PCSK9 gene is partially described as reference sequence NG_009061.1 in the NCBI database (ncbi.nlm.nih.gov), which is incorporated herein by reference. The PCSK9 locus has 12 exons and produces 3636 The bp mRNA encodes a 692-amino acid protein, which, after synthesis, undergoes an autocatalytic cleavage reaction to remove the predomain, resulting in an activated protein with 540 amino acids. The predomain remains attached to the catalytic domain and resistin-like domain, possibly because the predomain acts as a chaperone protein and promotes folding and secretion (Seidah, NG et al., Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci USA) 100(3):928 (2003)). Secretory proprotein convertase neuronal apoptosis-regulating convertase 1 (NARC-1): liver regeneration and neuronal differentiation (Seidah, NG et al.). This protein, also known as neuronal apoptosis-regulating convertase, is a serine protease belonging to the proteinase K subfamily of subtilisinases.

[0155] The human PCSK9 gene (HGNC:20001) encodes a sequence containing...

[0156] The protein (Q8NBP7) of (SEQ ID NO: 1823).

[0157] III. Proteins that catalyze death in repressor systems

[0158] In some embodiments, the DNA-binding protein used for the long-term repressor fusion protein of this disclosure is a zinc finger (ZF) or TALE (transcription activator-like effector) protein, which can bind to the PCSK9 target nucleic acid but does not cleave the target nucleic acid.

[0159] In some embodiments, the DNA-binding protein is a class 1 or class 2 CRISPR protein that catalyzes cell death. 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 the cell-catalyzing Cas9. In another embodiment, the class 2 CRISPR protein is selected from the group consisting of type II, type V, or type VI proteins. 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 catalyzing cell death through a specific mutation, as described herein. In another embodiment, the class 2 CRISPR type V protein is the cell-catalyzing CasX protein.

[0160] The CasX proteins disclosed herein, including dCasX, contain the following domains: non-target chain binding (NTSB) domain, target chain loading (TSL) domain, helical I domain, helical II domain, oligonucleotide binding domain (OBD) and RuvC domain, and in some cases, the domains can be further divided into subdomains, as listed in Table 1.

[0161] In the context of this disclosure, the CasX used in the system is catalytically dead (dCasX); this is achieved by introducing a mutation at a selected position in the RuvC sequence, as described below.

[0162] a. Referencing the CaSX protein

[0163] This disclosure provides a naturally occurring CasX protein (referred to herein as the "reference CasX protein") which is subsequently modified to produce the engineered dCasX of this disclosure. For example, the reference CasX protein can be isolated from naturally occurring prokaryotes such as species of the class Delta Proteobacteria, phylum Planctomycetes, or provisionally phylum Morulatum. The reference CasX protein (which may be interchangeably referred to herein as the reference CasX polypeptide) is a class 2 type V CRISPR / Cas endonuclease belonging to the CasX protein family (which may be interchangeably referred to herein as Cas12e), which interacts with guide RNA to form a ribonucleoprotein (RNP) complex.

[0164] In some cases, the reference CasX protein is isolated from or derived from the class Deltaproteobacter and contains the sequence of SEQ ID NO: 1.

[0165] In some cases, the reference CAX protein is isolated from or derived from the phylum Planctomycetes and contains the sequence of SEQ ID NO: 2.

[0166] In some cases, the reference CasX protein is isolated from or derived from the provisional phylum Candidatus Sungbacteria and contains the sequence of SEQ ID NO: 3.

[0167] b. Class 1 or 2 CRISPR proteins that catalyze cell death

[0168] In the long-term repressor fusion protein and gene repressor system comprising the present disclosure, the catalytically dead class 1 or class 2 CRISPR protein is catalytically dead because it cannot cleave DNA, but retains the ability to bind to target nucleic acids when complexed with 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 selected domains. The present disclosure provides catalytically dead CasX variants (which are interchangeably referred to herein as “dCasX variants” or “dCasX variant proteins”) wherein the catalytically dead CasX variants comprise multiple modifications in the RuvC domain relative to the catalytically dead version of a reference CasX protein comprising the sequences of SEQ ID NO: 1-3 (described above). In some embodiments, the catalytically dead reference CasX protein comprises substitutions relative to residues 672, 769, and / or 935 of SEQ ID NO: 1. In some embodiments, the catalytically dead reference CasX protein comprises substitutions relative to D672A, E769A, and / or D935A of SEQ ID NO: 1. In other embodiments, the catalytic death reference CasX protein comprises substitutions at amino acids 659, 756, and / or 922 relative to SEQ ID NO: 2. In some embodiments, the catalytic death reference CasX protein comprises substitutions at D659A, E756A, and / or D922A relative to SEQ ID NO: 2. Exemplary RuvC domains of the dCasX of this disclosure comprise 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, having one or more amino acid modifications relative to the RuvC cleavage domain sequence, wherein the dCasX variant exhibits one or more improved properties relative to the properties of the reference dCasX. In further embodiments, the catalytic death CasX variant protein comprises all or part of the deletion of the RuvC domain of the reference CasX protein. It should be understood that the same aforementioned substitutions or omissions can be similarly introduced into CasX variants known in the art, thereby producing dCasX variants (see, for example, exemplary sequences in WO2022120095A1 and US11,560,555, which are incorporated herein by reference).

[0169] In some embodiments, a long-term repressor fusion protein comprising a dCasX variant having a linked repressor domain exhibits at least one improved property compared to a long-term repressor fusion protein comprising a reference dCasX protein comprising a substantially linked repressor domain. All dCasX variants that improve one or more functions or properties of a long-term repressor fusion protein comprising a dCasX variant having a linked repressor domain compared to a long-term repressor fusion protein comprising a reference dCasX protein are contemplated within the scope of this disclosure. In some embodiments, the modification is a mutation of one or more amino acids of the reference dCasX, rather than those mutations that cause catalytic death of dCasX. For example, the dCasX variant may comprise one or more amino acid substitutions, insertions, deletions, or exchanges, or any combination thereof, relative to the reference dCasX protein sequence. In the substitutions described herein, any amino acid may be substituted with any other amino acid. The substitution may be a conserved substitution (e.g., a basic amino acid is substituted with another basic amino acid). The substitution may be a non-conserved substitution (e.g., a basic amino acid is substituted with an acidic amino acid or vice versa). For example, proline in the reference dCasX protein can be substituted for any one of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine to produce the disclosed dCasX variant protein. In some embodiments, the dCasX variant exhibits improved properties compared to the reference dCasX. Exemplary improved properties of dCasX variant embodiments include, but are not limited to, improved variant folding, increased binding affinity to target nucleic acids, improved ability to utilize broader PAM sequences in transcriptional repression and / or binding of target nucleic acids, improved target DNA unwinding, increased target strand loading, increased binding of non-target DNA strands, improved protein stability, increased ability to complex with gRNA, increased binding affinity to gRNA, improved protein:gRNA (RNP) complex stability, and, in the presence of the linked repressor domain and when complexed as an RNP, increased repressor activity, improved repressor specificity to target nucleic acids, reduced off-target repression, and an increased percentage of eukaryotic genome that can be effectively repressed and / or epigenetically modified. In some embodiments, the improvements of the dCasX variant relative to the reference dCasX protein are characterized by an improvement of at least about 1.1 to about 100,000-fold.In some embodiments, the improvement of the dCasX variant relative to the reference dCasX protein is characterized by an improvement of at least about 1.1 to about 10,000 times, an improvement of at least about 1.1 to about 1,000 times, an improvement of at least about 1.1 to about 500 times, an improvement of at least about 1.1 to about 400 times, an improvement of at least about 1.1 to about 300 times, an improvement of at least about 1.1 to about 200 times, an improvement of at least about 1.1 to about 100 times, an improvement of at least about 1.1 to about 50 times, an improvement of at least about 1.1 to about 40 times, an improvement of at least about 1.1 to about 30 times, an improvement of at least about 1.1 to about 20 times, an improvement of at least about 1.1 to about 10 times, and an improvement of at least about 1.1 to about 9 times. The improvements are described as follows: at least about 1.1 to about 8 times, at least about 1.1 to about 7 times, at least about 1.1 to about 6 times, at least about 1.1 to about 5 times, at least about 1.1 to about 4 times, at least about 1.1 to about 3 times, at least about 1.1 to about 2 times, at least about 1.1 to about 1.5 times, at least about 1.5 to about 3 times, at least about 1.5 to about 4 times, at least about 1.5 to about 5 times, at least about 1.5 to about 10 times, at least about 5 to about 10 times, at least about 10 to about 20 times, at least 10 to about 30 times, at least 10 to about 50 times, or at least 10 to about 100 times. In some embodiments, the improvement of the dCasX variant relative to the reference dCasX protein is characterized by an improvement of at least about 10 to about 1000 times. Further disclosures regarding the characteristics of the improvement are described below.

[0170] In other embodiments, modification is the substitution of one or more domains of a reference dCasX with one or more domains from a different CasX. In some embodiments, insertion includes inserting a portion or all of a domain from a different CasX protein. Mutations can occur in any one or more domains of the dCasX variant and can include, for example, partial or complete deletion of one or more domains, or substitution, deletion, or insertion of one or more amino acids in any domain. Domains of the dCasX protein include non-target strand binding (NTSB) domains, target strand loading (TSL) domains, helical I domains, helical II domains, oligonucleotide binding domains (OBD), and RuvC DNA cleavage domains, which may further include subdomains described below.

[0171] In some embodiments, the dCasX variant protein contains 800 to 1100 amino acids or 900 to 1000 amino acids.

[0172] Compared to equivalent assay systems containing fusion proteins of a reference dCasX protein and gRNA, long-term repressor fusion proteins comprising the dCasX variant of this disclosure and the linked repressor domain exhibit enhanced, efficient binding to target nucleic acids when complexed with gRNA into an RNP, utilizing and binding to a PAM TC motif, including a PAM sequence selected from TTC, ATC, GTC, or CTC. In the foregoing, the PAM sequence is located at at least one nucleotide 5' of the non-target strand of a protospacer that is identical to the target sequence of the gRNA.

[0173] In some embodiments, RNPs comprising a long-term repressor fusion protein including a dCasX variant having a linked repressor domain and a gRNA of the present disclosure at a concentration of 20 pM or less are capable of binding to double-stranded DNA targets with an efficiency of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. In one embodiment, compared to RNPs in equivalent assay systems comprising a reference dCasX protein including a linked repressor domain and a dCasX variant and a gRNA variant, the RNP comprising a long-term repressor fusion protein including a reference dCasX protein including a linked repressor domain exhibits greater binding to the target sequence in the target nucleic acid, wherein the PAM sequence of the target nucleic acid is TTC. In another embodiment, compared to an RNP in a comparable assay system that includes a long-term repressor fusion protein and gRNA comprising a reference dCasX protein having a linked repressor domain, an RNP comprising a long-term repressor fusion protein and gRNA comprising a linked repressor domain exhibits a greater binding affinity for the target sequence in the target nucleic acid, wherein the PAM sequence of the target nucleic acid is ATC. In another embodiment, compared to an RNP in a comparable assay system that includes a reference dCasX protein having a linked repressor domain and gRNA, an RNP comprising a long-term repressor fusion protein and gRNA comprising a linked repressor domain, an RNP comprising a long-term repressor fusion protein and gRNA comprising a linked repressor domain exhibits a greater binding affinity for the target sequence in the target nucleic acid, wherein the PAM sequence of the target nucleic acid is CTC. In another embodiment, compared to an equivalent long-term repressor fusion protein and gRNA comprising a reference dCasX protein with a linked repressor domain in an equivalent assay system, an RNP comprising a long-term repressor fusion protein and gRNA variant of a dCasX variant exhibits greater binding affinity for the target sequence in the target nucleic acid, wherein the PAM sequence of the target nucleic acid is GTC. In other embodiments, compared to an equivalent repressor fusion protein and gRNA comprising a reference dCasX protein with a linked repressor domain in an equivalent assay system, an RNP comprising a repressor fusion protein and gRNA comprising a reference dCasX protein with a linked repressor domain, an RNP comprising a repressor fusion protein and gRNA variant of a dCasX variant exhibits greater binding affinity for the target sequence in the target nucleic acid, wherein the PAM sequence of the target nucleic acid is GTC, TTC, ATC, or CTC. In the foregoing embodiments, the increased binding affinity to one or more PAM sequences is at least 1.5 times or more the binding affinity of the RNP of any of the reference dCasX proteins (modified by SEQ ID NO: 1-3) having the linked repressor domain and the gRNAs of SEQ ID NO: 1731-1743 in Table 8 to the PAM sequences.

[0174] c. dCasX variant proteins with domains derived from proteins from multiple sources

[0175] In some embodiments, this disclosure provides chimeric dCasX variant proteins for long-term repressor fusion proteins.

[0176] As used herein, a "chimeric dCasX" protein refers to both a catalytically dead CasX protein containing at least two domains from different sources and a catalytically dead CasX protein containing at least one domain that is itself chimeric. Thus, in some embodiments, a chimeric dCasX protein is a protein comprising at least two domains isolated from 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, a chimeric dCasX protein is a protein containing at least one domain as a chimeric domain; for example, in some embodiments, a portion of the domain contains substitutions from different CasX proteins (from a reference CasX protein or another dCasX protein).

[0177] In some embodiments, at least one chimeric domain may be any of the NTSB, TSL, helix I, helix II, OBD, or RuvC domains as described herein. In the case of split or discontinuous domains such as helix I, RuvC, and OBD, a portion of the discontinuous domain may be replaced by a corresponding portion from any other source. In some embodiments, the helix I-II domain (sometimes referred to as helix Ia) of dCasX derived from SEQ ID NO: 2 is replaced by the corresponding helix I-II sequence from SEQ ID NO: 1, thereby producing a chimeric dCasX protein.

[0178] In some embodiments, the helical I-II and NTSB domains of dCasX derived from SEQ ID NO: 2 are replaced by the corresponding helical I-II and NTSB sequences from SEQ ID NO: 1, thereby producing a chimeric dCasX protein.

[0179] Chimeric dCasX variant proteins may comprise the NTSB, TSL, helix II, helix I-II, helix II, OBD-I, and OBD-II domains from the CasX protein of SEQ ID NO: 2, and the RuvC-I and / or RuvC-II domains from the CasX protein of SEQ ID NO: 1, or vice versa, wherein mutations or other sequence alterations are introduced to produce a variant of catalytic death with improved variant properties 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, the 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, the dCasX for the long-term repressor fusion protein comprises an NTSB domain and a helical I-II domain from SEQ ID NO: 1 and a helical II domain from SEQ ID NO: 2; the latter being a chimeric domain. It should be understood that the dCasX variant has additional amino acid changes at selected positions (relative to the reference sequence) and that the resulting chimeric dCasX protein has improved characteristics relative to the reference dCasX protein. The sequences in Table 2 having the NTSB domain and helical I-II domains from SEQ ID NO: 1 and the helical II 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 NO: 9-11), 522-527 (SEQ ID NO: 12-17), 532 (SEQ ID NO: 22), 593 (SEQ ID NO: 25), 676 (SEQ ID NO: 28, with L169K substitution in the NTSB domain), and 812 (SEQ ID NO: 29). The coordinates of the CasX domains in the reference CasX proteins of SEQ ID NO: 1 and SEQ ID NO: 2 are provided in Table 1 below. Those skilled in the art will understand that the domain boundaries indicated in Table 1 below are approximate, and protein fragments whose boundaries differ from those given in the table by one, two, or three amino acids may have the same activity as the domains described below.

[0180] Table 1: Domain coordinates in the reference CAX protein

[0181]

[0182] In some embodiments, the dCasX variant protein in the long-term repressor fusion protein of this disclosure comprises a sequence selected from the group consisting of SEQ ID NO: 4-29 as shown in Table 2, wherein the sequence comprises a RuvC domain including one or more mutations that inactivate the cleavage activity of the RuvC domain; in other embodiments, the dCasX variant protein in the long-term repressor fusion protein of this disclosure comprises a sequence consisting of SEQ ID NO: 4-29 as shown in Table 2. The sequences of 4-29 are at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 89%, 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%, or at least about 99.5% identical. In some embodiments, the dCasX variant protein comprises a RuvC domain including one or more mutations that inactivate the cleavage activity of the RuvC domain. In some embodiments, the long-term repressor fusion protein comprising dCasX retains the ability to form an RNP with gRNA. In a particular embodiment, the dCasX variant protein used in the long-term repressor fusion protein of the gene repressor system of this disclosure comprises the sequence of SEQ ID NO: 4 (dCasX 491). In another particular embodiment, the dCasX variant protein used in the long-term repressor fusion protein of the gene repressor system of this disclosure comprises the sequence of SEQ ID NO: 6 (dCasX 515). In yet another particular embodiment, the dCasX variant protein used in the long-term repressor fusion protein of the gene repressor system of this disclosure comprises the sequence of SEQ ID NO: 29 (dCasX 812).

[0183] Table 2: dCasX variant sequences

[0184]

[0185] d. Affinity for gRNA

[0186] In some embodiments, a long-term repressor fusion protein comprising a dCasX with a linked repressor domain exhibits improved affinity for gRNA compared to a comparable long-term repressor fusion protein comprising a reference dCasX protein having a corresponding linked repressor domain, thereby enabling the formation of a ribonucleoprotein complex (RNP). For example, the increased affinity of the long-term repressor fusion protein for gRNA can enhance the affinity of K for generating the RNP complex. d The lower Kc of the long-term repressor fusion protein relative to the reference dCasX protein and the linked repressor domain can, in some cases, lead to the formation of a more stable ribonucleoprotein complex. In some embodiments, the Kc of the long-term repressor fusion protein relative to the reference dCasX protein and the linked repressor domain is lower than that of the gRNA. d The binding affinity of the long-term repressor fusion protein containing the dCasX variant to gRNA was increased by at least about 1.1 to about 10 times compared to the corresponding repressor fusion protein containing the catalytic death variant of the reference CasX protein of SEQ ID NO: 2. In some embodiments, the binding affinity of the long-term repressor fusion protein containing the dCasX variant to gRNA was increased by about 1.1 to about 10 times.

[0187] In some embodiments, the increased affinity of the long-term repressor fusion protein for gRNA results in increased stability of the ribonucleoprotein complex upon delivery to mammalian cells, including in vivo delivery to a subject. This increased stability can affect the function and utility of the complex in the subject's cells and produce improved pharmacokinetic properties in the blood upon delivery to the subject. In some embodiments, the increased affinity of the long-term repressor fusion protein and, consequently, the increased stability of the ribonucleoprotein complex allow for the delivery of lower doses of the long-term repressor fusion protein to a subject or cells while still retaining the desired activity; for example, in vivo or in vitro gene repression and / or epigenetic modifications. The increased ability to form RNPs and maintain their stable form can be assessed using in vitro assays known in the art.

[0188] In some embodiments, when both the long-term repressor fusion protein and the gRNA remain within the RNP complex, the higher affinity (tighter binding) of the dCasX variant of the long-term repressor fusion protein to the gRNA allows for a greater number of transcriptional repression and / or epigenetic modification events. The increased transcriptional repression events can be assessed using the assays described herein.

[0189] Methods for measuring the binding affinity of long-term repressor fusion proteins to gRNA include in vitro methods using purified long-term repressor fusion proteins and gRNA. If the gRNA or long-term repressor fusion protein is labeled with a fluorophore, the binding affinity of the long-term repressor fusion protein can be measured by fluorescence polarization. Alternatively or additionally, binding affinity can be measured by biomembrane interferometry, electrophoretic mobility shift assay (EMSA), or filter binding. Other standard techniques for quantifying the absolute affinity of RNA-binding proteins (such as the dCasX protein of this disclosure) for specific gRNAs (such as reference gRNAs and their variants) include, but are not limited to, isothermal calorimetry (ITC) and surface plasmon resonance (SPR).

[0190] e. Improved specificity for target nucleic acid sequences

[0191] In some embodiments, a long-term repressor fusion protein containing a dCasX variant protein with a linked repressor domain exhibits improved specificity for target nucleic acid sequences complementary to the target sequence of the gRNA, relative to a reference dCasX protein with a linked repressor domain. As used herein, “specificity,” sometimes referred to as “target specificity,” refers to the degree to which the CRISPR / Cas system ribonucleoprotein complex binds to off-target sequences that are similar to but not identical to the target nucleic acid sequence; for example, a long-term repressor fusion protein containing an RNP with a higher degree of specificity than a reference dCasX RNP with a linked repressor domain will exhibit reduced off-target methylation of the sequence. The reduced specificity and potentially harmful off-target effects of the long-term repressor fusion protein can contribute to achieving an acceptable therapeutic index for use in mammalian subjects. Without wishing to be bound by theory, amino acid changes in the helical I and II domains that increase the specificity of the long-term repressor fusion protein to the target nucleic acid chain are possible, and thereby can increase the overall specificity of the long-term repressor fusion protein to the target nucleic acid. In some embodiments, amino acid alterations that increase the specificity of the long-term repressor fusion protein to the target nucleic acid may also reduce the affinity of the long-term repressor fusion protein for DNA, but the overall benefit and safety of the composition are enhanced.

[0192] f. Repressor fusion proteins containing heterologous proteins

[0193] Within the scope of this disclosure, long-term repressor fusion proteins comprising a heterologous protein fused to a long-term repressor fusion protein are also contemplated for use in systems of this disclosure. This includes long-term repressor fusion proteins comprising an N-terminal or C-terminal fusion variant of a heterologous protein or its domain. In some embodiments, the long-term repressor fusion protein is fused to one or more proteins or their domains having the activity of interest.

[0194] In some cases, heteropeptides (fusion couplers) used with long-term repressor fusion proteins provide subcellular localization, i.e., the heteropeptides contain subcellular localization sequences (e.g., nuclear localization signals (NLS) for targeting the nucleus, sequences that decouple the fusion protein from the nucleus, nuclear export sequences (NES), sequences that retain the fusion protein in the cytoplasm, mitochondrial localization signals for targeting mitochondria, chloroplast localization signals for targeting chloroplasts, ER retention signals, etc.).

[0195] In some cases, the long-term repressor fusion protein includes a nuclear localization signal (NLS) (fused to said NLS). In some cases, the 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, or 8 or more NLS. In some cases, one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) are localized at or near the N-terminus and / or C-terminus of the long-term repressor fusion protein (e.g., within its 20 amino acids). In some cases, one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) are localized at or near the N-terminus of the long-term repressor fusion protein (e.g., within its 20 amino acids). In some cases, one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) are localized at or near the C-terminus of the long-term repressor fusion protein (e.g., within its 20 amino acids). In some cases, one or more NLSs (three or more, four or more, or five or more NLSs) are located at or near both the N-terminus and C-terminus of the long-term repressor fusion protein (e.g., within 20 amino acids). In some cases, a single NLS is located at the N-terminus and a single NLS is located at the C-terminus of the long-term repressor fusion protein. Those skilled in the art will understand that NLSs at or near the N-terminus or C-terminus of a protein can be located 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-terminus or C-terminus. In some embodiments, the NLS attached to the N-terminus of dCasX or the long-term repressor fusion protein is the same as the NLS attached to the C-terminus. In other embodiments, the NLS attached to the N-terminus of dCasX or the long-term repressor fusion protein is different from the NLS attached to the C-terminus. Representative conformations of long-term repressor fusion proteins with NLSs are shown. Figure 61In some embodiments, an NLS suitable for use with a long-term repressor fusion protein in a system of this disclosure comprises a sequence having at least about 85%, at least about 90%, or at least about 95% identity with or identical to sequences derived from: an NLS of the SV40 viral large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 30); an NLS derived from a nucleoside (e.g., a nucleoside bilateral NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 31); or a c-MYC NLS having the amino acid sequences PAAKRVKLD (SEQ ID NO: 32) or RQRRNELKRSP (SEQ ID NO: 33). In some embodiments, the NLS and short peptide linker attached to the N-terminus of the long-term repressor fusion protein is the sequence PKKKRKVSR (SEQ ID NO: 34). In some embodiments, the NLS and short peptide linker attached 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 short peptide linker attached to the C-terminus of the long-term repressor fusion protein are sequences TSPKKKRKV (SEQ ID NO: 3273). In some embodiments, the NLS attached to the N-terminus of the long-term repressor fusion protein is selected from the group consisting of N-terminal sequences as shown in Table 3. In some embodiments, the NLS comprises sequences selected from the group consisting of SEQ ID NOs: 30-97, 3273, and 3298. In some embodiments, the NLS comprises sequences selected from the group consisting of SEQ ID NOs: 34-97. In some embodiments, the NLS attached to the C-terminus of the long-term repressor fusion protein is selected from the group consisting of C-terminal sequences as shown in Table 4. In some embodiments, NLS suitable for use with the long-term repressor fusion protein in the system of this disclosure comprises sequences having at least about 80%, at least about 90%, or at least about 95% identity with one or more sequences in Table 3 or Table 4, or identical sequences thereto. Those skilled in the art will understand that any NLS sequence listed in Tables 3 and 4 may be fused to or near the N-terminus or C-terminus of the long-term repressor fusion protein described herein.

[0196] Table 3: N-terminal NLS amino acid sequence

[0197]

[0198] Table 4: C-terminal NLS amino acid sequences

[0199]

[0200] In some embodiments, one or more NLS are linked to a long-term repressor fusion protein or an adjacent NLS having an optional adaptor peptide. In some embodiments, the adapter 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 (SEQ ID NO: 104), GGSSG (SEQ ID NO: 105), GGSGG (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: 110), and PPPGPPP (SEQ ID NO: 111). 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), GGSGSGG (SEQ ID NO: 121), SSGNSNANSRGPSFSSGLV PLSLRGSH (SEQ ID NO: 122), GGPSSGAPPPSGGSPAGSPTSTEEGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE (SEQ ID NO: 123) and GGSGGG (SEQ ID NO: 124), where n is 1 to 5.

[0201] Generally, NLS (or multiple NLS) possess sufficient strength to drive the accumulation of long-term repressor fusion proteins in the nucleus of eukaryotic cells. Detection of nuclear accumulation can be performed using any suitable technique. For example, detectable markers can be fused with long-term repressor fusion proteins, allowing visualization of their intracellular location. The nucleus can also be isolated from the cell, and its contents can then be analyzed using any suitable method for protein detection, such as immunohistochemistry, Western blotting, or enzyme activity assays. Nuclear accumulation can also be determined indirectly.

[0202] IV. Long-term repressor domain fusion protein

[0203] This disclosure provides a system for long-term fusion proteins comprising DNA-binding proteins linked to multiple repressor domains, wherein the system is capable of binding to and repressing transcription of the PCSK9 gene, including through epigenetic modifications of the target nucleic acid. Exemplary DNA-binding proteins for fusion proteins include zinc finger (ZF), TALE (transcription activator-like effector) proteins, and CRISPR proteins that catalyze cell death.

[0204] In some embodiments, this disclosure provides a system for a long-term repressor fusion protein comprising a CRISPR protein, such as dCasX, that catalytically kills a target nucleic acid (gRNA) upon complexation with a guide ribonucleic acid (gRNA) containing a target sequence complementary to the target nucleic acid sequence of PCSK9, and wherein the system is capable of binding to the target nucleic acid of PCSK9 and repressing or silencing transcription of the PCSK9 gene. Examples of gene repression processes that reduce transcription include, but are not limited to, gene repression processes that inhibit the formation of the transcription initiation complex, gene repression processes that reduce the rate of transcription initiation, gene repression processes that reduce the rate of transcriptional elongation, gene repression processes that reduce the sustained synthetic capacity of transcription, and gene repression processes that antagonize transcriptional activation (e.g., by blocking the binding of transcriptional activators). For example, gene repression can constitute prevention of activation and suppression of expression below existing levels. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription; the latter may be caused by epigenetic modifications of the target nucleic acid.

[0205] Among repressor domains capable of repressing or silencing genes, the Krüppel-associated box (KRAB) repressor domain is the most powerful in the human genome system (Alerasool, N. et al., An efficient KRAB domain for CRISPRi applications. Nature Methods 17:1093 (2020)). KRAB-like domains are present in approximately 400 human zinc finger-based transcription factors that, upon binding of their linked dCasX to target nucleic acids, recruit additional repressor domains, such as, but not limited to, Trim28 (also known as Kap1 or Tif1-β). These repressor domains then assemble into protein complexes with chromatin regulators such as CBX5 / HP1α and SETDB1, inducing transcriptional repression of genes, but in a finite-time manner. Representative non-limiting examples of KRAB domains applicable to the systems of this disclosure include ZIM3 (SEQ ID NO: 129) and ZNF10 (SEQ ID NO: 128). This disclosure provides additional repressor domains from human origins, as well as a repressor domain from a non-human origin having significantly different sequences (referred to herein as “RD1”), which has been found to induce enhanced transcriptional repression compared to ZIM3 and ZNF10 when integrated into long-term repressor fusion protein constructs as described herein.

[0206] In some embodiments, this disclosure provides a system in which the modification conferred by using the LTRP:gRNA system is epigenetic, and therefore the silencing of the PCSK9 gene can be inherited through mechanisms other than edited DNA replication. As used herein, “epigenetic modification” means modification of DNA or DNA-associated histones, rather than alteration of the DNA sequence itself (e.g., substitution, deletion, or rearrangement), wherein the modification is performed by direct modification of system components or indirectly by recruiting one or more additional cellular components, but wherein the DNA target nucleic acid sequence itself is not edited to alter the sequence. For example, DNA methyltransferase 3A (DNMT3A) (or its catalytic domain) directly modifies DNA by methylation, while KRAB recruits the KAP-1 / TIF1β co-repressor complex, which acts as an effective transcriptional repressor, and can further recruit factors associated with DNA methylation and the 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 AcidsRes. 43(3): 1549 (2015)). Furthermore, the non-catalytically active DNMT3L cofactor, together with the cell's endogenous DNMT1, helps to establish a heritable methylation pattern after DNA replication.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 acting as a self-repressive domain for methyltransferases; and 2) it specifically interacts with the unmethylated histone H3 tail at lysine (K)4, leading to preferential DNA methylation that binds to the unmethylated chromatin H3 tail 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 Acid Research 38:4246(2010)). In some embodiments, including the ADD domain enhances transcriptional repression of the target gene when compared to an otherwise equivalent LTRP lacking the ADD domain. In other embodiments, the inclusion of the ADD domain enhances the specificity of transcriptional repression of the target gene when compared to an otherwise equivalent LTRP lacking the ADD domain. Supporting data for the foregoing are provided in the examples and in WO2023049742A2, which is incorporated herein by reference.

[0207] In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein linked to a first repressor domain, a second repressor domain, a third repressor domain, and a fourth repressor domain, wherein each repressor domain is distinct, 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 the first repressor domain, the second repressor domain, the third repressor domain, and the fourth repressor domain, wherein each repressor domain is distinct. In any of the foregoing embodiments, when the DNA-binding domain comprises a CRISPR protein that catalyzes cell death, the fusion protein is capable of forming an RNP with the systemic gRNA that binds to the target nucleic acid.

[0208] In some embodiments, the DNA-binding protein comprises a TALE that can bind to a target nucleic acid but does not cleave the target nucleic acid. In some embodiments, the DNA-binding protein comprises a zinc finger protein modified to bind to a target nucleic acid but not cleave the target nucleic acid. In some embodiments, the DNA-binding protein comprises a catalytically dead CRISPR protein that can bind to a target nucleic acid but does not cleave the target nucleic acid and can complex with gRNA to form an RNP. 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 (hereinafter referred to as "DNMT3A") from the DNMT3A protein as a second domain, and a DNMT3L interaction domain (hereinafter referred to as "DNMT3L") from the DNMT3L protein as a third domain. In some embodiments, the long-term repressor fusion protein comprises a catalytically dead CRISPR protein sequence, RD1, DNMT3A as a second domain, DNMT3L as a third domain, and an ATRX-DNMT3-DNMT3L domain (hereinafter referred to as "ADD") from the DNMT3A protein as a fourth domain. In some embodiments, the long-term repressor fusion protein comprises dCasX and further comprises the first and second NLSs described herein, as well as one or more adaptor peptides. In some embodiments, the long-term repressor fusion protein is capable of forming an RNP with gRNA that binds to the target nucleic acid. It has been found that when configured in the long-term repressor fusion protein relative to dCasX in a selected orientation, the use of the aforementioned domains can result in significant epigenetic modifications of the PCSK9 target nucleic acid upon complexation with gRNA having a target sequence complementary to the defined region of the PCSK9 gene, and the combination of repressor domains acts synchronously, thereby producing an additive or synergistic effect of transcriptional silencing of the target gene, depending on the conformation.

[0209] This article provides representative amino acid sequences of the components used in the long-term repressor fusion protein construct.

[0210] In some embodiments, the dCasX of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 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%, or at least about 99% identity with it. In some embodiments, the dCasX of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 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%, or at least about 99% identity with it. In some embodiments, the dCasX of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 4.

[0211] In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 128-1726, 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 with it. In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 128-1726. 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% sequence identity with it. 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 NO: 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 with it. 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 NO: 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 NO: 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 with it. 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 NO: 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 NO: 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 least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. 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 NO: 130-138. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the 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 with it. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the 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 with it. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 135. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the 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 with it. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 130. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 131. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 132. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 133. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 134.In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 135. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 136. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 137. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 138.

[0212] In some embodiments, the second repressor domain of the long-term repressor fusion protein is DNMT3A, which comprises the 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%, or at least about 99% identity with it. In some embodiments, the second repressor domain of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 126.

[0213] In some embodiments, the third repressor domain of the long-term repressor fusion protein is DNMT3L, which contains the 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 with it.

[0214] In some embodiments, an optional fourth repressor domain of the long-term repressor fusion protein 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 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 with it. In some embodiments, an optional fourth repressor domain of the long-term repressor fusion protein comprises the sequence of SEQ ID NO: 125. In some embodiments, the C-terminus of the ADD is linked to the N-terminus of the DNMT3A. It has been found that the addition of an ADD to a long-term repressor fusion protein comprising RD1, DNMT3A, and DNMT3L significantly enhances or increases the long-term repression and / or epigenetic modification of the target nucleic acid, as well as the specificity of the repression, compared to a long-term repressor fusion protein lacking an ADD. Exemplary data on improved repression and specificity of long-term repressor fusion proteins containing ADD are presented in examples and described in WO2023049742A2, which is incorporated herein by reference. In some embodiments, the fusion protein comprises one or more adaptor peptides selected from the group consisting of SEQ ID NO: 98-124, 3278-3289 (exemplary sequences are shown in Tables 5 and 58), and one or more NLS sequences selected from the group consisting of SEQ ID NO: 30-97, 32998, and 3299. Exemplary conformations of long-term repressor fusion proteins containing ADD are presented in Figure 61 middle.

[0215] In some embodiments, the long-term repressor fusion protein comprises DNMT3A, DNMT3L, a DNA-binding protein, and RD1 from the N-terminus to the C-terminus. In some embodiments, the long-term repressor fusion protein comprises ADD, DNMT3A, DNMT3L, a DNA-binding protein, and RD1 from the N-terminus to the C-terminus. In some embodiments, the DNA-binding protein may be a zinc finger, TALE, or a CRISPR protein that catalyzes cell death. In some embodiments, the long-term repressor fusion protein comprises NLS at the N-terminus, C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between DNMT3A and DNMT3L, between DNMT3L and the DNA-binding protein, and / or between the DNA-binding protein and RD1.

[0216] In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein, RD1, DNMT3A, and DNMT3L from its N-terminus to its C-terminus. In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein, RD1 and ADD, DNMT3A, and DNMT3L from its N-terminus to its C-terminus. In some embodiments, the DNA-binding protein may be a zinc finger, TALE, or a CRISPR protein that catalyzes death. In some embodiments, the long-term repressor fusion protein comprises an NLS at its N-terminus. In some embodiments, the long-term repressor fusion protein comprises an NLS between RD1 and DNMT3A. In some embodiments, the long-term repressor fusion protein comprises an NLS at its N-terminus, C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between the N-terminal NLS and the DNA-binding protein, between the DNA-binding protein and RD1, between RD1 and DNMT3A, or optionally between ADD, and / or between DNMT3A and DNMT3A.

[0217] In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein, DNMT3A, DNMT3L, and RD1 from its N-terminus to its C-terminus. In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein, ADD, DNMT3A, DNMT3L, and RD1 from its N-terminus to its C-terminus. In some embodiments, the DNA-binding protein may be a zinc finger, TALE, or a CRISPR protein that catalyzes death. In some embodiments, the long-term repressor fusion protein comprises an NLS at its N-terminus, C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between the N-terminal NLS and the DNA-binding protein, between the DNA-binding protein and DNMT3A, or optionally between ADD, between DNT3A and DNMT3L, and / or between DNMT3L and RD1.

[0218] In some embodiments, the long-term repressor fusion protein comprises RD1, DNMT3A, DNMT3L, and a DNA-binding protein from the N-terminus to the C-terminus. In some embodiments, the long-term repressor fusion protein comprises RD1, ADD, DNMT3A, DNMT3L, and a DNA-binding protein from the N-terminus to the C-terminus. In some embodiments, the DNA-binding protein may be a zinc finger, TALE, or a CRISPR protein that catalyzes cell death. In some embodiments, the long-term repressor fusion protein comprises NLS at the N-terminus, C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between RD1 and DNMT3A, or optionally between ADD, between DNMT3A and DNMT3L, between DNMT3L and the DNA-binding protein, and / or between the DNA-binding protein and the C-terminal NLS.

[0219] In some embodiments, the long-term repressor fusion protein comprises DNMT3A, DNMT3L, RD1, and a DNA-binding protein from the N-terminus to the C-terminus. In some embodiments, the long-term repressor fusion protein comprises ADD, DNMT3A, DNMT3L, RD1, and a DNA-binding protein from the N-terminus to the C-terminus. In some embodiments, the DNA-binding protein may be a zinc finger, TALE, or a CRISPR protein that catalyzes cell death. In some embodiments, the long-term repressor fusion protein comprises NLS at the N-terminus, C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between DNMT3A and DNMT3L, between DNMT3L and RD1, between RD1 and the DNA-binding protein, and / or between the DNA-binding protein and the C-terminal NLS.

[0220] In some embodiments, the long-term repressor fusion protein comprises DNMT3A, DNMT3L, RD1, a DNA-binding protein, and a second RD1 from the N-terminus to the C-terminus. In some embodiments, the long-term repressor fusion protein comprises ADD, DNMT3A, DNMT3L, RD1, a DNA-binding protein, and a second RD1 from the N-terminus to the C-terminus. In one of the foregoing embodiments, the second RD1 may be sequence-identical to the first RD1. In another of the foregoing embodiments, the second RD1 may be sequence-different from the first RD1. In some embodiments, the DNA-binding protein may be a zinc finger, TALE, or a CRISPR protein that catalyzes cell death. In some embodiments, the long-term repressor fusion protein comprises NLS at the N-terminus, C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between DNMT3A and DNMT3L, between DNMT3L and RD1, between RD1 and the DNA-binding protein, between the DNA-binding protein and the second RD1, and / or between the second RD1 and the C-terminal NLS.

[0221] In some cases, the long-term repressor fusion protein further comprises one or more NLS. In some embodiments, the long-term repressor fusion protein comprises, from its N-terminus to its C-terminus, the conformations 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, TALE, or a CRISPR protein that catalyzes cell death. In some embodiments, the long-term repressor fusion protein is capable of binding to and repressing or silencing transcription of the PCSK9 gene.

[0222] In some embodiments of the long-term repressor fusion protein, one or more adaptor 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, from the N-terminus to the C-terminus, the NLS-ADD-DNMT3A-adaptor 2-DNMT3L-adaptor 1-adaptor 3A-DNA binding protein-adaptor 3B-RD1-NLS (configuration 1). In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, the NLS-adaptor 3A-DNA binding protein-adaptor 3B-RD1-NLS-adaptor 1-ADD-DNMT3A-adaptor 2-DNMT3L (configuration 2). In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, the NLS-adaptor 3A-DNA binding protein-adaptor 1-ADD-DNMT3A-adaptor 2-DNMT3L-adaptor 3B-RD1-NLS (configuration 3). In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, the NLS-RD1-connector 3A-ADD-DNMT3A-connector 2-DNMT3L-connector 1-DNA binding protein-connector 3B-NLS configuration (configuration 4). In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, the NLS-ADD-DNMT3A-connector 2-DNMT3L-connector 3A-RD1-connector 1-DNA binding protein-connector 3B-NLS configuration (configuration 5). In some embodiments, the DNA binding protein of the aforementioned configurations may be a zinc finger, TALE, or a CRISPR protein that catalyzes cell death. Schematic diagrams of the configurations are depicted in... Figure 61In some embodiments of the LTRP in configurations 1-5, the NLS may comprise sequences selected from the group consisting of SEQ ID NOs: 30-97, 3298, and 3299 (Tables 3 and 4), and the adapter sequence may comprise sequences independently selected from the group consisting of SEQ ID NOs: 98-124 and 3278-3289 (representative adapters shown in Tables 5 and 58). In some embodiments, the NLS may comprise the sequence of SEQ ID NO: 30, and the adapter sequence 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 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 with it. 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 with it. 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 with it. In some embodiments, the fourth repressor is an ADD domain comprising the sequence of SEQ ID NO: 125, or a sequence that is 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% identical to it. In some embodiments, the long-term repressor fusion protein is capable of binding to and repressing or silencing the PCSK9 gene target nucleic acid.

[0223] In some embodiments, this 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 operatively linked to a DNA-binding protein; for example, a zinc finger, TALE, or a catalytic death CRISPR protein. In some embodiments, the DNA-binding protein comprises a sequence selected from the group consisting of SEQ ID NO: 4-29, or a catalytic death CasX having sequence identity with a 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%. In some embodiments, the two RD1s have identical sequences. In other embodiments, the two RD1s comprise different sequences. In some embodiments, the two RD1s are located at the N-terminus of the DNA-binding protein. In some embodiments, the two RD1s are located at the C-terminus of the DNA-binding protein. In some embodiments, one RD1 is located at the N-terminus of the DNA-binding protein, and another RD1 is located at the C-terminus of the DNA-binding protein.

[0224] In some embodiments, the long-term repressor fusion protein comprises two RD1s. In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, the conformation (conformation 6a) of NLS-ADD-DNMT3A-linker 2-DNMT3L-linker 3A-RD1a-linker 1-DNA binding protein-linker 3B-RD1a-linker 4-NLS, wherein the RD1a sequences are identical (see [link to previous embodiment]). Figure 61 (Schematic diagram of a long-term repressor fusion protein). In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, the conformation (conformation 6b) of NLS-ADD-DNMT3A-linker 2-DNMT3L-linker 3A-RD1a-linker 1-DNA binding protein-linker 3B-RD1b-linker 4-NLS, wherein RD1a and RD1b sequences are different (see [reference]). Figure 61 (Illustrative diagram of a long-term repressor fusion protein). In some embodiments, the DNA-binding protein may be a zinc finger, TALE, or a CRISPR protein that catalyzes cell death. In some embodiments, the long-term repressor protein examples in this paragraph are capable of binding to and repressing or silencing the expression of the PCSK9 gene.

[0225] In some embodiments, this disclosure provides a long-term repressor fusion protein of configuration 6a, wherein the two RD1 sequences are identical. In some embodiments of the long-term repressor fusion protein of configuration 6a, the DNA-binding protein comprises the sequence of SEQ ID NO: 4, or dCasX 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 with it, wherein the first and second copies of RD1a comprise sequences selected from the group consisting of SEQ ID NO: 130-1726, or sequences 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 with it, and the second repressor domain is comprising SEQ ID NO: The third repressor is a sequence comprising SEQ ID NO: 126, or a DNMT3A containing 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 with it. The fourth repressor is a sequence comprising SEQ ID NO: 127, or a DNMT3L containing 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 with it. The NLS comprises sequences of SEQ ID NO: 30-97 (Tables 3 and 4), the L1 connector comprising the sequence of SEQ ID NO: 123, the L2 connector comprising the sequence of SEQ ID NO: 122, the L3A connector comprising the sequence of SEQ ID NO: 124, the L3B connector comprising the sequence of SEQ ID NO: 120, and the L4 connector comprising the 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 adapter sequence is 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 RD1 are identical, each of RD1 containing 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%, or at least about 95% identity with it. A schematic diagram of configuration 6a is shown. Figure 61 In some embodiments, the long-term repressor fusion protein of configuration 6a can form an RNP with the gRNA of this disclosure and can bind to and repress or silence the gene target nucleic acid.

[0226] In some embodiments of the long-term repressor fusion protein of configuration 6b, wherein the two RD1 sequences are distinct, the DNA-binding protein comprises the sequence of SEQ ID NO: 4, or a dCasX 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 with it, the first copy (RD1a) of the first repressor domain comprises a sequence selected from the group consisting of SEQ ID NO: 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 with it, and the second copy (RD1b) of the first repressor domain comprises a distinct sequence selected from the group consisting of SEQ ID NO: 130-1726, or sequences 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 with them, the second repressor domain being a sequence containing SEQ ID NO: 126, or DNMT3A containing 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 with them, the third repressor being a sequence containing SEQ ID NO: The fourth repressor is a sequence comprising SEQ ID NO: 127, or a sequence variant thereof 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. The NLS comprises a sequence independently selected from the group consisting of: SEQ ID NO: 30-97 of Tables 3 and 4, the L1 connector comprising the sequence of SEQ ID NO: 123, the L2 connector comprising the sequence of SEQ ID NO: 122, and the L3A connector comprising the sequence of SEQ ID NO: 123. The L3B connector contains the sequence of SEQ ID NO: 124, and the sequence of SEQ ID NO: 120 is also present.The L4 connector comprises the 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 connector sequence is selected from the group consisting of SEQ ID NO: 98-124, 3278-3289 (exemplary sequences shown in Tables 5 and 58). In some embodiments of the long-term repressor fusion protein of configuration 6b, wherein the two copies of RD1 are distinct, RD1a comprises the 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 with it, and RD1b comprises the 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 with it. In some embodiments of the long-term repressor fusion protein of configuration 6b, wherein the two copies of RD1 are different, RD1a contains the 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 with it, and RD1b contains the 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 with it. In some embodiments of the long-term repressor fusion protein of configuration 6b, wherein the two copies of RD1 are different, RD1a contains the 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 with it, and RD1b contains the 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 with it. In some embodiments of the conformation 6b long-term repressor fusion protein, wherein the two copies of RD1 are distinct, RD1a contains the 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 with it, and RD1b contains the 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 with it. In some embodiments of the long-term repressor fusion protein of configuration 6b, wherein the two copies of RD1 are different, RD1a contains the 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 with it, and RD1b contains the 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 with it. In some embodiments of the conformation 6b long-term repressor fusion protein, wherein the two copies of RD1 are distinct, RD1a comprises the 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 with it, and RD1b comprises the 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 with it. In some embodiments of the conformation 6b long-term repressor fusion protein, wherein the two copies of RD1 are distinct, RD1a and RD1b are independently selected from the group consisting of the following sequences: SEQ ID NO: 132-134 and 136-138. In some embodiments of the long-term repressor fusion protein of configuration 6b, the two copies of RD1 are distinct, with RD1a and Rd1b independently selected from the group consisting of sequences SEQ ID NO: 130, 131, and 135. A schematic diagram of configuration 6b is shown below. Figure 61 In some embodiments, the long-term repressor fusion protein can form an RNP with the gRNA of this disclosure and can bind to the target nucleic acid of a gene and repress or silence the target nucleic acid.

[0227] Table 5: Exemplary linker amino acid sequences of long-term repressor fusion proteins

[0228]

[0229] In some embodiments of a long-term repressor fusion protein comprising dCasX and optionally ADD and configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NO: 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 about 99% identity with it. In some embodiments of a long-term repressor fusion protein comprising dCasX and optionally ADD and configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NO: 22836-22855. In some embodiments of a long-term repressor fusion protein comprising dCasX and optionally ADD and configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NO: 22838 and 22847. In some embodiments comprising dCasX and optionally ADD and configured as conformation 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NO: 22839 and 22848. In some embodiments comprising dCasX and optionally ADD and configured as conformation 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NO: 22840 and 22849.

[0230] In some embodiments of a system comprising a DNA-binding protein and configured to conformate to the group consisting of conformations 1, 2, 3, 4, 5, 6a, and 6b described above, the long-term repressor fusion protein comprises dCasX and is capable of complexing with a gRNA having a target sequence complementary to the PCSK9 target nucleic acid in the cell to form an RNP, wherein, upon binding of the RNP to the PCSK9 target nucleic acid in the 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 in cells within a cell population is repressed by 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. In some embodiments, transcriptional repression is measured in vitro in assays, including cell-based assays, when compared to untreated cells or cells treated with a comparable system containing non-target spacers. In some embodiments, transcriptional repression is measured in vivo in cells obtained from a subject in which the long-term repressor fusion protein and a gRNA having a targeting sequence complementary to the PCSK9 target nucleic acid of the gene in the cell are administered; as a protein and gRNA, or as a nucleic acid (e.g., gRNA and mRNA encoding the long-term repressor fusion protein), wherein the subject is selected from the group consisting of mice, rats, pigs, non-human primates, and humans.

[0231] Most preferably, PCSK9 gene repression completely inhibits gene expression, making the gene product undetectable. However, those skilled in the art will understand that complete inhibition remains useful and desirable for various applications. In some embodiments, when measured in in vitro assays, including cell-based assays, the repression of PCSK9 gene transcription by the systemic embodiments lasts 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. In some embodiments, when a composition comprising the system of this disclosure is administered at a therapeutically effective dose, the repression of PCSK9 gene transcription by the systemic composition lasts 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 the target cells of the subject. In some embodiments, the subject is selected from the group consisting of mice, rats, pigs, non-human primates, and humans. In some embodiments, when used in the LTRP:gRNA system of the embodiment, the use of LTRPs of configurations 1, 4, 5 and 6 results in off-target methylation or off-target activity of 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 than about 0.5% or less than about 0.1% in the cells.

[0232] In some embodiments, the repression of transcription of the PCSK9 gene in cells treated with the LTRP:gRNA system of the embodiment is heritable and stable through one or more cell divisions. In some embodiments, the repression of transcription is stable through 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more cell divisions.

[0233] V. mRNA composition encoding long-term repressor fusion protein

[0234] On the other hand, this disclosure relates to messenger RNA (mRNA) compositions comprising sequences of individual components and the full-length mRNA sequence of a long-term repressor domain fusion protein construct of this disclosure. In some embodiments, the mRNA composition is useful for transcriptional repression and epigenetic modification of genes (e.g., the PCSK9 gene) when used to express the long-term repressor fusion protein. In some cases, the mRNA is designed for use in certain delivery formulations; for example, nanoparticles, such as synthetic nanoparticles or lipid nanoparticles (LNPs). This disclosure also provides methods for designing the mRNA sequence of the mRNA used in the composition. In some cases, the mRNA encoding the long-term repressor fusion protein can be co-formulated in nanoparticles (e.g., LNPs) with gRNA containing a targeting sequence complementary to the sequence of the PCSK9 gene. Upon delivery to target cells via LNP, the long-term repressor domain fusion protein is expressed from the mRNA and can be complexed with the gRNA to form an RNP capable of binding to the PCSK9 target nucleic acid. In other cases, the mRNA and gRNA encoding the long-term repressor domain fusion protein can be formulated in individual nanoparticles and delivered individually or as a mixture.

[0235] In some embodiments, the mRNA compositions of this disclosure have been modified to produce one or more improved features relative to unmodified mRNA encoding the same long-term repressor protein, and may therefore have a positive impact on the efficacy of mRNA-based delivery. Exemplary improved features of the mRNAs described herein compared to unmodified mRNA include, but are not limited to, improved expression upon delivery to cells, reduced immunogenicity, increased stability, and enhanced manufacturability. In some cases, the improved features resulting from the modification of the mRNA relative to unmodified mRNA are at least about 1.1 to about 100,000 times better. In some embodiments, the improvement of the modified mRNA relative to the unmodified mRNA is characterized by an improvement of at least about 1.1 to about 10,000-fold, an improvement of at least about 1.1 to about 1,000-fold, an improvement of at least about 1.1 to about 500-fold, an improvement of at least about 1.1 to about 400-fold, an improvement of at least about 1.1 to about 300-fold, an improvement of at least about 1.1 to about 200-fold, an improvement of at least about 1.1 to about 100-fold, an improvement of at least about 1.1 to about 50-fold, an improvement of at least about 1.1 to about 40-fold, an improvement of at least about 1.1 to about 30-fold, an improvement of at least about 1.1 to about 20-fold, an improvement of at least about 1.1 to about 10-fold, and an improvement of at least about 1.1 to about 9-fold. The improvements are described as follows: at least about 1.1 to about 8 times, at least about 1.1 to about 7 times, at least about 1.1 to about 6 times, at least about 1.1 to about 5 times, at least about 1.1 to about 4 times, at least about 1.1 to about 3 times, at least about 1.1 to about 2 times, at least about 1.1 to about 1.5 times, at least about 1.5 to about 3 times, at least about 1.5 to about 4 times, at least about 1.5 to about 5 times, at least about 1.5 to about 10 times, at least about 5 to about 10 times, at least about 10 to about 20 times, at least 10 to about 30 times, at least 10 to about 50 times, or at least 10 to about 100 times. In some embodiments, the improvement of the modified mRNA relative to the unmodified mRNA is characterized by an improvement of at least about 10 to about 1000 times.

[0236] When delivering mRNA encoding the protein of interest, optimization of the coding sequence and untranslated region (UTR) can be particularly important compared to the DNA template to be transcribed into mRNA. DNA templates have a long lifespan, can replicate, and can produce numerous RNA transcripts within their lifetime. For DNA templates, transcription efficiency and pre-mRNA processing are major determinants of protein expression levels. In contrast, mRNAs typically have a much shorter half-life, approximately a few hours, because they are readily degraded in the cytoplasm and cannot produce many more copies of themselves. Therefore, mRNA stability and translation efficiency may be determinants of protein expression levels in mRNA-based delivery, and thus, specific sequences of the UTR and coding sequence that determine mRNA stability and translation efficiency can be enhanced to improve the efficacy of mRNA-based delivery.

[0237] a. 5' cap

[0238] In some embodiments of the mRNA disclosed herein, the mRNA includes a 5' cap connected to the 5' UTR of the mRNA sequence of any of the embodiments described herein. In some embodiments, the 5' cap is a 7-methylguanylic acid cap. In some embodiments, the 5' cap contains m7G(5')ppp(5')mAG. In other embodiments, the 5' cap contains m7G(5')ppp(5'(A, G(5')ppp(5')A, or G(5')ppp(5')G. Exemplary caps are known in the art and are described, for example, in WO 2017 / 053297, the contents of which are incorporated herein by reference.

[0239] b. 5' Untranslated Region (UTR)

[0240] The 5' UTR of mRNA molecules can be a key determinant of mRNA stability and the efficiency of its translation into proteins. Specifically, the 5' UTR, which binds to the 5' cap structure, acts as a binding site and recruitment platform for the pre-translational initiation complex, as well as a source of additional regulatory proteins that can have a positive or negative impact on translation. The structure within the 5' UTR can enhance translation by recruiting initiation factors or other proteins or RNA factors, reduce translation by physically blocking ribosome binding and scanning, and promote mRNA stability by influencing both hydrolysis and nuclease digestion.

[0241] Exemplary 5' UTR sequences for the mRNAs used in this disclosure are provided in Table 6. Table 6 lists the RNA sequences and RNA sequences in which uridine is replaced with N1-methyl-pseudouridine.

[0242] Table 6: 5' UTR Sequence

[0243]

[0244] 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 with it. 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 with it. 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.

[0245] c. 3' UTR

[0246] 3' UTR sequences can influence mRNA stability and translation efficiency, and thus determine subcellular localization and tissue-specific expression. Factors influencing these properties include microRNA binding sites, AU-rich elements of arrays recruiting RNA-binding proteins, Pumilio binding elements, and other binding sites of RNA-binding proteins. While many of these interactions with the 3' UTR are known to adversely affect stability or expression, some can enhance translation. Due to the differential expression of microRNAs and RNA-binding proteins, the effects of 3' UTR sequences can be highly cell-type specific, providing an opportunity to engineer tissue-specific expression into therapeutic mRNAs. In some embodiments, the 3' UTR used in the mRNAs of this disclosure is the mouse 3' UTR. In some embodiments, the 3' UTR is the mouse HBA gene 3' UTR as shown in Table 7.

[0247] Exemplary 3' UTR sequences of this disclosure are provided in Table 7. Table 7 lists RNA sequences and RNA sequences in which uridine is replaced with N1-methyl-pseudouridine.

[0248] Table 7: 3' UTR Sequence

[0249]

[0250] 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 with it. 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 with it. 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.

[0251] d. poly(A) sequence

[0252] Including a 3' poly(A) tail in the mRNA sequence can contribute to mRNA stability and translation efficiency. Generally, longer poly(A) tails are associated with increased mRNA stability, thereby allowing for translation and promoting high protein expression.

[0253] In some embodiments, the mRNA of this 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 the mRNA of this disclosure comprises 80 adenine nucleotides. In some embodiments, the poly(A) sequence comprises a nucleic acid sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 3057). In some embodiments, the poly(A) sequence contains the nucleic acid sequence AAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 3307).

[0254] e. Sequence modifications of mRNA

[0255] In some embodiments, the mRNA sequences disclosed herein are modified to enhance expression in target cells by codon optimization of the sequence encoding a long-term repressor protein using one or more parameters. Non-limiting examples of such parameters include codon usage in human host cells (e.g., utilizing a codon adaptation index (CAI)), minimizing rare codons, utilizing a codon usage table derived from a biologic intended for use as a therapeutic agent, utilizing an mRNA stability index, or reducing GC content. Methods of codon optimization and codon usage in various organisms are known in the art. See, for example, www.genscript.com / tools / codon-frequency-table.

[0256] In some embodiments, this document provides mRNA sequences for long-term repressor protein constructs, codon-optimized for expression in human cells. In other embodiments, various naturally occurring or modified nucleosides can be used to generate modified mRNA according to this disclosure. In some embodiments, the mRNA is or comprises a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynylcytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazolamidine). The mRNA contains glycosides, 7-denitroguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methyl-pseuuridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., thiophosphates and 5'-N-phosphoramide bonds). In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues may include, for example, 5-methylcytidine (“5 mC”), pseudouridine (“ψU”), and / or 2-thiouridine (“2 sU”). In certain embodiments, one or more or all uridine residues of the mRNA disclosed herein are replaced with 1-methyl-pseuuridine. In some embodiments, all uridine residues of the mRNA disclosed herein are replaced with 1-methyl-pseuuridine. See, for example, U.S. Patent No. 8,278,036 or WO2011012316 (incorporated herein by reference), 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 the uridine nucleoside in the mRNA sequence is replaced by N1-methylpseudouridine.

[0257] In some embodiments, the improvement resulting from modifications to mRNA, such as codon optimization and / or substitution of modified nucleotides in the sequence, relative to unmodified mRNA, is characterized by an improvement of at least about 1.1 to about 100,000-fold. In some embodiments, the improvement of modified mRNA relative to unmodified mRNA is characterized by improvements of at least about 1.1 to about 10,000-fold, at least about 1.1 to about 1,000-fold, at least about 1.1 to about 500-fold, at least about 1.1 to about 400-fold, at least about 1.1 to about 300-fold, at least about 1.1 to about 200-fold, at least about 1.1 to about 100-fold, at least about 1.1 to about 50-fold, at least about 1.1 to about 40-fold, at least about 1.1 to about 30-fold, at least about 1.1 to about 20-fold, at least about 1.1 to about 10-fold, and at least about 1.1 to about 9-fold. The improvements are described as follows: at least about 1.1 to about 8 times, at least about 1.1 to about 7 times, at least about 1.1 to about 6 times, at least about 1.1 to about 5 times, at least about 1.1 to about 4 times, at least about 1.1 to about 3 times, at least about 1.1 to about 2 times, at least about 1.1 to about 1.5 times, at least about 1.5 to about 3 times, at least about 1.5 to about 4 times, at least about 1.5 to about 5 times, at least about 1.5 to about 10 times, at least about 5 to about 10 times, at least about 10 to about 20 times, at least 10 to about 30 times, at least 10 to about 50 times, or at least 10 to about 100 times. In some embodiments, the improvement of the modified mRNA relative to the unmodified mRNA is characterized by an improvement of at least about 10 to about 1000 times.

[0258] f. LTRP mRNA component sequence

[0259] This disclosure provides mRNAs containing sequences encoding components used in the long-term repressor fusion proteins of this disclosure. In some embodiments, the mRNA contains sequences encoding DNA-binding proteins, including TALE, ZF, and CRISPR proteins that catalyze death. In some embodiments, the mRNA contains the sequence encoding SEQ ID NO: 3274 of 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 therewith. In some embodiments, the mRNA contains the sequence encoding SEQ ID NO: 3276 of 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 therewith. In some embodiments, the mRNA comprises the sequence encoding dCasX 491 (SEQ ID NO: 4) of SEQ ID NO: 3275, 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 with it. In some embodiments, the mRNA comprises the sequence encoding dCasX 676 (SEQ ID NO: 28) of SEQ ID NO: 22736, 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 with it.

[0260] In some embodiments, the mRNA sequence encoding dCasX 491 (SEQ ID NO: 4) has a pseudouridine nucleoside of one or more of the uridines in the substitution sequence (SEQ ID NO: 22729). In some embodiments, the mRNA sequence encoding dCasX 515 (SEQ ID NO: 6) has a pseudouridine nucleoside of one or more of the uridines in the substitution sequence (SEQ ID NO: 22728). In some embodiments, the mRNA sequence encoding dCasX 676 (SEQ ID NO: 28) has a pseudouridine nucleoside of one or more of the uridines in the substitution sequence. In some embodiments, the mRNA sequence encoding dCasX812 (SEQ ID NO: 29) has a pseudouridine nucleoside of one or more of the uridines in the substitution sequence (SEQ ID NO: 22730 or 22731).

[0261] In some embodiments, this disclosure provides an mRNA sequence encoding the RD1 domain. In some embodiments, the mRNA sequence encoding 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 with it. In some embodiments, the mRNA sequence encoding RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 3120, 22804, and 3334-6527. In another embodiment, the mRNA sequence encoding RD1 comprises sequences selected from the group consisting of SEQ ID NOs: 3334-3342 and 4931-4939, or sequences 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 with them. In another embodiment, the mRNA sequence encoding RD1 comprises sequences selected from the group consisting of SEQ ID NOs: 3334-3342 and 4931-4939. In another embodiment, the mRNA sequence encoding RD1 comprises the 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 with it. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3334 or SEQ ID NO: 4931. In another embodiment, the mRNA sequence encoding RD1 comprises the 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 with it. In another embodiment, the sequence encoding the mRNA of RD1 comprises the sequence of SEQ ID NO: 3335 or SEQ ID NO: 4932.In another embodiment, the mRNA sequence encoding RD1 comprises the 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 with it. In another embodiment, the mRNA sequence encoding RD1 comprises the sequence of SEQ ID NO: 3336 or SEQ ID NO: 4933. In another embodiment, the mRNA sequence encoding RD1 comprises the 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 with it. In another embodiment, the sequence of the mRNA encoding RD1 comprises the sequence of SEQ ID NO: 3337 or SEQ ID NO: 4934. In another embodiment, the sequence of the mRNA encoding RD1 comprises the 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 with it. In another embodiment, the sequence of the mRNA encoding RD1 comprises the sequence of SEQ ID NO: 3338 or SEQ ID NO: 4935. In another embodiment, the sequence encoding the mRNA of RD1 comprises the 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 with it. In another embodiment, the sequence encoding the mRNA of RD1 comprises the sequence of SEQ ID NO: 3339 or SEQ ID NO: 4936. In another embodiment, the sequence encoding the mRNA of RD1 comprises the 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 with it.In another embodiment, the sequence of the mRNA encoding RD1 comprises the sequence of SEQ ID NO: 3340 or SEQ ID NO: 4937. In another embodiment, the sequence of the mRNA encoding RD1 comprises the 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 with it. In another embodiment, the sequence of the mRNA encoding RD1 comprises the sequence of SEQ ID NO: 3341 or SEQ ID NO: 4938.

[0262] In some embodiments, the mRNA comprises a sequence encoding a second repressor domain. In some embodiments, the second repressor domain comprises DNMT3A. In some embodiments, the sequence of the mRNA encoding DNMT3A comprises the 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%, or at least about 99% identity with it. In some embodiments, the sequence of the mRNA encoding DNMT3A comprises the sequence of SEQ ID NO: 3128 or SEQ ID NO: 3331.

[0263] In some embodiments, the mRNA comprises a sequence encoding a third repressor domain. In some embodiments, the third repressor domain comprises DNMT3L. In some embodiments, the sequence of the mRNA encoding DNMT3L comprises the 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%, or at least about 99% identity with it. In some embodiments, the sequence of the mRNA encoding DNMT3L comprises the sequence SEQ ID NO: 3119 or SEQ ID NO: 3332.

[0264] In some embodiments, the mRNA comprises a sequence encoding a fourth repressor domain. In some embodiments, the fourth repressor domain comprises an ADD. In some embodiments, the sequence of the mRNA encoding an ADD comprises the 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%, or at least about 99% identity with it. In some embodiments, the sequence of the mRNA encoding an ADD comprises the sequence of SEQ ID NO: 3296 or SEQ ID NO: 22726.

[0265] In some embodiments, the mRNA disclosed herein comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises GCCACCAUGG (SEQ ID NO: 22805). In some embodiments, the mRNA disclosed herein comprises a Kozak sequence and two bases upstream of the NLS (to generate 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.

[0266] 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 with it. 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 example embodiments in which 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.

[0267] In some embodiments, the mRNA comprises a sequence encoding a linker for a 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%, or at least about 95% identity with it. 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 embodiments in which the long-term repressor fusion protein comprises more than one linker, the sequence encoding the linker is 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%, or at least about 95% identity with it.

[0268] In some embodiments, the mRNA comprises a sequence encoding a long-term repressor fusion protein of the conformation provided herein. In some embodiments, the mRNA encodes a long-term repressor fusion protein of conformation 1, and the mRNA comprises a sequence 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%, or at least about 99% identity with such sequences. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 6529-8134, 9741-11347, 14628-16233, and 17841-19446.

[0269] In some embodiments, the mRNA comprises a sequence encoding a long-term repressor fusion protein of configuration 1, and also comprises a sequence encoding RD1, including the sequence of SEQ ID NO: 130. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 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 with that sequence. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 6531, 9744, 14630, or 17843. In some embodiments, the mRNA comprises a sequence encoding a long-term repressor fusion protein of configuration 1, and also comprises a sequence encoding RD1, including SEQ ID NO: 131. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 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 with it. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 6529, 9742, 14628, or 17841. In some embodiments, the mRNA comprises the sequence encoding a long-term repressor fusion protein of conformation 1, and the mRNA comprises the sequence encoding RD1 of SEQ ID NO: 132. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 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 with it. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 6535, 9748, 14634, or 17847. In some embodiments, the mRNA comprises the sequence encoding a long-term repressor fusion protein of conformation 1, and comprises the sequence encoding RD1 of SEQ ID NO: 133. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 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 with it.In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 6536, 9749, 14635, or 17848. In some embodiments, the mRNA comprises the sequence encoding a long-term repressor fusion protein of configuration 1, and the mRNA comprises the sequence encoding RD1 of SEQ ID NO: 134. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 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 with it. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 6534, 9747, 14633, or 17846. In some embodiments, the mRNA comprises the sequence encoding a long-term repressor fusion protein of configuration 1, and comprises the sequence encoding RD1 of SEQ ID NO: 135. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 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 with it. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 6530, 9743, 14629, or 17842. In some embodiments, the mRNA comprises the sequence encoding a long-term repressor fusion protein of conformation 1, and comprises the sequence encoding RD1 of SEQ ID NO: 136. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 6537, 9750, 14636, or 17849, 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 with it. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 6537, 9750, 14636, or 17849. In some embodiments, the mRNA comprises the sequence encoding a long-term repressor fusion protein of conformation 1, and comprises the sequence encoding RD1 of SEQ ID NO: 137. In some embodiments, the mRNA comprises a sequence of SEQ ID NO: 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 with it.In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 6533, 9746, 14632, or 17845. In some embodiments, the mRNA comprises the sequence encoding a long-term repressor fusion protein of conformation 1, and comprises the sequence encoding RD1 of SEQ ID NO: 138. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 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 with it. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 6532, 9745, 14631, or 17844.

[0270] This disclosure provides an mRNA encoding a long-term repressor fusion protein of configuration 5. In some embodiments, the mRNA comprises a sequence 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 with it. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 8135-9740, 11348-12953, 16234-17839, and 19447-21052.

[0271] This disclosure provides an mRNA encoding a long-term repressor fusion protein of configuration 6a, wherein the encoded RD1 is identical. In some embodiments, the mRNA comprises sequences selected from the group consisting of SEQ ID NOs: 12954-14553 and 21053-22652, or sequences 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 with those sequences. In some embodiments, the mRNA comprises sequences selected from the group consisting of SEQ ID NOs: 12954-14553 and 21053-22652.

[0272] This disclosure provides an mRNA encoding a long-term repressor fusion protein of conformation 6b, wherein the encoded RD1 is distinct. In some embodiments, the mRNA comprises a sequence 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 with it. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 14554-14626 and 22653-22725.

[0273] In some embodiments encoding the mRNA of the long-term repressor fusion protein of the LTRP:gRNA system disclosed herein, the mRNA is expressed when the system is delivered to target cells, and the long-term repressor fusion protein is able to complex with the gRNA and bind to the target DNA, thereby causing repression or silencing of the transcription of the PCSK9 gene in the cell.

[0274] VI. Systematic Guided Nucleic Acids

[0275] On the other hand, this disclosure relates to specially designed guide RNA (gRNA) comprising a scaffold and a linked target sequence complementary to (and thus capable of hybridizing with) the target nucleic acid sequence of the PCSK9 gene. The gRNA described herein can be used in conjunction with long-term repressor proteins and systems containing them to repress the transcription of the PCSK9 target nucleic acid in eukaryotic cells. As used herein, the term "gRNA" encompasses naturally occurring molecules and gRNA variants, including chimeric gRNA variants comprising domains from different gRNAs. The gRNA of this disclosure comprises a scaffold and a target sequence complementary to the cell's target nucleic acid, linked to the 3' end of the scaffold.

[0276] In some embodiments, a system comprising mRNA encoding a long-term repressor fusion protein includes a dCasX protein and one or more gRNAs. When the dCasX protein is expressed in transfected cells, it forms a ribonucleoprotein (RNP) complex with the gRNA, which can target and bind to specific sites within 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 complementary to the target nucleic acid sequence, while the long-term repressor fusion protein of the system provides site-specific activity, such as binding and repression of the target sequence by association with the gRNA to a target site within the target nucleic acid sequence (e.g., stabilization at the target site).

[0277] Examples of gRNAs, mRNAs, and gRNA modulators for repressing and / or epigenetic modification of PCSK9 target nucleic acids are described below.

[0278] a. Reference gRNA and gRNA variants

[0279] As used herein, “reference gRNA” refers to a CRISPR guide ribonucleic acid containing a wild-type sequence of a naturally occurring gRNA. In some embodiments, the gRNA scaffold of this disclosure may be subjected to one or more mutagenesis methods, such as those described in WO2023235818A2, WO2022120095A1, and WO2020247882A1 (incorporated herein by reference), which may include deep mutation evolution (DME), deep mutation scanning (DMS), error-prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, domain exchange, or chemical modification to produce one or more gRNA variants that have enhanced or altered properties relative to the modified gRNA scaffold. The activity of the gRNA scaffold from which the gRNA variant is derived can be used as a benchmark for comparing the activity of the gRNA variants, thereby measuring improvements in the function or other properties of the gRNA scaffold.

[0280] Table 8 provides the sequences of the reference gRNA tracer and scaffold sequence. In some embodiments, this disclosure provides gRNA variants having a scaffold comprising a sequence having one or more nucleotide modifications relative to the reference gRNA sequence of any one of SEQ ID NOs: 1731-1743 in Table 8.

[0281] Table 8: Reference gRNA tracr and scaffold sequences

[0282]

[0283] b. gRNA domains and their functions

[0284] The gRNA of this disclosure comprises two segments: a targeting sequence and a protein-binding segment. The targeting segment of the gRNA comprises a nucleotide sequence (interchangeably referred to as a spacer, target, or targeting sequence) that is complementary (and thus hybridizes with) a specific sequence (target site) within a target nucleic acid sequence (e.g., a double-stranded target DNA strand, target ssRNA, target ssDNA, etc.), which will be described more fully below. The targeting sequence of the gRNA is capable of binding to the target nucleic acid sequence, which, in the context of this disclosure, includes coding sequences, complement of coding sequences, non-coding sequences, and accessory elements. The protein-binding segment of the gRNA (or “activator” or “protein-binding sequence”) interacts (e.g., binds) with the dCasX protein as a complex, thereby forming an RNP (described more fully below). As used herein, “scaffold” refers to all parts of the guide other than the targeting sequence, which comprises several regions, described more fully below. The properties and characteristics of wild-type and variant CasX gRNAs are described in WO2020247882A1, US20220220508A1 and WO2022120095A1 (incorporated hereby by application).

[0285] In the case of reference gRNA, gRNA naturally exists as a dual guide RNA (dgRNA), which has double-strand-forming segments in both the target and activator portions, said segments being complementary to each other and hybridizing to form a double-stranded double (dsRNA double-stranded gRNA). The terms “target” or “target RNA” are used herein to refer to crRNA-like molecules (crRNA: “CRISPR RNA”) of CasX dual guide RNA (and therefore CasX single guide RNA when the “activator” and “target” are linked together; for example, linked together by an intermediate nucleotide). crRNA has a 5' region that allows the target sequence to be tracrRNA followed by nucleotide recombination. In the case of gRNA used in the systems of this disclosure, the scaffold is designed such that the activator and target portions are covalently linked to each other (rather than hybridizing) and comprise a single molecule, and may be referred to as “single-molecule gRNA,” “single guide RNA,” “single-molecule guide RNA,” or “sgRNA.” The gRNA variants of this disclosure used in said systems are all single-molecule versions.

[0286] In summary, the assembled gRNA of this disclosure comprises different structured regions or domains: an RNA triplet, a scaffold stem loop, an extended stem loop, a pseudoknot, and a target sequence, which, in embodiments of this disclosure, is complementary to the sequence of the target nucleic acid and is located at the 3' end of the gRNA. The RNA triplet, scaffold stem loop, pseudoknot, and extended stem loop, as well as the unstructured triplet loop bridging portions of the triplet together, are referred to as the "scaffold" of the gRNA. In some cases, the scaffold stem further comprises a vesicle. In other cases, the scaffold further comprises a triplet loop region. In still other cases, the scaffold further comprises a 5' unstructured region. In some embodiments, the gRNA scaffold of this disclosure for use in the LTRP:gRNA system comprises a scaffold stem loop having the sequence CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1822) or a sequence having at least 1, 2, 3, 4, or 5 mismatches therewith.

[0287] Each of the structured domains contributes to establishing the overall RNA fold of the guide and maintaining its functionality; specifically, the ability to properly complex with the dCasX protein. For example, the guide scaffold stem interacts with the helical I domain of the dCasX protein, while residues within the triplet, triplet loop, and pseudoknot stem interact with the OBD of the dCasX protein. In summary, these interactions confer the guide's ability to bind to LTRPs and form stable RNPs, while the spacer (or target sequence) guides and determines the specificity of RNP binding to a specific sequence of DNA.

[0288] Site-specific binding of a target nucleic acid sequence (e.g., genomic DNA) via LTRP using a dCasX protein can occur at one or more locations (e.g., the sequence of a PCSK9 target nucleic acid) determined by the base pairing complementarity between the target sequence of the gRNA and the target nucleic acid sequence. Thus, for example, the gRNA of this disclosure has a sequence complementary to the target nucleic acid and can therefore hybridize with the target nucleic acid, said sequence being adjacent to a TC protospacer adjacent motif (PAM) motif or a PAM sequence such as ATC, CTC, GTC, or TTC. Because the target sequence of the guide sequence hybridizes with the target nucleic acid sequence, the user can modify the target sequence to hybridize with a specific target nucleic acid sequence, taking into account the position of the PAM sequence. In some embodiments, to design the target sequence, the target nucleic acid contains a PAM sequence located at the 5' of the target sequence, wherein at least one nucleotide separates the PAM from the first nucleotide of the target nucleic acid that is complementary to the nucleotide of the target sequence. This feature distinguishes the system described herein from the cCas9 system and makes the system of this disclosure capable of modifying different locations in a DNA sequence compared to the Cas9 system. In some embodiments, the PAM is located on the non-target strand (i.e., the strand complementary to the target nucleic acid) of the target region. In some embodiments, the target sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence that differs from the ATC PAM sequence by one nucleotide. In some embodiments, the target sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence that differs from the CTC PAM sequence by one nucleotide. In some embodiments, the target sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence that differs from the GTC PAM sequence by one nucleotide. In some embodiments, the target sequence of the gRNA is complementary to a PCSK9 target nucleic acid sequence that differs from the TTC PAM sequence by one nucleotide. By selecting the target sequence of the gRNA, the LTRP:gRNA system described herein can be used to repress a defined region of one or more target nucleic acid sequences at a specific location within the target nucleic acid. In some embodiments, the target sequence of the gRNA has 15 to 22 consecutive nucleotides. In some embodiments, the target sequence has 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides. In some embodiments, the target sequence consists of 22 consecutive nucleotides. In some embodiments, the target sequence consists of 21 consecutive nucleotides. In some embodiments, the target sequence consists of 20 consecutive nucleotides. In some embodiments, the target sequence consists of 19 consecutive nucleotides. In some embodiments, the target sequence consists of 18 consecutive nucleotides. In some embodiments, the target sequence consists of 17 consecutive nucleotides. In some embodiments, the target sequence consists of 16 consecutive nucleotides. In some embodiments, the target sequence consists of 15 consecutive nucleotides.By selecting the target sequence of the gRNA, the restricted region of the PCSK9 target nucleic acid sequence, which is described in this paper as LTRP:gRNA systemic repression and / or epigenetic modification, can be used.

[0289] The gene repressor system disclosed herein can be designed to target the PCSK9 gene seeking transcriptional repression, or any region of the PCSK9 gene or a region of the PCSK9 gene, or any region of the gene or a region of the gene. When the entire gene is to be repressed, this disclosure envisions a guide designed with a target sequence complementary to a sequence covering or near the transcription start site (TSS). TSS selection occurs at different locations within the promoter region, depending on the promoter sequence and the initiator-substrate concentration. The core promoter acts as a binding platform for transcriptional mechanisms, containing Pol II and its associated universal transcription factor (GTF) (Haberle, V. et al., Eukaryotic core promoters and the functional basis of transcription initiation (Nature Reviews Mol Cell Biol 19(10):621 (2018))). The variability in TSS selection has been proposed to involve DNA 'splitting' and 'anti-splitting', characterized by: (i) forward and reverse movement of the RNA polymerase front relative to the DNA, but not involving movement of the trailing edge, and (ii) expansion and contraction of the transcription vesicle. In some embodiments, the target nucleic acid sequence bound by the RNP of the LTRP:gRNA system is within 1.5 kb of the transcription start site (TSS) in the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the system's RNP is within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb, or 1.5 kb upstream of the TSS of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the system's RNP is located within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb, or 1.5 kb downstream of the TSS of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the system's RNP is located within 1.5 kb upstream to 1.5 kb downstream, 1 kb upstream to 1 kb downstream, 500 bp upstream to 500 bp downstream, 300 bp upstream to 300 bp downstream, or 100 bp upstream to 100 bp downstream of the TSS of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the system's RNP is located within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb, or 1.5 kb of the enhancer of the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the RNP of the system disclosed herein is located within 1 kb of the 3' to 5' untranslated region of the PCSK9 gene.In other embodiments, the target nucleic acid sequence bound by the RNP of the system is within the open reading frame of the PCSK9 gene, including introns (if any). In some embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of an exon of the PCSK9 gene. In a particular embodiment, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of exon 1 of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of an intron of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of an intron-exon junction of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of a regulatory element of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of an intergenic region of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system disclosed herein is complementary to the sequence of an exon junction, intron, and / or regulatory element of the PCSK9 gene. In cases where the target sequence is complementary to the regulatory element sequence, 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 containing cis-regulatory elements. Promoter regions are intended to encompass nucleotides within 5 kb of the start of the coding sequence, or, in the case of gene enhancer elements or conserved elements, can be thousands, hundreds of thousands, or even millions of bp away from the coding sequence of the PCSK9 gene. In the foregoing, the target is a target whose coding PCSK9 gene is intended to be repressed so that the PCSK9 gene product is not expressed or is expressed at a low level in the cell. In some embodiments, when the RNP of the system of this disclosure binds to the binding site of the target nucleic acid, the system is capable of repressing transcription of the PCSK9 gene at the 5' of the RNP binding site. In other embodiments, when the RNP of the system binds to the binding site of the target nucleic acid, the system is capable of repressing transcription of the PCSK9 gene at the 3' of the RNP binding site.

[0290] In some embodiments, the target nucleic acid comprises a PAM sequence located at the 5' of the target sequence, wherein at least one nucleotide separates the PAM from the first nucleotide of the target sequence. In some embodiments, the PAM is located on the non-target strand of the target region, i.e., on the strand complementary to the target nucleic acid, and is shown below as Table 9, representing the target sequences for PCSK9 target nucleic acids to be linked with the gRNA scaffold of this disclosure; for example, 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.

[0291] In some embodiments, the target sequence of the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 1824-2544, 2672, 2675, 2694, and 2714, as shown in Table 9. In some embodiments, the target sequence of the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments, the target sequence of the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 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 NO: 1855, 1867, and 1869. In a particular embodiment, the targeting sequence of the gRNA consists of the sequence of SEQ ID NO: 1855. In another particular embodiment, the targeting sequence of the gRNA consists of the sequence of SEQ ID NO: 1867. In yet another particular embodiment, the targeting sequence of the gRNA consists of the sequence of SEQ ID NO: 1869. In some embodiments, the aforementioned targeting sequence is linked to a gRNA scaffold of gRNA174 (SEQ ID NO: 1744), 235 (SEQ ID NO: 1745), or 316 (SEQ ID NO: 1746), or a chemically modified version thereof.

[0292] Table 9: PCSK9-specific targeting sequences using long-term repressor fusion proteins

[0293]

[0294] c. gRNA modification

[0295] On the other hand, this disclosure relates to gRNAs (sometimes referred to herein as gRNA variants) that contain modifications to a reference gRNA relative to a gRNA from which they are derived. The gRNAs can be used in the long-term gene repressor fusion protein systems described herein. In some embodiments, the gRNA variants used in the systems of this disclosure contain one or more nucleotide substitution, insertion, deletion, or exchange or substitution domains relative to the gRNA sequence of this disclosure that improve characterization. Exemplary regions and exchange regions or domains for modification include RNA triplet, pseudoknot, scaffold stem-loop, and elongated stem-loop. In some embodiments, the gRNA variants of this disclosure contain at least a first exchange region from a different gRNA, thereby producing a chimeric gRNA. A representative example of such chimeric gRNAs is guide 316 (SEQ ID NO: 1746), in which the elongated stem-loop of gRNA scaffold 235 (SEQ ID NO: 1745) is replaced by an elongated stem-loop of gRNA scaffold 174 (SEQ ID NO: 1744), wherein the resulting 316 variant retains the ability to form RNPs with long-term repressor fusion proteins and exhibits improved characteristics compared to parental 235 when evaluated in in vitro or in vivo assays under comparable conditions.

[0296] When gRNA scaffold variants are compared to their derived gRNA scaffolds, all gRNAs that possess one or more improved functions, features, or added new functions while retaining the functional property of complexing with long-term repressor fusion proteins and guiding the ribonucleoprotein whole RNP complex to PCSK9 target nucleic acids are considered to be within the scope of this disclosure. In some embodiments, the gRNA has improved features selected from the group consisting of: increased pseudoknot stem stability, increased triplet region stability, increased scaffold stem stability, elongated stem stability, reduced off-target folding intermediates, increased binding affinity to long-term repressor fusion proteins, and increased repressive activity when complexing with long-term repressor fusion proteins, or any combination thereof. In some cases, the improved features are evaluated in in vitro assays, including the assays described in the examples. In other cases, the improved features are evaluated in vivo.

[0297] Table 10 provides exemplary gRNA variant scaffold sequences of this disclosure, which can be used as gRNA scaffolds or for generating gRNAs for use in the LTRP:gRNA system of this disclosure. In some embodiments, the gRNA variant scaffold for the system comprises sequences selected from the group consisting of SEQ ID NO: 1744-1746, or sequences 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%, or at least about 99% sequence identity with the gRNA variant, wherein the gRNA variant retains the ability to form RNPs with the long-term repressor fusion protein of this disclosure. In some embodiments, the gRNA variant scaffold for the system comprises sequences selected from the group consisting of SEQ ID NO: 1744-1746. In some embodiments, the gRNA variant scaffold for the system comprises SEQ ID NO: 1744. In some embodiments, the gRNA variant scaffold for the system comprises SEQ ID NO: 1745. In some embodiments, the gRNA variant scaffold used in the system comprises SEQ ID NO: 1746. It should be understood that in those embodiments in which the vector comprises a DNA-coding sequence of gRNA, thymine (T) bases may replace uracil (U) bases in any of the gRNA sequence embodiments described herein. Similarly, any RNA sequence disclosed herein may be encoded by DNA, wherein uracil bases are replaced by thymine. In some embodiments, this disclosure provides chemically modified gRNA variants of Table 10 described below. In some embodiments, the gRNA comprises a scaffold containing the sequences SEQ ID NO: 1744-1746 and is chemically modified.

[0298] Table 10: gRNA scaffold sequences

[0299]

[0300] Other gRNA variants for use in the systems of this disclosure are selected from the group consisting of SEQ ID NO: 1747-1821. In some embodiments, the gRNA comprises a scaffold containing the sequence of SEQ ID NO: 1747-1821 and is chemically modified.

[0301] Guide scaffolds can be prepared by several methods, including recombination or solid-phase RNA synthesis. However, when using solid-phase RNA synthesis, the length of the scaffold can affect manufacturability, with longer lengths leading to increased manufacturing costs, reduced purity and yield, and a higher rate of synthesis failure. For use in particulate formulations such as lipid nanoparticle (LNP) formulations, solid-phase RNA synthesis of the scaffold is preferred to produce the quantities required for commercial development. While previous experiments have identified enhanced properties of gRNA scaffold 235 (SEQ ID NO: 1745) compared to gRNA scaffold 174 (SEQ ID NO: 1744), its increased length (in nucleotides) makes it less desirable for use in LNP formulations due to limitations in synthetic preparation. Therefore, alternative sequences have been sought. In some embodiments, this disclosure provides gRNA variant scaffolds with improved manufacturability compared to gRNA scaffolds from which they are derived. In some embodiments, this disclosure provides gRNAs wherein the gRNA scaffold and the linked target sequence have sequences of 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 its derived 235 scaffold. The 316 gRNA scaffold was designed where the scaffold 235 sequence was modified by domain exchange, wherein an extended stem-loop of scaffold 174 replaced the extended stem-loop of scaffold 235, resulting in a chimeric gRNA scaffold 316 with a sequence containing 89 nucleotides, compared to the 99 nucleotides of gRNA scaffold 235: ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG (SEQ ID NO: 1746). The resulting 316 scaffold has the additional advantage that the extended stem-loop does not contain CpG motifs, thus conferring an enhanced property that reduces the likelihood of triggering an immune response. In some embodiments, the shorter sequence length of the 316 scaffold imparts an improved fidelity to the ability to synthesize guides with correct and complete sequences, as well as an enhanced ability to successfully integrate into LNPs. In some embodiments, this disclosure provides chemically modified gRNA316 variants as described below.

[0302] d. Chemically modified gRNA

[0303] In some embodiments, the gRNA has 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 the substitution of a phosphate-thioester bond between two or more nucleotides of the sequence. In some embodiments, the chemical modification is the substitution of a phosphate-thioester bond between two or more nucleotides at each end of the gRNA. In some embodiments, the gRNA contains the substitution of a phosphate-thioester bond between two or more nucleotides located at the 5' end, 3' end, or 1, 2, 3, or 4 nucleotides from the two ends of the gRNA. In some embodiments, the gRNA contains one or more nucleotides to which a 2'O-methyl group is added. In some embodiments, the addition of a 2'O-methyl group modifies one or more nucleotides located at the 5' end, 3' end, or 1, 2, 3, or 4 nucleotides from the two ends of the gRNA. In some embodiments, the first 1, 2, or 3 nucleotides of the 5' end of the scaffold (i.e., A, C, and U in the cases of gRNA 174, 235, and 316) are modified by adding a 2' O-methyl group, and each modified nucleotide is linked to an adjacent nucleotide via a phosphate thioester bond. Similarly, the last 1, 2, or 3 nucleotides of the 3' end of the target sequence linked to the 3' end of the scaffold are similarly modified. In some embodiments, this disclosure provides chemical modifications for gRNA selected from the group consisting of sequences of SEQ ID NO: 2948-2956, 2958-2966, and 2968-2976, as shown in Table 22, or sequences 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 with them. In any of the foregoing embodiments, the 20 nucleotides at the 3' end of the sequence are replaced by a targeting sequence complementary to the 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 chemical modifications at the 3' end of the gRNA. In some embodiments, the 20 nucleotides at the 3' end of the sequence are replaced by a targeting sequence complementary to the PCSK9 target nucleic acid sequence, said targeting sequence being 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 chemical modifications at the 3' end of the gRNA.In some embodiments, the 20 nucleotides at the 3' end of the sequence are replaced by a targeting sequence complementary to the PCSK9 target nucleic acid sequence, the targeting sequence being selected from the group consisting of SEQ ID NO: 1855, 1867, and 1869, but including a chemical modification at the 3' end of the gRNA. In some embodiments, the chemically modified gRNA comprises a sequence selected from the group consisting of SEQ ID NO: 2948-2956, 2958-2966, and 2968-2976, wherein the 20 nucleotides at the 3' end are replaced by a targeting sequence selected from the group consisting of SEQ ID NO: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714, but having a chemical modification at the 3' end. In some embodiments, the chemically modified gRNA comprises the sequence of SEQ ID NO: 2968, wherein the 20 nucleotides at the 3' end are replaced by a target sequence selected from the group consisting of SEQ ID NOs: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714, but with a chemical modification at the 3' end. In some embodiments, the chemically modified gRNA comprises the sequence of SEQ ID NO: 2968, wherein the 20 nucleotides at the 3' end are replaced by a target sequence selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869, but with a chemical modification at 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 comprises a sequence selected from the group consisting of SEQ ID NOs: 22788-22790. Schematic diagrams of the structures of gRNA variants 174, 235, and 316 are shown below. Figure 7-9 As shown, and chemically modified versions of gRNA variants 235 and 316 in Figure 10-14 As shown in the figure. In some embodiments, chemically modified gRNAs exhibit improved stability compared to unmodified gRNAs.

[0304] e. Formation of complexes with repressor fusion proteins

[0305] When components of the system are delivered or expressed in target cells, the gRNA variant is capable of complexing with the long-term repressor fusion protein to form an RNP and binding to the target nucleic acid of the PCSK9 gene. In some embodiments, the ability of the gRNA variant to form an RNP complex with the long-term repressor fusion protein is improved compared to a reference gRNA. In some embodiments, the improved ribonucleoprotein complex formation can improve the efficiency of assembling functional RNPs therewith. 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 the gRNA variant and its target sequence, as well as the long-term repressor fusion protein of this disclosure, are capable of gene repression of the target nucleic acid.

[0306] VII. Polynucleotides and Carriers

[0307] This disclosure provides polynucleotides encoding long-term repressor fusion proteins and / or gRNAs that are effective in repression and epigenetic modification of the PCSK9 gene. This disclosure also provides polynucleotides encoding mRNAs encoding long-term repressor fusion proteins, for example, DNA polynucleotides encoding the corresponding mRNAs.

[0308] The long-term repressor fusion protein or the mRNA encoding the long-term repressor fusion protein disclosed herein can be prepared in vitro using conventional methods as known in the art. Various commercial synthetic apparatuses are available, such as automated synthesizers provided by Applied Biosystems, Inc., Beckman Coulter, etc. Using the synthesizer, naturally occurring amino acids or nucleotides (where applicable) can be substituted with non-natural amino acids or nucleotides. Specific sequences and preparation methods will be determined based on convenience, cost-effectiveness, desired purity, etc. gRNA can also be synthesized; for example, using a T7 RNA polymerase system known in the art.

[0309] Long-term repressor fusion proteins and / or gRNAs can also be prepared by recombining polynucleotide sequences encoding any of the long-term repressor fusion proteins or gRNAs described herein using standard recombination techniques known in the art, and by integrating the encoding gene into an expression vector suitable for host cells. To produce the encoded long-term repressor fusion proteins and / or gRNAs of any of the embodiments described herein, the method includes transforming suitable host cells with an expression vector containing the encoding polynucleotides, and culturing host cells under conditions that allow or permit the expression or transcription of the resulting long-term repressor fusion protein or gRNA of any of the embodiments described herein in the transformed host cells, the transformed host cells being recovered by the methods described herein or by standard purification methods known in the art or as described in the examples. Standard recombination techniques in molecular biology are used to prepare the polynucleotides and expression vectors of this disclosure.

[0310] The long-term repressor fusion protein and / or gRNA of this disclosure can also be isolated and purified according to conventional methods of recombinant synthesis. Lysates can be prepared from the expression host, and the lysates are purified using high-performance liquid chromatography (HPLC), size exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. For most fractions, the composition used will contain 50% by weight or more of the desired product, more typically 75% by weight or more, preferably 95% by weight or more, and for therapeutic purposes, typically 99.5% by weight or more, depending on contaminants associated with the preparation of the product and its purification methods. Percentages will generally be based on total protein. Thus, in some cases, the long-term repressor fusion protein or gRNA of this 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 from contaminants or other macromolecules, etc.).

[0311] Additionally, this disclosure provides vectors comprising polynucleotides encoding long-term repressor fusion proteins and gRNAs as described herein. In some cases, when the LTRP:gRNA system is delivered as a repressor fusion protein and gRNA or RNP, the vector is used to express and recover the CasX and gRNA components of said system. In other cases, the vector is used to deliver polynucleotides encoding target cells to repress and / or epigenetically modify target nucleic acids, as described more fully below. In some embodiments, the sequence encoding the long-term repressor fusion protein and the gRNA are templated on the same vector. In some embodiments, the sequence encoding the long-term repressor fusion protein and the gRNA are templated on different vectors. Suitable vectors are described, for example, in WO2023235818A2, WO2022120095A1, and WO2020247882A1 (incorporated herein by reference). As described in WO2023235818A2, WO2022120095A1 and WO2020247882A1, depending on the host / vector system used, any element from a number of suitable transcription and / or translation control elements can be used in the expression vector, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc.

[0312] This disclosure provides a polynucleotide sequence encoding a long-term repressor fusion protein of any of the embodiments described herein. In some embodiments, this disclosure provides a DNA sequence encoding a long-term repressor fusion protein for use in a vector. In some embodiments, this disclosure provides an mRNA sequence encoding a long-term repressor fusion protein of any of the embodiments described herein for delivery to a particle system in cells. In some embodiments, this disclosure provides an mRNA sequence encoding a long-term repressor fusion protein of any of the embodiments described herein for delivery to cells in an LNP particle formulation. In one particular embodiment, this disclosure provides a gRNA and an mRNA sequence encoding a long-term repressor fusion protein of any of the embodiments described herein for delivery to cells in an LNP particle formulation. In some embodiments, this disclosure provides a separate polynucleotide sequence encoding a gRNA variant of any of the embodiments described herein, having a linked targeting sequence complementary to a PCSK9 target nucleic acid sequence.

[0313] In some embodiments, this disclosure relates to methods for generating a polynucleotide sequence encoding a long-term repressor fusion protein or gRNA (including variants thereof) of any of the embodiments described herein, and methods for expressing a protein or RNA transcribed from the polynucleotide sequence. Generally, the methods include generating a polynucleotide sequence encoding a long-term repressor fusion protein or gRNA of any of the embodiments described herein, and integrating the encoding gene into an expression vector. In some embodiments, the vector is designed to transduce cells to repress and / or epigenetically modify the PCSK9 target nucleic acid. Such vectors may include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, plasmids, microcircles, nanoparticles, DNA vectors, and RNA vectors. In other embodiments, the expression vector is designed to generate a long-term repressor fusion protein, mRNA, or gRNA encoding the long-term repressor fusion protein in a cell-free system or host cells. To produce the encoded long-term repressor fusion protein or gRNA of any of the embodiments described herein in host cells, the method includes transforming suitable host cells with an expression vector containing a encoding polynucleotide, and culturing the host cells under conditions that enable or allow 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 cells, thereby producing the long-term repressor fusion protein or gRNA, wherein the transformed host cells are recovered by the method described herein (e.g., in examples below) or by standard purification methods known in the art. Standard recombinant techniques in molecular biology are used to prepare the polynucleotides and expression vectors of this disclosure.

[0314] According to this disclosure, nucleic acid sequences encoding long-term repressor fusion proteins or gRNAs of any of the embodiments described herein are used to generate recombinant DNA molecules that guide expression in suitable host cells. Several cloning strategies are suitable for carrying out this disclosure, among which many cloning strategies are used to generate constructs or complements thereof comprising genes encoding long-term repressor fusion proteins or gRNAs of this disclosure. In some embodiments, cloning strategies are used to generate genes encoding constructs comprising nucleotides encoding long-term repressor fusion proteins or gRNAs. In some embodiments, the gene, for example as part of a vector, is used to transform host cells for gene expression, such as the expression of long-term repressor fusion proteins or gRNAs.

[0315] In one method, a construct containing a DNA sequence encoding a long-term repressor fusion protein or gRNA is first prepared. Exemplary methods for preparing such constructs are described in the examples. The construct is then used to generate an expression vector suitable for transforming a host cell (such as a prokaryotic or eukaryotic host cell) to express and recover the protein construct, in the case of a long-term repressor fusion protein or gRNA. If desired, the host cell is *Escherichia coli*. In other embodiments, the host cell is a eukaryotic cell. The eukaryotic host cell may be selected from young hamster kidney fibroblast (BHK) cells, human embryonic kidney 293 (HEK293), human embryonic kidney 293T (HEK293T), NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, NIH3T3 cells, CV-1 (monkey) (COS) from sources with SV40 genetic material, HeLa, Chinese hamster ovary (CHO), yeast cells, or other eukaryotic cells known in the art suitable for producing recombinant products. Exemplary methods for generating expression vectors, transformation of host cells, and expression and recovery of long-term repressor fusion proteins or gRNAs are described in the examples.

[0316] Genes encoding long-term repressor fusion proteins or gRNA constructs can be synthesized entirely in one or more steps or prepared by synthesis in combination with enzymatic processes such as restriction enzyme-mediated cloning, PCR, and overlap extension, including the methods described more fully in the examples. The methods disclosed herein can be used, for example, to ligate polynucleotide sequences encoding various components into genes with desired sequences. Genes encoding polypeptide compositions are assembled from oligonucleotides using standard gene synthesis techniques.

[0317] In some embodiments, the nucleotide sequence encoding the long-term repressor fusion protein is codon-optimized. This type of optimization may require mutations in the encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, codons can change, but the encoded protein remains unchanged. For example, if the intended target cell for the long-term repressor fusion protein is a human cell, a human codon-optimized nucleotide sequence encoding the long-term repressor fusion protein can be used. As another non-limiting example, if the intended host cell is a mouse cell, a mouse codon-optimized nucleotide sequence encoding the long-term repressor fusion protein can be generated. Gene design can be performed using algorithms that optimize codon usage and the amino acid composition of the host cell suitable for use in the production of the long-term repressor fusion protein or gRNA. In one method of this disclosure, a library of polynucleotides encoding components of a construct is generated and then assembled, as described above. The resulting gene is then assembled, and the resulting gene is used to transform host cells and produce and recover the long-term repressor fusion protein or gRNA composition to evaluate its properties or for modification of PCSK9 target nucleic acids, as described herein.

[0318] In some embodiments, the nucleotide sequence encoding gRNA is operatively linked to a control element, such as a transcriptional control element, like a promoter. In some embodiments, the nucleotide sequence encoding a long-term repressor fusion protein is operatively linked to a control element, such as a transcriptional control element, like a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulated promoter. In some cases, the promoter is an inducible promoter. In some 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 the target cell type or target cell population. For example, in some cases, the transcriptional control element may function in eukaryotic cells (e.g., hepatocytes or hepatic sinusoidal endothelial cells).

[0319] Non-limiting examples of Pol II promoters operatively linked to polynucleotides encoding the long-term repressor fusion proteins disclosed herein include, but are not limited to: EF-1α, EF-1α core promoter, Jens Tornoe (JeT), promoters from cytomegalovirus (CMV), CMV Immediate Early (CMVIE), CMV enhancers, herpes simplex virus (HSV) thymidine kinase, early and late simian virus 40 (SV40), SV40 enhancers, long terminal repeats (LTRs) from retroviruses, mouse metallothionein-I, adenovirus major late promoter (Ad MLP), full-length CMV promoters, minimal CMV promoters, chicken β-actin promoters (CBA), CBA mixtures (CBh), chicken β-actin promoters with cytomegalovirus enhancers (CB7), chicken β-actin promoters and rabbit β-globin splice acceptor site fusion (CAG), and other sarcoma viruses. Virus (RSV) promoter, HIV-Ltr promoter, hPGK promoter, HSV TK promoter, 7SK promoter, Mini-TK promoter, human synaptic protein I (SYN) promoter confers neuron-specific expression, β-actin promoter, supercore promoter 1 (SCP1), Mecp2 promoter for selective expression in neurons, minimal IL-2 promoter, Rous sarcoma virus enhancer / promoter (single), splenic focal formation virus long terminal repeat (LTR) promoter, TBG promoter, promoter from the gene for binding human thyroxine (liver-specific), PGK promoter, human ubiquitin C promoter (UBC), UCOE promoter (promoter of HNRPA2B1-CBX3), synthetic CAG promoter, histone H2 promoter, histone H3 promoter, U1a1 small nuclear RNA promoter (226 nt), U1b2 small nuclear RNA promoter (246 nt) The promoters include 26, GUSB promoter, CBh promoter, Rhodopsin (Rho) promoter, Splenic Focal Formation Virus (SFFV) promoter, Human H1 promoter (H1), POL1 promoter, TTR minimal enhancer / promoter, β-kinin promoter, Mouse Mammary Tumor Virus Long Terminal Repeat (LTR) promoter, Human Eukaryotic Initiation Factor 4A (EIF4A1) promoter, ROSA26 promoter, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, tRNA promoter, and truncated versions and sequence variants thereof. In a particular embodiment, the Pol II promoter is EF-1α, wherein the promoter enhances the transfection efficiency of CRISPR nucleases in long-term culture, transgene transcription or expression, the proportion of expression-positive clones, and the copy number of free vectors.

[0320] Non-limiting examples of Pol III promoters operatively linked to polynucleotides encoding gRNA variants disclosed herein include, but are not limited to, U6, miniature U6, truncated U6 promoters, 7SK and H1 variants, BiH1 (bidirectional H1 promoter), BiU6, Bi7SK, BiH1 (bidirectional U6, 7SK, and H1 promoters), gorilla U6, rhesus monkey U6, human 7SK, human H1 promoters, and their truncated versions and sequence variants. In the foregoing embodiments, the pol III promoter enhances gRNA transcription. In a specific embodiment, the Pol III promoter is U6, wherein said promoter enhances gRNA expression. Experimental details and data using such promoters are provided in the examples.

[0321] The selection of appropriate vectors and promoters is entirely within the skill of a person skilled in the art, as it involves controlling expression. Expression vectors may also contain ribosome-binding sites for translation initiation and transcription terminators. Expression vectors may also include appropriate sequences for amplifying expression. Expression vectors may also include nucleotide sequences encoding protein tags (e.g., 6xHis tags, hemagglutinin tags, fluorescent proteins, etc.), which can be fused with long-term repressor fusion proteins, thus producing chimeric proteins for purification or detection.

[0322] The recombinant expression vectors of this disclosure may also include elements that promote robust expression of the proteins and gRNAs of this disclosure. For example, the recombinant expression vector may include one or more of the following: a poly(A) signal, an intron sequence, or a post-transcriptional regulatory element such as the marmot hepatitis post-transcriptional regulatory element (WPRE). Exemplary poly(A) sequences include a short hGH poly(A) signal, an HSV TK poly(A) signal, a synthetic poly(A) signal, a poly(A) sequence with an SphI restriction site between two 60A segments (SEQ ID NO: 3307), an SV40 poly(A) signal, a β-globin poly(A) signal, etc. Those skilled in the art will be able to select appropriate elements to include in the recombinant expression vectors described herein.

[0323] The polynucleotide or gRNA sequence encoding the long-term repressor fusion protein can be cloned individually into an expression vector. The selection of a suitable vector and promoter is entirely within the skill of a person skilled in the art, as it involves controlling expression, for example, for repressing expression and / or epigenetic modification of the PCSK9 gene. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include suitable sequences for amplifying expression.

[0324] Nucleic acid sequences are inserted into vectors using various methods. Generally, DNA is inserted into appropriate restriction endonuclease sites using techniques known in the art. Vector components typically include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. The construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art. Such techniques are well known in the art and are described in detail in scientific and patent literature. Various vectors are publicly available. For example, vectors may be in the form of plasmids, granules, viral particles, or bacteriophages that can readily undergo recombinant DNA procedures, and the choice of vector will generally depend on the host cell into which it will be introduced. Thus, a vector may be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid. Alternatively, a vector may be a vector that integrates into the host cell genome upon introduction into the host cell and replicates along with one or more chromosomes into which it is integrated. Once introduced into a suitable host cell, the expression of a 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 transtranscribed mRNA of a long-term repressor fusion protein can be detected and / or quantified using probes complementary to any region of the polynucleotide via conventional hybridization assays (e.g., Northern ink dot assay), amplification procedures (e.g., RT-PCR), SAGE (U.S. Patent No. 5,695,937), and array-based techniques (see, for example, U.S. Patent Nos. 5,405,783, 5,412,087, and 5,445,934).

[0325] In some embodiments, vectors are created for the transcription of long-term repressor fusion proteins and the expression and recovery of the resulting encoding mRNA. In some embodiments, mRNA is generated via in vitro transcription (IVT) using a PCR product or a linearized plasmid DNA template and T7 RNA polymerase, wherein the plasmid contains a T7 promoter. If a PCR product is used, the DNA sequence encoding a candidate mRNA is cloned into a plasmid containing a T7 promoter, wherein the plasmid DNA template is linearized and then used for an IVT reaction to express the mRNA. Exemplary methods for generating such vectors and for generating and recovering mRNA are provided in the examples below.

[0326] VIII. Particles used for delivering the LTRP:gRNA system

[0327] On the other hand, this disclosure provides particulate compositions for delivering the LTRP:gRNA system to cells or subjects for transcriptional repression or silencing of the PCSK9 gene. Particulates contemplated within the scope of this disclosure include, but are not limited to, nanoparticles such as synthetic nanoparticles, polymer nanoparticles, lipid nanoparticles, viral particles, and virus-like particles. As described herein, the particles of this disclosure may encapsulate a payload, such as a gRNA variant, optionally combined with mRNA encoding a long-term repressor fusion protein of any of the embodiments described herein. Alternatively or additionally, the particles of this disclosure may encapsulate a payload of a gRNA variant and a long-term repressor fusion protein, for example, when associated as a ribonucleoprotein (RNP) complex. In some embodiments, the particles are synthetic nanoparticles encapsulating a payload of a gRNA variant and mRNA encoding a long-term repressor fusion protein of any of the embodiments described herein. In some embodiments, the synthetic nanoparticles comprise biodegradable polymer nanoparticles (PNPs). In some embodiments, materials used to generate biodegradable polymeric nanoparticles (PNPs) 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(α-caprolactone) (PCL), cyclodextrin, and natural polymers such as chitosan, albumin, gelatin, and alginate, which are the most commonly used polymers for synthesizing PNPs (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 encapsulating gRNA variants and mRNA encoding the long-term repressor fusion protein of any of the embodiments described herein, as described more fully below. In other embodiments, the particles are lipid nanoparticles (LNPs) that encapsulate a gRNA variant and mRNA encoding a long-term repressor fusion protein of any of the embodiments described herein in separate particles, which are co-formulated into a mixture for administration, as described more fully below. In other embodiments, the particles are lipid nanoparticles that encapsulate a gRNA variant and mRNA encoding a long-term repressor fusion protein of any of the embodiments described herein, and the two types of particles are administered separately.

[0328] a. Lipid nanoparticles (LNP)

[0329] On the other hand, this disclosure provides lipid nanoparticles (LNPs) for delivering the LTRP:gRNA system of this disclosure to cells or subjects for transcriptional repression of the PCSK9 gene. In some embodiments, the LNPs of this disclosure are tissue or organ (e.g., liver) specific, have excellent biocompatibility, and can be delivered to systems with high efficiency, and therefore can be effectively used for transcriptional repression of the PCSK9 gene.

[0330] This disclosure further provides LNP compositions and pharmaceutical compositions comprising the various LNPs described herein.

[0331] Nucleic acid polymers, in their native form, are typically unstable in biological fluids and cannot penetrate the cytoplasm of target cells, thus necessitating delivery systems. Lipid nanoparticles (LNPs) have been shown to be useful for protecting nucleic acids and delivering them to tissues and cells. Furthermore, compared to DNA vectors, using mRNA to encode long-term repressor fusion proteins in LNPs eliminates the possibility of undesirable genome integration. Additionally, mRNA efficiently transfects both mitotic and non-mitotic cells because it does not need to enter the nucleus, as it functions within the cytoplasmic compartment. Therefore, LNPs, as a delivery platform, offer the additional advantage of being able to co-decode both mRNA and gRNA encoding long-term repressor fusion proteins into a single LNP particle.

[0332] Therefore, in various embodiments, this disclosure covers lipid nanoparticles and compositions that can be used for a variety of purposes, including the in vitro and in vivo delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells. In some embodiments, this disclosure covers methods of treating or preventing a disease or condition of a subject by contacting lipid nanoparticles with the subject in need, the lipid nanoparticles being encapsulated with or associated with a suitable therapeutic agent through various physical, chemical, or electrostatic interactions between one or more lipid components used in the composition to prepare an LNP. In some embodiments, the suitable therapeutic agent comprises an LTRP:gRNA system as described herein.

[0333] In some embodiments, lipid nanoparticles can be used to deliver nucleic acids, including, for example, mRNA and gRNA variants encoding long-term repressor fusion proteins of the present disclosure. In some embodiments, the LNP comprises mRNA containing a sequence encoding a long-term repressor fusion protein selected from the group consisting of: SEQ ID NO: 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 with it. In some embodiments, the LNP comprises an mRNA including a sequence selected from the group consisting of: SEQ ID NO: 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 with it. In some embodiments, the LNP comprises mRNA including sequences selected from the group consisting of: SEQ ID NOs: 6529-6537, 9742-9750, 14628, 14636, and 17841-17849, or sequences 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 with them. In some embodiments, the LNP comprises mRNA including sequences selected from the group consisting of: SEQ ID NOs: 6529-6537, 9742-9750, 14628, 14636, and 17841-17849. In some embodiments, the LNP comprises mRNA including sequences selected from the group consisting of SEQ ID NOs: 6531, 9744, 14630, and 17843, or sequences 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 with them. In some embodiments, the LNP comprises mRNA including sequences selected from the group consisting of SEQ ID NOs: 6531, 9744, 14630, and 17843.In some embodiments, the LNP comprises mRNA including sequences selected from the group consisting of SEQ ID NOs: 6529, 9742, 14628, and 17841, or sequences having sequence identity of 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%. In some embodiments, the LNP comprises mRNA including sequences selected from the group consisting of SEQ ID NOs: 6529, 9742, 14628, and 17841. In some embodiments, the LNP comprises mRNA including sequences selected from the group consisting of SEQ ID NOs: 6530, 9743, 14629, and 17842, or sequences 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 with them. In some embodiments, the LNP comprises mRNA including sequences selected from the group consisting of SEQ ID NOs: 6530, 9743, 14629, and 17842. In some embodiments, the LNP comprises a gRNA including a scaffold sequence selected from the group consisting of SEQ ID NOs: 1744-1746, 2948-2956, 2958-2966, and 2968-2974, and a linked target sequence complementary to the PCSK9 gene (in the case of SEQ ID NOs: 2948-2956, 2958-2966, and 2968-2974, the 20 non-target nucleotides at the 3' end are replaced with a target sequence complementary to the PCSK9 gene, but the chemical modification at the 3' end is retained). In some embodiments, the LNP comprises a gRNA including a scaffold sequence selected from the group consisting of SEQ ID NOs: 2278-22803. In some embodiments, the LNP comprises a gRNA including a sequence selected from the group consisting of SEQ ID NOs: 2278-22790. In some embodiments, this disclosure provides an LNP in which gRNA and mRNA encoding a long-term repressor fusion protein are integrated into a single LNP particle. In other embodiments, this disclosure provides LNPs in which gRNAs that can be formulated together in different ratios for administration and mRNAs encoding long-term repressor fusion proteins are integrated into separate LNP populations.

[0334] The lipid nanoparticles and lipid nanoparticle compositions of this disclosure can be used to repress the expression of desired proteins in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more ionizable lipids as described herein, wherein the lipid nanoparticles encapsulate or associate with nucleic acids expressed to produce the desired protein (e.g., messenger RNA encoding a long-term repressor fusion protein). In some embodiments, the lipid nanoparticles and compositions can be used to repress the expression of target genes in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more cationic lipids as described herein, wherein the lipid nanoparticles encapsulate or associate with one or more nucleic acids of the LTRP:gRNA system of this disclosure. The lipid nanoparticles and compositions of embodiments of this disclosure can also be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA) alone or in combination, such as to provide the desired effect of co-localizing different nucleic acids (e.g., mRNA encoding a suitable gene repressor factor or enzyme and gRNA for a target gene).

[0335] In some embodiments, the LNPs and LNP compositions described herein comprise at least one cationic lipid, at least one conjugated lipid, at least one steroid or a derivative thereof, at least one additional lipid, or any combination thereof. Alternatively, the lipid compositions disclosed herein may comprise ionizable lipids, such as ionizable cationic lipids, accessory lipids (typically phospholipids), cholesterol, and polyethylene glycol-lipid conjugates (PEG-lipids), to improve colloidal stability in biological environments by, for example, reducing the specific uptake of plasma proteins and forming a hydration layer over nanoparticles. Such lipid compositions can be formulated in typical molar ratios of 50:10:37-39:1.5-2.5 or 20-50:8-65:25-40:1-2.5, and variations can be made to tailor individual properties.

[0336] The LNPs and LNP compositions disclosed herein are configured to protect the encapsulated payload of the system disclosed herein and deliver the payload to tissues and cells, whether in vitro or in vivo. Various embodiments of the LNPs and LNP compositions disclosed herein are further described in detail herein.

[0337] cationic lipids

[0338] In some embodiments, the LNPs and LNP compositions of this disclosure comprise at least one cationic lipid. The term "cationic lipid" refers to a lipid species having 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 pKa of the ionizable cationic lipid is less than 7, such that the LNPs and LNP compositions achieve efficient encapsulation of the payload at relatively low pH. In some embodiments, the pKa of the cationic lipid is 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. In some embodiments, the cationic lipid can be protonated at pH below the pKa of the cationic lipid, and the cationic lipid can be substantially neutral at pH above the pKa. The LNPs and LNP compositions can be safely delivered in vivo to target organs (e.g., liver, lung, heart, spleen, and to tumors) and / or cells (hepatocytes, LSECs, cardiomyocytes, cancer cells, etc.) and, following endocytosis, exhibit a positive charge to release the encapsulated payload through electrostatic interactions with anionic proteins of the endosome membrane.

[0339] Early formulations of permanently charged cationic lipids (LNPs) were developed to produce positively surface-charged LNPs that have been shown to be toxic in vivo and rapidly cleared by phagocytes. By converting them into ionizable cationic lipids carrying tertiary amines, specifically ionizable cationic lipids with pKa <7, the LNPs achieve efficient encapsulation of nucleic acid polymers at low pH through electrostatic interactions with the negatively charged phosphate backbone of mRNA. This also makes the system primarily neutral at physiological pH, thus mitigating the problems associated with permanently charged cationic lipids.

[0340] As used herein, "ionizable lipid" means an amine-containing lipid that can be readily protonated, and, for example, the ionizable lipid can be a lipid whose charge state changes according to the ambient pH. Ionizable lipids can be protonated (positively charged) at pH levels below the pKa of cationic lipids, and can be substantially neutral at pH levels above the pKa. In one example, an LNP may comprise protonated ionizable lipids and / or neutral ionizable lipids. In some embodiments, the pKa of the LNP is 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7. The pKa of the LNP can affect the in vivo stability and release of the nucleic acid payload of the LNP in target cells or organs. In some embodiments, LNPs having the aforementioned pKa range can be safely delivered in vivo to target organs (e.g., liver, lung, heart, spleen, and to tumors) and / or target cells (hepatocytes, LSECs, cardiomyocytes, cancer cells, etc.) and, following endocytosis, exhibit a positive charge to release the encapsulated payload through electrostatic interaction with anionic proteins of the endosome membrane.

[0341] Ionizable lipids are ionizable compounds that generally have properties similar to lipids and can play a role in efficiently encapsulating nucleic acid payloads within LNPs through electrostatic interactions with nucleic acids (e.g., mRNA of this disclosure).

[0342] Depending on the type of amines and tails contained in the ionizable lipids, (i) nucleic acid encapsulation efficiency; (ii) PDI (polydispersity index); and / or (iii) nucleic acid delivery efficiency to tissues and / or cells of the organ constituting the LNP (e.g., hepatocytes or sinusoidal endothelial cells in the liver). In some embodiments, the ionizable lipids are ionizable cationic lipids and comprise approximately 46 mol% to approximately 66 mol% of the total lipids present in the particles.

[0343] LNPs containing ionizable lipids including amines may have one or more of the following properties: (1) the ability to encapsulate nucleic acids with high efficiency; (2) uniformly sized prepared particles (or with low PDI values); and / or (3) excellent nucleic acid delivery efficiency to organs such as liver, lung, heart, spleen, bone marrow, and to tumors and / or cells constituting such organs (e.g., hepatocytes, LSECs, cardiomyocytes, cancer cells, etc.).

[0344] In certain embodiments, the cationic lipid form plays a crucial role in both nucleic acid encapsulation via electrostatic interactions and intracellular release via disruption of the endosome membrane. The nucleic acid payload is encapsulated within the LNP via ionic interactions formed between it and the positively charged cationic lipid. Non-limiting examples of the cationic lipid components used in the LNPs of this disclosure are selected from DLin-MC3-DMA (4-(dimethylamino)butanoic acid heptadecane-6,9,28,31-tetraen-19-yl ester), DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), TNT (1,3,5-triazine-2,4,6-trione), and TT (N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide). Non-limiting examples of accessory lipids used in the LNPs disclosed herein are selected from: DSPC (1,2-distearyl-sn-glycerol-3-phosphate choline), POPC (2-oleoyl-1-palmitoyl-sn-glycerol-3-phosphate choline), and DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)DOPG, 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 1,2-dilauroyl-sn-glycerol-3-phosphate choline (DLPC), sphingolipids, and ceramides. Cholesterol and PEG-DMG ((R)-2,3-bis(octadecyloxy)propyl-1-(methoxy polyethylene glycol 2000) carbamate), PEG-DSG (1,2-distearyl-rac-glycerol-3-methylpolyoxyethylene glycol 2000) or DSPE-PEG2k (1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)-2000]) are components in the LNP of this disclosure for the stability, cycling and size of the LNP.

[0345] In some embodiments, the cationic lipids in the LNP of this disclosure comprise tertiary amines. In some embodiments, the tertiary amine comprises an alkyl chain linked to the N-terminus of the tertiary amine having an ether bond. In some embodiments, the alkyl chain comprises a C12-C30 alkyl chain having 0 to 3 double bonds. In some embodiments, the alkyl chain comprises a C16-C22 alkyl chain. In some embodiments, the alkyl chain comprises a C18 alkyl chain. Numerous cationic lipids and related analogues have been described in U.S. Patent Publications Nos. 20060083780, 20060240554, 20110117125, 20190336608, 20190381180, and 20200121809; U.S. Patents 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 The contents of document No. 96 / 10390 are incorporated herein by reference in their entirety.

[0346] In some embodiments, the cationic lipids in the LNP of this disclosure may comprise, for example, one or more ionizable cationic lipids, wherein the ionizable cationic lipids are dialkyl lipids. In other embodiments, the ionizable cationic lipids are trialkyl lipids.

[0347] In some embodiments, the cationic lipids in the LNP of this disclosure are selected from: 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleoyl-4-(3-dimethylamino) 2,2-Dilinoleoyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleoyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleoyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K- MPZ), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl... DLin-3-dimethylaminopropane (DLinDAP), DLin-S-DMA, DLin-2-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), DLin-TMA.Cl, and DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazine)propane (DLin-MPZ), 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyloxy-N,N-dimethyl... DODMA, 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleoyloxy)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-Cho) l), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[2-(spermine-formamide)ethyl]-N,N-dimethyl-1-propionitrile trifluoroacetate (DOSPA), bis(octadecylamide)spermine glyceryl ester (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[ 5'-(cholest-5-en-3-β-oxy)-3'-oxaproloxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), and any combination thereof.

[0348] In some embodiments, the cationic lipids in the LNP of this disclosure are selected from methyl heptanetridec-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (1,3,5-triazine-2,4,6-trione) (TNT), N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT), and any combination thereof.

[0349] In some embodiments, the N / P ratio (nitrogen from cations / ionizable lipids and phosphate from nucleic acids) in the LNP of this disclosure ranges from about 3:1 to 7:1, or about 4:1 to 6:1, or 3:1, or 4:1, or 5:1, or 6:1, or 7:1.

[0350] Conjugated lipids

[0351] In some embodiments, the LNPs and LNP compositions of this disclosure comprise at least one conjugated lipid. In some embodiments, the conjugated lipid may be selected from polyethylene glycol (PEG)-lipid conjugates, polyamide (ATTA)-lipid conjugates, cationic polymer-lipid conjugates (CPL), and any combination thereof. In some cases, the conjugated lipid may inhibit the aggregation of the LNPs of this disclosure.

[0352] In some embodiments, the conjugated lipids of the LNP disclosed herein comprise PEGylated lipids. The terms “polyethylene glycol (PEG)-lipid conjugate,” “PEGylated lipid,” “lipid-PEG conjugate,” “lipid-PEG,” “PEG-lipid,” “PEG-lipid,” or “lipid-PEG” are used interchangeably herein and refer to lipids attached to a polyethylene glycol (PEG) polymer, which is a hydrophilic polymer. PEGylated lipids contribute to the stability of the LNP and the LNP composition and reduce LNP aggregation.

[0353] When PEG-lipids can form surface lipids, the size of LNPs can be readily altered by changing the ratio of surface (PEG) lipids to core (ionizable cationic) lipids. In some embodiments, the PEG-lipids of the LNPs of this disclosure can be varied by about 1 to 5 mol% to modify particle properties such as size, stability, and cycling time.

[0354] Lipid-PEG conjugates contribute to the stability of nanoparticles in serum within LNPs and prevent aggregation between nanoparticles. Additionally, lipid-PEG conjugates can protect nucleic acids (such as mRNA encoding the long-term repressor fusion protein of this disclosure or gRNA of this disclosure) from enzyme degradation during in vivo delivery of nucleic acids, and enhance the stability of nucleic acids in vivo and increase the half-life of the delivered nucleic acids encapsulated in nanoparticles. Examples of PEG-lipid conjugates include, but are not limited to, PEG-DAG conjugates, PEG-DAA conjugates, and mixtures thereof. In some embodiments, the PEG-lipid conjugates are selected from the group consisting of PEG-diacylglycerol (PEG-DAG) conjugates, PEG-dialkyloxypropyl (PEG-DAA) conjugates, PEG-phospholipid conjugates, PEG-ceramide (PEG-Cer) conjugates, and mixtures thereof.

[0355] In some embodiments, the PEGylated lipids of the LNP disclosed herein are selected from PEG-ceramide, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-dialkoxypropylcarbamate, PEG-phosphatidylethanolamine, PEG-phospholipid, PEG-succinate diacylglycerol and any combination thereof.

[0356] In some embodiments, the PEGylated lipid of the LNP disclosed herein is PEG-dialkyloxypropyl. In some embodiments, the PEGylated lipid is selected from PEG-decyloxypropyl (C10), PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmitoyloxypropyl (C16), PEG-distearyloxypropyl (C18), and any combination thereof.

[0357] In other embodiments, the lipid-PEG conjugate of the LNP disclosed herein may be a PEG conjugated with phospholipids, such as phosphatidylethanolamine (PEG-PE); a PEG conjugated with ceramides (PEG-CER, ceramide-PEG conjugate, ceramide-PEG, cholesterol or PEG conjugated with its derivatives, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE (DSPE-PEG) and mixtures thereof, and may be, for example, C16-PEG2000 ceramide (N-palmitoyl-sphingosine-1-{succinoyl[methoxy(polyethylene glycol)2000]}), DMG-PEG 2000, 14:0 PEG2000 PE).

[0358] In some embodiments, the PEGylated lipids of the LNP disclosed herein are selected from 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol, 4-O-(2',3'-di(tetradecyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate, 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, and any combination thereof.

[0359] In some embodiments, the PEGylated lipids of the LNP disclosed herein are selected from mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol (PEG-C-DOMG), 1-[8'-(1,2-dimyristoyl-3-propoxy)-formamide-3',6'-dioxaoctyl]carbamoyl-w-methyl-poly(ethylene glycol) (2KPEG-DMG) and any combination thereof.

[0360] In some embodiments, PEG is directly attached to the lipids of the PEGylated lipid. In other embodiments, PEG is attached to the lipids of the PEGylated lipid via a connector portion selected from ester-free or ester-containing connector portions. Non-limiting examples of ester-free connector portions include amides (-C(O)NH-), aminos (-NR-), carbonyls (-C(O)-), carbamates (-NHC(O)O-), ureas (-NHC(O)NH-), disulfides (-SS-), ethers (-O-), succinoyl groups (-(O)CCH2CH2C(O)-), succinamide groups (-NHC(O)CH2CH2C(O)NH-), ethers, disulfides, and combinations thereof. For example, the connector may contain both carbamate and amide connector portions. Non-limiting examples of ester-containing connector portions include carbonates (-OC(O)O-), succinoyl groups, phosphate esters (-O-(O)POH-O-), sulfonates, and combinations thereof.

[0361] The average molecular weight of the PEG portion of the PEGylated lipid of the LNP disclosed herein can range from about 550 Daltons to about 10,000 Daltons. In some embodiments, the average molecular weight of the PEG portion is 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 1,750 Daltons to about 2,000 Daltons.

[0362] In some embodiments, the conjugated lipids (e.g., PEGylated lipids) comprise about 1 mol% to about 60 mol%, about 2 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 20 mol% of the total lipids present in the LNP and / or LNP composition. In some embodiments, the conjugated lipids comprise about 0.5 mol% to about 3 mol% of the total lipids present in the particles.

[0363] In another embodiment, the conjugated lipids of the LNP of this disclosure (e.g., PEGylated lipids) account for at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 mol% of the total lipids present in the LNP and / or LNP composition, or an intermediate range of any of the foregoing.

[0364] For the lipids in the lipid-PEG conjugates of the LNP of this disclosure, any lipid capable of binding to polyethylene glycol can be used without limitation, and phospholipids and / or cholesterol, which are other elements of the LNP, can also be used. In some embodiments, the lipids in the lipid-PEG conjugates can be ceramides, dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s-DAG), distearate phosphatidylcholine (DSPC), distearate phosphatidylethanolamine (DSPE), or cholesterol, but are not limited thereto.

[0365] In the lipid-PEG conjugates of the LNP disclosed herein, PEG can be directly conjugated to the lipid or linked to the lipid via a linker portion. Any linker portion suitable for binding PEG to the lipid can be used, and includes, for example, ester-free and ester-containing linker portions. The ester-free linker portions include, but are not limited to, amide groups (-C(O)NH-), amino groups (-NR-), carbonyl groups (-C(O)-), urethane groups (-NHC(O)O-), urea groups (-NHC(O)NH-), disulfides (-SS-), ethers (-O-), succinyl groups (-(O)CCH2CH2C(O)-), succinimide groups (-NHC(O)CH2CH2C(O)NH-), ethers, disulfides, and combinations thereof (e.g., linkers containing both urethane and amide linker portions). Ester-containing linker portions include, for example, carbonates (-OC(O)O-), succinyl groups, phosphate esters (-O-(O)POH-O-), sulfonates, and combinations thereof, but are not limited thereto.

[0366] steroids

[0367] In some embodiments, the LNP and LNP composition disclosed herein comprise at least one steroid or a derivative thereof. In some embodiments, the steroid comprises cholesterol. In some embodiments, the LNP and LNP composition comprises a cholesterol derivative selected from: cholesterolanol, cholesterolanone, cholesterolenone, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, and any combination thereof.

[0368] In some embodiments, the steroids (e.g., cholesterol) of the LNP of this disclosure account for about 1 mol% to about 60 mol%, about 2 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 20 mol% of the total lipids present in the LNP and / or the LNP composition. In other embodiments, the steroids (e.g., cholesterol) of the LNP of this disclosure account for at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol% of the total lipids present in the LNP and / or the LNP composition, or an intermediate range of any of the foregoing.

[0369] Other lipids

[0370] In some embodiments, the LNP and LNP composition disclosed herein include at least one additional lipid. In some embodiments, the additional lipid is a non-cationic lipid selected from anionic lipids, neutral lipids, or both. In some embodiments, the additional lipid comprises at least one phospholipid. In some embodiments, the phospholipid is selected from anionic phospholipids, neutral phospholipids, or both. The phospholipids of the elements of the LNP and LNP composition can serve to cover and protect the core of the LNP formed by the interaction of cationic lipids and nucleic acids in the LNP, and can promote cell membrane permeation and endosome escape during intracellular delivery of nucleic acids by binding to the phospholipid bilayer of the target cell. Phospholipids that can promote LNP fusion with cells may include, but are not limited to, any phospholipid selected from the group described below.

[0371] In some embodiments, the LNP and the LNP composition comprise at least one phospholipid selected from, but not limited to, the following: dipalmitoyl-phosphatidylcholine (DPPC), distearyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylethanolamine (DOPE), dioleoyl-phosphatidylcholine (DOPC), dioleoyl-phosphatidylglycerol (DOPG), palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), palmitoyl-oleoyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-phosphatidylglycerol (DPPG), dimyristoyl-phosphatidylethanolamine (… DMPE), distearate-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, ditransoleoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lecithinylcholine (EPC), phosphatidylethanolamine (PE), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycerol-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-sn-glycerol-3-[phospho-L-serine], and any combination thereof. In one instance, LNPs containing DOPE can be effective in mRNA delivery (exhibiting excellent drug delivery efficacy).

[0372] In some embodiments, the additional lipids (e.g., phospholipids) of the LNP of this disclosure account for about 1 mol% to about 60 mol%, about 2 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 20 mol% of the total lipids present in the LNP and / or the LNP composition. In other embodiments, the additional lipids (e.g., phospholipids) of the LNP of this disclosure account for at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol% of the total lipids present in the LNP and / or the LNP composition, or an intermediate range of any of the foregoing.

[0373] It should be understood that the total lipids present in LNP and / or LNP compositions comprise a combination of cationic lipids or ionizable cationic lipids, conjugated lipids (e.g., PEGylated lipids), steroids (e.g., cholesterol) and other lipids (e.g., phospholipids).

[0374] LNPs and / or LNP compositions can be prepared by dissolving total lipids (or a portion thereof) in an organic solvent (e.g., ethanol), followed by mixing with a payload (e.g., the system's nucleic acid) dissolved in an acidic buffer (e.g., pH 4) via a micromixer. At this pH, the cationic lipids are positively charged and interact with the negatively charged nucleic acid polymer. Upon dialyzing with a neutral buffer, the resulting nanostructure containing the nucleic acid is converted to a neutral LNP, which can then be followed by removal of the organic solvent (e.g., ethanol) and exchange of the LNP for a physiologically relevant buffer. In contrast to conventional bilayer liposome structures, the LNPs and / or LNP compositions thus formed possess a unique electronically dense nanostructured core, in which the cationic lipids are organized into reverse micelles surrounding the encapsulated payload. In another embodiment, the LNPs can form bubble-like structures with the nucleic acid along a non-electronically dense lipid core in an aqueous bag.

[0375] b. Properties of lipid nanoparticles

[0376] In some embodiments, the LNP and / or the LNP composition comprises about 50 mol% to about 85 mol% of cationic lipids or ionizable cationic lipids, about 0.5 mol% to about 10 mol% of conjugated lipids (e.g., PEGylated lipids), about 0.5 mol% to about 10 mol% of steroids (e.g., cholesterol), and about 5 mol% to about 50 mol% of other lipids (e.g., phospholipids). In some embodiments, the LNP and / or the LNP composition comprises about 50 mol% to about 85 mol% of cationic lipids or ionizable cationic lipids, about 0.5 mol% to about 5 mol% of conjugated lipids (e.g., PEGylated lipids), about 0.5 mol% to about 5 mol% of steroids (e.g., cholesterol), and about 5 mol% to about 20 mol% of other lipids (e.g., phospholipids).

[0377] In some embodiments, the LNP and / or LNP compositions disclosed herein comprise a cationic lipid:another lipid (e.g., phospholipid):steroid (e.g., cholesterol):conjugated lipid (e.g., PEGylated lipid) in a molar ratio of 20 to 50:10 to 30:30 to 60:0.5 to 5, 25 to 45:10 to 25:40 to 50:0.5 to 3, 25 to 45:10 to 20:40 to 55:0.5 to 3, or 25 to 45:10 to 20:40 to 55:1.0 to 1.5.

[0378] In some embodiments, the total lipids:load ratio (mass / mass) of the LNP and / or LNP compositions disclosed herein is from about 1 to about 100. In some embodiments, the total lipids:load ratio is from 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 lipids:load ratio is from about 5 to about 15, for example, from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or a range in between.

[0379] In some embodiments, the LNP of this disclosure comprises a total lipid:nucleic acid mass ratio of about 5:1 to about 15:1. In some embodiments, the weight ratio of cationic lipids and nucleic acids contained 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.

[0380] In some embodiments, the LNP disclosed herein may comprise 20 to 50 parts by weight of cationic lipids, 10 to 30 parts by weight of phospholipids, 20 to 60 parts by weight (or 20 to 60 parts by weight) of cholesterol, and 0.1 to 10 parts by weight (or 0.25 to 10 parts by weight, 0.5 to 5 parts by weight) of a lipid-PEG conjugate. Alternatively, based on the total nanoparticle weight, the LNP may comprise 20% to 50% by weight of cationic lipids, 10% to 30% by weight of phospholipids, 20% to 60% by weight (or 30% to 60% by weight) of cholesterol, and 0.1% to 10% by weight (or 0.25% to 10% by weight, 0.5% to 5% by weight) of a lipid-PEG conjugate. As an alternative, based on the total nanoparticle weight, the LNP may contain 25% to 50% cationic lipids, 10% to 20% phospholipids, 35% to 55% cholesterol, and 0.1% to 10% (or 0.25% to 10% and 0.5% to 5% lipid-PEG conjugates) of lipids.

[0381] In some embodiments, the average diameter of the LNP disclosed herein is approximately 20 nm to 200 nm, 20 nm to 180 nm, 20 nm to 170 nm, 20 nm to 150 nm, 20 nm to 120 nm, 20 nm to 100 nm, 20 nm to 90 nm, 30 nm to 200 nm, 30 nm to 180 nm, 30 nm to 170 nm, 30 nm to 150 nm, 30 nm to 120 nm, 30 nm to 100 nm, 30 nm to 90 nm, 40 nm to 200 nm, 40 nm to 180 nm, 40 nm to 170 nm, 40 nm to 150 nm, 40 nm to 120 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 50 nm to 200 nm, 50 nm to 180 nm, 50 nm to 170 nm, 50 nm to 200 nm, 50 nm to 180 nm, 50 nm to 170 nm, 50 nm to 170 nm, 50 nm to 18 ... nm to 150 nm, 50 nm to 120 nm, 50 nm to 100 nm, 50 nm to 90 nm, 60 nm to 200 nm, 60 nm to 180 nm, 60 nm to 170 nm, 60 nm to 150 nm, 60 nm to 120 nm, 60 nm to 100 nm, 60 nm to 90 nm, 70 nm to 200 nm, 70 nm to 180 nm, 70 nm to 170 nm, 70 nm to 150 nm, 70 nm to 120 nm, 70 nm to 100 nm, 70 nm to 90 nm, 80 nm to 200 nm, 80 nm to 180 nm, 80 nm to 170 nm, 80 nm to 150 nm, 80 nm to 120 nm, 80 nm to 100 nm, 80 nm to 90 nm, 90 nm to 200 nm, 90 nm to 180 nm, 90 nm to 170 nm nm, 90 nm to 150 nm, 90 nm to 120 nm, or 90 nm to 100 nm, or any intermediate range of the foregoing.

[0382] In some embodiments, the LNPs and / or LNP compositions of this disclosure have a positive charge at an acidic pH and can encapsulate the payload (e.g., a therapeutic agent, such as the LTRP:gRNA system, or a polynucleotide encoding it) by electrostatic charge generated by the negative charge of the payload (e.g., a therapeutic agent). The term “encapsulation” refers to a mixture of lipids that surround and encapsulate the payload (e.g., a therapeutic agent) under physiological conditions, thereby forming an LNP. As used herein, the term “encapsulation efficiency” is the amount of payload (e.g., a therapeutic agent) encapsulated by the LNP divided by the total amount of payload (e.g., a therapeutic agent) used to load the payload (e.g., a therapeutic agent) into the LNP. The encapsulation efficiency of the LNPs and / or LNP compositions can 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 LNP and / or the LNP composition is about 80% to 99%, about 85% to 98%, about 88% to 95%, or about 90% to 95%, or the payload (e.g., a systemic nucleic acid) can be completely encapsulated within the lipid portion of the LNP composition, thereby protecting it from enzymatic degradation. In some embodiments, after exposing the LNP and / or the LNP composition to a nuclease at 37°C for at least about 20, 30, 45, or 60 minutes or 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, the payload (e.g., a therapeutic agent) is substantially undegraded. In some embodiments, the payload (e.g., a systemic nucleic acid) is compounded with the lipid portion of the LNP and / or the LNP composition. The LNP and / or LNP compositions of this disclosure are non-toxic to mammals such as humans.

[0383] The term "fully encapsulated" indicates that the payload (e.g., systemic nucleic acid) in the LNP and / or LNP composition does not significantly degrade after exposure to conditions that degrade cell-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., systemic nucleic acid) in the LNP and / or LNP composition is degraded under conditions that would degrade 100% of the unencapsulated payload. "Fully encapsulated" also indicates that the LNP and / or LNP composition is serum stable and does not disintegrate into its component parts after in vivo administration.

[0384] In some embodiments, the amount of LNP and / or LNP composition having an effective payload (e.g., a therapeutic agent) encapsulated therein is about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 8 ... 5% to about 95%, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, 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 any intermediate range of the foregoing.

[0385] In some embodiments, the amount of payload (e.g., nucleic acid) encapsulated within the LNP and / or LNP composition is about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, or about 85%. The percentage is approximately 95%, approximately 90% to approximately 95%, approximately 30% to approximately 90%, approximately 40% to approximately 90%, approximately 50% to approximately 90%, approximately 60% to approximately 90%, approximately 70% to approximately 90%, approximately 80% to approximately 90%, or at least approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any intermediate range of the foregoing.

[0386] In some embodiments, the nucleic acids of this disclosure, such as mRNA and / or gRNA encoding a long-term repressor fusion protein, may be provided in a solution mixed with a lipid solution, such that the nucleic acids can be encapsulated in lipid nanoparticles. Suitable nucleic acid solutions may be any aqueous solution containing nucleic acids to be encapsulated at various concentrations. For example, suitable nucleic acid solutions may contain one or more nucleic acids at concentrations of 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 mRNA encoding a long-term repressor fusion protein, and a suitable mRNA solution may contain mRNA concentrations 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, and 0.05-0.6 mg / ml. 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 mRNA concentrations of 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 gRNA concentrations of 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.

[0387] In some embodiments, the average diameter of the LNP can be 20 nm to 200 nm, 20 nm to 180 nm, 20 nm to 170 nm, 20 nm to 150 nm, 20 nm to 120 nm, 20 nm to 100 nm, 20 nm to 90 nm, 30 nm to 200 nm, 30 nm to 180 nm, 30 nm to 170 nm, 30 nm to 150 nm, 30 nm to 120 nm, 30 nm to 100 nm, 30 nm to 90 nm, 40 nm to 200 nm, 40 nm to 180 nm, 40 nm to 170 nm, 40 nm to 150 nm, 40 nm to 120 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 50 nm to 200 nm, 50 nm to 180 nm, 50 nm to 170 nm, 50 nm to 150 nm. nm, 50 nm to 120 nm, 50 nm to 100 nm, 50 nm to 90 nm, 60 nm to 200 nm, 60 nm to 180 nm, 60 nm to 170 nm, 60 nm to 150 nm, 60 nm to 120 nm, 60 nm to 100 nm, 60 nm to 90 nm, 70 nm to 200 nm, 70 nm to 180 nm, 70 nm to 170 nm, 70 nm to 150 nm, 70 nm to 120 nm, 70 nm to 100 nm, 70 nm to 90 nm, 80 nm to 200 nm, 80 nm to 180 nm, 80 nm to 170 nm, 80 nm to 150 nm, 80 nm to 120 nm, 80 nm to 100 nm, 80 nm to 90 nm, 90 nm to 200 nm, 90 nm to 180 nm, 90 nm to 170 nm, 90 LNPs can be sized to facilitate delivery to liver tissue, hepatocytes, and / or LSECs (hepatic sinusoidal endothelial cells) in the ranges of 150 nm, 90 nm to 120 nm, or 90 nm to 100 nm. LNPs can also be sized for easy delivery to organs or tissues, including but not limited to the liver, lungs, heart, and spleen. When the size of an LNP is smaller than the above ranges, stability may be difficult to maintain due to the excessive increase in surface area, and thus delivery to the target tissue and / or drug efficacy may be reduced. LNPs can specifically target liver tissue. Without being bound by theory, it is thought that one mechanism by which LNPs can be used to deliver therapeutic agents is by mimicking the metabolic behavior of natural lipoproteins, and therefore LNPs can be efficiently delivered to the subject through lipid metabolism processes carried out by the liver.During the delivery of therapeutic agents to hepatocytes and / or LSECs (hepatic sinusoidal endothelial cells), the diameter of the fenestrations guiding from the sinusoidal lumen to the hepatocytes and LSECs is about 140 nm in mammals and about 100 nm in humans. Therefore, LNP compositions for therapeutic agent delivery having LNPs with diameters in the above range can have excellent delivery efficiency to hepatocytes and LSECs compared to LNPs with diameters outside the above range.

[0388] According to one example, the LNP in the LNP composition may comprise a cationic lipid:phospholipid:cholesterol:lipid-PEG conjugate in the ranges described above or in molar ratios of 20 to 50:10 to 30:30 to 60:0.5 to 5, 25 to 45:10 to 25:40 to 50:0.5 to 3, 25 to 45:10 to 20:40 to 55:0.5 to 3, or 25 to 45:10 to 20:40 to 55:1.0 to 1.5. LNPs containing components in molar ratios within the above ranges may exhibit excellent delivery efficiency as therapeutic agents specific to the cells of target organs.

[0389] In some respects, LNPs exhibit a positive charge under acidic pH conditions by displaying pKas of 5 to 8, 5.5 to 7.5, 6 to 7, or 6.5 to 7, and can readily encapsulate nucleic acids with high efficiency by forming complexes with therapeutic agents such as negatively charged nucleic acids through electrostatic interactions. In this case, LNPs can be effectively used as compositions for intracellular or in vivo delivery of therapeutic agents (e.g., nucleic acids).

[0390] In this paper, "encapsulation" or "encapsulation" refers to the effective delivery of a therapeutic agent, i.e., by surrounding the therapeutic agent with the particle surface and / or embedding the therapeutic agent within the particle. Encapsulation efficiency refers to the amount of therapeutic agent encapsulated in the LNP relative to the total amount of therapeutic agent used to prepare the LNP.

[0391] The encapsulation of nucleic acids in the composition of 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 in the composition encapsulating nucleic acids. In some embodiments, the encapsulation of nucleic acids in the composition of the LNP is such that the encapsulation of nucleic acids in the LNP is 80% to 99%, 80% to 97%, 80% to 95%, 85% to 95%, 87% to 95%, 90% to 95%, 91% or more to 95% or less, 91% or more to 94% or less, greater than 91% to 95% or less, 92% to 99%, 92% to 97%, or 92% to 95%. In some embodiments, the mRNA and gRNA encoding the long-term repressor fusion protein of any embodiment of the embodiments of this disclosure are completely encapsulated in the LNP.

[0392] Nucleic acids can be delivered to target organs via LNPs, including but not limited to the liver, lungs, heart, spleen, and tumors. One example of an LNP is liver tissue-specific and exhibits excellent biocompatibility, and can efficiently deliver nucleic acids from the composition, thus making it effective for use in related technological fields such as lipid nanoparticle-mediated gene therapy. In a particular embodiment, the target cells to which the nucleic acids are to be delivered via an LNP according to one example may be hepatocytes and / or in vivo LSECs. In other embodiments, this disclosure provides LNPs formulated for delivering nucleic acids of the embodiments to ex vivo cells.

[0393] This disclosure provides a pharmaceutical composition comprising a plurality of LNPs and a pharmaceutically acceptable carrier, wherein the plurality of LNPs comprise nucleic acids, such as mRNA and / or gRNA variants encoding long-term repressor fusion proteins as described herein.

[0394] In some embodiments, the LNP containing one or more nucleic acids has an electron-dense core.

[0395] This disclosure provides an LNP comprising one or more nucleic acids, said nucleic acids comprising: (a) mRNA and / or gRNA variants of the long-term repressor fusion protein described herein encoding such protein; (b) one or more cationic lipids or ionizable cationic lipids or salts thereof, said one or more cationic lipids or ionizable cationic lipids or salts thereof comprising about 50 mol% to about 85 mol% of the total lipids present in the LNP; (c) one or more non-cationic lipids, said one or more non-cationic lipids comprising about 13 mol% to about 49.5 mol% of the total lipids present in the LNP; and (d) one or more conjugated lipids that inhibit the aggregation of the LNP, said one or more conjugated lipids comprising about 0.5 mol% to about 2 mol% of the total lipids present in the particle. In another embodiment, this disclosure provides an LNP comprising one or more nucleic acids, said nucleic acids comprising: (a) mRNA and / or gRNA variants encoding a long-term repressor fusion protein as described herein; (b) one or more cationic lipids or ionizable cationic lipids or salts thereof, said one or more cationic lipids or ionizable cationic lipids or salts thereof comprising about 22 mol% to about 85 mol% of the total lipids present in the LNP; (c) one or more non-cationic / phospholipids, said one or more non-cationic / phospholipids comprising about 10 mol% to about 70 mol% of the total lipids present in the LNP; (d) 15 mol% to about 50 mol% of sterols; and (d) 1 mol% to about 5 mol% of lipid-PEG or lipid-PEG-peptide in the particle. In some 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.

[0396] This disclosure provides an LNP comprising one or more nucleic acids, said nucleic acids comprising: (a) mRNA encoding a long-term repressor fusion protein as described herein; (b) a cationic lipid or a salt thereof, said cationic lipid or salt thereof comprising about 52 mol% to about 62 mol% of the total lipids present in the LNP; (c) a mixture of phospholipids and cholesterol or derivatives thereof, said mixture of phospholipids and cholesterol or derivatives thereof comprising about 36 mol% to about 47 mol% of the total lipids present in the LNP; and (d) a PEG-lipid conjugate comprising about 1 mol% to about 2 mol% of the total lipids present in the LNP. In a particular embodiment, the formulation is a four-component system comprising about 1.4 mol% of a PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 57.1 mol% of a cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 7.1 mol% of DPPC (or DSPC) and about 34.3 mol% of cholesterol (or a derivative thereof). In some embodiments, the LNP comprises mRNA and gRNA encoding the CasX described herein.

[0397] In other embodiments, the LNP comprising one or more nucleic acids comprises: (a) mRNA and / or gRNA encoding a long-term repressor fusion protein of any of the embodiments described herein; (b) a cationic lipid or a salt thereof, said cationic lipid or salt thereof comprising about 46.5 mol% to about 66.5 mol% of the total lipids present in the LNP; (c) cholesterol or a derivative thereof, said cholesterol or a derivative thereof comprising about 31.5 mol% to about 42.5 mol% of the total lipids present in the LNP; and (d) a PEG-lipid conjugate comprising about 1 mol% to about 2 mol% of the total lipids present in the LNP. In a particular embodiment, the formulation is a phospholipid-free three-component system and comprises about 1.5 mol% of a PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 61.5 mol% of a cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, and about 36.9 mol% of cholesterol (or a derivative thereof). In some embodiments, the LNP comprises mRNA and gRNA encoding the CasX described herein.

[0398] Other formulations are described in PCT Publication WO 2009 / 127060 and U.S. Patent Publications US 2011 / 0071208 A1 and US 2011 / 0076335 A1, the disclosures of which are incorporated herein by reference in their entirety.

[0399] In other embodiments, the LNP comprising one or more nucleic acids comprises: (a) mRNA and gRNA encoding a long-term repressor fusion protein of any of the embodiments described herein; (b) one or more cationic lipids or ionizable cationic lipids or salts thereof, said one or more cationic lipids or ionizable cationic lipids or salts thereof comprising about 2 mol% to about 50 mol% of the total lipids present in the LNP; (c) one or more non-cationic lipids or ionizable cationic lipids, said one or more non-cationic lipids or ionizable cationic lipids comprising about 5 mol% to about 90 mo...

Claims

1. A system for transcriptional repression of the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene, the system comprising: (a) Guide RNA (gRNA) containing a target sequence complementary to the target nucleic acid sequence of the PCSK9 gene, and (b) mRNA encoding a long-term repressor fusion protein (LTRP), wherein the LTRP comprises: a. Catalytically dead CasX (dCasX); b. DNA methyltransferase (DNMT) 3A catalytic domain (DNMT3A); c. DNMT3-like interaction domain (DNMT3L); and d. First repressor domain (RD1). The LTRP is capable of forming a ribonucleoprotein (RNP) with the gRNA.

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

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

4. The system of claim 3, wherein the LTRP further includes an additional RD1 at the C end of the dCasX.

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

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

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

8. The system according to any one of claims 1 to 7, wherein the LTRP comprises one or more adapter peptides.

9. The system of claim 8, wherein at least one of the one or more adapter peptides comprises a sequence independently selected from the group consisting of: SEQ ID NO: 98-124 and 3278-3289.

10. The system according to any one of claims 1 to 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 NO: 30-97.

12. The system according to claim 10 or claim 11, wherein at least one of the one or more NLSs 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 NLSs 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 according to any one of claims 1 to 2 or 8 to 15, wherein the LTRP comprises, from the N-terminus to the C-terminus, a. First NLS; b. The DNMT3A; c. First linker peptide; d. The DNMT3L; e. Second adaptor peptide; f. Third connector peptide; g. The dCasX mentioned above; h. Fourth linker peptide; i. The aforementioned RD1, and j. 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 NO: 22838-22846.

18. The system according to any one of claims 7 to 15, wherein the LTRP comprises, from the N-terminus to the C-terminus, a. First NLS; b. The aforementioned ADD; c. The DNMT3A; d. First linker peptide; e. The DNMT3L; f. Second adaptor peptide; g. Third linker peptide; h. The dCasX; i. Fourth linker peptide; j. The aforementioned RD1, and k. 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 NO: 22847-22855.

20. The system according to any one of claims 1, 3, or 8 to 15, wherein the LTRP comprises, from the N-terminus to the C-terminus, a. First NLS; b. The DNMT3A; c. First linker peptide; d. The DNMT3L; e. Third linker peptide; f. The aforementioned RD1; g. Second adaptor peptide; h. The dCasX; i. Fourth linker peptide, and j. Second NLS.

21. The system according to any one of claims 7 to 15, wherein the LTRP comprises, from the N-terminus to the C-terminus, a. First NLS; b. The aforementioned ADD; c. The DNMT3A; d. First linker peptide; e. The DNMT3L; f. Second adaptor peptide; g. The RD1 h. Third linker peptide; i. The dCasX mentioned above; j. Fourth linker peptide, and k. Second NLS.

22. The system according to any one of claims 7 to 15, wherein the LTRP comprises, from the N-terminus to the C-terminus, a. First NLS; b. The aforementioned ADD; c. The DNMT3A; d. First linker peptide; e. The DNMT3L; f. Second adaptor peptide; g. The RD1 h. Third linker peptide; i. The dCasX mentioned above; j. Fourth linker peptide; k. Another RD1; l. The fifth joint, and m. Second NLS.

23. The system of claim 22, wherein the RD1 and the other RD1 sequence are identical.

24. The system of claim 22, wherein the RD1 and the other RD1 sequence are different.

25. The system according to any one of claims 1 to 24, wherein the dCasX comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

26. The system of claim 25, wherein the dCasX comprises the 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 with it.

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

4.

28. The system according to any one of claims 1 to 27, wherein the RD1 comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

29. The system according to any one of claims 1 to 27, wherein the RD1 comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

30. The system according to any one of claims 1 to 27, wherein the RD1 comprises the 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

31. The system according to any one of claims 1 to 27, wherein the RD1 comprises the 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

32. The system according to any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 132, 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 with it.

33. The system according to any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 133, 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 with it.

34. The system according to any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 134, 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 with it.

35. The system according to any one of claims 1 to 27, wherein the RD1 comprises the 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

36. The system according to any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 136, 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 with it.

37. The system according to any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 137, 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 with it.

38. The system according to any one of claims 1 to 27, wherein the RD1 comprises the sequence of SEQ ID NO: 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 with it.

39. The system according to any one of claims 7 to 15, 18, 19 or 19 to 38, wherein the ADD comprises 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

40. The system according to any one of claims 1 to 39, wherein the DNMT3A comprises 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

41. The system according to any one of claims 1 to 40, wherein the DNMT3L comprises 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

42. The system according to any one of claims 1 to 41, wherein the sequence encoding the dCasX comprises a sequence selected from the group consisting of SEQ ID NO: 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 with it.

43. The system according to any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

44. The system according to any one of claims 1 to 41, wherein the sequence encoding the RD1 comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

45. The system according to any one of claims 1 to 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 with it.

46. ​​The system according to any one of claims 1 to 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 with it.

47. The system according to any one of claims 1 to 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 with it.

48. The system according to any one of claims 1 to 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 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 with it.

49. The system according to any one of claims 1 to 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 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 with it.

50. The system according to any one of claims 1 to 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 with it.

51. The system according to any one of claims 1 to 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 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 with it.

52. The system according to any one of claims 1 to 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 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 with it.

53. The system according to any one of claims 1 to 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 with it.

54. The system according to any one of claims 1 to 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 with it.

55. The system according to any one of claims 1 to 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 with it.

56. The system according to any one of claims 7 to 15, 18, 19 or 21 to 55, wherein the sequence encoding the ADD comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

57. The system according to any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

58. The system according to any one of claims 1, 2, 8 to 17 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

59. The system according to any one of claims 1, 2, 8 to 17 or 25 to 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 with it.

60. The system according to any one of claims 1, 2, 8 to 17 or 25 to 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 with it.

61. The system according to any one of claims 1, 2, 8 to 17 or 25 to 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 with it.

62. The system according to any one of claims 1, 2, 8 to 17 or 25 to 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 with it.

63. The system according to any one of claims 1, 2, 8 to 17 or 25 to 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 with it.

64. The system according to any one of claims 1, 2, 8 to 17 or 25 to 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 with it.

65. The system according to any one of claims 1, 2, 8 to 17 or 25 to 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 with it.

66. The system according to any one of claims 1, 2, 8 to 17 or 25 to 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 with it.

67. The system according to any one of claims 1, 2, 8 to 17 or 25 to 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 with it.

68. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

69. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

70. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 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 with it.

71. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 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 with it.

72. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 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 with it.

73. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 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 with it.

74. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 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 with it.

75. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 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 with it.

76. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 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 with it.

77. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 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 with it.

78. The system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 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 with it.

79. The system according to any one of claims 1, 3, 8 to 15, 20 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

80. The system according to any one of claims 1, 3, 8 to 15, 20 or 25 to 55, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

81. The system according to any one of claims 1, 3, 7 to 15, 21 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

82. The system according to any one of claims 1, 3, 7 to 15, 21 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

83. The system according to any one of claims 1, 3 to 5, 7 to 15, 22, 23 or 25 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

84. The system according to any one of claims 1, 3 to 4, 6-15, 22 or 24 to 56, wherein the mRNA encoding the LTRP comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

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

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

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

88. The system according to any one of claims 1 to 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 the sequence of SEQ ID NO: 3300.

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

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

92. The system according to any one of claims 88 to 91, wherein the 5' cap is attached to the mRNA at the 5'.

93. The system according to any one of claims 87 to 92, wherein the mRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 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 with it.

94. The system according to any one of claims 87 to 92, wherein the mRNA comprises a sequence of SEQ ID NO: 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 with it.

95. The system according to any one of claims 87 to 92, wherein the mRNA comprises the 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 with it.

96. The system according to any one of claims 87 to 92, wherein the mRNA comprises the 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 with it.

97. The system according to any one of claims 87 to 92, wherein the mRNA comprises the 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 with it.

98. The system according to any one of claims 87 to 92, wherein the mRNA comprises the 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 with it.

99. The system according to any one of claims 87 to 92, wherein the mRNA comprises the 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 with it.

100. The system according to any one of claims 87 to 92, wherein the mRNA comprises the 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 with it.

101. The system according to any one of claims 87 to 92, wherein the mRNA comprises the 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 with it.

102. The system according to any one of claims 87 to 92, wherein the mRNA comprises the 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 with it.

103. The system according to any one of claims 87 to 92, wherein the mRNA comprises the 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 with it.

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

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

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

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

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

109. The system according to any one of claims 1 to 107, wherein the PCSK9 gene target nucleic acid sequence is located in the 3' untranslated region of the PCSK9 gene.

110. The system according to any one of claims 1 to 109, wherein the targeting sequence of said gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 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 according to any one of claims 1 to 109, wherein the targeting sequence of the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694 and 2714.

112. The system according to any one of claims 1 to 109, wherein the target sequence of said gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 1834, 1849, 1853, 1855-1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874 and 1875.

113. The system according to any one of claims 1 to 109, wherein the targeting sequence of said gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 1855, 1867 and 1869.

114. The system according to any one of claims 110 to 113, wherein the target sequence of said gRNA comprises a sequence wherein 1, 2, 3, 4 or 5 nucleotides are removed from the 3' end of said sequence.

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

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

117. The system according to any one of claims 1 to 116, wherein the gRNA comprises a scaffold comprising a sequence selected from the group consisting of: SEQ ID NO: 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%, or at least about 99% sequence identity with it.

118. The system according to any one of claims 1 to 116, wherein the gRNA comprises a scaffold comprising the sequence of SEQ ID NO: 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%, or at least about 99% sequence identity with it.

119. The system according to any one of claims 1 to 118, wherein the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 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%, or at least about 99% sequence identity with it.

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

121. The system of claim 120, wherein the chemical modification of the gRNA comprises adding 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 at one, two, three or four nucleotides from the 5' end, 3' end or both ends of the gRNA are modified by adding a 2'O-methyl group.

123. The system according to any one of claims 120 to 122, wherein the chemical modification of the gRNA comprises substitution of phosphate thioester bonds between two or more nucleotides of the gRNA.

124. The system of claim 123, wherein the chemical modification comprises the substitution of a phosphate thioester bond between two or more nucleotides located at one, two, three or four nucleotides from the 5' end, 3' end or both ends of the gRNA.

125. The system according to any one of claims 104 to 124, wherein the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 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%, or at least about 99% sequence identity with it.

126. The system according to any one of claims 104 to 124, wherein the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 22788-22803.

127. The system according to any one of claims 104 to 124, wherein the gRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 22788-22790.

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

129. A lipid nanoparticle (LNP) comprising mRNA according to any one of claims 87 to 103 and gRNA according to any one of claims 120 to 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 auxiliary phospholipids, one or more polyethylene glycol (PEG) modified lipids and cholesterol or derivatives thereof.

131. The LNP according to any one of claims 128 to 130, wherein the LNP comprises ionizable lipids, cofactor phospholipids, polyethylene glycol (PEG) modified lipids, and cholesterol or derivatives thereof.

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

133. A pharmaceutical composition comprising: a. The system according to any one of claims 1 to 127; or b. The LNP according to any one of claims 128 to 132, And one or more pharmaceutically suitable excipients.

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

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

136. The pharmaceutical composition according to claim 134 or claim 135, wherein the pharmaceutical composition is formulated for administration via a route selected from the group consisting of: intravenous, intra-arterial, portal vein injection, intraperitoneal, intramuscular, intravenous, intracisional, intrathecal, intracranial, intralumbar, intraocular, subcutaneous, and oral routes.

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

138. The method of claim 137, wherein the 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 in the population.

139. The method of claim 137 or claim 138, wherein, compared with untreated cells, the transcription of the PCSK9 gene in the cells of the population is reduced 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%.

140. The method according to any one of claims 137 to 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 cell is a human cell.

143. The method according to any one of claims 140 to 142, wherein the eukaryotic cells are selected from the group consisting of: hepatocytes, intestinal cells, kidney cells, central nervous system cells, smooth muscle cells, macrophages, and arterial wall cells.

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

145. The method according to any one of claims 137 to 143, wherein the repression of the PCSK9 gene in the cell population occurs in vivo in the subject.

146. The method of claim 145, wherein the subject is selected from the group consisting of rodents, mice, rats and non-human primates.

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

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

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

150. The method according to any one of claims 137 to 149, wherein the inhibition is reversible.

151. The method of claim 150, wherein the inhibition is reversible by using 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 PCSK9-related disease in a subject of need, the method comprising administering a therapeutically effective dose of the following: a. The system according to any one of claims 1 to 127; b. The LNP according to any one of claims 128 to 132; or c. The pharmaceutical composition according to any one of claims 133 to 136, This is used to treat the aforementioned PCSK9-related diseases.

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 via a route selected from the group consisting of: intravenous, intra-arterial, portal vein injection, intraperitoneal, intramuscular, intravenous, intracisional, intrathecal, intracranial, intralumbar, intraocular, subcutaneous, and oral routes.

157. The method according to any one of claims 154 to 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, decreased high-density lipoprotein levels, hepatic steatosis, coronary heart disease, local ischemia, stroke, peripheral vascular disease, thrombosis, type 2 diabetes, hypertension, atherosclerosis, obesity, aortic stenosis, elevated PCSK9 levels, or combinations thereof.

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

159. The method according to any one of claims 154 to 158, wherein the method results in improvement of at least two clinically relevant endpoints, said at least two clinically relevant endpoints being selected from the group consisting of: changes in LDL-cholesterol relative to baseline, reduction in plaque atherosclerosis volume, reduction in coronary plaque, reduction in atherosclerotic cardiovascular disease (ASCVD), cardiovascular death, non-fatal myocardial infarction, ischemic stroke, non-fatal stroke, coronary revascularization, unstable angina, and visual acuity.

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

161. The kit of claim 160, comprising a buffer, excipients, nuclease inhibitors, protease inhibitors, liposomes, therapeutic agents, labels, label visualization reagents, instructions for use, or any combination thereof.

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

163. The composition according to claim 162, used to prepare a medicament for treating PCSK9-related diseases in subjects of need.

164. The composition of claim 162, for treating PCSK9-related disease in a subject of need.

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