Compositions and methods for targeting PCSK9

Abstract

This specification provides a gene repressor system comprising a fusion protein, such as a fusion protein containing a non-catalyzed class 2, type V CRISPR protein useful for repressing the precursor protein-converting enzyme subtilisin / kexin type 9 (PCSK9) gene, and a guide nucleic acid (gRNA). Methods for constructing and using such a system for repressing PCSK9 transcription are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest to U.S. Provisional Patent Application No. 63 / 492,978, filed on 29 March 2023, and U.S. Provisional Patent Application No. 63 / 505,888, filed on 2 June 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (SCRB_058_02WO_SeqList_ST26.xml, size: 141,865,746 bytes, created: March 26, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] In mammals, cholesterol is transported within lipoproteins via emulsification. Lipoprotein particles are classified based on their density: low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), high-density lipoprotein (HDL), and chylomicrons. Surface LDL receptors are internalized during cholesterol absorption. Cholesterol-rich cells block their LDL receptor synthesis to prevent the uptake of new cholesterol in LDL particles. Conversely, LDL receptor synthesis is promoted when cells are deficient in cholesterol. If the process is not regulated, excess LDL particles travel through the bloodstream without being taken up by LDL receptors. LDL particles in the blood are oxidized and taken up by macrophages, which then fill up and form foam cells. These foam cells can become trapped in the walls of blood vessels and contribute to the formation of atherosclerotic plaques, which are one of the main causes of heart attacks, strokes, and other serious medical problems.

[0004] Hepatic protein precursor protein convertase subtilisin / kexin type 9 (PCSK9) is a secreted, spherical, autoactivating serine protease that binds to the low-density lipoprotein receptor (LDL-R) during endocytosis of LDL particles, preventing the recycling of LDL-R to the cell surface and resulting in reduced LDL-cholesterol clearance. PCSK9 binds to LDL-R (via its EGF-A domain) and prevents conformational changes in the receptor-ligand complex, instead redirecting LDL-R to lysosomes. Since the receptor for low-density lipoprotein particles (LDL) typically transports thousands of lipid molecules (including cholesterol) per particle in the extracellular fluid, blocking or inhibiting the function of PCSK9 can reduce LDL particle concentration by promoting LDL-R-mediated clearance of LDL cholesterol. PCSK9 is primarily expressed in the liver, intestines, kidneys, and central nervous system, but it is also highly expressed in the arterial walls of endothelial cells, smooth muscle cells, and macrophages, and has local effects that can regulate vascular homeostasis and atherosclerosis.

[0005] PCSK9 is a member of the progenitor protein-converting enzyme (PC) family, and its gene is mutated in approximately 2%–3% of individuals with familial hypercholesterolemia (FH) (Sepideh Mikaeeli, S., et al. Functional analysis of natural PCSK9 mutants in modern and archaic humans. FEBS J. 2019 Aug 6. doi:10.1111 / febs.15036). Researchers have identified several PCSK9 mutations that cause the high cholesterol genotype (hypercholesterolemia). These mutations alter a single amino acid in the PCSK9 protein. Researchers describe mutations that cause hypercholesterolemia as "gain-of-function" because they appear to enhance the activity of the PCSK9 protein or give the protein a new, atypical function (Blesa, S., et al. A New PCSK9 Gene Promoter Variant Affects Gene Expression and Causes Autosomal Dominant Hypercholesterolemia. J. Clin. Endocrinol. & Metab. 93:3577 (2008)). Overactive PCSK9 protein substantially reduces the number of low-density lipoprotein receptors on the surface of hepatocytes. Because there are fewer receptors to remove low-density lipoprotein from the blood, people with gain-of-function mutations in the PCSK9 gene have very high blood cholesterol levels. Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by elevated low-density lipoprotein (LDL) cholesterol levels and carries a high risk of early cardiovascular disease. Approximately 10 mutations in PCSK9 have been identified as causes of the disease in different populations. All known mutations in PCSK9 that cause hypercholesterolemia result in increased enzymatic activity of this protease (Bleasa, S., 2008). Furthermore, PCSK9 mutations can lead to autosomal dominant familial hypobetalipoproteinemia, which can result in hepatic steatosis, cirrhosis, and other disorders.

[0006] The emergence of CRISPR / Cas systems and the programmable nature of these minimal systems has facilitated their use as versatile technologies for genome manipulation and engineering. However, current methods for producing PCSK9 protective variants and loss-of-function mutants in vivo have been ineffective because they require the modification of a large number of cells to regulate cholesterol levels. Other concerns include the off-target effects, genomic instability, or oncogenic modifications that can be caused by genome editing, as well as the lack of a safe delivery mode for repressor protein systems. Furthermore, in certain disease manifestations, gene silencing, or suppression, is preferred over gene editing. The ability to catalytically inactivate CRISPR nucleases such as Cas9 and CasX has been demonstrated (WO2020247882A1 and US20200087641A1, incorporated herein by reference), making these systems an attractive platform for the production of fusion proteins with gene-silencing repressor domains. While specific repressor systems have been described, there is a need for additional gene repressor systems that are optimized and / or offer improvements over earlier generation gene repressor systems, such as Cas9-based systems, for use in various therapeutic, diagnostic, and research applications. Therefore, there is a need for improved compositions and methods for modulating PCSK9. [Overview of the project]

[0007] This disclosure provides a system comprising or encoding a fusion protein containing a DNA-binding and linked repressor domain used for repression and / or epigenetic modification of a progenitor protein-converting enzyme subtilisin / kexin type 9 (PCSK9) gene-targeted nucleic acid sequence. In some embodiments, the fusion protein comprises a DNA-binding protein containing a non-catalyzed CRISPR protein, such as a class 2, type V CRISPR protein, and a guide nucleic acid containing a targeting sequence complementary to the PCSK9 gene-targeted nucleic acid sequence. The protein and guide nucleic acid can be modified for passive entry into target cells and are useful in various ways for PCSK9 repression, and these methods are also provided. This disclosure also provides vectors and lipid nanoparticles (LNPs) encoding or encapsulating the fusion protein and guide nucleic acid components for delivery of the system to cells for transcriptional repression of the PCSK9 gene-targeted nucleic acid sequence. This disclosure also provides methods for modifying the PCSK9 gene-targeted nucleic acid sequence. This disclosure also provides methods for treating subjects with PCSK9-related diseases. In some embodiments, the compositions and methods, though not limited thereto, are useful in subjects with metabolic disorders such as familial hypercholesterolemia, familial hypobetalipoproteinemia, or elevated cholesterol levels.

[0008] In another embodiment, this specification provides a system comprising a PCRK9 repressor system for use in the manufacture of a pharmaceutical product for treating a PCSK9-related disease in a subject requiring treatment for the PCSK9-related disease, or a vector comprising or encoding a PCSK9 repressor system.

[0009] Further features and advantages of certain embodiments of this disclosure will become more readily apparent in the following description of the embodiments and their drawings, and from the claims.

[0010] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. The contents of WO2020 / 247882, WO2020 / 247883, WO2021 / 113772, WO2022 / 120095, WO2022 / 125843, WO2022 / 261150, WO2022 / 261149, WO2023 / 049872, WO2023 / 049742, and WO2023 / 240162 disclosing CasX variants and gRNA variants and methods for their delivery, as well as WO2021 / 142342 disclosing compositions and methods for modifying PCSK9, are incorporated herein by reference in their entirety.

[0011] Understanding the features and benefits of this disclosure will be facilitated by referring to the following diagram. [Brief explanation of the drawing]

[0012] [Figure 1] This diagram shows schematic representations of various configurations of a repressor fusion protein incorporating 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 a nuclear localization signal. [Figure 2] This bar graph shows the quantification of secreted PCSK9 levels at 6, 18, and 36 days after transfection in Huh7 cells lipofected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3, paired with the indicated targeted gRNA described in Example 1. Secreted PCSK9 levels were normalized to the total cell number. Naive, untreated cells served as experimental controls. [Figure 3]This bar graph shows the quantification of normalized secreted PCSK9 levels 4 days after transfection in HepG2 cells lipofected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3, paired with the indicated targeted gRNAs, as described in Example 2. Secreted PCSK9 levels were normalized relative to the total cell number. Naive, untreated cells served as experimental controls. [Figure 4] This bar graph shows the quantification of normalized secreted PCSK9 levels 4 days after transfection in Huh7 cells lipofected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3, paired with the indicated targeted gRNAs, as described in Example 2. Secreted PCSK9 levels were normalized relative to the total cell number. Naive, untreated cells served as experimental controls. [Figure 5] This bar graph shows the quantification of normalized secreted PCSK9 levels 4 days after transfection in Hep3B cells lipofected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3, paired with the indicated targeted gRNAs, as described in Example 2. Secreted PCSK9 levels were normalized relative to the total cell number. Naive, untreated cells served as experimental controls. [Figure 6] This bar graph shows the quantification of secreted PCSK9 levels at 4, 14, and 27 days after transfection in Huh7 cells lipofected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3, paired with the indicated targeted gRNAs, as described in Example 2. The quantification of secreted PCSK9 levels is shown compared to the secretion level detected in naive, untreated cells at day 4. [Figure 7] This is a schematic diagram of gRNA scaffold variant 174 (SEQ ID NO: 1744) described in Example 3. Structural motifs are highlighted. [Figure 8]This is a schematic diagram of gRNA scaffold variant 235 (SEQ ID NO: 1745) described in Example 3. The highlighted structural motifs are the same as those in Figure 7. The differences between variant 174 and variant 235 lie in the elongation stem motif and several single-nucleotide changes (indicated by asterisks). Variant 316 retains the short elongation stem from variant 174 but possesses the four substitutions found in scaffold 235. [Figure 9] This is a schematic diagram of gRNA scaffold variant 316 (SEQ ID NO: 1746) described in Example 3. The highlighted structural motif is the same as in Figure 7. Variant 316 retains the short elongated stem from variant 174 (Figure 7) but possesses the four substitutions found in scaffold 235 (Figure 8). [Figure 10] This is a schematic diagram showing versions 1-3 of chemical modifications performed on gRNA scaffold variant 235, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are shown as white circles, and 2'OMe-modified ribonucleotides are shown as black circles. Phosphothioate bonds are indicated by an asterisk (*) below or next to the bond. For the v2 profile, the addition of three 3'uracils (3'UUU) is annotated with a "U" in the corresponding circle. [Figure 11] This is a schematic diagram showing versions 4–6 of chemical modifications performed on gRNA scaffold variant 235, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are indicated by white circles, and 2'OMe-modified ribonucleotides are indicated by black circles. Phosphothioate bonds are indicated by an asterisk (*) below or next to the bond. [Figure 12] This is a schematic diagram showing versions 7–9 of chemical modifications performed on gRNA scaffold variant 316, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are indicated by white circles, and 2'OMe-modified ribonucleotides are indicated by black circles. Phosphothioate bonds are indicated by an asterisk (*) below or next to the bond. [Figure 13]Schematic diagrams showing versions 1 - 3 of the chemical modifications performed on gRNA scaffold variant 316 described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are shown as white circles and 2’OMe modified ribonucleotides are shown as black circles. Phosphorothioate bonds are indicated by * below or next to the bond. [Figure 14] Schematic diagrams showing versions 4 - 6 of the chemical modifications performed on gRNA scaffold variant 316 described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are shown as white circles and 2’OMe modified ribonucleotides are shown as black circles. Phosphorothioate bonds are indicated by * below or next to the bond. [Figure 15] Plot showing the correlation between the indel rate (shown as the editing rate) at the PCSK9 locus measured by next-generation sequencing (NGS) (x-axis) and the secreted PCSK9 level (ng / mL) detected by enzyme-linked immunosorbent assay (ELISA) (y-axis) in HepG2 cells lipofected with the indicated scaffold variants and spacers in combination with CasX 491 mRNA-containing PCSK9-targeting gRNA as described in Example 3. [Figure 16] Plot showing the quantification of the percentage of B2M knockout in HepG2 cells co-transfected with 100 ng of CasX 491 mRNA and either end-modified (v1) or unmodified (v0) B2M-targeting gRNA having the indicated dose of spacer 7.37 as described in Example 3. The editing level was determined by flow cytometry as the population of cells in which surface presentation of the HLA complex was lost due to successful editing at the B2M locus. [Figure 17] Plot showing the results of an editing assay measured as the indel rate detected by NGS at the human B2M locus in HepG2 cells treated with the indicated doses of LNPs formulated with CasX 491 mRNA and the indicated B2M-targeting gRNA as described in Example 3. [Figure 18]A plot showing the quantification of the percentage of B2M knockout in HepG2 cells treated with the indicated doses of LNP formulated with CasX 491 mRNA and the indicated B2M-targeting gRNA as described in Example 3. The editing level was determined by flow cytometry as the population of cells that did not have surface presentation of the HLA complex due to successful editing at the B2M locus. [Figure 19] A plot showing the results of an editing assay measured as the indel rate detected by NGS at the mouse ROSA26 locus in Hepa1-6 cells treated with the indicated doses of LNP formulated with CasX 676 mRNA #2 and the indicated ROSA26-targeting gRNA having either a v1 or v5 modification profile as described in Example 3. [Figure 20] A plot showing the quantification of the editing percentage measured as the indel rate detected by NGS at the ROSA26 locus in mice treated with LNP formulated with CasX 676 mRNA #2 and the indicated chemically modified ROSA26-targeting gRNA as described in Example 3. [Figure 21] A bar graph showing the results of an editing assay measured as the indel rate detected by NGS at the mouse PCSK9 locus in mice treated with LNP formulated with CasX 676 mRNA #1 and the indicated chemically modified PCSK9-targeting gRNA as described in Example 3. Untreated mice served as experimental controls. [Figure 22] A diagram of the secondary structure of guide RNA scaffold 235 (SEQ ID NO: 1745) focusing on the region having a CpG motif as described in Example 6. The CpG motifs in (1) pseudoknot stem, (2) scaffold stem, (3) extension stem bubble, (4) extension step, and (5) extension stem loop are structurally labeled. [Figure 23] A diagram of the CpG-reducing mutations introduced into each of five regions in the coding sequence of the guide RNA scaffold as described in Example 6. [Figure 24]This document provides the results of editing experiments in which AAV vectors with various CpG reduction or CpG depletion guide RNA scaffolds, as described in Example 6, were used to edit the B2M locus in induced neurons. The AAV vectors were administered at an infection multiplicity (MOI) of 4e3. The bars represent the mean ± SD of two copies per sample. "No Tx" indicates a non-transduction control, and "NT" indicates a control with a non-targeting spacer. [Figure 25] This provides the results of editing experiments in which AAV vectors with various CpG reduction or CpG depletion guide RNA scaffolds, as described in Example 6, were used to edit the B2M locus in induced neurons. The AAV vectors were administered at an MOI of 3e3. The bars show the mean ± SD of two copies per sample. "No Tx" indicates a non-transduction control. [Figure 26] This provides the results of editing experiments in which AAV vectors with various CpG reduction or CpG depletion guide RNA scaffolds, as described in Example 6, were used to edit the B2M locus in induced neurons. The AAV vectors were administered at an MOI of 1e3. The bars show the mean ± SD of two copies per sample. "No Tx" indicates a non-transduction control. [Figure 27] This provides the results of editing experiments in which AAV vectors with various CpG reduction or CpG depletion guide RNA scaffolds, as described in Example 6, were used to edit the B2M locus in induced neurons. The AAV vectors were administered at an MOI of 3e2. The bars show the mean ± SD of two replicates per sample. "No Tx" indicates a non-transduction control. [Figure 28] A schematic diagram of the five components of a long-term repressor protein (LTRP, also referred to herein as "repressor fusion protein") fusion protein having a repressor molecule linked to CasX without catalytic activity is shown. D3A and D3L represent DNA methyltransferase 3-alpha (DNMT3A) and DNMT3A-like protein (DNMT3L), respectively. L1-L4 are linkers. NLS is a nuclear localization signal. [Figure 29]Schematic diagrams of various LTRP#5 architectures incorporating additional DNMT3A domains, as described in Example 7, are shown. The additional DNMT3A domains were the ADD domain of DNMT3A ("D3A ADD") and the PWWP domain of DNMT3A (Pro-Trp-Trp-Pro motif) ("D3A PWWP"). "D3A endo" encodes the endogenous sequence that occurs between the DNMT3A PWWP domain and the ADD domain. "D3A CD" and "D3L ID" represent the catalytic domain of DNMT3A and the interaction domain of DNMT3L, respectively. "L1~L3" are linkers. "NLS" is a nuclear localization signal. See Table 13 for the LTRP sequences. [Figure 30] The results of a time-course experiment comparing the B2M inhibitory activity (expressed as the percentage of HLA-negative cells) of the indicated LTRP-ZIM3 and its variants using B2M-targeted gRNAs with spacer 7.37, as described in Example 7, are shown. Data are presented as mean with standard deviation, N=3. CD=DNMT3A catalytic domain. [Figure 31] Figure 30 shows the results of the same time-course experiment, but with the B2M inhibitory activity of the indicated LTRP-ZIM3 variant using B2M-targeted gRNA with spacer 7.160, as described in Example 7. Data are expressed as mean with standard deviation, N=3. [Figure 32] Figure 30 shows the results of the same time-course experiment, but with the B2M inhibitory activity of the indicated LTRP-ZIM3 variant using B2M-targeted gRNA with spacer 7.165, as described in Example 7. Data are expressed as mean with standard deviation, N=3. [Figure 33] The results of the same time-series experiment shown in Figure 30 are presented, but as described in Example 7, they show the B2M inhibitory activity of the LTRP-ZIM3 variant using untargeted gRNA. Data are expressed as mean with standard deviation, N=3. [Figure 34]This is a violin plot of the percentage of CpG methylation of the downstream CpG site of the VEGFA locus transcription start site for each of the three B2M-targeted and non-targeted gRNAs described in Example 7. [Figure 35] This is a scatter plot showing the relative activity (average percentage of HLA-negative cells on day 21 for spacer 7.160) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 7 for spacer 7.160) for the LTRP5-ZIM3 variant described in Example 7. [Figure 36] A schematic diagram of the general architecture of LTRP molecules having ADD domains of LTRP configurations #1, #4, and #5 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 a nuclear localization signal. See Table 11 for LTRP sequences. [Figure 37] The results of a time-course experiment comparing the B2M inhibitory activity (expressed as the percentage of HLA-negative cells) of LTRPs having a ZIM3-KRAB domain with configurations #1, #4, or #5, with or without a DNMT3A ADD domain, when paired with a B2M-targeting gRNA having spacer 7.160 as described in Example 8 are shown. Data are presented as mean with standard deviation, N=3. "NT" is gRNA with a non-targeting spacer. [Figure 38] This plot shows the results of the same time-course experiment as shown in Figure 37, but illustrates the B2M inhibitory activity of ZNF10 with or without the DNMT3A ADD domain or LTRP#5 with the ZIM3-KRAB domain, paired with the B2M-targeted gRNA having spacer 7.160 as described in Example 8. Data are expressed as mean with standard deviation, N=3. "NT" is gRNA with a non-targeting spacer. [Figure 39]This plot shows the results of the same time-course experiment as shown in Figure 37, but illustrates the B2M inhibitory activity of LTRP5-ZIM3 with or without the DNMT3A ADD domain, paired with the B2M-targeting gRNA with the indicated spacer, as described in Example 8. Data are expressed as mean with standard deviation, N=3. "NT" is the gRNA with the untargeting spacer. [Figure 40] This plot shows the B2M inhibitory activity results 27 days post-transfection for LTRPs having either ZNF10 or a ZIM3-KRAB domain with configuration #1 of the shown gRNA, with or without the DNMT3A ADD domain, as described in Example 8. Data are expressed as mean with standard deviation, N=3. "NT" is a gRNA with a non-targeting spacer. [Figure 41] This plot shows the B2M inhibitory activity results 27 days post-transfection for LTRPs having either ZNF10 or a ZIM3-KRAB domain with configuration #4 of the shown gRNA, with or without the DNMT3A ADD domain, as described in Example 8. Data are expressed as mean with standard deviation, N=3. "NT" is a gRNA with a non-targeting spacer. [Figure 42] This plot shows the B2M inhibitory activity results 27 days post-transfection for LTRPs having either ZNF10 or a ZIM3-KRAB domain with configuration #5 of the shown gRNA, with or without the DNMT3A ADD domain, as described in Example 8. Data are expressed as mean with standard deviation, N=3. "NT" is a gRNA with a non-targeting spacer. [Figure 43]This plot shows the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus 5 days post-transfection for LTRPs having either ZNF10 or a ZIM3-KRAB domain with configuration #1 of the shown gRNA with or without the DNMT3A ADD domain, as described in Example 8. The data are expressed as the mean percentage of CpG methylation at CpG sites near the VEGFA locus, with the standard error of the mean also shown, N=3. "NT" is a gRNA with a non-targeting spacer. [Figure 44] This plot shows the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus 5 days post-transfection for LTRPs having either ZNF10 or a ZIM3-KRAB domain with configuration #4 of the shown gRNA, with or without the DNMT3A ADD domain, as described in Example 8. The data are expressed as the mean percentage of CpG methylation at CpG sites near the VEGFA locus, with the standard error of the mean also shown, N=3. "NT" is a gRNA with a non-targeting spacer. [Figure 45] This plot shows the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus 5 days post-transfection for LTRPs having either ZNF10 or a ZIM3-KRAB domain with configuration #5 of the shown gRNA, with or without the DNMT3A ADD domain, as described in Example 8. The data are expressed as the mean percentage of CpG methylation at CpG sites near the VEGFA locus, with the standard error of the mean also shown, N=3. "NT" is a gRNA with a non-targeting spacer. [Figure 46]This is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules having ZIM3-KRAB domains with configurations #1, #4, and #5 for the B2M-targeted gRNA having spacer 7.160 as described in Example 8. [Figure 47] This is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules having ZNF10-KRAB domains with configurations #1, #4, and #5 for B2M-targeted gRNA with spacer 7.160 as described in Example 8. [Figure 48] This is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules having ZIM3-KRAB domains with configurations #1, #4, and #5 for the B2M-targeted gRNA having spacer 7.37 as described in Example 8. [Figure 49] This is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules having ZNF10-KRAB domains with configurations #1, #4, and #5 for the B2M-targeted gRNA having spacer 7.37 as described in Example 8. [Figure 50] This is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules having ZIM3-KRAB domains with configurations #1, #4, and #5 for the B2M-targeted gRNA having spacer 7.165 as described in Example 8. [Figure 51]This is a dot plot showing the relative activity (average percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) of LTRP molecules having ZNF10-KRAB domains with configurations #1, #4, and #5 for the B2M-targeted gRNA having spacer 7.165 as described in Example 8. [Figure 52] This plot shows the percentage of HEK293T cells transfected with plasmids encoding the indicated CasX or LTRP:gRNA constructs, expressing B2M 6 days after treatment with various concentrations of the DNMT1 inhibitor 5-azadC, as described in Example 9. [Figure 53] This plot juxtaposes the quantification of B2M suppression in HEK293T cells transfected with plasmids encoding the indicated CasX or LTRP:gRNA constructs described in Example 9 and cultured for 58 days with the quantification of B2M reactivation upon treatment of cells transfected with 5-azadC. [Figure 54] A schematic diagram of the LTRP5 molecule without the ADD domain of DNMT3A, as described in Example 11, is shown. "D3A CD" and "D3L ID" represent the catalytic domain of DNMT3A and the interaction domain of DNMT3L, respectively. "L1", "L2", "L3A", and "L3B" are linkers. "NLS" is a nuclear localization signal. "RD1" represents the repressor domain. [Figure 55] This bar graph shows the results of a time-course experiment comparing the level of B2M suppression (expressed as the mean percentage of HLA-negative cells) in HEK293T cells transfected with LTRP5 variant plasmids containing linker sets 1-11, as described in Example 13. Data from each time point (days 8, 15, and 45) are superimposed and expressed as the mean with standard deviation, N=3. Untargeted (NT) spacers were included as experimental controls. [Figure 56]This bar graph shows the results of a time-series experiment comparing the suppression levels of target 1 (expressed as the percentage of total cells with target 1 knockdown) of LTRP5 variants containing linker sets 1-11, as measured with HEK293T, as described in Example 13. Data from each time point (days 8, 15, and 45) are superimposed and shown as the mean with standard deviation, N=3. [Figure 57] This bar graph shows the results of a time-course experiment comparing the suppression levels of target 2 (expressed as the percentage of total cells with target 2 knockdown) of LTRP5 variants containing linker sets 1-11, as measured with HEK293T, as described in Example 13. Data from each time point (days 8, 15, and 45) are superimposed and presented as the mean with standard deviation, N=3. A non-targeting (NT) spacer was included as an experimental control. [Figure 58] This bar graph shows the results of a time-course experiment comparing the level of B2M suppression (expressed as the mean percentage of HLA-negative cells) in HEK293T cells transfected with LTRP5 variant plasmids containing linker sets 12-28, as described in Example 13. Data from each time point (days 7 and 17) are superimposed and shown as the mean with standard deviation, N=3. A non-targeting (NT) spacer was included as an experimental control. [Figure 59] This bar graph shows the results of a time-series experiment comparing the suppression levels of target 1 (expressed as the percentage of total cells with target 1 knockdown) of LTRP5 variants containing linker sets 12-28, as measured with HEK293T, as described in Example 13. Data from each time point (days 7 and 17) are superimposed and presented as the mean with standard deviation, N=3. A non-targeting (NT) spacer was included as an experimental control. [Figure 60]This bar graph shows the results of a time-series experiment comparing the suppression levels of target 2 (expressed as the percentage of total cells with target 2 knockdown) of LTRP5 variants containing linker sets 12-28, as measured with HEK293T, as described in Example 13. Data from each time point (days 7 and 17) are superimposed and presented as the mean with standard deviation, N=3. A non-targeting (NT) spacer was included as an experimental control. [Figure 61] This diagram shows schematic representations of various configurations of LTRP molecules containing 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," "L2," "L3A," "L3B," and "L4" are linkers. "NLS" is a nuclear localization signal. "RD1" represents the repressor domain, and "RD1a" and "RD1b" represent repressor domain variants. [Figure 62] This is a violin plot where each point represents the mean methylation percentage at individual CpGs. The median methylation is shown by a dashed line, and the upper and lower quartiles are shown by dotted lines. Proximal DNA methylation at transcription start sites (TSS) was measured by amplicon enzyme methylation sequencing (EM-seq) from homogenized liver extract gDNA from N=3 mice sacrificed on days 7, 14, and 42 post-treatment, as described in Example 14. [Figure 63] This is a violin plot where each point represents the mean methylation percentage at individual CpGs. The median methylation is shown by a dashed line, and the upper and lower quartiles are shown by dotted lines. Proximal DNA methylation at the transcription start site (TSS) was measured by amplicon enzyme methylation sequencing (EM-seq) from homogenized liver extract gDNA from N=3 mice sacrificed 7 days after treatment, as described in Example 14. [Modes for carrying out the invention]

[0013] While exemplary embodiments are shown and described herein, it will be apparent to those skilled in the art that only one such embodiment is provided. Numerous variations, modifications, and substitutions will be conceivable to those skilled in the art without departing from the spirit of the invention. It should be understood that various alternatives to the embodiments specifically described herein may be employed when practicing the embodiments of this disclosure.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this embodiment, but preferred methods and materials are described below. In case of any inconsistency, the patent specification containing the definitions shall prevail. In addition, the materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting. Numerous variations, modifications, and substitutions will be conjured here upon those skilled in the art without departing from the spirit of this disclosure and the claims.

[0015] definition As used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple referents unless the context otherwise clearly determines. For example, a reference to "a host cell" includes two or more such host cells, a reference to "a dCasX protein" includes one or more dCasX proteins, a reference to "a nucleic acid sequence" includes one or more nucleic acid sequences, and so on.

[0016] Where used herein, the term “about” may vary to some extent depending on the context in which it is understood and used by those skilled in the art. Where the usage of the term “about” is not obvious to those skilled in the art, judging from the context in which it is used, “about” means up to ±10% of a particular term.

[0017] As will be understood by those skilled in the art, for all purposes, the entire scope disclosed herein also includes any possible sub-scopes and combinations thereof. Furthermore, as will be understood by those skilled in the art, the scope includes each individual member. Thus, for example, a group having 1 to 3 members refers to a group having 1, 2, or 3 members. Similarly, a group having 1 to 5 members refers to a group having 1, 2, 3, 4, or 5 members, and so on.

[0018] The term "those combinations" includes all possible combinations of the elements referred to by that term.

[0019] As used herein, the term “exemplary” means an example or illustration and is not intended to imply any preference or value.

[0020] As used herein, the term “CasX protein” refers to a family of proteins, and includes, for example, all naturally occurring CasX proteins ("reference CasX") as well as CasX proteins with sequence modifications, such as dCasX, which have one or more improved features compared to the CasX protein from which they are derived, as fully described herein below.

[0021] As used interchangeably herein, the terms “polynucleotide” and “nucleic acid” refer to polymeric forms of nucleotides of any length, which are either 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 or biochemically modified, unnatural, or derivatized nucleotide bases.

[0022] The terms "hybridable" or "complementary" are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) contains a sequence of nucleotides that, under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength, allows for non-covalent binding to another nucleic acid in a sequence-specific and antiparallel manner (i.e., the nucleic acid specifically binds to its complementary nucleic acid), i.e., forming Watson-Crick base pairs and / or G / U base pairs, or "annealing," or "hybridizing." It is understood that a polynucleotide sequence does not need to be 100% complementary to its target nucleic acid sequence in order to be specifically hybridizable, but may have at least about 70%, at least about 80%, at least about 90%, or at least about 95% sequence identity and still be able to hybridize to the target nucleic acid. Furthermore, a polynucleotide may hybridize across one or more segments such that intervening or adjacent segments do not participate in hybridization events (e.g., loop or hairpin structures, "bulges," "bubbles," etc.). Therefore, those skilled in the art will understand that while individual bases within a sequence may not be complementary to another sequence, the sequence as a whole is still considered complementary.

[0023] For the purposes of this disclosure, “gene” includes the DNA region encoding a gene product (e.g., protein, RNA), and all DNA regions that regulate the production of the gene product, regardless of whether such regulatory sequences are adjacent to the coding and / or transcription sequences. Therefore, a gene may include, but is not limited to, translation regulatory sequences such as promoter sequences, terminators, ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus regulatory regions. The coding sequence encodes the gene product during transcription or transcription and translation, and the coding sequences in this disclosure may include fragments and do not need to include a full-length open reading frame. A gene may include both the transcribed strand and a complementary strand containing an anticodon.

[0024] The term "downstream" refers to a nucleotide sequence located at the 3' position of a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence relates to the sequence following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0025] The term "upstream" refers to a nucleotide sequence located 5' of the reference nucleotide sequence. In certain embodiments, the upstream nucleotide sequence refers to a sequence located 5' of the coding region or transcription start site. For example, most promoters are located upstream of the transcription start site.

[0026] With respect to polynucleotide or amino acid sequences, the term “adjacent to” refers to sequences that are adjacent to or adjacent to each other within the polynucleotide or polypeptide. Those skilled in the art will understand that two sequences considered adjacent to each other may still contain a limited number of intervening sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or amino acids.

[0027] The term “regulatory element” is used herein interchangeably with the term “regulatory sequence” and is intended to include promoters, enhancers, and other expression regulatory elements. It will be understood that the selection of an appropriate regulatory element depends on whether the encoded component to be expressed (e.g., protein or RNA) or nucleic acid requires different polymerases or contains multiple components that are not intended to be expressed as a fusion protein.

[0028] The term “associated element” is used herein interchangeably with the term “associated sequence” and is intended to include coding and non-coding sequences that enhance expression, nucleic acid transport, or mRNA or protein function, and is intended to include, among other things, polyadenylation signals (poly(A) signals), enhancer elements, introns, post-transcriptional regulators (PTREs), nuclear localization signals (NLSs), deaminases, DNA glycosylase inhibitors, additional promoters, factors that stimulate CRISPR-mediated homology-directed repair (e.g., in cis or trans), autocleavage sequences, and fusion domains, e.g., fusion domains fused to CRISPR proteins. It will be understood that the selection of one or more appropriate associated elements depends on whether the encoded component to be expressed (e.g., protein or RNA), or whether the nucleic acid requires different polymerases or includes multiple components not intended to be expressed as a fusion protein.

[0029] The term “promoter” refers to a DNA sequence containing a transcription initiation site and additional sequences for polymerase binding and transcription promotion. Exemplary eukaryotic promoters include elements such as TATA boxes and / or B-recognition elements (BREs) that assist or promote the transcription and expression of associated transcriptionable polynucleotide sequences and / or genes (or transgenes). Promoters can be produced synthetically or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can be located proximal or distal to the gene being transcribed. Promoters can also include chimeric promoters, which include combinations of two or more heterologous sequences to confer a particular characteristic. Promoters in this disclosure may include variants of promoter sequences that are known or similar in composition to, but not identical to, other promoters provided herein. Promoters can be classified according to criteria such as constitutive, developmental, tissue-specific, and inducible, which affect the expression pattern of associated codes or transcriptionable sequences or genes operably linked to the promoter. Promoters can also be classified according to their strength. When used in relation to 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.

[0030] The promoters of this disclosure may be polymerase II (Pol II) promoters. Polymerase II transcribes all protein-coding genes and many non-coding genes. Typical Pol II promoters include 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 basal transcription factors. The promoter may contain one or more core promoter elements, such as a TATA box, BRE, initiator (INR), motif 10 elements (MTE), downstream core promoter element (DPE), and downstream core element (DCE), although core promoters lacking these elements are also known in the art. All Pol III promoters are assumed to be within the scope of this disclosure.

[0031] The promoters of this disclosure may be polymerase III (Pol III) promoters. Pol III transcribes DNA to synthesize small ribosomal RNAs such as 5S rRNA, tRNA, and other small RNAs. Typical Pol III promoters use internal regulatory sequences (sequences within the transcription region of a gene) to support transcription, but upstream elements such as TATA boxes may also be used. All Pol III promoters are assumed to be within the scope of this disclosure.

[0032] The term "enhancer" refers to a regulatory DNA sequence that, when bound by a specific protein called a transcription factor, modulates the expression of the associated gene. Enhancers can be located in the intron of a gene, or at the 5' or 3' 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 bp away from the promoter). A single gene may be regulated by one or more enhancers, all of which are assumed to be within the scope of this disclosure.

[0033] As used herein, “post-transcriptional regulatory elements (PTREs),” such as hepatitis PTREs, refer to DNA sequences that, upon transcription, generate tertiary structures capable of exhibiting post-transcriptional activity to enhance or promote the expression of related genes operably ligated to them.

[0034] "Operatively coupled" refers to the juxtaposition of two or more components (such as array elements) such that both components function properly and at least one of the components can mediate the function exerted on the other component, for example, at least one of the promoter and code arrays. Those skilled in the art will understand that the two components do not need to be physically coupled in order to be operationally coupled.

[0035] In the context of this disclosure, and with respect to genes, the terms “repress,” “suppression,” “repression,” “transcriptional repression,” “inhibition of gene expression,” “downregulation,” and “silencing” are used interchangeably herein to refer to the inhibition or blockage of the transcription of a gene or a portion thereof. Thus, gene repression can result in a reduction in the production of gene products. Examples of gene repression processes that reduce transcription include, but are not limited to, inhibiting the formation of the transcription initiation complex, reducing the transcription initiation rate, reducing the transcription elongation rate, reducing the forward momentum of transcription, and antagonizing transcriptional activation (e.g., by blocking the binding of transcription activators). Gene repression can constitute, for example, the prevention of activation and the inhibition of expression below existing levels. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription, the latter of which may result from epigenetic modifications of a gene.

[0036] The terms “repressor” and “repressor domain” are used interchangeably and refer to polypeptide factors that act as DNA regulators, inhibiting, repressing, or blocking DNA transcription, thereby resulting in repression of gene expression. In the context of this disclosure, ligation of a repressor domain to a DNA-binding protein can prevent transcription from a promoter or inhibit gene expression when bound to a target nucleic acid. While we do not wish to be constrained by theory, transcriptional repressors can function through a variety of mechanisms, including physically blocking the passage of RNA polymerase 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 or regulating nucleosome remodeling through histone deacetylation, or preventing enhancer-promoter interactions, thereby resulting in gene silencing or a reduction in gene expression levels.

[0037] "Long-term repressor fusion protein" or "LTRP" is used herein interchangeably with "repressor fusion protein" and refers to a fusion protein comprising a DNA-binding protein (or DNA-binding domain of a protein) fused to one or more domains capable of repressing the transcription of a target nucleic acid sequence. Optionally, the long-term repressor fusion proteins of this disclosure may include 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.

[0038] As used herein, “LTRP:gRNA system” is a system for transcriptional repression comprising a long-term repressor fusion protein containing a non-catalyzed CRISPR protein and one or more linked repressor domains, and a guide nucleic acid (gRNA) that binds to the non-catalyzed CRISPR protein. For clarity, the system also comprises any coding DNA, RNA, or vector, etc., that may be used to produce the long-term repressor fusion protein and gRNA components of the system.

[0039] As used herein, “non-catalytic DNA-binding protein” refers to a protein or protein domain that can bind to DNA but cannot nick or cleave it. As used herein, “non-catalytic CRISPR protein” refers to a CRISPR protein that lacks endonuclease activity. Those skilled in the art will understand that a CRISPR protein may lack catalytic activity but can still perform additional protein functions such as DNA binding. Similarly, “non-catalytic CasX” refers to a CasX protein that lacks endonuclease activity but can still perform additional protein functions such as DNA binding.

[0040] As used herein, “recombinant” means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps, resulting in a construct having a structural code or non-coding sequence that is distinguishable from endogenous nucleic acids found in the natural system. Generally, DNA sequences encoding structural codes can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide synthetic nucleic acids that can be expressed from recombinant transcription units contained in cells or cell-free transcription and translation systems. Such sequences can typically be provided in the form of an open reading frame uninterrupted by internal non-coding sequences or introns present in eukaryotic genes. Genomic DNA containing the relevant sequences can also be used to form recombinant genes or transcription units. The non-coding DNA sequence may be located at 5' or 3' of the open reading frame, and such sequences may not interfere with the manipulation or expression of the coding region, but in practice may act to regulate the production of desired products by various mechanisms (see “enhancers” and “promoters” above).

[0041] The terms “recombinant polynucleotides” or “recombinant nucleic acids” refer to those that do not exist naturally, for example, those created by the artificial combination of two originally separate amino acid sequence segments through human intervention. This artificial combination is often achieved either by chemical synthesis or by artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques. Such combinations are typically performed to replace codons with redundant codons encoding the same or conserved amino acids, typically while introducing or removing sequence recognition sites. Alternatively, this is carried out to join nucleic acid segments of desired function together to produce a desired combination of function. This artificial combination is often achieved either by chemical synthesis or by artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques.

[0042] Similarly, the terms “recombinant polypeptide” or “recombinant protein” refer to polypeptides or proteins that do not exist in nature and are created, for example, by human intervention, through the artificial combination of two originally separate amino acid sequence segments. Therefore, for example, a protein containing heterologous amino acid sequences is recombinant.

[0043] As used herein, “lipid nanoparticles” or “LNP” refers to particles having at least one dimension of about nanometers (e.g., 1 to 1,000 nm), comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, helper phospholipids, and PEG-modified lipids) and cholesterol. Specific components of LNPs are described more thoroughly below. Lipid nanoparticles may be included in formulations that can be used to deliver activators or therapeutic agents, such as nucleic acids (e.g., mRNA), to a target site of interest (e.g., cells, tissues, organs, tumors, etc.). Lipid nanoparticles of this disclosure may include nucleic acids. Such lipid nanoparticles typically include neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Activators or therapeutic agents, such as nucleic acids, may be encapsulated in the lipid portion of the lipid nanoparticle, or in an aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, thereby protecting them from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cell, such as adverse immune responses.

[0044] As used herein, “lipid encapsulation” refers to lipid nanoparticles that provide an activator or therapeutic agent, such as nucleic acid (e.g., mRNA, gRNA, or both mRNA and gRNA), fully encapsulated, partially encapsulated, or both. Nucleic acid (e.g., mRNA) can be fully encapsulated within lipid nanoparticles.

[0045] As used herein, “lipoproteins,” such as VLDL, LDL, and HDL, refer to a group of proteins found in serum, plasma, and lymph that function in lipid transport. The chemical composition of each lipoprotein differs, for example, in that HDL has a higher ratio of protein to lipid, while VLDL has a lower ratio of protein to lipid.

[0046] As used herein, “atherosclerosis” refers to the hardening of arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. These fatty deposits, consisting mainly of cholesterol and other fats, calcium and scar tissue, damage the inner lining of the arteries and are called “atheroma” or “plaque.”

[0047] As used herein, “coronary artery disease (CHD)” means narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis.

[0048] As used herein, “dyslipidemia” refers to a disorder of lipid and / or lipoprotein metabolism, including the overproduction or deficiency of lipids and / or lipoproteins. Dyslipidemia may manifest as elevated levels of lipids such as chylomicrons, cholesterol, and triglycerides, as well as elevated levels of lipoproteins such as low-density lipoprotein (LDL) cholesterol.

[0049] As used herein, “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 typically quantified in mg / dL or nmol / L. “Serum HDL-C” and “Plasma HDL-C” refer to HDL-C in serum and plasma, respectively.

[0050] As used herein, “low-density lipoprotein cholesterol (LDL-C)” means cholesterol supported in low-density lipoprotein particles. The concentration of LDL-C in serum (or plasma) is typically quantified in mg / dL or nmol / L. “Serum LDL-C” and “plasma LDL-C” mean LDL-C in serum and plasma, respectively.

[0051] As used herein, “hypercholesterolemia” means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL cholesterol and VLDL cholesterol, as defined in the guidelines of the National Cholesterol Education Program (NCEP) Expert Panel Report on the Detection, Assessment and Treatment of High Cholesterol in Adults (see Arch.Int.Med.148:36(1988)).

[0052] As used herein, “dyslipidemia” or “hyperlipidemia” refers to a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition exhibits abnormally high concentrations of fat. The lipid fractions in circulating blood are cholesterol, low-density lipoproteins, very low-density lipoproteins, chylomicrons, and triglycerides. The Fredrickson classification of dyslipidemia is based on patterns of TG and cholesterol-rich lipoprotein particles measured by electrophoresis or ultracentrifugation and is commonly used to characterize the main causes of dyslipidemia, such as hypertriglyceridemia.

[0053] As used herein, “triglyceride” or “TG” means a lipid or neutral fat consisting of glycerol combined with three fatty acid molecules.

[0054] As used herein, “hypertriglyceridemia” means a condition characterized by elevated triglyceride levels. Its etiology includes primary (i.e., genetic causes) and secondary (other underlying causes such as diabetes mellitus, metabolic syndromes / insulin resistance, obesity, physical inactivity, cigarette smoking, excessive alcohol consumption, and diets very high in carbohydrates) factors, or most frequently, a combination of both.

[0055] As used herein, “diabetes mellitus” or “diabetes” is a syndrome characterized by abnormally high blood glucose levels (hyperglycemia) resulting from metabolic disorders and insufficient levels of insulin or decreased insulin sensitivity. Characteristic symptoms include excessive urine production due to high blood glucose levels (polyuria), excessive thirst and increased fluid intake in an attempt to compensate for increased urination (hyperthirst), blurred vision due to the effects of hyperglycemia on the optics of the eye, unexplained weight loss, and lethargy.

[0056] As used herein, “diabetic dyslipidemia” or “type 2 diabetes with dyslipidemia” means a condition characterized by type 2 diabetes, decreased HDL-C, elevated triglycerides (TG), and elevated small, high-density LDL particles.

[0057] As used herein, the term “to bring into contact” means to establish a physical connection between two or more entities. For example, bringing a target nucleic acid into contact with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid share a physical connection, for example, they can hybridize if their sequences share sequence similarity.

[0058] "Dissociation constant" or "K" d The term "L" is used interchangeably and refers to the affinity between ligand "L" and protein "P," i.e., how tightly the ligand binds to a particular protein. This is represented by formula K. d It can be calculated using =[L][P] / [LP], where [P], [L], and [LP] represent the molar concentrations of the protein, ligand, and complex, respectively.

[0059] As used herein, the term "knockdown" refers to the reduction of expression of a gene or its gene product. As a result of gene knockdown, protein activity or function may be reduced, or protein levels may be reduced or eliminated.

[0060] A polynucleotide or polypeptide has a certain degree of "sequence similarity" or "sequence identity" with another polynucleotide or polypeptide, meaning that when aligned, the proportion of bases or amino acids is identical and that they are in the same relative positions when comparing the two sequences. Sequence similarity (sometimes referred to as similarity rate, identity rate, or homology) can be determined in several different ways. To determine sequence similarity, sequences can be aligned using methods and computer programs known in the art, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov / BLAST. The complementarity rate between specific stretches of nucleic acid sequences within a nucleic acid can be determined using any convenient method. Examples of methods include using the BLAST program (a basic local sorting 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 the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), for example, by using the default settings that employ the Smith and Waterman algorithm (Adv.Appl.Math., 1981, 2, 482-489).

[0061] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include encoded and unencoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone. The terms include, but are not limited to, fusion proteins having heterologous amino acid sequences.

[0062] A "vector" or "expression vector" is a replicon such as a plasmid, phage, virus, or cosmid, which may contain another DNA segment, i.e., an expression cassette, resulting in the replication or expression of the other DNA segment in a cell.

[0063] As used herein, the terms “naturally occurring,” “unmodified,” or “wild-type,” applied to nucleic acids, polypeptides, cells, or organisms, refer to nucleic acids, polypeptides, cells, or organisms found in nature.

[0064] As used herein, “mutation” means an 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.

[0065] As used herein, the term “isolated” is intended to describe polynucleotides, polypeptides, or cells that are in an environment different from the environment in which they naturally occur. Isolated recombinant host cells may exist in a mixed population of recombinant host cells.

[0066] As used herein, “host cell” means a eukaryotic cell, a prokaryotic cell, or a cell derived from a multicellular organism (e.g., a cell line) cultured as a single-cell entity, the eukaryotic or prokaryotic cell being used as a recipient of nucleic acid (e.g., an AAV vector), and including offspring of the original cell that have been genetically modified by the nucleic acid. Offspring of a single cell may not necessarily be completely identical to the original parent in morphology or in genomics 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.

[0067] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains consists of serine and threonine; the group of amino acids with amide-containing side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0068] As used herein, “treatment” or “to treat” are interchangeable herein and refer to an approach to obtain a beneficial or desired outcome, including but not limited to therapeutic benefits and / or preventive benefits. Therapeutic benefits mean the eradication or improvement of the underlying disease or condition being treated. Therapeutic benefits may also be achieved by the eradication or improvement of one or more symptoms, or by improvement of one or more clinical parameters related to the underlying disease, such that improvement is observed in the subject, even though the subject may still have the underlying disease.

[0069] As used herein, the terms “therapeutic dose” and “therapeutic size” refer to a certain amount of a drug or bioagent, alone or as part of a composition, that, when administered in a single or repeated dose to a subject such as a human or experimental animal, is capable of producing any detectable and beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition. Such an effect does not need to be absolute to be beneficial.

[0070] As used herein, “administer” means a method of giving a certain dose of a compound (e.g., a composition of this disclosure) or a composition (e.g., a pharmaceutical composition) to a subject.

[0071] The "subjects" are mammals. Mammals include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats, and other rodents.

[0072] The term "low-density lipoprotein (LDL)" refers to one of five major groups of lipoproteins ranging from the lowest density (low weight-to-volume particle ratio) to the highest density (high weight-to-volume particle ratio): chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL). Lipoproteins transport lipids (fat) throughout the body in the extracellular fluid, thereby facilitating the transport of fat to the cell body via receptor-mediated endocytosis. LDL particles have a diameter of approximately 220–275 angstroms.

[0073] The "low-density lipoprotein (LDL) receptor" refers to an 839-amino acid receptor protein (after removal of a 21-amino acid signal peptide) that mediates the endocytosis of cholesterol-rich LDL particles. It is a cell surface receptor that recognizes the apolipoprotein B100 and apoE proteins found in chylomicron remnants and very low-density LDL 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 in the NCBI database (ncbi.nlm.nih.gov) as reference sequence NG_009060.1, which is incorporated herein by reference.

[0074] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent that each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0075] I. General Methods The practice of this invention employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, unless otherwise indicated, and these are referenced in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001) and Short Protocols in Molecular Biology, 4 thThis information can be found in standard textbooks such as Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), and these disclosures are incorporated herein by reference.

[0076] Where a range of values ​​is provided, it is understood that, unless the context otherwise explicitly determines, each intermediary value up to one-tenth of the lower limit unit is included between the upper and lower limits of that range, and between any other listed values ​​or intermediary values ​​within that listed range. The upper and lower limits of these smaller ranges may independently be included within smaller ranges and are included by any specifically excluded limits within the listed range. If a listed range includes one or both limits, it also includes ranges that exclude one or both of those included limits.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure pertains. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials relating to those cited in those publications.

[0078] For clarity, it will be understood that certain features of the Disclosure described in relation to separate embodiments may be provided in combination in a single embodiment. In other cases, for brevity, various features of the Disclosure described in relation to a single embodiment may also be provided separately or in any preferred secondary combination. All combinations of embodiments relating to the Disclosure are specifically encompassed by the Disclosure and are intended to be disclosed herein in the same way that any combination may be disclosed individually and expressly. In addition, all secondary combinations of various embodiments and their elements are also specifically encompassed by the Disclosure and are disclosed herein in the same way that any such secondary combination may be disclosed herein individually and expressly.

[0079] II. Systems for epigenetic modification and repression of the PCSK9 gene In a first aspect, the Disclosure provides a system comprising or encoding a long-term repressor fusion protein ("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 modification. The Disclosure also provides a system comprising or encoding an LTRP. In some cases, the system is designed to repress the transcription of the PCSK9 gene in mutant eukaryotic cells.

[0080] As used herein, “System” is used interchangeably with “Composition.” This disclosure also provides nucleic acids encoding the Systems provided herein. Also provided herein are methods for constructing the Systems, as well as methods for using the Systems, including methods for gene repression and / or epigenetic modification and methods for treating PCSK9-related diseases. For clarity, the term “System” also includes any coding DNA, RNA, or vector, etc., that may be used to produce the repressor fusion proteins and gRNA components of the Systems.

[0081] In some embodiments, the DNA-binding protein for use in the long-term repressor fusion protein of this disclosure comprises a DNA-binding domain that binds to but does not cleave a target nucleic acid, and is also referred to herein as a zinc finger (ZF) or TALE (transmission activator-like effector) protein. The DNA-binding domain of TALE consists of a tandem arrangement of customizable monomers 33-34 amino acid (aa) long and can theoretically be assembled to recognize any gene sequence according to a recognition code in which one repeat binds to one base pair (Jain, S., et al. TALEN outperforms Cas9 in editing heterochromatin target sites. Nat.Commun. 12:606 (2021)). The specificity of TALE for binding to DNA arises from two polymorphic amino acids, so-called repeat variable duodecimal residues (RVDs) located at positions 12 and 13 of the repeat unit. By rearranging the repeat, the DNA-binding specificity of TALE can be arbitrarily altered. Zinc finger proteins are transcription factors, and each finger recognizes 3-4 bases of DNA. By mixing and matching these finger modules, ZFs can be customized for target sequences. An example ZF capable of binding to the PCSK9 gene is described in WO2018049009A2.

[0082] In some embodiments, the DNA-binding protein for use in long-term repressor fusion proteins is a non-catalyzed class 1 or class 2 CRISPR protein. Non-catalyzed CRISPR proteins are also referred to in the art as "catalyzably inactive" CRISPR proteins. In one embodiment, the class 2, type II protein is a non-catalyzed 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 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 which case catalytic activity is not associated with any particular mutation. The CRISPR-based system further comprises a guide nucleic acid, such as a guide ribonucleic acid (gRNA), having a targeting sequence complementary to the target sequence of the PCSK9 gene that is bound and repressed by a complex of a fusion protein (CRISPR protein and linked repressor domain) and gRNA.

[0083] In some embodiments, the Disclosure provides systems comprising or encoding a long-term repressor fusion protein comprising a CasX CRISPR nuclease protein without catalytic activity and a linked repressor domain, and a guide ribonucleic acid (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence of the PCSK9 gene targeting transcriptional repression, silencing, or downregulation, as well as nucleic acids encoding the long-term repressor fusion protein and / or gRNA. In some embodiments, the system comprises a long-term repressor fusion protein and gRNA as a gene repressor pair capable of forming a ribonucleoprotein (RNP) complex and binding to the PCSK9 target nucleic acid ("LTRP:gRNA system"). In other embodiments, the Disclosure provides systems of nucleic acids encoding a long-term repressor fusion protein and gRNA. In yet other embodiments, the Disclosure provides systems of gRNA and mRNA encoding a long-term repressor fusion protein for use in specific particle formulations (e.g., LNPs) described herein.

[0084] This specification also provides methods for producing long-term repressor fusion proteins and gRNAs, as well as methods for using the LTRP:gRNA system, including gene repression and / or epigenetic modification of the PCSK9 gene, and methods for treating PCSK9-related diseases or disorders. The DNA-binding proteins (e.g., dCasX) and the ligated repressor domains and gRNA components of the LTRP:gRNA system and their characteristics, as well as the modes of delivery and methods for using the system for repression, downregulation, or silencing of the PCSK9 gene, are described in detail below.

[0085] In some embodiments, this disclosure provides systems specifically designed to repress or silence the transcription of the PCSK9 gene. In some cases, the system is designed to repress the transcription of the PCSK9 gene in eukaryotic cells having a gain-of-function mutation. In some cases, the system is designed to repress the transcription of the wild-type PCSK9 gene in eukaryotic cells. Alternatively, the system is designed to repress the transcription of a mutant allele of the PCSK9 gene in eukaryotic cells. In general, any portion of the PCSK9 gene can be targeted using programmable systems and methods provided herein, which are described more fully herein.

[0086] The PCSK9 gene encodes subtilisin / kexin 9 ("PCSK9"), a precursor protein convertase that binds to receptors for low-density lipoprotein particles (LDL) for the transport of LDL into cells. The PCSK9 gene encompasses a sequence in the human genome (GRCh38 / hg38) chr1:55,039,476–55,064,853 (the notation refers to chromosome 1 (chr1), starting at 55,039,476 bp and ending at 55,064,853 bp on chromosome 1 (Homo sapiens Updated Annotation Release 109.20190905,GRCh38.p13) (NCBI)). The human PCSK9 gene is partially described in the NCBI database (ncbi.nlm.nih.gov) as reference sequence NG_009061.1, which is incorporated herein by reference. The PCSK9 gene locus has 12 exons that produce a 3636 bp mRNA encoding a 692-amino acid protein. After synthesis, it undergoes an autocatalytic cleavage reaction that cleaves the prodomain, resulting in an activated protein with 540 amino acids. The prodomain remains attached to the catalytic domain and resistin-like domain, which is likely because the prodomain functions as a chaperone, promoting folding and secretion (Seidah, NG et al., Proc Natl Acad Sci USA 100(3):928(2003)). Secretory convertase precursor protein, neuronal apoptosis regulatory convertase 1 (NARC-1): liver regeneration and neural differentiation (Seidah NG, et al.). This protein, also called neuronal apoptosis regulatory convertase, is a serine protease belonging to the subfamily of subtilase proteases K.

[0087] The human PCSK9 gene (HGNC:20001) is, It encodes a protein (Q8NBP7) with the sequence (SEQ ID NO: 1823).

[0088] III. Non-catalytically active proteins for use in repressor systems In some embodiments, the DNA-binding protein for use in the long-term repressor fusion protein of this disclosure is a zinc finger (ZF) or TALE (transmission activator-like effector) protein that can bind to but cannot cleave PCSK9 target nucleic acids.

[0089] In some embodiments, the DNA-binding protein is a non-catalytically active class 1 or class 2 CRISPR protein. In some embodiments, the DNA-binding protein is a class 2, type II CRISPR protein. In one embodiment, the class 2, type II protein is a non-catalytically active 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, and in all cases, it is non-catalytically active due to specific mutations as described herein. In another embodiment, the class 2 CRISPR type V protein is a CasX protein that does not exhibit catalytic activity.

[0090] CasX proteins, including dCasX, contain the following domains: non-target chain binding (NTSB) domain, target chain loading (TSL) domain, helix I domain, helix II domain, oligonucleotide binding (OBD) domain, and RuvC domain. In some cases, these domains are further classified into subdomains listed in Table 1.

[0091] In the context of this disclosure, CasX for use in the system is achieved by mutations introduced at a select position in the RuvC sequence, without catalytic activity (dCasX), as described below.

[0092] a. Reference CasX protein This disclosure provides a naturally occurring CasX protein (referred to herein as the “reference CasX protein”) which was 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 Deltaproteobacteria, Plantomycetes, or Candidatus Sungbacteria. The reference CasX protein (referred to herein interchangeably as the reference CasX polypeptide) is a class 2, type V CRISPR / Cas endonuclease belonging to the CasX (referred to interchangeably as Cas12e) family of proteins that interact with a guide RNA to form a ribonucleoprotein (RNP) complex.

[0093] In some cases, the reference CasX protein is isolated from or derived from Deltaproteobacter and contains the sequence of Sequence ID No. 1.

[0094] In some cases, the reference CasX protein is isolated from or derived from Plantomycetes and contains the sequence of SEQ ID NO: 2.

[0095] In some cases, the reference CasX protein is isolated from or derived from Candidatus Sungbacteria and contains the sequence of Sequence ID No. 3.

[0096] b. Class 1 or Class 2 CRISPR proteins that do not exhibit catalytic activity. In the long-term repressor fusion proteins and gene repressor systems comprising the same, non-catalyzed class 1 or class 2 CRISPR proteins are non-catalyzed in that they cannot cleave DNA, but they retain the ability to bind to a target nucleic acid when complexed with guide RNA (gRNA). The disclosure provides non-catalyzed variants of class 1 or class 2 CRISPR proteins, the non-catalyzed variants comprising multiple modifications in a selected domain. The disclosure provides non-catalyzed variants (hereinafter interchangeably referred to as “dCasX variants” or “dCasX variant proteins”), the non-catalyzed CasX variants comprising multiple modifications in the RuvC domain compared to a non-catalyzed version of a reference CasX protein comprising the sequences of SEQ ID NOs. 1–3 (above). In some embodiments, the non-catalyzed reference CasX protein comprises substitutions at residues 672, 769, and / or 935 with respect to SEQ ID NOs. 1. In some embodiments, the non-catalyzed reference CasX protein includes substitutions to D672A, E769A, and / or D935A with reference to SEQ ID NO: 1. In other embodiments, the non-catalyzed reference CasX protein includes substitutions to amino acids 659, 756, and / or 922 with reference to SEQ ID NO: 2. In some embodiments, the non-catalyzed reference CasX protein includes substitutions to D659A, E756A, and / or D922A with reference to SEQ ID NO: 2. The exemplary RuvC domain of dCasX in this disclosure includes 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, and has one or more amino acid modifications to the RuvC cleavage domain sequence, and the dCasX variant exhibits one or more improved features compared to the reference dCasX. In further embodiments, the CasX variant protein lacking catalytic activity comprises the deletion of all or part of the RuvC domain of the reference CasX protein.The same aforementioned substitutions or deletions can similarly be introduced into CasX variants known in the Art, resulting in dCasX variants, as will be understood (see, for example, WO2022120095A1 and US11,560,555 incorporated herein by reference for exemplary sequences).

[0097] In some embodiments, a long-term repressor fusion protein comprising a dCasX variant having a linked repressor domain exhibits at least one improved feature compared to a long-term repressor fusion protein comprising a reference dCasX protein having an equivalent linked repressor domain. All dCasX variants that improve one or more functions or features of a long-term repressor fusion protein comprising a dCasX variant protein having a linked repressor domain compared to a long-term repressor fusion protein comprising a reference dCasX protein are assumed to be within the scope of this disclosure. In some embodiments, the modification is a mutation of one or more amino acids in the reference dCasX other than causing loss of catalytic activity of dCasX. For example, a dCasX variant may comprise one or more amino acid substitutions, insertions, deletions, or swapped domains relative to the reference dCasX protein sequence, or any combination thereof. Any amino acid may be substituted for any other amino acid in the substitutions described herein. Substitutions may be conserved substitutions (e.g., one basic amino acid is substituted for another basic amino acid). The substitutions may be non-conservative substitutions (e.g., a basic amino acid being substituted for an acidic amino acid, or vice versa). For example, proline in the reference dCasX protein may be substituted for any of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine to produce the dCasX variant proteins of this disclosure. In some embodiments, the dCasX variant exhibits improved properties compared to the reference dCasX.Examples of improved features of dCasX variant embodiments include improved folding of the variant, increased binding affinity to target nucleic acids, improved ability to utilize a wider range of PAM sequences in transcriptional repression and / or binding to target nucleic acids, improved unwinding of target DNA, increased loading of the target strand, increased binding of the non-target strand of DNA, improved protein stability, increased ability to complex with gRNA, increased binding affinity to gRNA, improved stability of the protein:gRNA(RNP) complex, and, if having a linked repressor domain and complexing as RNP, increased repressor activity, improved repressor specificity to target nucleic acids, reduced off-target repression, and an increased proportion of eukaryotic genomes that can be efficiently repressed and / or epigenetically modified. In some embodiments, the improved features of the dCasX variant are improved by at least about 1.1 to about 100,000 times compared to the reference dCasX protein.In some embodiments, the improved features of the dCasX variant are improved by at least approximately 1.1 to 10,000 times compared to the reference dCasX protein, at least approximately 1.1 to 1,000 times, at least approximately 1.1 to 500 times, at least approximately 1.1 to 400 times, at least approximately 1.1 to 300 times, at least approximately 1.1 to 200 times, at least approximately 1.1 to 100 times, at least approximately 1.1 to 50 times, at least approximately 1.1 to 40 times, at least approximately 1.1 to 30 times, at least approximately 1.1 to 20 times, at least approximately 1.1 to 10 times, at least approximately 1.1 to 9 times, and less The improvement is at least 1.1 to 8 times, at least 1.1 to 7 times, at least 1.1 to 6 times, at least 1.1 to 5 times, at least 1.1 to 4 times, at least 1.1 to 3 times, at least 1.1 to 2 times, at least 1.1 to 1.5 times, at least 1.5 to 3 times, at least 1.5 to 4 times, at least 1.5 to 5 times, at least 1.5 to 10 times, at least 5 to 10 times, at least 10 to 20 times, at least 10 to 30 times, at least 10 to 50 times, or at least 10 to 100 times. In some embodiments, the improved features of the dCasX variant are at least 10 to 1000 times improved compared to the reference dCasX protein. Additional disclosures regarding the improved features are described below in this specification.

[0098] In other embodiments, a modification is the substitution of one or more domains of a reference dCasX with one or more domains derived from a different CasX. In some embodiments, an insertion includes the insertion of some or all of a domain from a different CasX protein. A mutation may occur in any one or more domains of a dCasX variant and may include, for example, the deletion of some or all of one or more domains, or the substitution, deletion, or insertion of one or more amino acids in any domain. The domains of the dCasX protein include a non-target chain binding (NTSB) domain, a target chain loading (TSL) domain, a helix I domain, a helix II domain, an oligonucleotide binding domain (OBD), and a RuvC DNA cleavage domain, which may further include the subdomains described below.

[0099] In some embodiments, the dCasX variant protein contains 800 to 1100 amino acids, or 900 to 1000 amino acids.

[0100] The long-term repressor fusion protein comprising the dCasX variant and the linked repressor domain of this disclosure exhibits enhanced ability to efficiently bind to target nucleic acids when complexed with gRNA as an RNP, utilizing a PAM TC motif containing a PAM sequence selected from TTC, ATC, GTC, or CTC, compared to the RNP of the fusion protein and gRNA containing the reference dCasX protein in a comparative assay system. As described above, the PAM sequence is located at least one nucleotide 5' on the non-target strand of the protospacer, which is identical to the targeting sequence of the gRNA.

[0101] In some embodiments, RNPs comprising a long-term repressor fusion protein and gRNA containing a dCasX variant having a linked repressor domain of the present disclosure can bind 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% at concentrations of 20 pM or less. In one embodiment, RNPs comprising a long-term repressor fusion protein and gRNA variant containing a dCasX variant having a linked repressor domain show greater binding of the target sequence in the target nucleic acid compared to an equivalent RNP comprising a reference dCasX protein having a linked repressor domain and gRNA in an equivalent assay system, where the PAM sequence of the target nucleic acid is TTC. In another embodiment, an RNP of a long-term repressor fusion protein and gRNA variant containing a dCasX variant having a ligated repressor domain exhibits greater binding affinity to a target sequence in a target nucleic acid compared to an RNP containing an equivalent long-term repressor fusion protein and gRNA containing a reference dCasX protein having a ligated repressor domain, in an equivalent assay system, where the PAM sequence of the target nucleic acid is ATC. In another embodiment, an RNP of a long-term repressor fusion protein and gRNA variant containing a dCasX variant having a ligated repressor domain exhibits greater binding affinity to a target sequence in a target nucleic acid compared to an RNP containing an equivalent long-term repressor fusion protein and gRNA containing a reference dCasX protein having a ligated repressor domain, in an equivalent assay system, where the PAM sequence of the target nucleic acid is CTC. In another embodiment, RNPs of long-term repressor fusion proteins and gRNA variants containing a dCasX variant having a ligated repressor domain exhibit greater binding affinity to the target sequence in the target nucleic acid in an equivalent assay system compared to equivalent RNPs of long-term repressor fusion proteins and gRNAs containing a reference dCasX protein having a ligated repressor domain, where the PAM sequence of the target nucleic acid is GTC.In other embodiments, RNPs of repressor fusion proteins and gRNAs containing dCasX variants having a ligated repressor domain exhibit greater binding affinity to target sequences in target nucleic acids in an equivalent assay system compared with RNPs containing equivalent long-term repressor fusion proteins and gRNAs containing a reference dCasX protein having a ligated repressor domain, where the PAM sequence of the target nucleic acid is GTC, TTC, ATC, or CTC. In the embodiments described above, the increase in binding affinity to one or more PAM sequences is at least 1.5 times compared to the binding affinity of RNPs to gRNAs of sequence numbers 1731-1743 in Table 8 to any one of the reference dCasX proteins having a ligated repressor domain (modified from SEQ ID NOs. 1-3) and the PAM sequences.

[0102] c. dCasX variant protein having domains derived from multiple source proteins In certain embodiments, the disclosure provides a chimeric dCasX variant protein for use in long-term repressor fusion proteins.

[0103] As used herein, “chimeric dCasX protein” refers to both a non-catalyzed CasX protein containing at least two domains from different sources, and a non-catalyzed CasX protein containing at least one domain that is itself a chimeric protein. Thus, in some embodiments, a chimeric dCasX protein contains at least two domains isolated or derived from different sources, such as two different naturally occurring CasX proteins (e.g., two different CasX reference proteins). In other embodiments, a chimeric dCasX protein contains at least one domain that is a chimeric domain, and in some embodiments, for example, a portion of the domain includes substitution from a different CasX protein (a reference CasX protein, or another dCasX protein).

[0104] In some embodiments, at least one chimeric domain may be one of the NTSB, TSL, helix I, helix II, OBD, or RuvC domains described herein. In the case of segmented or discontinuous domains such as helix I, RuvC, and OBD, a portion of the discontinuous domain may be replaced with a corresponding portion from any other source. In some embodiments, the helix I-II domain (also called helix Ia) of the dCasX variant derived from SEQ ID NO: 2 is replaced with the corresponding helix I-II sequence derived from SEQ ID NO: 1, resulting in a chimeric dCasX protein.

[0105] In some embodiments, the helical I-II domain and NTSB domain of the dCasX variant derived from SEQ ID NO: 2 are replaced with the corresponding helical I-II sequence and NTSB sequence derived from SEQ ID NO: 1, resulting in a chimeric dCasX protein.

[0106] The chimeric dCasX variant protein may include the NTSB, TSL, helix II, helix I-II, helix II, OBD-I, and OBD-II domains derived from the CasX protein of SEQ ID NO: 2, and the RuvC-I and / or RuvC-II domains derived from the CasX protein of SEQ ID NO: 1, or vice versa, and mutations or other sequence modifications may be introduced to produce a non-catalyzed variant with improved variant characteristics relative to the reference dCasX protein. As an example, the chimeric RuvC domain includes amino acids 660-823 of SEQ ID NO: 1 and amino acids 921-978 of SEQ ID NO: 2. As an alternative example, the chimeric RuvC domain includes amino acids 647-810 of SEQ ID NO: 2 and amino acids 934-986 of SEQ ID NO: 1. In certain embodiments, dCasX for use in long-term repressor fusion proteins comprises the NTSB domain and helix I-II domain from SEQ ID NO: 1, and the helix II domain from SEQ ID NO: 2, the latter being a chimeric domain, and it is understood that the dCasX variant has additional amino acid changes at a selected position (compared to the reference sequence), and the resulting chimeric dCasX protein has improved properties compared to the reference dCasX protein. Sequences in Table 2 that have the NTSB domain and helix I-II domain from SEQ ID NO: 1, and the helix 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 NOs: 9-11), 522-527 (SEQ ID NOs: 12-17), 532 (SEQ ID NO: 22), 593 (SEQ ID NO: 25), 676 (SEQ ID NO: 28, with an L169K substitution in the NTSB domain), and 812 (SEQ ID NO: 29). Table 1 below provides the coordinates of the CasX domain in the reference CasX protein of Sequence ID No. 1 and Sequence ID No. 2. Those skilled in the art will understand that the domain boundaries shown in Table 1 below are approximate, and that protein fragments whose boundaries differ by one, two, or three amino acids from those shown in the table below may have the same activity as the domains described below. Table 1: Domain coordinates in the reference CasX protein [Table 1] *Amino acid position

[0107] In some embodiments, the dCasX variant protein used in the long-term repressor fusion protein of this disclosure comprises a sequence selected from the group consisting of SEQ ID NOs. 4 to 29 listed in Table 2, wherein the sequence comprises a RuvC domain containing one or more mutations that inactivate the cleavage activity of the RuvC domain. In other embodiments, the dCasX variant protein used in the long-term repressor fusion protein of this disclosure comprises a sequence selected from the group consisting of SEQ ID NOs. 4 to 29 listed in Table 2, which is at least approximately 70% identical, at least approximately 75% identical, at least approximately 80% identical, and at least approximately 81% identical. The sequences are identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, and at least about 99.5% identical. In some embodiments, the dCasX variant protein contains a RuvC domain with one or more mutations that inactivate the cleavage activity of the RuvC domain. In some embodiments, the long-term repressor fusion protein containing dCasX retains the ability to form RNPs with gRNA. In certain embodiments, the dCasX variant protein used in the long-term 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 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 fusion protein of the gene repressor system of this disclosure comprises the sequence of SEQ ID NO: 29 (dCasX 812). Table 2: dCasX variant sequences [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14]

[0108] Affinity for d.gRNA In some embodiments, long-term repressor fusion proteins containing dCasX having a linked repressor domain exhibit improved affinity for gRNA compared to equivalent long-term repressor fusion proteins containing a reference dCasX protein with a corresponding linked repressor domain, leading to the formation of ribonucleoprotein complexes (RNPs). The increased affinity of long-term repressor fusion proteins for gRNA is, for example, lower K for RNP complex production. d This can result in the formation of a more stable ribonucleoprotein complex, in some cases. In some embodiments, the K of the long-term repressor fusion protein to the gRNA d The binding affinity is increased by at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100-fold compared to the reference dCasX protein and the linked repressor domain. In some embodiments, the long-term repressor fusion protein containing the dCasX variant has a binding affinity to gRNA that is increased by about 1.1 to about 10-fold compared to the corresponding repressor fusion protein containing a variant of the reference CasX protein of SEQ ID NO: 2 that does not have catalytic activity.

[0109] In some embodiments, increased affinity of the long-term repressor fusion protein to gRNA results in increased stability of the ribonucleoprotein complex when delivered to mammalian cells, including in vivo delivery to the target. This increased stability affects the function and utility of the complex in the target cells and may result in improved pharmacokinetic properties in the blood when delivered to the target. In some embodiments, increased affinity of the long-term repressor fusion protein, and the resulting increased stability of the ribonucleoprotein complex, allows for lower doses of the long-term repressor fusion protein delivered to the target or cells, while still possessing the desired activity, such as gene repression and / or epigenetic modification in vivo or in vitro. Increased ability to form RNPs and maintain them in a stable form can be evaluated using in vitro assays known in the art.

[0110] In some embodiments, the higher affinity (stronger binding) of the long-term repressor fusion protein to the gRNA of the dCasX variant protein allows for a greater amount of transcriptional repression and / or epigenetic modification events if both the long-term repressor fusion protein and the gRNA remain in the RNP complex. The increase in transcriptional repression events can be evaluated using the assays described herein.

[0111] Methods for measuring the binding affinity of a long-term repressor fusion protein to a gRNA include in vitro methods using purified long-term repressor fusion proteins and gRNAs. If the gRNA or long-term repressor fusion protein is tagged with a fluorophore, the binding affinity to the long-term repressor fusion protein may be measured by fluorescence polarization. Alternatively or additionally, binding affinity may be measured by biolayer interferometry, electrophoretic mobility shift assay (EMSA), or filter binding. Additional standard techniques for quantifying the absolute affinity of an RNA-binding protein, such as the dCasX protein of this disclosure, to a specific gRNA, such as a reference gRNA and its variants, include, but are not limited to, isothermal calorimetry (ITC) and surface plasmon resonance (SPR).

[0112] e. Improvement of specificity for target nucleic acid sequences In some embodiments, long-acting repressor fusion proteins containing a dCasX variant protein with a linked repressor domain exhibit improved specificity to target nucleic acid sequences complementary to the targeting sequence of the gRNA compared to a reference dCasX protein with a linked repressor domain. "Specificity," sometimes also referred to as "target specificity" as used herein, refers to the degree to which the CRISPR / Cas system ribonucleoprotein complex binds to off-target sequences similar to, but not identical to, the target nucleic acid sequence. For example, an RNP containing a long-acting repressor fusion protein with higher specificity exhibits reduced off-target methylation of the sequence compared to an RNP of a reference dCasX with a linked repressor domain. The specificity of long-acting repressor fusion proteins, and the reduction of potentially harmful off-target effects, may contribute to achieving an acceptable therapeutic index for use in mammalian subjects. While not wishing to be constrained by theory, amino acid changes in the helix I and II domains that enhance the specificity of long-acting repressor fusion proteins to the target nucleic acid chain may potentially enhance the specificity of long-acting repressor fusion proteins to the entire target nucleic acid. In some embodiments, amino acid modifications that increase the specificity of the long-term repressor fusion protein to the target nucleic acid may also result in a decrease in the affinity of the long-term repressor fusion protein to DNA, but the overall benefits and safety of the composition are enhanced.

[0113] f. Repressor fusion proteins containing heterologous proteins Furthermore, within the scope of this disclosure, long-term repressor fusion proteins are also envisioned, which include heterologous proteins fused to long-term repressor fusion proteins for use in the systems of this disclosure. These include long-term repressor fusion proteins that include N-terminal or C-terminal fusion to heterologous proteins or their domains. In some embodiments, the long-term repressor fusion protein is fused to one or more proteins or their domains having different desired activities.

[0114] In some cases, the heterologous polypeptide (fusion partner) for use with the long-term repressor fusion protein provides intracellular localization, i.e., the heterologous polypeptide contains intracellular localization sequences (e.g., nuclear localization signals (NLS) for targeting the nucleus, sequences for keeping the fusion protein outside the nucleus, nuclear export sequences (NES), sequences for keeping the fusion protein in the cytoplasm, mitochondrial localization signals for targeting mitochondria, chloroplast localization signals for targeting chloroplasts, ER retention signals, etc.).

[0115] In some cases, the long-term repressor fusion protein contains (is fused to) a nuclear localization signal (NLS). In some cases, the long-term repressor fusion protein is fused to two or more, three or more, four or more, or five or more, six or more, seven or more, or eight or more NLSs. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near the N-terminus and / or C-terminus of the long-term repressor fusion protein (e.g., within 20 amino acids). In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near the N-terminus of the long-term repressor fusion protein (e.g., within 20 amino acids). In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near the C-terminus of the long-term repressor fusion protein (e.g., within 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, one NLS is located at the N-terminus of the long-term repressor fusion protein and one NLS is located at the C-terminus. Those skilled in the art will understand that the NLSs at or near the N-terminus or C-terminus of a protein may be within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of the N-terminus or C-terminus. In some embodiments, the NLS ligated to the N-terminus of dCasX or the long-term repressor fusion protein is identical to the NLS ligated to the C-terminus. In other embodiments, the NLS ligated to the N-terminus of dCasX or the long-term repressor fusion protein is different from the NLS ligated to the C-terminus. A typical configuration of a long-term repressor fusion protein having an NLS is shown in Figure 61.In some embodiments, NLSs suitable for use in long-term repressor fusion proteins in the systems of this disclosure include sequences having at least about 85%, at least about 90%, or at least about 95% identity, or being identical, to sequences derived from nucleoplasmin-derived NLSs (e.g., nucleoplasmin bipartite NLS having sequence KRPAATKKAGQAKKKK (SEQ ID NO: 31), c-MYC NLS having amino acid sequence PAAKRVKLD (SEQ ID NO: 32), or RQRRNELKRSP (SEQ ID NO: 33)). In some embodiments, the NLS and short peptide linker ligated to the N-terminus of the long-term repressor fusion protein is sequence PKKKRKVSR (SEQ ID NO: 34). In some embodiments, the NLS and short peptide linker ligated to the N-terminus of the long-term repressor fusion protein is sequence PKKKRKVSRVNGSGSGGG (SEQ ID NO: 3298). In some embodiments, the NLS and short peptide linker ligated to the C-terminus of the long-term repressor fusion protein are sequence TSPKKKRKV (SEQ ID NO: 3273). In some embodiments, the NLS ligated to the N-terminus of the long-term repressor fusion protein is selected from the group consisting of N-terminal sequences listed in Table 3. In some embodiments, the NLS includes sequences selected from the group consisting of SEQ ID NOs: 30-97, 3273, and 3298. In some embodiments, the NLS includes sequences selected from the group consisting of SEQ ID NOs: 34-97. In some embodiments, the NLS ligated to the C-terminus of the long-term repressor fusion protein is selected from the group consisting of C-terminal sequences listed in Table 4. In some embodiments, an NLS suitable for use in the long-term repressor fusion protein in the system of this disclosure includes sequences that have at least about 80%, at least about 90%, or at least about 95% identity with, or are identical to, one or more sequences in Table 3 or Table 4. Those skilled in the art will understand that any of the NLS sequences listed in Tables 3 and 4 may be fused to, or proximal to, either the N-terminus or C-terminus of the long-term repressor fusion proteins described herein. Table 3: N-terminal NLS amino acid sequence [Table 15] [Table 16] *Bold residues are NLS residues, and non-bold residues are linkers. Table 4: C-terminal NLS amino acid sequence [Table 17] [Table 18]

[0116] In some embodiments, one or more NLSs are linked to adjacent NLSs by a long-term repressor fusion protein or an arbitrary linker peptide. In some embodiments, the linker peptides are 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), GSGSG (SEQ ID NO: 105), GSGGG (SEQ ID NO: 106), GGGSG (SEQ ID NO: 107), GSSSG (SEQ ID NO: 108), GPGP (SEQ ID NO: 109), GGP, PPP, VPPP, PPAPPA (SEQ ID NO: 110), PPPG (SEQ ID NO: 111), PPPGPPP (SEQ ID NO: 112), PPP(GGGS)n (SEQ ID NO: 113), (GGGS)nPPP (SEQ ID NO: 114), Selected from the group consisting of AEAAAKEAAAKEAAAKA (sequence number 115), VPPPGGGSGGGSGGGS (sequence number 116), TGGGPGGGAAAGSGS (sequence number 117), GGGSGGGSGGGSPPP (sequence number 118), TPPKTKRKVEFE (sequence number 119), GGSGGGS (sequence number 120), GGSGGGG (sequence number 121), SSGNSNANSRGPSFSSGLVPLSLRGSH (sequence number 122), GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE (sequence number 123), and GGSGGG (sequence number 124), where n is 1 to 5.

[0117] Generally, NLS (or multiple NLS) are strong enough to promote the accumulation of long-term repressor fusion proteins in the nucleus of eukaryotic cells. Detection of nuclear accumulation can be carried out by any suitable technique. For example, a detectable marker can be fused to the long-term repressor fusion protein to visualize its location within the cell. The cell nucleus can also be isolated from the cell, and its contents can then be analyzed by any suitable process for detecting proteins, such as immunohistochemistry, Western blotting, or enzyme activity assays. Nuclear accumulation can also be determined indirectly.

[0118] IV. Long-term repressor domain fusion protein In some embodiments, the disclosure provides a system of long-term repressor fusion proteins comprising DNA-binding proteins linked to multiple repressor domains, the system capable of binding to a target nucleic acid of PCSK9 and repressing the transcription of the PCSK9 gene, including by epigenetic modification of the target nucleic acid. Exemplary DNA-binding proteins for use in fusion proteins include zinc finger (ZF), TALE (transmission activator-like effector) proteins, and CRISPR proteins without catalytic activity.

[0119] In some embodiments, the disclosure provides a long-term repressor fusion protein comprising a non-catalyzed CRISPR protein, such as dCasX, linked to multiple repressor domains, which, when complexed with a guide ribonucleic acid (gRNA) containing a targeting sequence complementary to the target nucleic acid sequence of PCSK9, can bind to the target nucleic acid of PCSK9, repressing and silencing the transcription of the PCSK9 gene. Examples of gene repression processes that reduce transcription include, but are not limited to, inhibiting the formation of the transcription initiation complex, reducing the transcription initiation rate, reducing the transcription elongation rate, reducing the transcriptional processing capacity, and antagonizing transcriptional activation (e.g., by blocking the binding of transcription activators). Gene repression may constitute, for example, prevention of activation and inhibition of expression below existing levels. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription, the latter of which may result from epigenetic modifications of the target nucleic acid.

[0120] Among repressor domains capable of repressing or silencing genes, the Kruppel-associated box (KRAB) repressor domain is one of the most potent in the human genome system (Alerasool, N., et al. An efficient KRAB domain for CRISPRi applications. Nat. Methods 17:1093 (2020)). Domains like KRAB are present in approximately 400 transcription factors of the human zinc finger protein system and, upon binding of dCasX linked to a target nucleic acid, can recruit additional repressor domains, such as Trim28 (also known as Kap1 or Tif1-beta), which then assemble a protein complex with chromatin regulators such as CBX5 / HP1α and SETDB1, which induce repression of gene transcription in a limited and transient manner. Representative non-exclusive examples of KRAB domains suitable for use in the systems of this disclosure include ZIM3 (SEQ ID NO: 129) and ZNF10 (SEQ ID NO: 128). This disclosure provides additional repressor domains of human origin, as well as repressor domains of non-human origin having a distinctly different sequence (referred to herein as RD1) that, when incorporated into long-term repressor fusion protein construct embodiments as described herein, have been found to result in enhanced transcriptional repression compared to ZIM3 and ZNF10.

[0121] In some embodiments, the disclosure provides a system in which the modification provided by the use of the LTRP:gRNA system is epigenetic, and therefore the silencing of the PCSK9 gene is heritable by a mechanism other than edited DNA replication. As used herein, “epigenetic modification” means a modification of either DNA or DNA-associated histones other than a change in the DNA sequence itself (e.g., substitution, deletion, or rearrangement), where the modification is either a direct modification by a component of the system or an indirect modification by the recruitment of one or more additional cellular components, but the DNA target nucleic acid sequence itself is not edited to change its sequence. For example, while DNA methyltransferase 3A (DNMT3A) (or its catalytic domain) directly modifies DNA by methylating it, KRAB acts as a potent transcriptional repressor, recruiting the KAP-1 / TIF1β corepressor complex, which can further recruit factors involved in DNA methylation and repressive chromatin formation, such as heterochromatin protein 1 (HP1), histone deacetylase, and histone methyltransferase (Ying, Y., et al. The Kruppel-associated box repressor domain induces reversible and irreversible regulation of endogenous mouse genes by mediating different chromatin states. Nucleic Acids Res. 43(3):1549 (2015)). Furthermore, the catalytically inactive DNMT3L cofactor, along with the cellular endogenous DNMT1, helps establish hereditary methylation patterns after DNA replication.The ATRX-DNMT3-DNMT3L domain (ADD) of DNMT3A is known to have two main functions: 1) allosterically regulates the catalytic activity of DNMT3A by functioning as a methyltransferase autoinhibitory domain, and 2) specifically interacts with histone H3 tails not methylated at lysine (K)4, resulting in preferential methylation of DNA bound to chromatin H3 tails not methylated at K4 (Zhang, Y., et al. Chromatin methylation activity of Dnmt3a and Dnmt3a / 3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Research 38:4246 (2010)). In some embodiments, inclusion of the ADD domain enhances the transcriptional repression of target genes when incorporated into an LTRP compared to other equivalent LTRPs lacking the ADD domain. In other embodiments, inclusion of the ADD domain enhances the specificity of transcriptional repression of target genes compared to other equivalent LTRPs lacking the ADD domain. The aforementioned supporting data is provided in the examples and in WO2023049742A2, which is incorporated herein by reference.

[0122] In some embodiments, the long-term repressor fusion protein comprises DNA-binding proteins linked to first, second, and third repressor domains, each of which is distinct, and the fusion protein can bind to the PCKS9 target nucleic acid. In some embodiments, the long-term repressor fusion protein comprises DNA-binding proteins linked to first, second, third, and fourth repressor domains, each of which is distinct. In any of the embodiments described above, the fusion protein can form an RNP complex with the gRNA of the system that binds to the target nucleic acid, if the DNA-binding domain contains a CRISPR protein without catalytic activity.

[0123] In some embodiments, the DNA-binding protein includes a TALE that can bind to the target nucleic acid but cannot cleave it. In some embodiments, the DNA-binding protein includes a zinc finger protein that can bind to the target nucleic acid but cannot cleave it. In some embodiments, the DNA-binding protein includes a non-catalyzed CRISPR protein that can complex with gRNA to form an RNP that can bind to the target nucleic acid but cannot cleave it. In some embodiments, the long-term repressor fusion protein includes a non-catalyzed CRISPR protein sequence, a first repressor domain (hereinafter referred to herein as "RD1"), a DNMT3A catalytic domain derived from the DNMT3A protein as a second domain (hereinafter referred to herein as "DNMT3A"), and a DNMT3L interaction domain derived from the DNMT3L protein as a third domain (hereinafter referred to herein as "DNMT3L"). In some embodiments, the long-term repressor fusion protein comprises a CRISPR protein sequence without catalytic activity, RD1, DNMT3A as a second domain, DNMT3L as a third domain, and an ATRX-DNMT3-DNMT3L domain (hereinafter referred to as ADD) derived 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 and one or more linker peptides described herein. In some embodiments, the long-term repressor fusion protein can form an RNP with a gRNA that binds to a target nucleic acid. The use of the aforementioned domains, when composed of selective orientation to dCasX in the long-term repressor fusion protein, has been found to result in significant epigenetic modification of the PCSK9 target nucleic acid when complexed with a gRNA having a targeting sequence complementary to a defined region of the PCSK9 gene, and the combination of repressor domains functions synchronously, resulting in additional or synergistic effects on the transcriptional silencing of the target gene depending on the configuration.

[0124] Representative amino acid sequences of components used in long-term repressor fusion protein constructs are provided herein.

[0125] In some embodiments, the dCasX of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs. 4 to 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 thereto. In some embodiments, the dCasX of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs. 4 to 29. In some embodiments, the dCasX of the long-term repressor fusion protein includes 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 thereto. In some embodiments, the long-term repressor fusion protein dCasX contains the sequence of SEQ ID NO: 4.

[0126] In some embodiments, RD1 of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs. 128 to 1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, RD1 of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs. 128 to 1726. In some embodiments, RD1 of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs. 128 or 129, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the RD1 of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs: 128 or 129. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs: 130 to 1726, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs: 130 to 1726. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs: 130-224, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identity thereto.In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs: 130-224. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs: 130-138, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs: 130-138. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes 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 thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes 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 thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein contains the sequence of SEQ ID NO: 135.In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes 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 thereto. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes the sequence of SEQ ID NO: 130. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes the sequence of SEQ ID NO: 131. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes the sequence of SEQ ID NO: 132. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes the sequence of SEQ ID NO: 133. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes the sequence of SEQ ID NO: 134. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes the sequence of SEQ ID NO: 135. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes the sequence of SEQ ID NO: 136. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes the sequence of SEQ ID NO: 137. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein includes the sequence of SEQ ID NO: 138.

[0127] In some embodiments, the second repressor domain of the long-term repressor fusion protein is DNMT3A containing 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 thereto. In some embodiments, the second repressor domain of the long-term repressor fusion protein contains the sequence of SEQ ID NO: 126.

[0128] 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 thereto.

[0129] In some embodiments, an optional fourth repressor domain of the long-term repressor fusion protein is ADD, which contains 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 thereto. In some embodiments, the optional fourth repressor domain of the long-term repressor fusion protein contains the sequence of SEQ ID NO: 125. In some embodiments, the C-terminus of ADD is ligated to the N-terminus of DNMT3A. In a surprising finding, the addition of ADD to a long-term repressor fusion protein containing RD1, DNMT3A, and DNMT3L was found to significantly enhance or increase 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 ADD. Exemplary data on the improved repression and specificity of long-term repressor fusion proteins containing ADD are presented in the examples, which are described in WO2023049742A2, incorporated herein by reference. In some embodiments, the fusion protein includes one or more linker peptides selected from the group consisting of SEQ ID NOs: 98-124, 3278-3289 (exemplary sequences are shown in Tables 5 and 58), and one or more NLSs containing sequences selected from the group consisting of SEQ ID NOs: 30-97, 32998, and 3299. An exemplary configuration of a long-term repressor fusion protein containing ADD is shown in Figure 61.

[0130] 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 without catalytic activity. In some embodiments, the long-term repressor fusion protein comprises an 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.

[0131] In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein, RD1, DNMT3A, and DNMT3L from the N-terminus to the C-terminus. In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein, RD1, ADD, DNMT3A, and DNMT3L 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 without catalytic activity. In some embodiments, the long-term repressor fusion protein comprises an NLS at the N-terminus. In some embodiments, the long-term repressor fusion protein comprises an NLS between RD1 and DNMT3A. In some embodiments, the long-term repressor fusion protein comprises an NLS at the 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.

[0132] In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein, DNMT3A, DNMT3L, and RD1 from the N-terminus to the C-terminus. In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein, ADD, DNMT3A, DNMT3L, 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 without catalytic activity. In some embodiments, the long-term repressor fusion protein comprises an NLS at the 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.

[0133] 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 without catalytic activity. In some embodiments, the long-term repressor fusion protein comprises an 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.

[0134] 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 without catalytic activity. In some embodiments, the long-term repressor fusion protein comprises an 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.

[0135] 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 embodiment described above, the second RD1 may have the same sequence as the first RD1. In another embodiment described above, the second RD1 may have a different sequence from the first RD1. In some embodiments, the DNA-binding protein may be a zinc finger, TALE, or a CRISPR protein without catalytic activity. 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 includes 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.

[0136] In some cases, the long-term repressor fusion protein also includes one or more NLSs. In some embodiments, the long-term repressor fusion protein includes the following configurations from N-terminus to C-terminus: 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 without catalytic activity. In some embodiments, the long-term repressor fusion protein can bind to the PCSK9 gene and repress or silence the transcription of the PCSK9 gene.

[0137] In some embodiments of the long-term repressor fusion protein, one or more linker peptides may be inserted between any two adjacent domains of the long-term repressor fusion protein. In some embodiments, the long-term repressor fusion protein comprises the configuration NLS-ADD-DNMT3A-linker2-DNMT3L-linker1-linker3A-DNA-binding protein-linker3B-RD1-NLS (configuration 1) from N-terminus to C-terminus. In some embodiments, the long-term repressor fusion protein comprises the configuration NLS-linker3A-DNA-binding protein-linker3B-RD1-NLS-linker1-ADD-DNMT3A-linker2-DNMT3L (configuration 2) from N-terminus to C-terminus. In some embodiments, the long-term repressor fusion protein comprises the configuration NLS-linker3A-DNA-binding protein-linker1-ADD-DNMT3A-linker2DNMT3L-linker3B-RD1-NLS (configuration 3) from N-terminus to C-terminus. In some embodiments, the long-term repressor fusion protein includes the configuration NLS-RD1-linker3A-ADD-DNMT3A-linker2-DNMT3L-linker1-DNA-binding protein-linker3B-NLS (configuration 4) from the N-terminus to the C-terminus. In some embodiments, the long-term repressor fusion protein includes the configuration NLS-ADD-DNMT3A-linker2-DNMT3L-linker3A-RD1-linker1-DNA-binding protein-linker3B-NLS (configuration 5) from the N-terminus to the C-terminus. In some embodiments, the DNA-binding protein in the above configurations may be a zinc finger, TALE, or a CRISPR protein without catalytic activity. A schematic diagram of the configurations shown in Figure 61. In some embodiments of the LTRP of configurations 1 to 5, the NLS may include sequences selected from the group consisting of sequence numbers 30 to 97, 3298, and 3299 (Tables 3 and 4), and the linker sequence may include sequences independently selected from the group consisting of sequence numbers 98 to 124 and 3278 to 3289 (typical linkers shown in Tables 5 and 58). In some embodiments, the NLS may include the sequence of sequence number 30, and the linker sequence may include sequences independently selected from the group consisting of sequence numbers 120 and 122 to 124.In other embodiments, the DNA-binding protein may be a dCasX sequence selected from the group consisting of SEQ ID NOs. 4 to 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 thereto. In some embodiments, the second repressor domain is a DNMT3A domain containing the sequence of SEQ ID NO. 126, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the third repressor is a DNMT3L domain containing the sequence of sequence number 127, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the fourth repressor is an ADD domain containing the sequence of sequence number 125, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the long-term repressor fusion protein can bind to the PCSK9 gene target nucleic acid and repress or silence the PCSK9 gene target nucleic acid.

[0138] In some embodiments, the disclosure provides a system comprising a long-term repressor fusion protein comprising two RD1 domains, a second repressor domain, a third repressor domain, and a fourth repressor domain operably linked to a DNA-binding protein, such as a zinc finger, TALE, or a non-catalyzed CRISPR protein. In other embodiments, the DNA-binding protein comprises a non-catalyzed CasX comprising a sequence selected from the group consisting of SEQ ID NOs: 4-29, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the sequences of the two RD1s are identical. In other embodiments, the two RD1s comprise different sequences. In some embodiments, two RD1s are located at the N-terminus of the DNA-binding protein. In some embodiments, 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 one RD1 is located at the C-terminus of the DNA-binding protein.

[0139] In some embodiments, the long-term repressor fusion protein contains two RD1s. In some embodiments, the long-term repressor fusion protein contains the following configuration from N-terminus to C-terminus: NLS-ADD-DNMT3A-linker2-DNMT3L-linker3A-a RD1a-linker1-DNA binding protein-linker3B-RD1a-linker4-NLS (composition 6a), where the RD1a sequences are identical (see Figure 61 for a schematic diagram of the long-term repressor fusion protein). In some embodiments, the long-term repressor fusion protein contains the following configuration from N-terminus to C-terminus: NLS-ADD-DNMT3A-linker2-DNMT3L-linker3A-a RD1a-linker1-DNA binding protein-linker3B-RD1b-linker4-NLS (composition 6b), where the RD1a and RD1b sequences are different (see Figure 61 for a schematic diagram of the long-term repressor fusion protein). In some embodiments, the DNA-binding protein may be a zinc finger, TALE, or a CRISPR protein without catalytic activity. In some embodiments, the long-term repressor protein embodiments described in the paragraph can bind to the PCSK9 gene and suppress or silence the expression of the PCSK9 gene.

[0140] In some embodiments, the Disclosure provides a long-term repressor fusion protein of configuration 6a, in which case the two RD1 sequences are identical. In some embodiments of the long-term repressor fusion protein of configuration 6a, the DNA-binding protein comprises a dCasX of a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto, and the first and second RD1a A copy of contains a sequence selected from the group consisting of SEQ ID NOs. 130 to 1726, or a sequence having at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, or at least approximately 95% identity therewith, and the second repressor domain contains the sequence of SEQ ID NO. 126, or at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 9 DNMT3A containing a sequence having 2%, 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, wherein the third repressor is the sequence of sequence number 127, or 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%, or at least about 9 DNMT3L containing a sequence variant having 7%, at least about 98%, or at least about 99% identity, and the fourth repressor is an ADD containing the sequence of SEQ ID NO: 125, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.The NLS contains a sequence independently selected from the group consisting of SEQ ID NOs. 30-97 (Tables 3 and 4), the L1 linker contains the sequence of SEQ ID NO. 123, the L2 linker contains the sequence of SEQ ID NO. 122, the L3A linker contains the sequence of SEQ ID NO. 124, the L3B linker contains the sequence of SEQ ID NO. 120, and the L4 linker contains 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 linker sequences are independently selected from the group consisting of SEQ ID NOs. 98-124 and 3278-3289 (exemplary sequences shown in Tables 5 and 58). In some embodiments of the long-term repressor fusion protein of configuration 6a, two copies of RD1 are identical, and each RD1 contains 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%, and at least about 95% identity thereto. A schematic diagram of configuration 6a is shown in 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 a gene target nucleic acid and repress or silence the gene target nucleic acid.

[0141] In some embodiments of the long-term repressor fusion protein of configuration 6b, the two RD1 sequences are different, and the DNA-binding protein includes 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 thereto. The first copy of the first repressor domain (RD1a) contains a sequence selected from the group consisting of SEQ ID NOs. 130-1726, or a sequence having at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, or at least approximately 95% identity thereto, and the second copy of the first repressor domain (RD1b) contains a different sequence selected from the group consisting of SEQ ID NOs. 130-1726, or a sequence having at least approximately 70%, at least approximately 80%, The second repressor domain contains a sequence that is identical to at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, or at least approximately 95%, and the second repressor domain contains a sequence that is identical to sequence number 126, or at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 9 DNMT3A containing a sequence with 9% identity, and DNMT3L containing a sequence variant having at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity thereto, and a fourth repressor containing the sequence of sequence number 125, or at least approximately 70% thereto,The ADD contains sequences having at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity, wherein the NLS contains a sequence independently selected from the group consisting of SEQ ID NOs. 30-97 in Tables 3 and 4, the L1 linker contains the sequence of SEQ ID NO. 123, the L2 linker contains the sequence of SEQ ID NO. 122, the L3A linker contains the sequence of SEQ ID NO. 124, the L3B linker contains the sequence of SEQ ID NO. 120, and the L4 linker contains 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 linker sequences are selected from the group consisting of SEQ ID NOs. 98-124 and 3278-3289 (exemplary sequences shown in Tables 5 and 58). In some embodiments of the long-term repressor fusion protein of configuration 6b, the two copies of RD1 are different, with RD1a comprising 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 thereto, and RD1b comprising 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 thereto. In some embodiments of the long-term repressor fusion protein of configuration 6b, the two copies of RD1 are different, with RD1a comprising 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 thereto, and RD1b comprising 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 it contains a sequence having at least about 95% identity. In some embodiments of the long-term repressor fusion protein of configuration 6b, the two copies of RD1 are different, with RD1a containing 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 thereto, and RD1b containing 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 thereto. In some embodiments of the long-term repressor fusion protein of configuration 6b, the two copies of RD1 are different, with RD1a comprising 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 thereto, and RD1b comprising 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 thereto. In some embodiments of the long-term repressor fusion protein of configuration 6b, the two copies of RD1 are different, with RD1a containing 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 thereto, and RD1b containing 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 thereto. In some embodiments of the long-term repressor fusion protein of configuration 6b,The two copies of RD1 are different, with RD1a containing 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 thereto, and RD1b containing 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 thereto. In some embodiments of the long-term repressor fusion protein of configuration 6b, the two copies of RD1 are different, and RD1a and RD1b are independently selected from the group consisting of sequences SEQ ID NOs: 132-134 and 136-138. In some embodiments of the long-term repressor fusion protein of configuration 6b, two copies of RD1 are different, and RD1a and Rd1b are independently selected from the group consisting of sequences 130, 131, and 135. A schematic diagram of configuration 6b is shown in 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 a target nucleic acid and repress or silence the target nucleic acid. Table 5: Exemplary linker amino acid sequences of long-term repressor fusion proteins [Table 19]

[0142] In some embodiments of the long-term repressor fusion protein comprising dCasX and optionally ADD as component 1, the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs. 22836 to 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 thereto. In some embodiments of the long-term repressor fusion protein comprising dCasX and optionally ADD as component 1, the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs. 22836 to 22855. In some embodiments of the long-term repressor fusion protein comprising dCasX and optionally ADD as component 1, the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs. 22838 and 22847. In some embodiments of the long-term repressor fusion protein comprising dCasX and optionally ADD, and configured as component 1, the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs: 22839 and 22848. In some embodiments of the long-term repressor fusion protein comprising dCasX and optionally ADD, and configured as component 1, the long-term repressor fusion protein includes a sequence selected from the group consisting of SEQ ID NOs: 22840 and 22849.

[0143] In some embodiments of the DNA-binding protein system, the system is configured as a configuration selected from the group consisting of configuration 1, configuration 2, configuration 3, configuration 4, configuration 5, configuration 6a, and configuration 6b, as described above, and the long-term repressor fusion protein comprises dCasX and can complex with a targeting sequence and gRNA complementary to the PCSK9 target nucleic acid in the cell to form an RNP, and upon binding of the RNP to the PCSK9 target nucleic acid in the cell, the target nucleic acid is epigenetically modified and the transcription of the PCSK9 gene is repressed. In some embodiments, the 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, PCSK9 gene transcription 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%, and at least about 80% or more of cells in a cell population. In some embodiments, transcriptional repression is assayed in in vitro assays, including cell-based assays, compared to untreated cells or cells treated with an equivalent system including a non-targeting spacer. In some embodiments, transcriptional repression is assayed in vivo in cells obtained from subjects who have been administered a long-term repressor fusion protein and a gRNA having a targeting sequence complementary to the PCSK9 target nucleic acid of a gene in the cell, either as a protein and / or gRNA, or as nucleic acid (e.g., gRNA and mRNA encoding the long-term repressor fusion protein). Subjects are selected from a group consisting of mice, rats, pigs, non-human primates, and humans.

[0144] Most preferably, PCSK9 gene repression results in complete inhibition of gene expression such that the gene product is undetectable. However, those skilled in the art will understand that incomplete inhibition may still be useful and desirable for various applications. In some embodiments, when assayed in an in vitro assay including a cell-based assay, the repression of PCSK9 gene transcription 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 the present disclosure is administered as a therapeutically effective dose, the repression of PCSK9 gene transcription by the system 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 interest. In some embodiments, the subjects are selected from the group consisting of mice, rats, pigs, non-human primates, and humans. In some embodiments, the use of LTRPs of configurations 1, 4, 5, and 6 results in off-target methylation or off-target activity in cells that is less than approximately 10%, less than approximately 9%, less than approximately 8%, less than approximately 7%, less than approximately 6%, less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 2%, less than approximately 1%, less than approximately 0.5%, or less than approximately 0.1% when used in an LTRP:gRNA system.

[0145] In some embodiments, the repression of PCSK9 gene transcription 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.

[0146] V. mRNA compositions encoding long-term repressor fusion proteins In another aspect, the disclosure relates to messenger RNA (mRNA) compositions comprising the sequences of individual components, as well as the full-length mRNA sequences of the long-term repressor domain fusion protein constructs of the disclosure. In some embodiments, the mRNA compositions are useful for transcriptional repression and epigenetic modification of a gene, such as the PCSK9 gene, when used to express a long-term repressor fusion protein. In some cases, the mRNA is designed for use in a particular delivery formulation, such as synthetic nanoparticles or lipid nanoparticles (LNPs). The disclosure also provides methods for designing mRNA sequences for use in compositions. In some cases, the mRNA encoding the long-term repressor fusion protein can be co-formulated with a gRNA containing a targeting sequence complementary to the PCSK9 gene sequence and a nanoparticle (such as an LNP). Upon delivery to target cells by the LNP, the long-term repressor domain fusion protein can be expressed from the mRNA and complexed with the gRNA as an RNP that can bind to the PCSK9 target nucleic acid. In other cases, the mRNA encoding the long-term repressor domain fusion protein and gRNA may be formulated into separate nanoparticles and delivered separately or as a mixture.

[0147] In some embodiments, the mRNA compositions of this disclosure are modified to yield one or more improved features compared to unmodified mRNA encoding the same long-term repressor protein, and thus may have a positive impact on the efficacy of mRNA-based delivery. Examples of improved features of mRNA described herein include, but are not limited to, improved expression upon delivery to cells, reduced immunogenicity, increased stability, and enhanced manufacturability compared to unmodified mRNA. In some cases, the modification of mRNA yields improved features that are at least about 1.1 to about 100,000 times better compared to unmodified mRNA. In some embodiments, the improved features of modified mRNA are improved by at least approximately 1.1 to approximately 10,000 times compared to unmodified mRNA, at least approximately 1.1 to approximately 1,000 times, at least approximately 1.1 to approximately 500 times, at least approximately 1.1 to approximately 400 times, at least approximately 1.1 to approximately 300 times, at least approximately 1.1 to approximately 200 times, at least approximately 1.1 to approximately 100 times, at least approximately 1.1 to approximately 50 times, at least approximately 1.1 to approximately 40 times, at least approximately 1.1 to approximately 30 times, at least approximately 1.1 to approximately 20 times, at least approximately 1.1 to approximately 10 times, at least approximately 1.1 to approximately 9 times, at least approximately The improvement is 1.1 to approximately 8 times, at least 1.1 to approximately 7 times, at least 1.1 to approximately 6 times, at least 1.1 to approximately 5 times, at least 1.1 to approximately 4 times, at least 1.1 to approximately 3 times, at least 1.1 to approximately 2 times, at least 1.1 to approximately 1.5 times, at least 1.5 to approximately 3 times, at least 1.5 to approximately 4 times, at least 1.5 to approximately 5 times, at least 1.5 to approximately 10 times, at least 5 to approximately 10 times, at least 10 to approximately 20 times, at least 10 to approximately 30 times, at least 10 to approximately 50 times, or at least 10 to approximately 100 times. In some embodiments, the improved features of modified mRNA are improved at least 10 to approximately 1000 times compared to unmodified mRNA.

[0148] Optimization of coding sequences and untranslated regions (UTRs) can be particularly significant when delivering mRNA encoding a target protein, as opposed to DNA templates transcribed into mRNA. DNA templates are long-lived, can replicate, and can produce many RNA transcripts throughout their lifetime. For DNA templates, the efficiency of transcription and mRNA precursor processing is a major determinant of protein expression levels. In contrast, mRNA generally has a much shorter half-life, measured in hours, as it is vulnerable to degradation within the cytoplasm and cannot produce many copies of itself. Therefore, mRNA stability and translation efficiency are critical determinants of protein expression levels for mRNA-based delivery, and thus, the effectiveness of mRNA-based delivery can be improved by enhancing specific sequences of the UTR and coding sequence that determine mRNA stability and translation efficiency.

[0149] a.5' cap In some embodiments of the mRNA described herein, the mRNA includes a 5' cap ligated at the 5' end to the 5' UTR of any of the mRNA sequences described herein. In some embodiments, the 5' cap is a 7-methylguanylate cap. In some embodiments, the 5' cap includes m7G(5')ppp(5')mAG. In other embodiments, the 5' cap includes 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 WO2017 / 053297, the contents of which are incorporated herein by reference.

[0150] b.5' Untranslated Region (UTR) The 5'UTR of an mRNA molecule can be a crucial determinant of both mRNA stability and how efficiently it is translated into protein. Specifically, the 5'UTR, in conjunction with the 5' cap structure, functions as a binding site and recruitment platform for pre-translational complexes and additional regulatory proteins that may positively or negatively influence translation. Structures within the 5'UTR can enhance translation by recruiting initiation factors or other protein or RNA factors, reduce translation by physically blocking ribosome binding and scanning, and contribute to mRNA stability by influencing both hydrolysis and nuclease digestion.

[0151] Table 6 provides exemplary 5'UTR sequences for use with the mRNAs of this disclosure. Table 6 lists the RNA sequences and the RNA sequences in which uridine has been replaced with N1-methyl-pseudridine. Table 6: 5'UTR sequences [Table 20] * "mψ" = N1-methyl-pseudolidine

[0152] In some embodiments, the 5'UTR includes the sequence of sequence number 22787, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 5'UTR includes the sequence of sequence number 22787. In some embodiments, the 5'UTR consists of the sequence of sequence number 22787. In some embodiments, the 5'UTR includes the sequence of sequence number 3300, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, the 5'UTR includes the sequence of sequence number 3300. In some embodiments, the 5'UTR consists of the sequence of sequence number 3300.

[0153] c.3'UTR 3'UTR sequences can influence mRNA stability and translation efficiency, and therefore may determine both intracellular localization and tissue-specific expression. Factors influencing these properties include microRNA binding sites, AU-rich elements that recruit various RNA-binding proteins, pumilio binding elements, and other binding sites for RNA-binding proteins. Many of these interactions with 3'UTRs are known to negatively affect stability or expression, while others can enhance translation. The effects of 3'UTR sequences can be more cell-type specific due to differential expression changes of microRNAs and RNA-binding proteins, providing an opportunity to manipulate tissue-specific expression of therapeutic mRNA. In some embodiments, the 3'UTR for use with the mRNAs of this disclosure is the mouse 3'UTR. In some embodiments, the 3'UTR is the mouse HBA gene 3'UTR shown in Table 7.

[0154] Exemplary 3'UTR sequences of this disclosure are provided in Table 7. Table 7 lists the RNA sequences and the RNA sequences in which uridine has been replaced with N1-methyl-pseudridine. Table 7: 3'UTR sequences [Table 21] * "mψ" = N1-methyl-pseudolidine

[0155] In some embodiments, 3'UTR includes the sequence of sequence number 3125, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, 3'UTR includes the sequence of sequence number 3125. In some embodiments, 3'UTR consists of the sequence of sequence number 3125. In some embodiments, 3'UTR includes the sequence of sequence number 3310, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity thereto. In some embodiments, 3'UTR includes the sequence of sequence number 3310. In some embodiments, 3'UTR consists of the sequence of sequence number 3310.

[0156] d. Poly(A) array Including a 3' poly(A) tail in mRNA sequences can contribute to mRNA stability and translation efficiency. Generally, longer poly(A) tails are associated with increased mRNA stability, thereby enabling their translation and promoting high protein expression.

[0157] In some embodiments, the mRNA of the Disclosure comprises a poly(A) sequence having at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides. In some embodiments, the poly(A) sequence of the mRNA of the Disclosure comprises 80 adenine nucleotides. In some embodiments, the poly(A) sequence comprises the nucleic acid sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (Sequence ID 3057). In some embodiments, the poly(O) sequence includes the nucleic acid sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (Sequence ID 3307).

[0158] Sequence modification of e mRNA In some embodiments, the mRNA sequences of this disclosure have been modified by codon optimization of the sequences encoding long-term repressor proteins using one or more parameters to enhance expression in target cells. Non-limiting examples of such parameters include codon usage frequency in human host cells (e.g., use of codon adaptation index (CAI)), minimization of rare codons, use of codon usage frequency tables derived from biologics intended for therapeutic use, use of mRNA stability indicators, or reduction of GC content. Methods for codon optimization and codon usage frequencies in various organisms are known in the art. See, for example, www.genscript.com / tools / codon-frequency-table.

[0159] In some embodiments, mRNA sequences of long-term repressor protein constructs codon-optimized for expression in human cells are provided herein. Various natural or modified nucleosides can be used to produce the modified mRNA according to this disclosure. In some embodiments, the mRNA may be a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); or a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-pripynyl-uridine, C5-pripynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deaza Azaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methylpseudridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite bonds), or comprising them. In some embodiments, mRNA comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues may include, for example, 5-methylcytidine ("5mC"), pseudouridine ("ψU"), and / or 2-thiouridine ("2sU"). In certain embodiments, one or more or all of the uridine residues in the mRNA of this disclosure are substituted with 1-methyl-pseudridine. In some embodiments, all of the uridine residues in the mRNA of this disclosure are substituted with 1-methyl-pseudridine. For example, see 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 with N1-methylpseudridine.

[0160] In some embodiments, modification of mRNA by codon optimization and / or substitution of modified nucleotides in the sequence results in property improvements of at least about 1.1 to about 100,000 times compared to unmodified mRNA. In some embodiments, the improved features of modified mRNA are improved by at least approximately 1.1 to approximately 10,000 times compared to unmodified mRNA, at least approximately 1.1 to approximately 1,000 times, at least approximately 1.1 to approximately 500 times, at least approximately 1.1 to approximately 400 times, at least approximately 1.1 to approximately 300 times, at least approximately 1.1 to approximately 200 times, at least approximately 1.1 to approximately 100 times, at least approximately 1.1 to approximately 50 times, at least approximately 1.1 to approximately 40 times, at least approximately 1.1 to approximately 30 times, at least approximately 1.1 to approximately 20 times, at least approximately 1.1 to approximately 10 times, at least approximately 1.1 to approximately 9 times, at least approximately The improvement is 1.1 to approximately 8 times, at least 1.1 to approximately 7 times, at least 1.1 to approximately 6 times, at least 1.1 to approximately 5 times, at least 1.1 to approximately 4 times, at least 1.1 to approximately 3 times, at least 1.1 to approximately 2 times, at least 1.1 to approximately 1.5 times, at least 1.5 to approximately 3 times, at least 1.5 to approximately 4 times, at least 1.5 to approximately 5 times, at least 1.5 to approximately 10 times, at least 5 to approximately 10 times, at least 10 to approximately 20 times, at least 10 to approximately 30 times, at least 10 to approximately 50 times, or at least 10 to approximately 100 times. In some embodiments, the improved features of modified mRNA are improved at least 10 to approximately 1000 times compared to unmodified mRNA.

[0161] f.LTRP mRNA component sequence This disclosure provides mRNAs containing sequences encoding components used in the long-term repressor fusion proteins of this disclosure. In some embodiments, the mRNAs contain sequences encoding DNA-binding proteins, including TALE, ZF, and CRISPR proteins without catalytic activity. In some embodiments, the mRNAs contain sequences of sequence number 3274 encoding dCasX 515 (sequence number 6), or sequences having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNAs contain sequences of sequence number 3276 encoding dCasX 812 (sequence number 29), or sequences having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA includes the sequence of sequence number 3275 encoding dCasX 491 (sequence number 4), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the mRNA includes the sequence of sequence number 22736 encoding dCasX 676 (sequence number 28), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0162] In some embodiments, the mRNA sequence encoding dCasX 491 (SEQ ID NO: 4) has one or more, or all, uridines substituted with pseudouridine nucleosides (SEQ ID NO: 22729). In some embodiments, the mRNA sequence encoding dCasX 515 (SEQ ID NO: 6) has one or more, or all, uridines substituted with pseudouridine nucleosides (SEQ ID NO: 22728). In some embodiments, the mRNA sequence encoding dCasX 676 (SEQ ID NO: 28) has one or more, or all, uridines substituted with pseudouridine nucleosides. In some embodiments, the mRNA sequence encoding dCasX 812 (SEQ ID NO: 29) has one or more, or all, uridines substituted with pseudouridine nucleosides (SEQ ID NO: 22730 or 22731).

[0163] In some embodiments, the disclosure provides mRNA sequences encoding the RD1 domain. In some embodiments, the mRNA sequence encoding RD1 includes a sequence selected from the group consisting of SEQ ID NOs: 3120, 22804, and 3334-6527, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity thereto. In some embodiments, the mRNA sequence encoding RD1 includes a sequence selected from the group consisting of SEQ ID NOs: 3120, 22804, and 3334-6527. In some embodiments, the mRNA sequence encoding RD1 includes a sequence selected from the group consisting of SEQ ID NOs. 3334-3342 and 4931-4939, or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA sequence encoding RD1 includes a sequence selected from the group consisting of SEQ ID NOs. 3334-3342 and 4931-4939. In another embodiment, the mRNA sequence encoding RD1 includes 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 thereto. In another embodiment, the mRNA sequence encoding RD1 includes the sequence of SEQ ID NO: 3334 or SEQ ID NO: 4931.In another embodiment, the mRNA sequence encoding RD1 includes 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 thereto. In another embodiment, the mRNA sequence encoding RD1 includes the sequence of SEQ ID NO: 3335 or SEQ ID NO: 4932. In another embodiment, the mRNA sequence encoding RD1 includes 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 thereto. In another embodiment, the mRNA sequence encoding RD1 includes the sequence of SEQ ID NO: 3336 or SEQ ID NO: 4933. In another embodiment, the mRNA sequence encoding RD1 includes 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 thereto. In another embodiment, the mRNA sequence encoding RD1 includes the sequence of SEQ ID NO: 3337 or SEQ ID NO: 4934. In another embodiment, the mRNA sequence encoding RD1 includes 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 thereto. In another embodiment, the mRNA sequence encoding RD1 includes the sequence of SEQ ID NO: 3338 or SEQ ID NO: 4935.In another embodiment, the mRNA sequence encoding RD1 includes 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 thereto. In another embodiment, the mRNA sequence encoding RD1 includes the sequence of SEQ ID NO: 3339 or SEQ ID NO: 4936. In another embodiment, the mRNA sequence encoding RD1 includes 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 thereto. In another embodiment, the mRNA sequence encoding RD1 includes the sequence of SEQ ID NO: 3340 or SEQ ID NO: 4937. In another embodiment, the mRNA sequence encoding RD1 includes 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 thereto. In another embodiment, the mRNA sequence encoding RD1 includes the sequence of SEQ ID NO: 3341 or SEQ ID NO: 4938.

[0164] In some embodiments, the mRNA includes a sequence encoding a second repressor domain. In some embodiments, the second repressor domain includes DNMT3A. In some embodiments, the mRNA sequence encoding DNMT3A includes 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 thereto. In some embodiments, the mRNA sequence encoding DNMT3A includes the sequence of SEQ ID NO: 3128 or SEQ ID NO: 3331.

[0165] In some embodiments, the mRNA includes a sequence encoding a third repressor domain. In some embodiments, the third repressor domain includes DNMT3L. In some embodiments, the mRNA sequence encoding DNMT3L includes the sequence of 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 thereto. In some embodiments, the mRNA sequence encoding DNMT3L includes the sequence of SEQ ID NO: 3119 or SEQ ID NO: 3332.

[0166] In some embodiments, the mRNA includes a sequence encoding a fourth repressor domain. In some embodiments, the fourth repressor domain includes ADD. In some embodiments, the mRNA sequence encoding ADD includes 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 thereto. In some embodiments, the mRNA sequence encoding ADD includes the sequence of SEQ ID NO: 3296 or SEQ ID NO: 22726.

[0167] In some embodiments, the mRNA of the Disclosure comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises GCCACCAUGG (SEQ ID NO: 22805). In some embodiments, the mRNA of the Disclosure comprises the 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.

[0168] In another embodiment, the mRNA includes an NLS. In some embodiments, the sequence encoding the NLS includes a sequence selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807, or a sequence having at least about 70%, at least about 80%, or at least about 90% identity thereto. In another embodiment, the mRNA includes a sequence selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807. In another embodiment, for example, in an embodiment in which the long-term repressor protein includes two or more NLSs, the mRNA includes two or more sequences independently selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807.

[0169] In some embodiments, the mRNA includes a sequence encoding a linker for a long-term repressor fusion protein. In some embodiments, the linker encoding sequence includes 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 thereto. In some embodiments, the linker encoding sequence includes a sequence selected from the group consisting of SEQ ID NOs. 3312-3327 and 22808-22828. In some embodiments, for example, in embodiments in which the long-term repressor protein includes two or more linkers, the linker encoding sequence is independently selected from the group consisting of SEQ ID NOs. 3312-3327 and 22808-22828, or is 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%, and at least about 95% identity with them.

[0170] In some embodiments, the mRNA includes a sequence encoding a long-term repressor fusion protein of the configuration provided herein. In some embodiments, the mRNA encodes a long-term repressor fusion protein of configuration 1, and the mRNA includes 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%, and at least about 99% identity thereto. In some embodiments, the mRNA includes a sequence selected from the group consisting of SEQ ID NOs. 6529-8134, 9741-11347, 14628-16233, and 17841-19446.

[0171] In some embodiments, the mRNA includes a sequence encoding the long-term repressor fusion protein of composition 1, and a sequence encoding RD1, which includes the sequence of SEQ ID NO: 130. In some embodiments, the mRNA includes the 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 thereto. In some embodiments, the mRNA includes the sequence of SEQ ID NO: 6531, 9744, 14630, or 17843. In some embodiments, the mRNA includes a sequence encoding the long-term repressor fusion protein of composition 1, and a sequence encoding RD1, which includes the sequence of SEQ ID NO: 131. In some embodiments, the mRNA includes 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 thereto. In some embodiments, the mRNA includes the sequence of SEQ ID NO: 6529, 9742, 14628, or 17841. In some embodiments, the mRNA includes the sequence encoding the long-term repressor fusion protein of composition 1, and the mRNA includes the sequence encoding RD1 of SEQ ID NO: 132. In some embodiments, the mRNA includes the sequence of SEQ ID NOs. 6535, 9748, 14634, or 17847, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA includes the sequence of SEQ ID NOs. 6535, 9748, 14634, or 17847.In some embodiments, the mRNA includes a sequence encoding the long-term repressor fusion protein of composition 1, and the mRNA includes a sequence encoding RD1 of sequence number 133. In some embodiments, the mRNA includes a sequence of sequence number 6536, 9749, 14635, or 17848, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA includes a sequence of sequence number 6536, 9749, 14635, or 17848. In some embodiments, the mRNA includes a sequence encoding the long-term repressor fusion protein of composition 1, and the mRNA includes a sequence encoding RD1 of sequence number 134. In some embodiments, the mRNA includes the sequence of SEQ ID NOs. 6534, 9747, 14633, or 17846, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA includes the sequence of SEQ ID NOs. 6534, 9747, 14633, or 17846. In some embodiments, the mRNA includes the sequence encoding the long-term repressor fusion protein of composition 1, and the sequence encoding RD1 of SEQ ID NOs. 135. In some embodiments, the mRNA includes 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 thereto. In some embodiments, the mRNA includes the sequence of SEQ ID NO: 6530, 9743, 14629, or 17842.In some embodiments, the mRNA includes a sequence encoding the long-term repressor fusion protein of composition 1, and a sequence encoding RD1 of sequence number 136. In some embodiments, the mRNA includes a sequence of sequence number 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 thereto. In some embodiments, the mRNA includes a sequence of sequence number 6537, 9750, 14636, or 17849. In some embodiments, the mRNA includes a sequence encoding the long-term repressor fusion protein of composition 1, and a sequence encoding RD1 of sequence number 137. In some embodiments, the mRNA includes the sequence of SEQ ID NOs. 6533, 9746, 14632, or 17845, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA includes the sequence of SEQ ID NOs. 6533, 9746, 14632, or 17845. In some embodiments, the mRNA includes the sequence encoding the long-term repressor fusion protein of composition 1, and the sequence encoding RD1 of SEQ ID NOs. 138. In some embodiments, the mRNA includes the sequence of SEQ ID NOs. 6532, 9745, 14631, or 17844, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA includes the sequence of SEQ ID NOs. 6532, 9745, 14631, or 17844.

[0172] This disclosure provides mRNA encoding a long-term repressor fusion protein of configuration 5. In some embodiments, the mRNA includes 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 thereto. In some embodiments, the mRNA includes a sequence selected from the group consisting of SEQ ID NOs: 8135-9740, 11348-12953, 16234-17839, and 19447-21052.

[0173] This disclosure provides mRNA encoding a long-term repressor fusion protein of configuration 6a, wherein the encoded RD1 is identical. In some embodiments, the mRNA includes a sequence selected from the group consisting of SEQ ID NOs: 12954-14553 and 21053-22652, or a sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the mRNA includes a sequence selected from the group consisting of SEQ ID NOs: 12954-14553 and 21053-22652.

[0174] This disclosure provides mRNA encoding a long-term repressor fusion protein of configuration 6b, wherein the encoded RD1 is different. 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 thereto. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 14554-14626 and 22653-22725.

[0175] In some embodiments of the mRNA encoding the long-term repressor fusion protein of the LTRP:gRNA system of this disclosure, upon delivery of the system to target cells, the mRNA is expressed, and the long-term repressor fusion protein can form a complex with the gRNA and bind to target DNA, resulting in repression or silencing of the transcription of the PCSK9 gene within the cell.

[0176] VI. System Guide Nucleic Acids In another aspect, the disclosure relates to a specially designed guide ribonucleic acid (gRNA) comprising a scaffold and a ligated targeting sequence complementary to (and thus hybridizable with) the target nucleic acid sequence of the PCSK9 gene. The gRNAs described herein can be used in conjunction with long-term repressor proteins and systems comprising 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 containing domains derived from different gRNAs. The gRNAs of the disclosure comprise a scaffold and a targeting sequence complementary to the target nucleic acid of the cell, ligated to the 3' end of the scaffold.

[0177] In some embodiments, a system comprising mRNA encoding a long-term repressor fusion protein comprises a dCasX protein and one or more gRNAs, and when the dCasX protein is expressed in transfected cells, it forms a ribonucleoprotein (RNP) complex with the gRNAs, which can target and bind to specific sites in the target nucleic acid sequence of the cell. The gRNAs provide target specificity to the complex by containing a targeting sequence (or spacer) having a nucleotide sequence complementary to the sequence of 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, which is induced to a target site within the target nucleic acid sequence (e.g., stabilized at the target site) upon binding to the gRNAs.

[0178] Embodiments of gRNA and mRNA and gRNA formulations for use in the suppression and / or epigenetic modification of PCSK9 target nucleic acids are described below.

[0179] a. Reference gRNA and gRNA variants As used herein, “reference gRNA” refers to a CRISPR guide ribonucleic acid containing the wild-type sequence of a naturally occurring gRNA. In some embodiments, the gRNA scaffolds of this disclosure may be subjected to one or more mutagenesis methods, such as those described in WO2023235818A2, WO2022120095A1 and WO2020247882A1, which are incorporated herein by reference, including deep mutagenesis (DME), deep mutagenesis scanning (DMS), error-prone PCR, cassette mutagenesis, random mutagenesis, alternating extension PCR, gene shuffling, domain swapping, or chemical modification, to produce one or more gRNA variants having enhanced or altered properties relative to the modified gRNA scaffold. The activity of the gRNA scaffold from which the gRNA variants are derived may be used as a benchmark to compare the activity of the gRNA variants to it, thereby measuring improvements in the function or other characteristics of the gRNA scaffold.

[0180] Table 8 provides reference gRNA tracr and scaffold sequences. In some embodiments, the disclosure provides gRNA variants in which the gRNA has a scaffold comprising a sequence having one or more nucleotide modifications to any one of the reference gRNA sequences from SEQ ID NOs. 1731 to 1743 in Table 8. Table 8: Reference gRNA tracr and scaffold sequence [Table 22]

[0181] b.gRNA domain and its function The gRNA of the system of this disclosure comprises two segments: a targeting sequence and a protein-binding segment. The targeting segment of the gRNA comprises a nucleotide sequence (referred to interchangeably with spacer, target factor, or targeting sequence) that is complementary to (and therefore hybridizes with) a specific sequence (target site) within the target nucleic acid sequence (e.g., a strand of double-stranded target DNA, target ssRNA, target ssDNA, etc.) as fully described below. In the context of this disclosure, the targeting sequence of the gRNA can bind to the target nucleic acid sequence, including coding sequences, complements of coding sequences, non-coding sequences, and associated elements. The protein-binding segment of the gRNA (or “activator” or “protein-binding sequence”) interacts with (e.g., binds to) the dCasX protein as a complex to form an RNP (more fully described below). As used herein, “scaffold” refers to all parts of the guide except for the targeting sequence, which consists of several regions fully described below. The properties and characteristics of both wild-type and variant CasX gRNAs are described in WO2020247882A1, US20220220508A1, and WO2022120095A1, which are incorporated herein by reference.

[0182] In the case of a reference gRNA, the gRNA naturally occurs as a dual guide RNA (dgRNA), and the target factor portion and the activator factor portion each have a double-stranded segment that hybridizes with each other to form a double-stranded duplex (dsRNA double-strand of gRNA). Herein, the terms “target factor” or “target factor RNA” are used to refer to the crRNA-like molecule (crRNA: “CRISPR RNA”) of the CasX dual guide RNA (and therefore, of the CasX single guide RNA when the “activator factor” and “target factor” are linked together, for example, by intervening nucleotides). The crRNA has a tracrRNA, followed by a 5' region that anneals with the nucleotides of the targeting sequence. For gRNAs intended for use in the systems of this disclosure, the scaffold is designed to consist of a single molecule, with the activator and target factors covalently bonded to each other (rather than hybridizing), and may be referred to as "single-molecule gRNA," "single-guide RNA," "single-molecule guide RNA," "single-molecule guide RNA," or "sgRNA." All gRNA variants of this disclosure intended for use in the systems are single-molecule versions.

[0183] In addition, the assembled gRNAs of this disclosure include distinctly different structured regions or domains, namely, RNA triple helix, scaffolding stem-loop, elongation stem-loop, pseudoknot, and, in embodiments of this disclosure, a targeting sequence complementary to the sequence of the target nucleic acid and located at the 3' end of the gRNA. The RNA triple helix, scaffolding stem-loop, pseudoknot, and elongation stem-loop, together with the unstructured triple-loop that cross-links the triple-helix portions, are referred to as the “scaffold” of the gRNA. In some cases, the scaffolding stem further includes a bubble. In other cases, the scaffold further includes a triple-loop region. In yet another case, the scaffold further includes a 5' unstructured region. In some embodiments, the gRNA scaffolds of this disclosure for use in an LTRP:gRNA system include a scaffolding stem-loop having the sequence CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1822), or a sequence having at least 1, 2, 3, 4, or 5 mismatches therewith.

[0184] Each of the structured domains contributes to establishing the global RNA folding of the guide and maintaining the guide's functionality, particularly its 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 triple helix, triple loop, and pseudoknot stem interact with the OBD of the dCasX protein. Collectively, these interactions confer the guide's ability to form an RNP that binds to the LTRP and maintains stability, while spacers (or targeting sequences) direct and define the specificity of the RNP for binding to specific sequences of DNA.

[0185] Site-specific binding of the target nucleic acid sequence (e.g., genomic DNA) by the dCasX protein of LTRP can occur at one or more positions (e.g., the sequence of the PCSK9 target nucleic acid) determined by the base pair complementarity between the targeting sequence of the gRNA and the target nucleic acid sequence. Therefore, for example, the gRNA of this disclosure is complementary to a target nucleic acid adjacent to a TC protospacer adjacency motif (PAM) motif or PAM sequence, e.g., a sequence complementary to ATC, CTC, GTC, or TTC, and can therefore hybridize with it. Since the targeting sequence of the guide sequence hybridizes with the sequence of the target nucleic acid sequence, the targeting sequence can be modified by the user to hybridize with a particular target nucleic acid sequence, insofar as the position of the PAM sequence is taken into consideration. In some embodiments, for the design of the targeting sequence, the target nucleic acid includes a PAM sequence located at 5' of the targeting sequence, where the PAM is separated by at least one nucleotide from the first nucleotide of the target nucleic acid that is complementary to the first nucleotide of the targeting sequence. This feature distinguishes the system described herein from the cCas9 system, resulting in the ability of the system to modify different locations in the DNA sequence compared to the Cas9 system. In some embodiments, the PAM is located on the non-target strand of the target region, i.e., the strand complementary to the target nucleic acid. In some embodiments, the gRNA targeting sequence is complementary to the PCSK9 target nucleic acid sequence located one nucleotide away from the ATC PAM sequence. In some embodiments, the gRNA targeting sequence is complementary to the PCSK9 target nucleic acid sequence located one nucleotide away from the CTC PAM sequence. In some embodiments, the gRNA targeting sequence is complementary to the PCSK9 target nucleic acid sequence located one nucleotide away from the GTC PAM sequence. In some embodiments, the gRNA targeting sequence is complementary to the PCSK9 target nucleic acid sequence located one nucleotide away from the TTC PAM sequence. By selecting the gRNA targeting sequence, a defined region of the target nucleic acid sequence or a sequence surrounding a specific location within the target nucleic acid can be suppressed using the LTRP:gRNA system described herein. In some embodiments, the gRNA targeting sequence has 15 to 22 consecutive nucleotides.In some embodiments, the targeting sequence has 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 21 consecutive nucleotides. In some embodiments, the targeting sequence consists of 20 consecutive nucleotides. In some embodiments, the targeting sequence consists of 19 consecutive nucleotides. In some embodiments, the targeting sequence consists of 18 consecutive nucleotides. In some embodiments, the targeting sequence consists of 17 consecutive nucleotides. In some embodiments, the targeting sequence consists of 16 consecutive nucleotides. In some embodiments, the targeting sequence consists of 15 consecutive nucleotides. By selecting the targeting sequence of the gRNA, a defined region of the PCSK9 target nucleic acid sequence can be repressed and / or epigenetically modified using the LTRP:gRNA system described herein.

[0186] The gene repressor systems of this disclosure may be designed to target any region of the PCSK9 gene or region of the PCSK9 gene whose transcriptional repression is desired, or any region proximal thereto. If the entire gene is to be repressed, the disclosure intends to design a guide that includes the transcription start site (TSS) or has a targeting sequence complementary to the sequence proximal thereto. TSS selection occurs at different locations within the promoter region depending on the promoter sequence and the concentration of the initiation substrate. The core promoter functions as a binding platform for the transcription mechanism, including Pol II and its associated general transcription factors (GTFs) (Haberle, V. et al. Eukaryotic core promoters and the functional basis of transcription initiation (Nat Rev Mol Cell)). Biol. 19(10):621(2018)). Variability in TSS selection has been proposed to involve DNA "scrunching" and "anti-scrunching," characterized by (i) forward and reverse movement of the leading edge rather than the trailing edge of RNA polymerase relative to DNA, and (ii) expansion and contraction of the transcription bubble. In some embodiments, the target nucleic acid sequence bound by the RNP of the LTRP:gRNA system is within 1 kilobase (kb) of the transcription start site (TSS) in the PCSK9 gene. In some embodiments, the target nucleic acid sequence bound by the RNP of the system is 20 bases upstream of the TSS in the PCSK9 gene. The target nucleic acid sequence is located within 1.5kb upstream to 1.5kb downstream, 1kb upstream to 1kb downstream, 500bp upstream to 500bp downstream, 300bp upstream to 300bp downstream, or 100bp upstream to 100bp downstream of the PCSK9 gene TSS.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 of the PCSK9 gene enhancer. In some embodiments, the target nucleic acid sequence bound by the system's RNP is within 1 kb of the 3'-5' untranslated region of the PCSK9 gene. In other embodiments, the target nucleic acid sequence bound by the system's RNP is within the open reading frame of the PCSK9 gene, including introns (if present). In some embodiments, the targeting sequence of the system's gRNA is complementary to the sequence of an exon of the PCSK9 gene. In certain embodiments, the targeting sequence of the system's gRNA is complementary to the sequence of exon 1 of the PCSK9 gene. In other embodiments, the targeting sequence of the system's gRNA is complementary to the sequence of an intron of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system of disclosure is complementary to the sequence of the intron-exon junction of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system of disclosure is complementary to the sequence of the regulatory element of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system of disclosure is complementary to the sequence of the intergenetic region of the PCSK9 gene. In other embodiments, the targeting sequence of the gRNA of the system of disclosure is complementary to the sequence of the junction of the exon, intron, and / or regulatory element of the PCSK9 gene. When the targeting sequence is complementary to the sequence of a regulatory element, such regulatory elements include, but are not limited to, regions containing promoter regions, enhancer regions, intergenetic regions, 5' untranslated regions (5'UTR), 3' untranslated regions (3'UTR), conserved elements, and cis-regulatory elements. The promoter region is intended to contain nucleotides within 5kb of the start of the coding sequence, or, in the case of a gene enhancer or conserved element, may be several thousand, several hundred thousand, or even several million bp away from the coding sequence of the PCSK9 gene.As described above, the target is intended to repress the target coding PCSK9 gene so that the PCSK9 gene product is not expressed in the cell or is expressed at a lower level. In some embodiments, when the RNP of the system of this disclosure binds to the binding site of the target nucleic acid, the system can repress the transcription of the PCSK9 gene 5' relative to 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 can repress the transcription of the PCSK9 gene 3' relative to the RNP binding site.

[0187] In some embodiments, the target nucleic acid includes a PAM sequence located at 5' of the targeting sequence, wherein the PAM is separated from the first nucleotide of the targeting sequence by at least one nucleotide. In some embodiments, the PAM is located on the non-target strand of the target region, i.e., the strand complementary to the target nucleic acid, and represents the targeting sequence of the PCSK9 target nucleic acid for ligation to the gRNA scaffolds of this disclosure, e.g., gRNA174 (SEQ ID NO: 1744), 235 (SEQ ID NO: 1745), 316 (SEQ ID NO: 1746), or chemically modified versions thereof, as shown in Table 9 below. In some embodiments, the PAM sequence is a TTC.

[0188] In some embodiments, the gRNA targeting sequence includes a sequence selected from the group consisting of SEQ ID NOs: 1824-2544, 2672, 2675, 2694, and 2714, as shown in Table 9. In some embodiments, the gRNA targeting sequence includes a sequence selected from the group consisting of SEQ ID NOs: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments, the gRNA targeting sequence includes a sequence selected from the group consisting of SEQ ID NOs: 1834, 1849, 1853, 1855-1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875. In some embodiments, the gRNA targeting sequence includes a sequence selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869. In certain embodiments, the gRNA targeting sequence consists of the sequence of SEQ ID NO: 1855. In another specific embodiment, the gRNA targeting sequence consists of the sequence of SEQ ID NO: 1867. In yet another specific embodiment, the gRNA targeting sequence consists of the sequence of SEQ ID NO: 1869. In some embodiments, the aforementioned targeting sequences are ligated to gRNA scaffolds of gRNA 174 (SEQ ID NO: 1744), 235 (SEQ ID NO: 1745), or 316 (SEQ ID NO: 1746), or chemically modified versions thereof. Table 9: PCSK9-specific targeting sequences using long-term repressor fusion proteins [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] Table 28 Table 29 Table 30 Table 31 Table 32 Table 33 Table 34 Table 35 Table 36 Table 37 Table 38 Table 39

[0189] c.gRNA modification In another embodiment, the disclosure relates to gRNAs (also referred to herein as gRNA variants) that include modifications to a reference gRNA from which the gRNA is derived. The gRNAs can be used in the long-term gene repressor fusion protein systems described herein. In some embodiments, gRNA variants for use in the systems of the disclosure include one or more nucleotide substitutions, insertions, deletions, or exchanges or substituted domains to the gRNA sequence of the disclosure that improve its properties. Examples of regions and exchange regions or domains for modification include RNA triple helices, pseudoknots, scaffolding stem-loops, and elongation stem-loops. In some embodiments, the gRNA variant includes at least a first exchange region from different gRNAs, resulting in a chimeric gRNA. A typical example of such chimeric gRNAs is guide 316 (sequence number 1746), in which the elongation stem loop of gRNA scaffold 235 (sequence number 1745) is replaced by the elongation stem loop of gRNA scaffold 174 (sequence number 1744). The resulting 316 variant retains the ability to form RNPs with long-term repressor fusion proteins and exhibits improved properties compared to parent 235 when evaluated in vitro or in vivo under equivalent conditions.

[0190] All gRNAs that possess one or more improved functions, features, or add one or more novel functions, while retaining the functional property of being able to complex with a long-term repressor fusion protein and induce a ribonucleoprotein holo RNP complex to a PCSK9 target nucleic acid when compared to the gRNA scaffold from which they originate, are assumed to be within the scope of this disclosure. In some embodiments, the gRNA has improved properties selected from the group consisting of increased pseudoknot stem stability, increased triple-stranded region stability, increased scaffold stem stability, elongation stem stability, reduced off-target folding intermediates, increased binding affinity to a long-term repressor fusion protein, and increased repressive activity when complexed with a long-term repressor fusion protein, or any combination thereof. In some cases, the improvement in features is evaluated by in vitro assays, including the assays of the Examples. In other cases, the improvement in features is evaluated in vivo.

[0191] Table 10 provides exemplary gRNA variant scaffold sequences of the present disclosure that can be used as gRNA scaffolds or for generating gRNAs for use in the LTRP:gRNA system of the present disclosure. In some embodiments, the gRNA variant scaffold for use in the system comprises a sequence selected from the group consisting of SEQ ID NOs: 1744-1746, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the gRNA variant retains the ability to form RNPs with the long-term repressor fusion protein of the present disclosure. In some embodiments, the gRNA variant scaffold for use in the system comprises a sequence selected from the group consisting of SEQ ID NOs: 1744-1746. In some embodiments, the gRNA variant scaffold for use in the system comprises SEQ ID NO: 1744. In some embodiments, the gRNA variant scaffold for use in the system includes SEQ ID NO: 1745. In some embodiments, the gRNA variant scaffold for use in the system includes SEQ ID NO: 1746. In these embodiments, where the vector includes the DNA coding sequence of the gRNA, it will be understood that the thymine (T) base may be substituted with the uracil (U) base of any of the gRNA sequence embodiments described herein. Similarly, any RNA sequence disclosed herein may be encoded by DNA in which the uracil base is substituted with thymine. In some embodiments, this disclosure provides the chemically modified gRNA variants of Table 10, which are described below. In some embodiments, the gRNA includes a scaffold containing the sequences of SEQ ID NOs: 1744-1746 and is chemically modified. Table 10: gRNA scaffold sequences [Table 40]

[0192] Additional gRNA variants intended for use in the systems of this disclosure are selected from the group consisting of SEQ ID NOs: 1747–1821. In some embodiments, the gRNAs are chemically modified and include a scaffold containing the sequences of SEQ ID NOs: 1747–1821.

[0193] Guide scaffolds can be prepared by several methods, including recombinant or solid-phase RNA synthesis. However, the length of the scaffold can affect manufacturability when using solid-phase RNA synthesis, with longer lengths resulting in increased manufacturing costs, decreased purity and yield, and a higher synthesis failure rate. When used in particulate formulations such as lipid nanoparticle (LNP) formulations, solid-phase RNA synthesis of the scaffold is preferred to produce the amount required for commercial development. Previous experiments confirmed that gRNA scaffold 235 (SEQ ID NO: 1745) has enhanced properties compared to gRNA scaffold 174 (SEQ ID NO: 1744), but its increased length (of nucleotides) made its use in LNP formulations undesirable due to synthetic manufacturing constraints. Therefore, alternative sequences were sought. In some embodiments, this disclosure provides gRNA variant scaffolds with improved manufacturability compared to the gRNA scaffolds from which they are derived. In some embodiments, the disclosure provides gRNAs in which the gRNA scaffold and the ligated targeting sequence have sequences of less than approximately 115 nucleotides, less than approximately 110 nucleotides, or less than approximately 100 nucleotides. In certain embodiments, the 316 gRNA scaffold (SEQ ID NO: 1746) has a shorter sequence compared to the 235 scaffold from which it is derived. The 316 gRNA scaffold was designed so that the scaffold 235 sequence is modified by a domain swap, in which the elongation stem loop of scaffold 174 replaces the elongation stem loop of scaffold 235, resulting in a chimeric gRNA scaffold 316ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGUGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG (SEQ ID NO: 1746) having a sequence with 89 nucleotides compared to the 99 nucleotides of gRNA scaffold 235. The resulting 316 scaffolds have a further advantage in that the elongated stem loops do not contain CpG motifs, which is an enhanced property that reduces the likelihood of inducing an immune response. In some embodiments, the shorter sequence length of the 316 scaffolds results in improved fidelity in the ability to synthesize guides with accurate and complete sequences, as well as an enhanced ability to be successfully incorporated into LNPs.In some embodiments, the present disclosure provides chemically modified gRNA316 variants as described below.

[0194] d. Chemically modified gRNA 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 in the sequence. In some embodiments, the chemical modification is the substitution of a phosphorothioate bond between two or more nucleotides in the sequence. In some embodiments, the chemical modification is the substitution of a phosphorothioate bond between two or more nucleotides at each end of the gRNA. In some embodiments, the gRNA includes the substitution of a phosphorothioate bond between two or more nucleotides located at the 5' end, 3' end, or 1, 2, 3, or 4 nucleotides from both ends of the gRNA. In some embodiments, the gRNA includes the addition of a 2'O-methyl group to one or more nucleotides in the gRNA. In some embodiments, one or more nucleotides located at the 5' end, 3' end, or 1, 2, 3, or 4 nucleotides from both ends of the gRNA are modified by the addition of a 2'O-methyl group. In some embodiments, the first one, two, or three nucleotides at the 5' end of the scaffold (i.e., A, C, and U in the case of gRNA 174, 235, and 316) are modified by the addition of a 2'O-methyl group, and each of the modified nucleotides is ligated to an adjacent nucleotide by a phosphorothioate bond. Similarly, the last one, two, or three nucleotides at the 3' end of the targeted sequence ligated to the 3' end of the scaffold are similarly modified. In some embodiments, the present disclosure provides chemically modified gRNAs selected from the group consisting of sequences SEQ ID NOs. 2948-2956, 2958-2966, and 2968-2976 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%, and at least about 99% sequence identity with them. In any of the above, 20 nucleotides at the 3' end of the sequence are replaced with a targeting sequence complementary to the PCSK9 target nucleic acid sequence, and 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, and includes chemical modifications at the 3' end of the gRNA.In some embodiments, 20 nucleotides on the 3' end of the sequence are replaced with a targeting sequence complementary to a PCSK9 target nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1834, 1849, 1853, 1855-1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875, but include chemical modifications on the 3' end of the gRNA. In some embodiments, 20 nucleotides on the 3' end of the sequence are replaced with a targeting sequence complementary to a PCSK9 target nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869, but include chemical modifications on the 3' end of the gRNA. In some embodiments, the chemically modified gRNA includes a sequence selected from the group consisting of sequences 2948-2956, 2958-2966, and 2968-2976, in which 20 nucleotides at the 3' end are replaced with a targeted sequence selected from the group consisting of sequences 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714, but which has a chemical modification at the 3' end. In some embodiments, the chemically modified gRNA includes a sequence of sequence 2968, in which 20 nucleotides at the 3' end are replaced with a targeted sequence selected from the group consisting of sequences 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714, but which has a chemical modification at the 3' end. In some embodiments, the chemically modified gRNA includes the sequence of SEQ ID NO: 2968, wherein the 20 nucleotides at the 3' end are replaced with a targeted sequence selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869, but the sequence has a chemical modification at the 3' end. In some embodiments, the modified gRNA includes a sequence selected from the group consisting of SEQ ID NOs: 22780 to 22803. In some embodiments, the modified gRNA includes a sequence selected from the group consisting of SEQ ID NOs: 22788 to 22790. Schematic diagrams of the structures of gRNA variants 174, 235, and 316 are shown in Figures 7 to 9, respectively, and chemically modified versions of gRNA variants 235 and 316 are shown in Figures 10 to 14.In some embodiments, gRNAs having chemical modifications exhibit improved stability compared to gRNAs without chemical modifications.

[0195] e. Complex formation with repressor fusion proteins Upon delivery or expression of the components of the system in target cells, the gRNA variant can complex with the long-term repressor fusion protein as an RNP and bind to the target nucleic acid of the PCSK9 gene. In some embodiments, the gRNA variant has an improved ability to form an RNP complex with the long-term repressor fusion protein compared to a reference gRNA. In some embodiments, the improvement in ribonucleoprotein complex formation can improve the efficiency with which a functional RNP is assembled. In some embodiments, more than 90%, more than 93%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the RNPs comprising the gRNA variant of the present disclosure and its target sequence and the long-term repressor fusion protein are eligible for gene silencing of the target nucleic acid.

[0196] VII. Polynucleotides and vectors In another aspect, the present disclosure relates to polynucleotides encoding long-term repressor fusion proteins and / or gRNAs that are useful for the repression and epigenetic modification of the PCSK9 gene. The present disclosure provides polynucleotides encoding mRNAs encoding long-term repressor fusion proteins, for example, DNA polynucleotides encoding the corresponding mRNAs.

[0197] The long-term repressor fusion proteins or mRNAs encoding long-term repressor fusion proteins of this disclosure may be prepared by in vitro synthesis using conventional methods known in the art. Various commercially available synthesizers, e.g., automated synthesizers from Applied Biosystems, Inc., Beckman, etc., are available. By using a synthesizer, naturally occurring amino acids or nucleotides (where applicable) may be substituted with non-natural amino acids or nucleotides. The specific sequence and preparation method will be determined by convenience, cost-effectiveness, required purity, etc. The gRNA may also be produced synthetically, for example, by using T7 RNA polymerase systems known in the art.

[0198] Long-term repressor fusion proteins and / or gRNAs may also be prepared by recombinantly producing polynucleotide sequences encoding any of the embodiments of the long-term repressor fusion proteins or gRNAs described herein, and incorporating the encoding gene into an expression vector suitable for host cells using recombinant techniques known in the art. For the production of any of the encoded long-term repressor fusion proteins and / or gRNAs described herein, the method comprises transforming suitable host cells with an expression vector containing the encoding polynucleotide, and culturing the host cells transformed with the obtained long-term repressor fusion proteins or gRNAs of any of the embodiments described herein, or under conditions that enable such expression or transcription, which are then recovered by the methods described herein, or by standard purification methods known in the art, or as described in the examples. The polynucleotides and expression vectors of this disclosure are prepared using standard recombinant techniques in molecular biology.

[0199] The long-term repressor fusion proteins and / or gRNAs of this disclosure may also be isolated and purified according to conventional recombinant synthesis methods. Lysates may be prepared and purified using high-performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the compositions used contain 50% by weight or more of the desired product, more commonly 75% by weight or more, preferably 95% by weight or more, and usually 99.5% by weight or more for therapeutic purposes, with respect to contaminants associated with the preparation and purification of the product. Typically, the percentages are based on total protein. Thus, in some cases, the long-term repressor fusion proteins or gRNAs of this disclosure are 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).

[0200] Furthermore, this disclosure provides vectors comprising polynucleotides encoding long-term repressor fusion proteins and gRNAs as described herein. In some cases, when the system is delivered as repressor fusion proteins and gRNAs, or RNPs, the vectors are used for the expression and restoration of CasX and gRNA components of the LTRP:gRNA system. In other cases, the vectors are used for the delivery of encoding polynucleotides to target cells for repression and / or epigenetic modification of target nucleic acids, as is fully described below. In some embodiments, the sequences encoding long-term repressor fusion proteins and gRNAs are encoded by the same vector. In some embodiments, the sequences encoding long-term repressor fusion proteins and gRNAs are templated on different vectors. Suitable vectors are described, for example, WO2023235818A2, WO2022120095A1 and WO2020247882A1, which are incorporated herein by reference. As described in WO2023235818A2, WO2022120095A1, and WO2020247882A1, depending on the host / vector system used, any of several suitable transcription and translational regulatory elements, including constitutive and inducible promoters, transcriptional enhancer elements, and transcriptional terminators, may be used in the expression vector.

[0201] This disclosure provides polynucleotide sequences encoding long-term repressor fusion proteins of any of the embodiments described herein. In some embodiments, this disclosure provides DNA sequences encoding long-term repressor fusion proteins for use in vectors. In some embodiments, this disclosure provides mRNA sequences encoding long-term repressor fusion proteins of any of the embodiments described herein for use in particle systems for delivery to cells. In some embodiments, this disclosure provides mRNA sequences encoding long-term repressor fusion proteins of any of the embodiments described herein for use in LNP particle formulations for delivery to cells. In certain embodiments, this disclosure provides gRNA and mRNA sequences encoding long-term repressor fusion proteins of any of the embodiments described herein for use in LNP particle formulations for delivery to cells. In some embodiments, this disclosure provides isolated polynucleotide sequences encoding gRNA variants of any of the embodiments described herein, having a ligated targeting sequence complementary to a PCSK9 target nucleic acid sequence.

[0202] In some embodiments, this disclosure relates to methods for generating polynucleotide sequences encoding long-term repressor fusion proteins or gRNAs of any of the embodiments described herein, and methods for expressing proteins or RNAs transcribed by polynucleotide sequences. Generally, the methods include generating polynucleotide sequences encoding long-term repressor fusion proteins or gRNAs of any of the embodiments described herein, and incorporating the encoding gene into an expression vector. In some embodiments, the vector is designed for transduction of cells for repression and / or epigenetic modification of PCSK9 target nucleic acids. Such vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, plasmids, minicircles, nanoplasmides, DNA vectors, and RNA vectors. In other embodiments, the expression vector is designed for the production of long-term repressor fusion proteins, mRNA encoding long-term repressor fusion proteins, or gRNAs in either a cell-free system or host cells. For the production of any of the encoded long-term repressor fusion proteins or gRNAs of the embodiments described herein in host cells, the method comprises transforming suitable host cells with an expression vector comprising the coding polynucleotide, culturing the transformed host cells under conditions that enable or allow the expression or transcription of any of the long-term repressor fusion proteins or gRNAs of the embodiments described herein in the transformed host cells, thereby producing long-term repressor fusion proteins or gRNAs, which are recovered by methods described herein (e.g., as described in the following examples) or by standard purification methods known in the art. The polynucleotides and expression vectors of this disclosure are prepared using standard recombination techniques in molecular biology.

[0203] According to this disclosure, nucleic acid sequences encoding long-term repressor fusion proteins or gRNAs of any embodiment described herein are used to generate recombinant DNA molecules that induce expression in suitable host cells. Several cloning strategies are suitable for carrying out this disclosure, many of which are used to generate constructs containing genes encoding the long-term repressor fusion proteins or gRNAs or their complements of this disclosure. In some embodiments, the cloning strategy is used to generate a gene encoding a construct containing nucleotides encoding the long-term repressor fusion proteins or gRNAs. In some embodiments, the gene is used, for example, as part of a vector to transform host cells for gene expression, e.g., expression of long-term repressor fusion proteins or gRNAs.

[0204] In one approach, 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 create an expression vector suitable for transforming host cells, such as prokaryotic or eukaryotic host cells, for the expression and recovery of the protein construct, in the case of a long-term repressor fusion protein or gRNA. If desired, the host cell is E. coli. In other embodiments, the cell is a eukaryotic cell. The eukaryotic host cells can be selected from baby hamster kidney fibroblast (BHK) cells, human embryonic kidney 293 (HEK293), human embryonic kidney 293T (HEK293T), NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, NIH3T3 cells, CV-1 (monkey) (COS) derived from the SV40 gene material, HeLa, Chinese hamster ovary (CHO), yeast cells, or other eukaryotic cells known in the art that are suitable for the production of recombinant products. Exemplary methods for generating expression vectors, transforming host cells, and expressing and recovering long-term repressor fusion proteins or gRNAs are described in the examples.

[0205] Genes encoding long-term repressor fusion proteins or gRNA constructs can be prepared in one or more steps either entirely synthetically or by synthesis combined with enzymatic processes such as restriction enzyme-mediated cloning, PCR, and overlap extension, including methods fully described in the examples. The methods disclosed herein can be used, for example, to ligate sequences of polynucleotides encoding various components into a gene of a desired sequence. Genes encoding polypeptide compositions are assembled from oligonucleotides using standard gene synthesis techniques.

[0206] 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, the codons can be altered, but the encoded protein remains immutable. For example, if the intended target cells of the long-term repressor fusion protein are human cells, a human codon-optimized long-term repressor fusion protein encoding nucleotide sequence can be used. As another non-limiting example, if the intended host cells are mouse cells, a mouse codon-optimized long-term repressor fusion protein encoding nucleotide sequence can be generated. Gene design can be carried out using an algorithm that optimizes the appropriate codon usage frequency and amino acid composition for the host cells used to produce the long-term repressor fusion protein or gRNA. In one method of this disclosure, a library of polynucleotides encoding the components of the construct is generated and then assembled, as described above. Subsequently, the resulting genes are assembled and used to transform host cells and produce and recover long-term repressor fusion proteins or gRNA compositions for characterization or modification of PCSK9 target nucleic acids, as described herein.

[0207] In some embodiments, the nucleotide sequence encoding the gRNA is operably ligated to a regulatory element, such as a transcriptional regulatory element, like a promoter. In some embodiments, the nucleotide sequence encoding the long-term repressor fusion protein is operably ligated to a regulatory element, such as a transcriptional regulatory element, like a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatory promoter. In some cases, the promoter is an inductive 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 regulatory element (e.g., the promoter) is functional in a target cell type or target cell population. For example, in some cases, the transcriptional regulatory element may be functional in eukaryotic cells, such as hepatocytes or hepatic sinusoidal endothelial cells.

[0208] Non-limiting examples of Pol II promoters operably linked to polynucleotides encoding the long-term repressor fusion proteins of this disclosure include EF-1 alpha, EF-1 alpha core promoter, Jens Tornoe (JeT), cytomegalovirus (CMV)-derived promoter, CMV very early (CMVIE), CMV enhancer, herpes simplex virus (HSV) thymidine kinase, early and late Simianvirus 40 (SV40), SV40 enhancer, retrovirus-derived long terminal repeat (LTR), mouse metallothionein-I, and adenovirus major late promoter (Ad MLP), CMV promoter full-length promoter, minimal CMV promoter, chicken β-actin promoter (CBA), CBA hybrid (CBh), chicken β-actin promoter with cytomegalovirus enhancer (CB7), chicken β-actin promoter and rabbit β-globin splice receptor site fusion (CAG), Roussarcoma virus (RSV) promoter, HIV-Ltr promoter, hPGK promoter, HSVTK promoter, 7SK promoter, Mini-TK promoter, human synapsin I (SYN) promoter conferring neuron-specific expression, beta-actin promoter, supercore promoter 1 (SCP1), Mecp2 promoter for selective expression in neurons, minimal IL-2 promoter, Roussarcoma virus enhancer / promoter (single), splenic fossaforming virus long-term repeat (LTR) promoter, TBG promoter, promoter from human thyroxine-binding globulin gene (liver-specific), PGK promoter, human ubiquitin C promoter (UBC), UCOE promoter (promoter for HNRPA2B1-CBX3), synthetic CAG promoter, histone H2 promoter, histone H3 promoter, U1a1 micronucleus R Examples of promoters include, but are not limited to, the NA promoter (226nt), U1a1 micronuclear RNA promoter (226nt), U1b2 micronuclear RNA promoter (246nt), GUSB promoter, CBh promoter, rhodopsin (Rho) promoter, silencing-prone splenic fossa-forming virus (SFFV) promoter, human H1 promoter (H1), POL1 promoter, TTR minimal enhancer / promoter, β-kinesin 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 the aforementioned shortened versions and sequence variants. In certain embodiments, the Pol II promoter is EF-1 alpha, and the promoter enhances transfection efficiency, CRISPR nuclease transgene transcription or expression, the percentage of expression-positive clones, and the copy number of episomal vectors in long-term culture.

[0209] Non-limiting examples of Pol III promoters ligated to polynucleotides encoding gRNA variants of this disclosure include, but are not limited to, U6, mini-U6, U6 truncated promoter, 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 promoter, and their truncated versions and sequence variants. In the embodiments described above, the Pol III promoter enhances gRNA transcription. In specific embodiments, the Pol III promoter is U6, and the promoter enhances gRNA expression. Experimental details and data for the use of such promoters are provided in the examples.

[0210] The selection of appropriate vectors and promoters is well within the normal level of technology in the art with respect to the control of expression. Expression vectors may also contain ribosome binding sites and transcription terminators for translation initiation. Expression vectors may also contain appropriate sequences for amplification of expression. Expression vectors may also contain nucleotide sequences encoding protein tags (e.g., 6xHis tags, hemagglutinin tags, fluorescent proteins, etc.) which can be fused to long-term repressor fusion proteins and thus result in chimeric proteins used for purification or detection.

[0211] The recombinant expression vectors of this disclosure may also contain elements that promote robust expression of the proteins and gRNAs of this disclosure. For example, the recombinant expression vectors may contain one or more post-transcriptional regulators such as polyadenylation (Poly(A)), intron sequences, or Woodchuck hepatitis post-transcriptional regulators (WPREs). Exemplary Poly(A) sequences include the hGH Poly(A) signal (short), HSV TK Poly(A) signal, synthetic polyadenylation signal, split Poly(A) sequence with an SphI restriction site between two 60A stretches (SEQ ID NO: 3307), SV40 Poly(A) signal, and β-globin Poly(A) signal. Those skilled in the art will be able to select elements suitable for inclusion in the recombinant expression vectors described herein.

[0212] Polynucleotides encoding long-term repressor fusion proteins or gRNA sequences can be individually cloned into expression vectors. The selection of appropriate vectors and promoters is well within the scope of those skilled in the art, as it relates, for example, to the repression of PCSK9 gene expression and / or the regulation of expression for epigenetic modification. Expression vectors may also contain ribosome-binding sites and transcriptional terminators for translation initiation. Expression vectors may also contain appropriate sequences for amplification of expression.

[0213] Nucleic acid sequences are inserted into vectors by various procedures. Generally, DNA is inserted into appropriate restriction endonuclease sites using techniques known in the art. Vector components generally include, but are not limited to, one or more of the following: signal sequences, replication origins, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. The construction of a suitable vector 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 adequately described in the scientific and patent literature. A variety of vectors are publicly available. Vectors may be in the form of plasmids, cosmids, viral particles, or phages that are conveniently provided for recombinant DNA procedures, and the choice of vector often depends on the host cell into which it is introduced. Thus, vectors may be autonomously replicating vectors, i.e., vectors that exist as extrachromosomal entities, and their replication is independent of chromosomal replication, such as plasmids. Alternatively, vectors may be integrated into the host cell genome when introduced into a host cell and replicate together with the chromosome into which they are integrated. Once introduced into suitable host cells, the expression of the long-term repressor fusion protein or gRNA can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of the long-term repressor fusion protein may be detected and / or quantified by conventional hybridization assays (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Patent No. 5,695,937), and array systems (see, for example, U.S. Patents No. 5,405,783, 5,412,087, and 5,445,934) using probes complementary to any region of the polynucleotide.

[0214] In some embodiments, vectors are constructed for the transcription of long-term repressor fusion protein genes, and for the expression and recovery of the resulting coding mRNA. In some embodiments, the mRNA is produced by PCR product or by in vitro transcription (IVT) using a linearized plasmid DNA template and T7 RNA polymerase, the plasmid containing the T7 promoter. When using a PCR product, the DNA sequence encoding the candidate mRNA is cloned into a plasmid containing the T7 promoter, the plasmid DNA template is linearized, and then used to carry out the IVT reaction for mRNA expression. Exemplary methods for producing such vectors, as well as mRNA production and recovery, are provided in the following examples.

[0215] VIII. LTRP: Delivery particles for gRNA systems In another embodiment, the Disclosure provides particle compositions for delivering an LTRP:gRNA system to a cell or subject for transcriptional repression or silencing of the PCSK9 gene. Particles assumed to be within the scope of the Disclosure include, but are not limited to, synthetic nanoparticles, polymer nanoparticles, lipid nanoparticles, viral particles, and virus-like particles. The particles of the Disclosure may encapsulate a payload, such as a gRNA variant described herein, in combination with mRNA encoding one of the long-term repressor fusion proteins of any of the embodiments described herein. Alternatively, or in addition, the particles of the 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 one of the gRNA variants and an mRNA encoding one of the embodiments described herein. In some embodiments, the synthetic nanoparticles include biodegradable polymer nanoparticles (PNPs). In some embodiments, materials for producing biodegradable polymer nanoparticles (PNPs) include polylactide, poly(lactic acid-co-glycolic acid) (PLGA), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), and poly(isohexyl cyanoacrylate), polyglutamic acid (PGA), poly(ε-caprolactone) (PCL), cyclodextrin, and natural polymers such as chitosan, albumin, gelatin, and alginates, which are the most commonly used polymers for PNP synthesis (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 a payload of mRNA encoding one of the gRNA variants and long-term repressor fusion proteins described herein, which are described in more detail below.In other embodiments, the particles are lipid nanoparticles (LNPs) that separately encapsulate the gRNA variant and mRNA encoding the long-term repressor fusion protein of any of the embodiments described herein, and are co-formulated as a mixture for administration, which is described more fully below. In other embodiments, the particles are lipid nanoparticles that separately encapsulate the gRNA variant and mRNA encoding the long-term repressor fusion protein of any of the embodiments, and the two types of particles are administered separately.

[0216] a. Lipid nanoparticles (LNPs) In another embodiment, the Disclosure provides lipid nanoparticles (LNPs) for delivering the LTRP:gRNA system of the Disclosure to cells or targets for transcriptional repression of the PCSK9 gene. In some embodiments, the LNPs of the Disclosure are tissue or organ specific (e.g., liver), possess excellent biocompatibility, can deliver the system with high efficiency, and can therefore be usefully used for transcriptional repression of the PCSK9 gene.

[0217] This disclosure further provides LNP compositions and pharmaceutical compositions comprising a plurality of LNPs as described herein.

[0218] Nucleic acid polymers, in their natural forms, are unstable in biological fluids and cannot penetrate the cytoplasm of target cells, thus requiring a delivery system. Lipid nanoparticles (LNPs) have proven useful for both the protection of nucleic acids and their delivery to tissues and cells. Furthermore, the use of mRNA in LNPs encoding long-term repressor fusion proteins eliminates the possibility of undesirable genomic integration compared to DNA vectors. Moreover, since mRNA exerts its function in the cytoplasmic compartment and does not require nuclear entry, it efficiently transfects both mitotic and non-mitotic cells. Therefore, LNPs as a delivery platform offer the additional advantage that both mRNA and gRNA encoding long-term repressor fusion proteins can be co-formulated into a single LNP particle.

[0219] Accordingly, in various embodiments, the Disclosure encompasses lipid nanoparticles and compositions that can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells both in vitro and in vivo. In certain embodiments, the Disclosure encompasses a method for treating or preventing a disease or disorder in a subject requiring treatment or prevention of the disease or disorder by encapsulating a suitable therapeutic agent, which is complexed via various physical, chemical, or electrostatic interactions between one or more lipid components used in the composition to produce LNPs, or by contacting the subject with associated lipid nanoparticles. In some embodiments, the suitable therapeutic agent includes the LTRP:gRNA system described herein.

[0220] In certain embodiments, lipid nanoparticles are useful for delivering nucleic acids, for example, mRNA encoding the long-term repressor fusion protein of the Disclosure, and gRNA variants. In some embodiments, the LNPs include mRNA comprising a sequence encoding a long-term repressor fusion protein selected from the group consisting of SEQ ID NOs. 6528-14626 (unmodified mRNA) and 14627-22725 (N1-methylpseudridine-modified mRNA), or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP includes mRNA containing a sequence selected from the group consisting of SEQ ID NOs. 6528-8134, 9741-11347, 14628-16233, and 17840-19446, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP includes mRNA containing a sequence selected from the group consisting of SEQ ID NOs. 6529-6537, 9742-9750, 14628, 14636, and 17841-17849, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP includes mRNA containing a sequence selected from the group consisting of SEQ ID NOs. 6529-6537, 9742-9750, 14628, 14636, and 17841-17849.In some embodiments, the LNP includes mRNA containing a sequence selected from the group consisting of SEQ ID NOs: 6531, 9744, 14630, and 17843, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP includes mRNA containing a sequence selected from the group consisting of SEQ ID NOs: 6531, 9744, 14630, and 17843. In some embodiments, the LNP includes mRNA containing a sequence selected from the group consisting of SEQ ID NOs: 6529, 9742, 14628, and 17841, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP includes mRNA containing a sequence selected from the group consisting of SEQ ID NOs: 6529, 9742, 14628, and 17841. In some embodiments, the LNP includes mRNA containing a sequence selected from the group consisting of SEQ ID NOs: 6530, 9743, 14629, and 17842, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the LNP includes mRNA containing a sequence selected from the group consisting of SEQ ID NOs: 6530, 9743, 14629, and 17842.In some embodiments, the LNP comprises a gRNA containing a scaffold sequence selected from the group consisting of SEQ ID NOs: 1744-1746, 2948-2956, 2958-2966, and 2968-2974, and a targeting sequence ligated to the PCSK9 gene sequence complementary to the PCSK9 gene sequence (in the case of SEQ ID NOs: 2948-2956, 2958-2966, and 2968-2974, 20 untargeted nucleotides on the 3' end are replaced with a targeting sequence complementary to the PCSK9 gene sequence, but the chemical modification on the 3' end is retained). In some embodiments, the LNP comprises a gRNA containing a sequence selected from the group consisting of SEQ ID NOs: 2278-22803. In some embodiments, the LNP comprises a gRNA containing a sequence selected from the group consisting of SEQ ID NOs: 2278-22790. In some embodiments, the disclosure provides an LNP in which a gRNA and an mRNA encoding a long-term repressor fusion protein are incorporated into a single LNP particle. In other embodiments, the disclosure provides LNPs in which a gRNA and mRNA encoding a long-term repressor fusion protein are incorporated into a separate population of LNPs, which can be formulated together in various ratios for administration.

[0221] The lipid nanoparticles and lipid nanoparticle compositions of this disclosure can be used to suppress the expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles containing one or more ionizable lipids as described herein, wherein the lipid nanoparticles encapsulate or associate with nucleic acids expressed to produce a 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 suppress the expression of a target gene both in vitro and in vivo by contacting cells with lipid nanoparticles containing 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 that suppresses a targeted gene. The lipid nanoparticles and compositions of embodiments of this disclosure may also be used separately or in combination for the co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA), which may be useful, for example, to provide effects requiring the co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene repressor or enzyme and gRNA for gene targeting).

[0222] In some embodiments, the LNPs and LNP compositions described herein include at least one cationic lipid, at least one conjugate lipid, at least one steroid or its derivative, at least one additional lipid, or any combination thereof. Alternatively, the lipid compositions of the Disclosure may include, for example, ionizable lipids such as ionizable cationic lipids, helper lipids (usually phospholipids), cholesterol, and polyethylene glycol lipid conjugates (PEG lipids) to improve colloidal stability in the biological environment by reducing the specific absorption of plasma proteins and forming a hydrate layer on 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 adjust individual properties.

[0223] The LNPs and LNP compositions of this disclosure are configured to protect and deliver the encapsulated payload of the systems of this disclosure to tissues and cells, both in vitro and in vivo. Various embodiments of the LNPs and LNP compositions of this disclosure are described in further detail herein.

[0224] Cationic lipids In some embodiments, the LNPs and LNP compositions of this disclosure include 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 having a net positive charge at a selected pH, such as physiological pH. In some embodiments, the ionizable cationic lipid has a pKa of less than 7 so that the LNPs and LNP compositions achieve efficient encapsulation of the payload at relatively low pH. In some embodiments, the cationic lipid has a pKa of 5–8, 5.5–7.5, 6–7, or 6.5–7. In some embodiments, the cationic lipid may be protonated at a pH below the pKa of the cationic lipid, and it may be substantially neutral at pH above the pKa. LNPs and LNP compositions can be safely delivered in vivo to target organs (e.g., liver, lungs, heart, spleen, and tumors) and / or cells (hepatocytes, LSECs, cardiac cells, cancer cells, etc.) and, after endocytosis, exhibit a positive charge for releasing the encapsulated payload via electrostatic interactions with anionic proteins of the endosomal membrane.

[0225] Initial formulations of LNPs utilizing permanently cationic lipids resulted in positively surface-charged LNPs that were proven toxic in vivo and were rapidly removed by phagocytic cells. By converting LNPs to tertiary amines, particularly ionizable cationic lipids with pKa less than 7, efficient encapsulation of nucleic acid polymers at low pH is achieved by electrostatically interacting with the negative charge of the mRNA phosphate backbone. This also results in a predominantly neutral system at physiological pH values, thus mitigating problems associated with permanently charged cationic lipids.

[0226] As used herein, “ionizable lipids” means amine-containing lipids that can be readily protonated, and which may be lipids whose charge state changes depending on the ambient pH. Ionizable lipids may be protonated (positively charged) at pH below the pKa of cationic lipids, and may be substantially neutral at pH above their pKa. In one example, LNPs may include protonated ionizable lipids and / or ionizable lipids that exhibit neutralization. In some embodiments, LNPs have pKas of 5–8, 5.5–7.5, 6–7, or 6.5–7. The pKa of an LNP may 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, lungs, heart, spleen, and tumors) and / or target cells (hepatocytes, LSECs, cardiac cells, cancer cells, etc.) and, after endocytosis, exhibit a positive charge for releasing the encapsulated payload via electrostatic interactions with anionic proteins of the endosomal membrane.

[0227] Ionizable lipids are ionizable compounds that generally have characteristics 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 as disclosed herein).

[0228] Depending on the type of amine and tail group contained in the ionizable lipid, the (i) nucleic acid encapsulation efficiency, (ii) PDI (polydispersion index), and / or (iii) nucleic acid delivery efficiency to tissues and / or cells constituting organs (e.g., hepatocytes or hepatic sinusoidal endothelial cells in the liver) of the LNP may differ. In certain embodiments, the ionizable lipid is an ionizable cationic lipid, comprising about 46 mol% to about 66 mol% of the total lipids present in the particles.

[0229] LNPs containing ionizable lipids including amines may have one or more of the following characteristics: (1) the ability to encapsulate nucleic acids with high efficiency, (2) uniform size of the prepared particles (or having a low PDI value), and / or (3) excellent nucleic acid delivery efficiency to organs such as the liver, lungs, heart, spleen, and bone marrow, as well as tumors, and / or cells constituting such organs (e.g., hepatocytes, LSECs, cardiac cells, cancer cells, etc.).

[0230] In certain embodiments, cationic lipid forms play a crucial role in both nucleic acid encapsulation via electrostatic interactions and intracellular release by disrupting the endosomal membrane. Nucleic acid payloads are encapsulated within LNPs by ionic interactions they form with positively charged cationic lipids. Non-limiting examples of cationic lipid components used in the LNPs of this disclosure are selected from DLin-MC3-DMA (heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate), DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), and TNT (1,3,5-triazinan-2,4,6-trione) and TT (N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide). Non-limiting examples of helper lipids used in the LNPs of this disclosure include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine), and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 1,2-dioleoyl-sn-glycero-3-phospho(1'-rac-glycerol)DOPG, 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), sphingolipids, and ceramides. Cholesterol and PEG-DMG((R)-2,3-bis(octadecyloxy)propyl-1-(methoxypolyethylene glycol 2000)carbamate), PEG-DSG(1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000), or DSPE-PEG2k(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) are components used in the LNPs of this disclosure for the stability, circulation, and size of the LNPs.

[0231] In some embodiments, the cationic lipid in the LNP of this disclosure comprises a tertiary amine. In some embodiments, the tertiary amine comprises an alkyl chain connected to the nitrogen of the tertiary amine having an ether linkage. In some embodiments, the alkyl chain comprises a C12-C30 alkyl chain having 0-3 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. Some cationic lipids and related analogues are described in U.S. Patent Publications 20060083780, 20060240554, 20110117125, 20190336608, 20190381180, and 20200121809, U.S. Patents 5,208,036, 5,264,618, 5,279,833, and 5,2 These disclosures are described in publications 83,185, 5,753,613, 5,785,992, 9,738,593, 10,106,490, 10,166,298, 10,221,127, and 11,219,634 of the PCT, as well as PCT Publication WO96 / 10390, the entirety of which is incorporated herein by reference.

[0232] In some embodiments, the cationic lipid in the LNP of the Disclosure may include, for example, one or more ionizable cationic lipids, the ionizable cationic lipids being dialkyl lipids. In other embodiments, the ionizable cationic lipids are trialkyl lipids.

[0233] In some embodiments, the cationic lipids in the LNPs of this disclosure are 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), and 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-di Oxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino (methylpepiazino)-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl L-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinole Oil-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N- Dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-C hol), N-(1,2-dimyrityloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propanaminonium trifluoroacetic acid (DOSPA), dioctadecylamide glycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDM) A) Selected from 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), and any combination thereof.

[0234] In some embodiments, the cationic lipids in the LNPs of the present disclosure are selected from heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (1,3,5-triazinan-2,4,6-trione) (TNT), N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT), and any combination thereof.

[0235] In some embodiments, the N / P ratio (nitrogen from cationic / ionizable lipids and phosphate from nucleic acids) in the LNPs of this disclosure is in the range of about 3:1 to 7:1, or about 4:1 to 6:1, or 3:1, 4:1, 5:1, 6:1, or 7:1.

[0236] Conjugate lipids In some embodiments, the LNPs and LNP compositions of the Disclosure include 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 can suppress aggregation of the LNPs of the Disclosure.

[0237] In some embodiments, the conjugated lipids of the LNPs of this disclosure include 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 conjugated to a polyethylene glycol (PEG) polymer, which is a hydrophilic polymer. Pegylated lipids contribute to the stability of LNPs and LNP compositions and reduce LNP aggregation.

[0238] Since PEG-lipids can form surface lipids, the size of LNPs can be easily 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 circulation time.

[0239] Lipid-PEG conjugates contribute to the particle stability of nanoparticles within LNPs in serum and play a role in preventing aggregation between nanoparticles. Furthermore, 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 degrading enzymes during in vivo delivery of nucleic acids, thereby enhancing the in vivo stability of nucleic acids and increasing the half-life of 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 certain embodiments, the PEG-lipid conjugate is selected from the group consisting of PEG-diacylglycerol (PEG-DAG) conjugate, PEG-dialkyloxypropyl (PEG-DAA) conjugate, PEG-phospholipid conjugate, PEG-ceramide (PEG-Cer) conjugate, and mixtures thereof.

[0240] In some embodiments, the pegylated lipids of the LNPs of this disclosure are selected from PEG-ceramide, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-dialkoxypropyl carbamate, PEG-phosphatidylethanolamine, PEG-phospholipids, PEG-diacylglycerol succinate, and any combination thereof.

[0241] In some embodiments, the pegylated lipid of the LNP of this disclosure is PEG-dialkyloxypropyl. In some embodiments, the pegylated lipid is selected from PEG-didecyloxypropyl (C10), PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), and any combination thereof.

[0242] In other embodiments, the lipid-PEG conjugate of the LNP of this disclosure may be phospholipid-conjugated PEG, such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide (PEG-CER, ceramide-PEG conjugate, ceramide-PEG, PEG conjugated to cholesterol or its derivatives, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE (DSPE-PEG), and mixtures thereof, for example, C16-PEG2000 ceramide (N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}), DMG-PEG2000, 14:0 PEG2000 PE.

[0243] In some embodiments, the pegylated lipids of the LNPs of the present disclosure are selected from 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol, 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, and any combination thereof.

[0244] In some embodiments, the pegylated lipids of the LNPs of this disclosure are selected from mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG), 1-[8'-(1,2-dimiristoyl-3-propanoxy)-carboxamide-3',6'-dioxaoctanyl]carbamoyl-w-methyl-poly(ethylene glycol) (2KPEG-DMG), and any combination thereof.

[0245] In some embodiments, PEG is directly bound to the lipids of the pegylated lipid. In other embodiments, PEG is bound to the lipids of the pegylated lipid by a linker moiety selected from ester-free or ester-containing linker moieties. Non-limiting examples of ester-free linker moieties include amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof. For example, the linker may contain carbamate linker moieties and amide linker moieties. Non-limiting examples of ester-containing linker moieties include carbonates (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonic acid esters, and combinations thereof.

[0246] The PEG portion of the pegylated lipids of the LNPs described herein may have an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In certain embodiments, the PEG portion may have an average molecular weight of 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.

[0247] In some embodiments, conjugated lipids (e.g., pegylated lipids) constitute 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 certain embodiments, the conjugated lipids constitute about 0.5 mol% to about 3 mol% of the total lipids present in the particles.

[0248] In additional embodiments, the conjugate lipids of the LNPs of this disclosure (e.g., pegylated lipids) constitute 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 LNPs and / or LNP composition, or any intermediate range as described above.

[0249] The lipids in the lipid-PEG conjugate of the LNPs of this disclosure may be any lipid that can be bound to polyethylene glycol, and other components of the LNP, such as phospholipids and / or cholesterol, may also be used. In some embodiments, the lipids in the lipid-PEG conjugate may be, but are not limited to, ceramide, dimyristoyl glycerol (DMG), succinoyl diacylglycerol (s-DAG), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine (DSPE), or cholesterol.

[0250] In the lipid-PEG conjugates of LNPs of this disclosure, PEG can be directly conjugated to the lipid or linked to the lipid via a linker moiety. Any linker moiety suitable for linking PEG to the lipid may be used, including, for example, ester-free linker moieties and ester-containing linker moieties. Ester-free linker moieties include, but are not limited to, amides (-C(O)NH-), aminos (-NR-), carbonyls (-C(O)-), carbamates (-NHC(O)O-), ureas (-NHC(O)NH-), disulfides (-SS-), ethers (-O-), succinyls (-(O)CCH2CH2C(O)-), succinamidyls (-NHC(O)CH2CH2C(O)NH-), ethers, disulfides, and combinations thereof (e.g., linkers containing both carbamate linker moieties and amide linker moieties). The ester-containing linker portion includes, but is not limited to, carbonate esters (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonic acid esters, and combinations thereof.

[0251] steroid In some embodiments, the LNPs and LNP compositions of the present disclosure comprise at least one steroid or a derivative thereof. In some embodiments, the steroid comprises cholesterol. In some embodiments, the LNPs and LNP compositions comprise cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and cholesterol derivatives selected from any combination thereof.

[0252] In some embodiments, the steroid of the LNP of the Disclosure (e.g., cholesterol) constitutes 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 other embodiments, the steroid of the LNP of the Disclosure (e.g., cholesterol) constitutes at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol%, or any intermediate range among those described above, of the total lipids present in the LNP and / or LNP composition.

[0253] Additional lipids In some embodiments, the LNPs and LNP compositions of this disclosure include at least one additional lipid. In some embodiments, the additional lipid is a noncationic lipid selected from anionic lipids, neutral lipids, or both. In some embodiments, the additional lipid includes at least one phospholipid. In some embodiments, the phospholipid is selected from anionic phospholipids, neutral phospholipids, or both. The phospholipids of the LNP and LNP composition elements may also play a role in covering and protecting the LNP core formed by the interaction of cationic lipids and nucleic acids in the LNP, and may facilitate cell membrane translocation and endosomal escape during intracellular delivery of nucleic acids by binding to the phospholipid bilayer of target cells. Phospholipids that can facilitate the fusion of LNPs into cells may include, but are not limited to, any of the phospholipids selected from the group described below.

[0254] In some embodiments, the LNP and LNP composition are, but are not limited to, dipalmitoyl-phosphatidylcholine (DPPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylethanolamine (DOPE), dioleoyl-phosphatidylcholine (DOPC), dioleoyl-phosphatidylglycerol (DOPG), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-phosphatidylglycerol (DPPG), dimyristoyl-phosphatidylethanolamine ( The LNP comprises at least one phospholipid selected from DMPE, distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), phosphatidylethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], and any combination thereof. For example, an LNP containing DOPE may be effective in mRNA delivery (excellent drug delivery efficacy).

[0255] In some embodiments, the additional lipids (e.g., phospholipids) of the LNPs of the Disclosure constitute 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 LNPs and / or LNP compositions. In other embodiments, the additional lipids (e.g., phospholipids) of the LNPs of the Disclosure constitute at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol%, or any intermediate range as described above, of the total lipids present in the LNPs and / or LNP compositions.

[0256] It will be understood that the total lipids present in LNPs and / or LNP compositions include a combination of cationic lipids or ionizable cationic lipids, conjugate lipids (e.g., pegylated lipids), steroids (e.g., cholesterol), and additional lipids (e.g., phospholipids).

[0257] LNPs and / or LNP compositions can be prepared by dissolving total lipids (or a portion thereof) in an organic solvent (e.g., ethanol) and subsequently mixing them with a payload (e.g., nucleic acids of the system) 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. The resulting nanostructures containing nucleic acids are then dialyzed against a neutral buffer to be converted to neutral LNPs, after which the buffer can be replaced with a physiologically relevant buffer for the LNPs by removing the organic solvent (e.g., ethanol). The LNPs and / or LNP compositions thus formed have a distinct electron-density nanostructure core, organized into inverse micelles around the payload containing the cationic lipids, in contrast to conventional bilayer liposome structures. In another embodiment, the LNPs may form bleb-like structures having nucleic acids in aqueous pockets along the electron-density lipid core.

[0258] b. Lipid nanoparticle properties In some embodiments, the LNP and / or 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 conjugate 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 additional lipids (e.g., phospholipids). In some embodiments, the LNP and / or 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 conjugate 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 additional lipids (e.g., phospholipids).

[0259] In some embodiments, the LNPs and / or LNP compositions of the present disclosure comprise cationic lipids: additional lipids (e.g., phospholipids): steroids (e.g., cholesterol): conjugate lipids (e.g., pegylated lipids) in molar ratios of 20-50:10-30:30-60:0.5-5, 25-45:10-25:40-50:0.5-3, 25-45:10-20:40-55:0.5-3, or 25-45:10-20:40-55:1.0-1.5.

[0260] In some embodiments, the LNPs and / or LNP compositions of this disclosure have a total lipid:payload ratio (mass / mass) of about 1 to about 100. In some embodiments, the total lipid:payload ratio is about 1 to about 50, about 2 to about 25, about 3 to about 20, about 4 to about 15, or about 5 to about 10. In some embodiments, the total lipid:payload ratio is about 5 to about 15, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or any intermediate range as described above.

[0261] In certain embodiments, the LNPs of this disclosure contain a total lipid:nucleic acid mass ratio of about 5:1 to about 15:1. In some embodiments, the weight ratio of cationic lipids to nucleic acids contained in the LNPs 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.

[0262] In some embodiments, the LNPs of the present disclosure 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 lipid-PEG conjugate. Alternatively, the LNPs may comprise, based on the total weight of nanoparticles, 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 lipid-PEG conjugate. As a further alternative, LNPs may contain, based on the total nanoparticle weight, 25–50% by weight of cationic lipids, 10–20% by weight of phospholipids, 35–55% by weight of cholesterol, and 0.1–10% by weight (or 0.25–10% by weight, 0.5–5% by weight) of lipid-PEG conjugates.

[0263] In some embodiments, the LNPs of this disclosure have wavelengths of approximately 20-200nm, 20-180nm, 20-170nm, 20-150nm, 20-120nm, 20-100nm, 20-90nm, 30-200nm, 30-180nm, 30-170nm, 30-150nm, 30-120nm, 30-100nm, 30-90nm, 40-200nm, 40-180nm, 40-170nm, 40-150nm, 40-120nm, 40-100nm, 40-90nm, 40-80nm, 40-70nm, 50-200nm, 50-180nm, 50-170nm, 50-150nm, 50-120nm, 50-100nm, It has an average diameter in the following ranges: 50-90nm, 60-200nm, 60-180nm, 60-170nm, 60-150nm, 60-120nm, 60-100nm, 60-90nm, 70-200nm, 70-180nm, 70-170nm, 70-150nm, 70-120nm, 70-100nm, 70-90nm, 80-200nm, 80-180nm, 80-170nm, 80-150nm, 80-120nm, 80-100nm, 80-90nm, 90-200nm, 90-180nm, 90-170nm, 90-150nm, 90-120nm, or 90-100nm, or an intermediate range among any of the above.

[0264] In some embodiments, the LNPs and / or LNP compositions of this disclosure have a positive charge at an acidic pH and can encapsulate a payload (e.g., a therapeutic agent, such as an LTRP:gRNA system, or a polynucleotide encoding it) by the static charge generated by the negative charge of the payload. The term "encapsulation" refers to the mixture of lipids forming an LNP that surrounds and embeds a payload (e.g., a therapeutic agent) under physiological conditions. 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 LNP. The encapsulation efficiency of the LNPs and / or LNP compositions may be 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 94% or higher, or 95% or higher. In other embodiments, the encapsulation efficiency of LNPs and / or LNP compositions is about 80%–99%, about 85%–98%, about 88%–95%, about 90%–95%, or the payload (e.g., nucleic acids of the system) can be completely encapsulated within the lipid portion of the LNP composition, thereby protecting it from enzymatic degradation. In some embodiments, the payload (e.g., therapeutic agent) remains substantially undegraded even after exposure of the LNPs and / or LNP compositions 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. In some embodiments, the payload (e.g., nucleic acids of the system) is complexed with the lipid portion of the LNPs and / or LNP compositions. The LNPs and / or LNP compositions of this disclosure are nontoxic to mammals such as humans.

[0265] The term "fully encapsulated" indicates that the payload (e.g., the nucleic acid in the system) in the LNP and / or LNP composition does not significantly degrade after exposure to conditions that degrade free DNA, RNA, or protein. In a fully encapsulated system, less than about 25%, more preferably less than about 10%, and most preferably less than 5% of the payload (e.g., the nucleic acid in the system) in the LNP and / or LNP composition is degraded by conditions that would degrade 100% of an unencapsulated payload. "Fully encapsulated" also indicates that the LNP and / or LNP composition are serum stable and do not degrade into their constituent parts upon in vivo administration.

[0266] In some embodiments, the amount of payload (e.g., therapeutic agent) encapsulated in the LNP and / or LNP composition is approximately 30% to 100%, approximately 40% to 100%, approximately 50% to 100%, approximately 60% to 100%, approximately 70% to 100%, approximately 80% to 100%, approximately 90% to 100%, approximately 30% to 95%, approximately 40% to 95%, approximately 50% to 95%, approximately 60% to 95%, approximately 70% to 95%, approximately 80% to 95%, approximately 85% 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 an intermediate range of any of the above.

[0267] In some embodiments, the amount of payload (e.g., nucleic acid) encapsulated in the LNP and / or LNP composition is approximately 30% to 100%, approximately 40% to 100%, approximately 50% to 100%, approximately 60% to 100%, approximately 70% to 100%, approximately 80% to 100%, approximately 90% to 100%, approximately 30% to 95%, approximately 40% to 95%, approximately 50% to 95%, approximately 60% to 95%, approximately 70% to 95%, approximately 80% to 95%, and approximately 85%. ~95%, ~90%~95%, ~30%~90%, ~40%~90%, ~50%~90%, ~60%~90%, ~70%~90%, ~80%~90%, or at least ~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 among the above.

[0268] In some embodiments, nucleic acids of the present disclosure, such as mRNA and / or gRNA encoding long-term repressor fusion proteins, may be provided in a solution that is mixed with a lipid solution so that the nucleic acids can be encapsulated in lipid nanoparticles. A suitable nucleic acid solution may be any aqueous solution containing nucleic acids to be encapsulated at various concentrations. For example, a suitable nucleic acid solution may contain 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 or higher. In some embodiments, the nucleic acid comprises mRNA encoding a long-term repressor fusion protein, and suitable mRNA solutions are approximately 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. It may contain mRNA at concentrations in the range of 0.1 mg / ml, 0.05-1.0 mg / ml, 0.05-0.9 mg / ml, 0.05-0.8 mg / ml, 0.05-0.7 mg / ml, 0.05-0.6 mg / ml, 0.05-0.5 mg / ml, 0.05-0.4 mg / ml, 0.05-0.3 mg / ml, 0.05-0.2 mg / ml, 0.05-0.1 mg / ml, 0.1-1.0 mg / ml, 0.2-0.9 mg / ml, 0.3-0.8 mg / ml, 0.4-0.7 mg / ml, or 0.5-0.6 mg / ml.In some embodiments, a suitable mRNA solution may contain mRNA at concentrations of up to approximately 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 at concentrations of up to approximately 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.

[0269] In some embodiments, LNPs are found in the following wavelength ranges: 20nm-200nm, 20-180nm, 20nm-170nm, 20nm-150nm, 20nm-120nm, 20nm-100nm, 20nm-90nm, 30nm-200nm, 30-180nm, 30nm-170nm, 30nm-150nm, 30nm-120nm, 30nm-100nm, 30nm-90nm, and 40 nm~200nm, 40~180nm, 40nm~170nm, 40nm~150nm, 40nm~120nm, 40nm~100nm, 40nm~90nm, 40nm~80nm, 4 0nm~70nm, 50nm~200nm, 50~180nm, 50nm~170nm, 50nm~150nm, 50nm~120nm, 50nm~100nm, 50nm~90nm, 6 0nm~200nm, 60~180nm, 60nm~170nm, 60nm~150nm, 60nm~120nm, 60nm~100nm, 60nm~90nm, 70nm~200nm , 70~180nm, 70nm~170nm, 70nm~150nm, 70nm~120nm, 70nm~100nm, 70nm~90nm, 80nm~200nm, 80~180nm, LNPs may have average diameters of 80nm–170nm, 80nm–150nm, 80nm–120nm, 80nm–100nm, 80nm–90nm, 90nm–200nm, 90–180nm, 90nm–170nm, 90nm–150nm, 90nm–120nm, or 90nm–100nm for easy delivery into liver tissue, hepatocytes, and / or LSECs (hepatic sinusoidal endothelial cells). LNPs can be sized for easy delivery into organs or tissues, including but not limited to the liver, lungs, heart, spleen, and tumors. If the size of the LNP is smaller than the above range, it becomes difficult to maintain stability as the surface area of ​​the LNP increases excessively, and therefore delivery to the target tissue and / or drug effect may be reduced. LNPs can specifically target liver tissue. While we do not wish to be bound by theory, one mechanism by which therapeutic agents can be delivered using LNPs is through mimicking the metabolic behavior of natural lipoproteins, and thus LNPs can be usefully delivered to the target through lipid metabolic processes performed by the liver.During the delivery of therapeutic agents to hepatocytes and / or LSECs (hepatic sinusoidal endothelial cells), the diameter of the window connecting the sinusoidal lumen to the hepatocytes and LSECs is approximately 140 nm in mammals and approximately 100 nm in humans. Therefore, an LNP composition for delivering therapeutic agents having LNPs with a diameter within the above range may have superior delivery efficiency to hepatocytes and LSECs compared to LNPs with a diameter outside the above range.

[0270] For example, the LNP in the LNP composition may contain cationic lipids:phospholipids:cholesterol:lipid-PEG conjugates within the above range, or in molar ratios of 20-50:10-30:30-60:0.5-5, 25-45:10-25:40-50:0.5-3, 25-45:10-20:40-55:0.5-3, or 25-45:10-20:40-55:1.0-1.5. LNPs containing components in molar ratios within the above ranges may have excellent delivery efficiency of therapeutic agents specific to cells of the target organ.

[0271] In certain embodiments, LNPs exhibit a pKa of 5-8, 5.5-7.5, 6-7, or 6.5-7, exhibiting a positive charge under acidic pH conditions. They readily form complexes with therapeutic agents such as negatively charged nucleic acids through electrostatic interactions, thereby enabling highly efficient encapsulation of nucleic acids. In such cases, LNPs can be usefully used as compositions for intracellular or in vivo delivery of therapeutic agents (e.g., nucleic acids).

[0272] In this specification, “encapsulation” or “inclusion” refers to the incorporation of an efficient delivery of a therapeutic agent, i.e., surrounding it with the surface of a particle and / or embedding it within the particle. Encapsulation efficiency means the content of the therapeutic agent encapsulated in the LNP relative to the total therapeutic agent content used in the preparation of the LNP.

[0273] The encapsulation of nucleic acids in LNPs of a composition may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 94% or more, or 95% or more of the LNPs in the composition encapsulating the nucleic acids. In some embodiments, the encapsulation of nucleic acids in LNPs of a composition may be 80% to 99%, 80% to 97%, 80% to 95%, 85% to 95%, 87% to 95%, 90% to 95%, 91% or more to 95%, 91% or more to 94%, greater than 91% to 95%, 92% to 99%, 92% to 97%, or 92% to 95% of the LNPs in the composition encapsulating the nucleic acids. In some embodiments, mRNA and gRNA encoding long-term repressor fusion proteins of any embodiment of the embodiments of this disclosure are completely encapsulated within the LNPs.

[0274] Target organs to which nucleic acids are delivered by LNPs include, but are not limited to, the liver, lungs, heart, spleen, and tumors. One example of an LNP is liver tissue specific, possesses excellent biocompatibility, and can deliver nucleic acids of its composition with high efficiency; therefore, it can be effectively used in related technological fields such as lipid nanoparticle-mediated gene therapy. In certain embodiments, the target cells to which nucleic acids are delivered by one example of an LNP may be hepatocytes and / or LSECs in vivo. In other embodiments, the present disclosure provides LNPs formulated for delivering nucleic acids of embodiments to cells ex vivo.

[0275] This disclosure provides a pharmaceutical composition comprising a plurality of LNPs, including nucleic acids such as mRNA and / or gRNA variants encoding the long-term repressor fusion proteins described herein, and a pharmaceutically acceptable carrier.

[0276] In certain embodiments, LNPs containing nucleic acids have a core with high electron density.

[0277] This disclosure provides an LNP comprising one or more nucleic acids, including (a) an mRNA and / or gRNA variant encoding a long-term repressor fusion protein as described herein; (b) 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 comprising about 13 mol% to about 49.5 mol% of the total lipids present in the LNP; and (d) one or more conjugate lipids that inhibit the aggregation of the LNP comprising about 0.5 mol% to about 2 mol% of the total lipids present in the particles. In another embodiment, the Disclosure provides an LNP comprising one or more nucleic acids, including (a) an mRNA and / or gRNA variant encoding a long-term repressor fusion protein as described herein; (b) one or more cationic 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 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) about 1 mol% to about 5 mol% of lipid-PEG or lipid-PEG-peptides in the particles. In certain embodiments, the long-term repressor fusion protein mRNA and gRNA may be present in the same nucleic acid-lipid particle or in different nucleic acid-lipid particles.

[0278] This disclosure provides an LNP comprising one or more nucleic acids, including (a) mRNA encoding a long-term repressor fusion protein as described herein; (b) a 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 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 certain embodiments, 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 salt thereof, about 7.1 mol% of DPPC (or DSPC), and about 34.3 mol% of cholesterol (or derivative thereof). In some embodiments, the LNP comprises mRNA and gRNA encoding CasX as described herein.

[0279] In other embodiments, a LNP comprising one or more nucleic acids comprises (a) mRNA and / or gRNA encoding a long-term repressor fusion protein of any embodiment described herein; (b) a 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 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 three-component system comprising a phospholipid-free PEG-lipid conjugate of about 1.5 mol% (e.g., PEG2000-C-DMA), about 61.5 mol% (e.g., DLin-K-C2-DMA) or a salt thereof, and about 36.9 mol% (cholesterol or a derivative thereof). In some embodiments, the LNP includes mRNA and gRNA encoding CasX as described herein.

[0280] Additional formulations are described in PCT Publication WO2009 / 127060, and U.S. Patent Publications 2011 / 0071208A1 and 2011 / 0076335A1, the disclosures thereof being incorporated herein by reference in their entirety.

[0281] In other embodiments, the LNP comprising one or more nucleic acids comprises (a) mRNA and gRNA variants 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 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 comprising about 5 mol% to about 90 mol% of the total lipids present in the LNP; and (d) one or more conjugate lipids that inhibit particle aggregation comprising about 0.5 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, the LNP comprises mRNA and gRNA encoding CasX as described herein.

[0282] In other embodiments, an LNP comprising one or more nucleic acids comprises (a) mRNA and / or gRNA encoding a long-term repressor fusion protein of any embodiment described herein; (b) a cationic lipid or salt thereof comprising about 30 mol% to about 50 mol% of the total lipids present in the LNP; (c) a mixture of phospholipids and cholesterol or derivatives thereof comprising about 47 mol% to about 69 mol% of the total lipids present in the LNP; and (d) a PEG-lipid conjugate comprising about 1 mol% to about 3 mol% of the total lipids present in the LNP. In a particular embodiment, the formulation is a four-component system comprising about 2 mol% of a PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 40 mol% of a cationic lipid (e.g., DLin-K-C2-DMA) or salt thereof, about 10 mol% of DPPC (or DSPC), and about 48 mol% of cholesterol (or a derivative thereof). In some embodiments, the LNP includes mRNA and gRNA encoding CasX as described herein.

[0283] In other embodiments, the LNP comprising o...

Claims

1. A system for repressing the transcription of the progenitor protein convertase subtilisin / kexin type 9 (PCSK9) gene, (a) A guide ribonucleic acid (gRNA) containing a targeting sequence complementary to the PCSK9 gene target nucleic acid sequence, (b) mRNA encoding a long-term repressor fusion protein (LTRP), wherein the LTRP is a. CasX without catalytic activity (dCasX), b. DNA methyltransferase (DNMT) 3A catalytic domain (DNMT3A), c. DNMT3-like interaction domain (DNMT3L), and d. mRNA containing the first repressor domain (RD1), A system in which the LTRP can form ribonucleoprotein (RNP) together with the gRNA.

2. The aforementioned LTRP extends from the N-terminus to the C-terminus, a. The DNMT3A mentioned above, b. The aforementioned DNMT3L, c. The dCasX and d. The system according to claim 1, comprising the RD1.

3. The aforementioned LTRP extends from the N-terminus to the C-terminus, a. The DNMT3A mentioned above, b. The aforementioned DNMT3L, c. The RD1 and d. The system according to claim 1, comprising the dCasX.

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

5. The system according to claim 4, wherein the arrangement of RD1 is the same.

6. The system according to claim 4, wherein the arrangement of RD1 is different.

7. The system according to any one of claims 1 to 6, wherein the LTRP includes a DNA DNMT3A ATRX-DNMT3-DNMT3L domain (ADD) ligated 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 linker peptides.

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

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

11. The system according to claim 10, wherein at least one of the one or more NLSs includes a sequence selected from the group consisting of sequence numbers 30 to 97.

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

13. The system according to claim 12, wherein the SV40 NLS includes the sequence of sequence number 30.

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

15. The system according to claim 14, wherein the c-MYC NLS includes the sequence of sequence number 32.

16. The aforementioned LTRP is located from the N-terminus to the C-terminus. a. The first NLS, b. The DNMT3A mentioned above, c. The first linker peptide, d. The aforementioned DNMT3L, e. Second linker peptide, f. The third linker peptide, g. The dCasX, h. The fourth linker peptide, i. The RD1 and j. The system according to any one of claims 1 to 2 and 8 to 15, comprising a second NLS.

17. The system according to claim 16, wherein the LTRP includes an array selected from the group consisting of sequence numbers 22836 and 22838 to 22846.

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

19. The system according to claim 18, wherein the LTRP includes an array selected from the group consisting of sequence numbers 22837 and 22847 to 22855.

20. The aforementioned LTRP is located from the N-terminus to the C-terminus. a. The first NLS, b. The DNMT3A mentioned above, c. The first linker peptide, d. The aforementioned DNMT3L, e. The third linker peptide, f. The aforementioned RD1, g. Second linker peptide, h. The aforementioned dCasX, i. The fourth linker peptide, and j. The system according to any one of claims 1, 3, and 8-15, comprising a second NLS.

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

22. The aforementioned LTRP is located from the N-terminus to the C-terminus. a. The first NLS, b. The ADD, c. The DNMT3A mentioned above, d. The first linker peptide, e. The aforementioned DNMT3L, f. The second linker peptide, g. The aforementioned RD1, h. The third linker peptide, i. The aforementioned dCasX, j. The fourth linker peptide, k. Additional RD1, l. The fifth linker, and m. The system according to any one of claims 7 to 15, comprising a second NLS.

23. The system according to claim 22, wherein the arrangement of the aforementioned RD1 and the additional RD1 are identical.

24. The system according to claim 22, wherein the arrangement of the RD1 and the additional RD1 are different.

25. The system according to any one of claims 1 to 24, wherein the dCasX includes a sequence selected from the group consisting of sequence numbers 4 to 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 therewith.

26. The system according to claim 25, wherein the dCasX includes the sequence of sequence number 4, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

27. The system according to claim 26, wherein the dCasX consists of the sequence of sequence number 4.

28. The system according to any one of claims 1 to 27, wherein RD1 includes a sequence selected from the group consisting of sequence numbers 128 to 1726, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

29. The system according to any one of claims 1 to 27, wherein RD1 includes a sequence selected from the group consisting of sequence numbers 130 to 138, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

30. The system according to any one of claims 1 to 27, wherein RD1 includes the sequence of sequence number 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 thereto.

31. The system according to any one of claims 1 to 27, wherein RD1 includes the sequence of sequence number 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 thereto.

32. The system according to any one of claims 1 to 27, wherein RD1 includes the sequence of sequence number 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 thereto.

33. The system according to any one of claims 1 to 27, wherein RD1 includes the sequence of sequence number 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 thereto.

34. The system according to any one of claims 1 to 27, wherein RD1 includes the sequence of sequence number 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 thereto.

35. The system according to any one of claims 1 to 27, wherein RD1 includes the sequence of sequence number 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 thereto.

36. The system according to any one of claims 1 to 27, wherein RD1 includes the sequence of sequence number 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 thereto.

37. The system according to any one of claims 1 to 27, wherein RD1 includes the sequence of sequence number 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 thereto.

38. The system according to any one of claims 1 to 27, wherein RD1 includes the sequence of sequence number 138, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

39. The system according to any one of claims 7 to 15, 18, 19 and 19 to 38, wherein the ADD includes the sequence of sequence number 125, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

40. The system according to any one of claims 1 to 39, wherein the DNMT3A includes the sequence of sequence number 126, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

41. The system according to any one of claims 1 to 40, wherein the DNMT3L includes the sequence of sequence number 127, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

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

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

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

45. The system according to any one of claims 1 to 41, wherein the sequence encoding RD1 includes a sequence selected from the group consisting of sequence number 3335 and sequence number 4932, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

46. The system according to any one of claims 1 to 41, wherein the sequence encoding RD1 includes a sequence selected from the group consisting of sequence number 3339 and sequence number 4936, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

47. The system according to any one of claims 1 to 41, wherein the sequence encoding RD1 includes a sequence selected from the group consisting of sequence number 3334 and sequence number 4931, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

48. The system according to any one of claims 1 to 41, wherein the sequence encoding RD1 includes a sequence selected from the group consisting of sequence number 3336 and sequence number 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 thereto.

49. The system according to any one of claims 1 to 41, wherein the sequence encoding RD1 includes a sequence selected from the group consisting of sequence number 3337 and sequence number 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 thereto.

50. The system according to any one of claims 1 to 41, wherein the sequence encoding RD1 includes a sequence selected from the group consisting of sequence number 3338 and sequence number 4935, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

51. The system according to any one of claims 1 to 41, wherein the sequence encoding RD1 includes a sequence selected from the group consisting of sequence number 3340 and sequence number 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 therewith.

52. The system according to any one of claims 1 to 41, wherein the sequence encoding RD1 includes a sequence selected from the group consisting of sequence number 3341 and sequence number 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 thereto.

53. The system according to any one of claims 1 to 41, wherein the sequence encoding RD1 includes a sequence selected from the group consisting of sequence number 3342 and sequence number 4939, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

78. The system according to any one of claims 1, 2, 7-15, 18, 19 and 25-56, wherein the mRNA encoding the LTRP includes a sequence selected from sequence number 9750 and sequence number 17849, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

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

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

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

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

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

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

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

86. The system according to 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 or all of the uridine residues of the mRNA are replaced with 1-methyl-pseudridine.

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 according to claim 88, wherein the 5'UTR includes the sequence of sequence number 3300.

90. The system according to claim 88 or 89, wherein the 3'UTR includes the sequence of sequence number 3310.

91. The system according to claims 88 to 90, wherein the poly(A) includes the sequence of sequence number 3057.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

107. The system according to any one of claims 1 to 103, wherein the PCSK9 gene target nucleic acid sequence is located 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 gene enhancer.

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

110. The target sequences of the gRNA are SEQ ID NOs: 1824-1881, 1883-1897, 1899-1934, 1937-1991, 1993-1999, 2001-2026, 2029-2057, 2059-2106, 2108-2114, 2116-2122, 2124-2139, 2141-2209, 2211-2212, 2214-2233, 2235-2252, 2254-2269, 2271-2 The system according to any one of claims 1 to 109, comprising an array selected from the group consisting of 281, 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 includes a sequence selected from the group consisting of SEQ ID NOs: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714.

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

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

114. The system according to any one of claims 110 to 113, wherein the targeting sequence of the gRNA has 1, 2, 3, 4, or 5 nucleotides removed from the 3' end of the 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 includes a scaffold stem loop containing the sequence CCAGCGGACUAUGUCGUAGUGG (Sequence ID 1822), or a sequence having one, two, three, four, or five mismatches therewith.

117. The system according to any one of claims 1 to 116, wherein the gRNA includes a scaffold containing a sequence selected from the group consisting of SEQ ID NOs: 1744 to 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%, and at least about 99% sequence identity thereto.

118. The system according to any one of claims 1 to 116, wherein the gRNA includes a scaffold containing the sequence of Sequence 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 thereto.

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

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

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

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

123. The system according to any one of claims 120 to 122, wherein the chemical modification to the gRNA includes substitution of phosphorothioate bonds between two or more nucleosides of the gRNA.

124. The system according to claim 123, wherein the chemical modification includes substitution of phosphorothioate bonds between two or more nucleotides located at 1, 2, 3, or 4 nucleotides from the 5' end, the 3' end, or both ends of the gRNA.

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

126. The system according to any one of claims 104 to 124, wherein the gRNA includes a sequence selected from the group consisting of sequence numbers 22788 to 22803.

127. The system according to any one of claims 104 to 124, wherein the gRNA includes a sequence selected from the group consisting of sequence numbers 22788 to 22790.

128. Lipid nanoparticles comprising the system according to any one of claims 1 to 127.

129. Lipid nanoparticles (LNPs) 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 according to claim 128 or 129, wherein the LNP comprises one or more components selected from the group consisting of ionizable lipids, one or more helper phospholipids, one or more polyethylene glycol (PEG) modified lipids, and cholesterol or derivatives thereof.

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

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

133. a. The system according to any one of claims 1 to 127, or b. An LNP according to any one of claims 128 to 132, A pharmaceutical composition comprising 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 according to claim 134, wherein the average diameter of the lipid nanoparticles in the plurality of lipid nanoparticles is about 20 nm to about 200 nm.

136. The pharmaceutical composition according to claim 134 or 135, wherein the pharmaceutical composition is formulated for a route of administration selected from the group consisting of intravenous, intraarterial, intraportal venous injection, intraperitoneal, intramuscular, intraventricular, intracisional, subarachnoid, intracranial, intralumbar, intraocular, subcutaneous, and oral routes.

137. A method for suppressing the transcription of the PCSK9 gene in a population of cells, wherein the method involves 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. A pharmaceutical composition according to any one of claims 133 to 136, or d. Introduction of two or more combinations of (a) to (c), A method comprising suppressing the transcription of the PCSK9 gene in the population of cells.

138. The method according to claim 137, wherein the transcription of the PCSK9 gene is suppressed 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%, or at least about 80% or more of the cells in the population.

139. The method according to claim 137 or 138, wherein the transcription of the PCSK9 gene 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% compared to untreated cells.

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

141. The method according to 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 according to 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 liver cells, 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 population of cells occurs in vitro or ex vivo.

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

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

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

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

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

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

151. The method according to claim 150, wherein the suppression is reversible by the use of a DNMT inhibitor.

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

153. The method according to claim 151 or 152, wherein the inhibitor of DNMT is selected from the group consisting of azacitidine, decitabine, clofarabine, and zebralin.

154. A method for treating PCSK9-related disease in a patient requiring treatment for PCSK9-related disease, wherein the therapeutic effective dose is 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. Administering the pharmaceutical composition according to any one of claims 133 to 136. A method including treating PCSK9-related disorders.

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

156. The method according to claim 154 or 155, wherein the LNP is administered by a route of administration selected from the group consisting of intravenous, intraarterial, intraportal venous injection, intraperitoneal, intramuscular, intraventricular, intracisional, subarachnoid, intracranial, intralumbar, intraocular, subcutaneous, and oral routes.

157. The method according to any one of claims 154 to 156, wherein the PCSK9-related disorder is autosomal dominant hypercholesterolemia (ADH), hypercholesterolemia, elevated total cholesterol levels, dyslipidemia, elevated low-density lipoprotein (LDL) levels, elevated LDL cholesterol levels, decreased high-density lipoprotein levels, fatty liver, coronary artery disease, ischemia, stroke, peripheral vascular disease, thrombosis, type 2 diabetes, hypertension, atherosclerosis, obesity, aortic stenosis, elevated PCSK9 levels, or a combination thereof.

158. The method according to any one of claims 154 to 157, wherein the method results in an improvement in at least one clinically relevant endpoint selected from the group consisting of a change from baseline in LDL cholesterol, a reduction in plaque atheroma volume, a reduction in coronary plaque, a 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 selected from the group consisting of a change from baseline in LDL cholesterol, a reduction in plaque atheroma volume, a reduction in coronary plaque, a 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 according to any one of claims 133 to 136, or a combination thereof, and a suitable container.

161. The kit according to claim 160, comprising a buffer, an excipient, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, instructions for use, or any combination thereof.

162. A composition, 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. A composition comprising the pharmaceutical composition described in any one of claims 133 to 136.

163. The composition according to claim 162, for use in the manufacture of a pharmaceutical for the treatment of PCSK9-related disease in subjects requiring treatment for PCSK9-related disease.

164. The composition according to claim 162, for use in the treatment of PCSK9-related disease in subjects requiring treatment for PCSK9-related disease.

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