WO2025231432A1 - In vivo gene editing with crispr systems - Google Patents

Abstract

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

Description

IN VIVO GENE EDITING WITH CRISPR SYSTEMS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to a U.S. provisional patent application Serial No. 63/642,275 filed on May 3, 2024 and incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] Not applicable

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 24, 2025, is named CBI058_30_SL.xml and is 943,237 bytes in size.

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

[0005] Gene therapy holds the promise of treating genetic diseases. The initial attempts to treat genetic diseases were limited to delivering a functional copy of the gene whose expression was diminished or abolished by a mutation. For example, the use of viral vectors capable of crossing the blood-brain barrier enabled successful delivery of functional gene copies to treat neuromuscular and central nervous system disorders. Approved therapies exist for spinal muscular atrophy, and treatments for Duchenne muscular dystrophy are in clinical trials. Viral gene delivery therapies are being developed for Alzheimer’s disease and Parkinson’s disease. AAV-based gene delivery is also used to treat retinal diseases. Approved therapy exists for Leber congenital amaurosis and gene delivery treatments are in development for retinitis pigmentosa.

[0006] In addition to enhancing or restoring gene function as described above, certain conditions can be treated by inhibiting or blocking gene function. Such intervention may not always be carried out via a simple delivery of a gene expression cassette. Some gene inhibiting therapies are now available or are in development. [0007] Hereditary amyloidogenic transthyretin amyloidosis is caused by instability and proteolysis of the mutant transthyretin protein resulting in deposition of amyloid and fatal damage of the peripheral nervous system. One of the approaches to treatment has been gene silencing with small interfering RNA (siRNAs) or anti-sense oligonucleotides (ASO). While showing promise, such therapies must be frequently readministered due to the short half-life of RNA molecules.

[0008] One of the factors affecting serum cholesterol levels is the protein PSCK9 that binds to LDL receptors and promotes their internalization and lysosomal degradation. Depletion of PSCK9 with monoclonal antibodies has been shown to increase numbers of LDL receptors on the surface of hepatocytes and lowering of serum cholesterol (LDL) levels. FDA- approved PSCK9 inhibiting antibodies REPATHA® and PRALUENT® require monthly or biweekly injections in order to maintain the cholesterol-lowering effect.

[0009] There is a need for safe, effective, and long-lasting gene therapies for genetic and chronic diseases.

SUMMARY OF THE INVENTION

[0010] In one embodiment, the invention is a method of modifying a sequence of a target nucleic acid in a somatic cell in a living organism, the method comprising: contacting the organism with (i) a nucleic acid coding for a CRISPR endonuclease and (ii) a guide polynucleotide comprising a targeting region capable of hybridizing to a target sequence within the target nucleic acid, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA), and wherein the CRISPR endonuclease binds the guide polynucleotide and cleaves the target nucleic acid and the sequence of the target nucleic acid is modified within the somatic cell. In some embodiments, both the targeting region and the activating region of the guide polynucleotide comprise DNA and ribonucleic acid (RNA). In some embodiments, at least one of the targeting region and the activating region further comprises a chemical modification, e.g., a deoxyribonucleotide, a phosphorothioate ribonucleotide, a phosphorothioate deoxyribonucleotide, and a 2’-O-methyl nucleotide. In some embodiments, the guide polynucleotide comprises a sequence selected from SEQ ID NOs: 63-82, 143-434, 10-36 and 83-140. [0011] In some embodiments, the CRISPR endonuclease is a CRISPR Class 2 endonuclease, e.g., Casl2a. In some embodiments, the CRISPR endonuclease comprises a nuclear localization signal (NLS), e.g., the NLS is selected from the group consisting of SV40 large T-antigen, nucleoplasmin, 53BP1, VACM-1/CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrpl, c-Myc, Mouse c-able IV, Mata2 and MINIYO. In some embodiments, the nucleoplasmin NLS conjugated to a linker to form SEQ ID NO: 2. In some embodiments, the Casl2a comprises the amino acid sequence of SEQ ID NO: 1.

[0012] In some embodiments, the nucleic acid coding for the CRISPR endonuclease is an mRNA. In some embodiments, the mRNA comprises codon optimization for optimizing mRNA expression in mammalian cells. In some embodiments, the mRNA comprises modifications minimizing immunogenicity in mammalian cells, e.g., a uridine modification (e.g., 5-methoxyuridine, 5-methyluridine, 5-carboxymethytl ester uridine, 2-thiouridine and pseudouridine and derivatives thereof) or a cytidine modification (e.g, 2-methoxy cytidine or its derivatives). In some embodiments, the 5 ’-cap comprises a chemical modification selected from a modification to the guanosine base, a modification to the ribose sugar moiety, and a modification or to the phosphate moiety, e.g., N7-methyl guanosine, 2’-O-methyl ribose, a- thiophosphate, a-methyl phosphate, boranophosphate and selenophosphate. In some embodiments, the mRNA comprises a 5’-cap having a formula selected from the group consisting of 3’G(5’)PPP-5’, N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G, N7-(4- bromophenoxy ethyl)-m3 '-OG(5 ')ppp ( 5 ') G, 3 ’ -O-Me-m7G(5 ’ )ppp(5 ')G) . and m7(3’OMeG)(5’)ppp(5’)m6(2’OMe)pG. In some embodiments, the mRNA further comprises a 5 ’-untranslated region (UTR) and a 3’-UTR comprising eukaryotic sequences.

[0013] In some embodiments, the target sequence is a gene sequence selected from the group consisting of an enhancer sequence, a promoter sequence, an exon sequence, and an intron sequence. In some embodiments, the gene is selected from TTR, PSCK9 and ANGPTL3. [0014] In some embodiments, the nucleic acid coding for the CRISPR endonuclease and the guide polynucleotide are present in a lipid nanoparticle (LNP). In some embodiments, lipid phase comprises an ionizable cationic lipid at about 46-50%, cholesterol at about 38-43%, a phospholipid at about 9-10%, and a polyethylene glycol (PEG) derivative at about 1-2%. In some embodiments, the lipid phase comprises 6-((2-hexyldecanoyl)oxy)-N-(6-((2- hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan- 1 -aminium (ALC-0315), cholesterol, l,2-Distearoyl-sn-glycero-3-PC (1,2-DSPC), and Methoxypolyethyleneglycoloxy(2000)-N,N- ditetradecyl acetamide (ALC-0159). In some embodiments, the lipid phase comprises ALC- 0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about 1-2%. In some embodiments, the lipid phase comprises 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM102), cholesterol, 1,2-DSPC, and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). In some embodiments, the lipid phase comprises SM102 at about 50%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and DMG-PEG2000 at about 1-2%. In some embodiments, the lipid phase comprises 4-(dimethylamino)-butanoic acid, (10Z, 13Z)-1 - (9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester (MC3), cholesterol, 1,2- DSPC, and DMG-PEG2000. In some embodiments, the lipid phase comprises MC3 at about 50%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and DMG-PEG2000 at about 1- 2%. In some embodiments, the LNP are characterized by encapsulation efficiency of 70-100%. In some embodiments, the LNP are characterized by poly dispersity index of 0-0.25. In some embodiments, the LNP are characterized by diameter of 65-100 nanometers.

[0015] In some embodiments, contacting the organism is via systemic administration. In some embodiments, the rate of sequence modification at a selected genomic locus of at least 65% in vitro or in vivo in experimental animals. In some embodiments, upon sequence modification, the rate of chromosomal translocations is undetectable by sequencing. In some embodiments, upon sequence modification, the rate of off-target sequence modification is undetectable by sequencing. In some embodiments, upon sequence modification, the rate of extra-organ sequence modification is no greater than 0.6%. In some embodiments, sequence modification results in reduced expression of a gene. In some embodiments, the step of assessing the reduction in the expression of the gene. In some embodiments, the gene is TTR and the assessing comprises assessing levels of TTR protein in the blood plasma. In some embodiments, the gene is PSCK9 and the assessing comprises assessing levels of PCSK9 protein or levels of LDL cholesterol in the blood plasma. In some embodiments, the gene is ANGPTL3 and the assessing comprises assessing levels of ANGPTL3 protein or levels of triglycerides or LDL in the blood plasma. In some embodiments, the method further comprises monitoring the patient for excessive immune response. In some embodiments, the method further comprises monitoring the patient for change in the function of the target organ. In some embodiments, the target organ is the liver and the change in the function is the change in the amount of liver-secreted enzymes.

[0016] In one embodiment, the invention is a therapeutic composition for modifying a sequence of a target nucleic acid in a somatic cell in a living organism the composition comprising a lipid nanoparticle (LNP) wherein the lipid phase comprises ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about l-2°/o, and the LNP contains a therapeutically effective amount of (i) a nucleic acid coding for a CRISPR endonuclease and (ii) a guide polynucleotide comprising a targeting region capable of hybridizing to a target sequence within the target nucleic acid, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA). In some embodiments, the therapeutically effective amount is between 0.7 mg/kg and 2 mg/kg of total nucleic acid, or between 1 mg/kg and 2 mg/kg of total nucleic acid, or between 30 mg and 80 mg of total nucleic acid, or between 55 mg and 80 mg of total nucleic acid. In some embodiments, the therapeutic composition further comprises one or more of excipient, antimicrobial agent, an antioxidant, a surfactant, and a freezing agent. In some embodiments, the LNP are characterized by encapsulation efficiency of 70-100%. In some embodiments, the LNP are characterized by polydispersity index of 0-0.25. In some embodiments, the LNP are characterized by diameter of 65-100 nanometers. In some embodiments, the therapeutically effective amount is capable of achieving the rate of sequence modification at a selected genomic locus of at least 65% in vitro or in vivo in experimental animals. In some embodiments, upon sequence modification, the rate of chromosomal translocations undetectable by sequencing. In some embodiments, upon sequence modification, the rate of off-target sequence modification is undetectable by sequencing. In some embodiments, upon sequence modification, the rate of extra-organ sequence modifications is no greater than 0.6%.

[0017] In one embodiment, the invention is a method of treating a disease or condition comprising a step of systemic administration to a patient of the composition described above, wherein the somatic cell is a hepatocyte. In some embodiments, the gene is selected from TTR, PSCK9 and ANGPTL3. In some embodiments, the target nucleic acid is within the TTR gene, and wherein the level of the TTR protein in the patient’s plasma is reduced. In some embodiments, the target nucleic acid is within the PCSK9 gene, and wherein the level of the PCSK9 protein or the level of LDL cholesterol in the patient’s plasma is reduced. In some embodiments, the target nucleic acid is within the ANGPTL3 gene, and wherein the level of the ANGPTL3 protein the level of triglycerides in the patient’s plasma is reduced.

[0018] In some embodiments, the invention is a method of making the therapeutic composition described above, the method comprising combining the lipid phase comprising ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC- 0159 at about 1-2%, and the therapeutically effective amount of (i) a nucleic acid coding for a CRISPR endonuclease and (ii) a guide polynucleotide comprising a targeting region capable of hybridizing to a target sequence within the target nucleic acid, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA). In some embodiments, the therapeutically effective amount is between 0.7 mg/kg and 2 mg/kg, or between 1 mg/kg and 2 mg/kg, or between 30 mg and 80 mg, or between 55 mg and 80 mg of total nucleic acid. In some embodiments, the method further comprises adding one or more of excipient, antimicrobial agent, an antioxidant, a surfactant, and a freezing agent. In some embodiments, the resulting LNP are characterized by encapsulation efficiency of 70-100%. In some embodiments, the resulting LNP are characterized by poly dispersity index of 0-0.25. In some embodiments, the resulting LNP are characterized by diameter of 65-100 nanometers.

[0019] In one embodiment, the invention is a method of modifying a sequence of two or more target nucleic acids in a somatic cell in a living organism, the method comprising: contacting the organism with lipid nanoparticles (LNP) enclosing: (i) a nucleic acid coding for a CRISPR endonuclease and (ii) two or more guide polynucleotides, each polynucleotide comprising a targeting region capable of hybridizing to a target sequence within one of the two or more target nucleic acids, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA), and wherein the CRISPR endonuclease binds the guide polynucleotide and cleaves each target nucleic acid and the sequence of each target nucleic acid is modified within the somatic cell. In some embodiments, the two or more guide polynucleotides target two or more target sequences located in the same gene. In some embodiments, the two or more guide polynucleotides target two or more target sequences located in two or more genes. In some embodiments, the same LNP encloses two or more guide polynucleotides. In some embodiments, each of the two or more guide polynucleotides is enclosed in a separate LNP and the method comprises administering a first LNP enclosing a first guide polynucleotide and a second LNP enclosing a second guide polynucleotide. In some embodiments, the first LNP and the second LNP are administered sequentially after an interval selected from 24, 48, 72 and 96 hours.

[0020] In one embodiment, the invention is a composition for modifying a sequence of two or more target nucleic acids in a somatic cell in a living organism, the composition comprising lipid nanoparticles (LNP) enclosing: (i) a nucleic acid coding for a CRISPR endonuclease and (ii) two or more guide polynucleotides, each polynucleotide comprising a targeting region capable of hybridizing to a target sequence within one of the two or more target nucleic acids, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA), and wherein the CRISPR endonuclease is capable of binds the guide polynucleotide and cleaving each target nucleic acid. In some embodiments, the two or more guide polynucleotides target two or more target sequences located in the same gene. In some embodiments, the two or more guide polynucleotides target two or more target sequences located in two or more genes. In some embodiments, the same LNP encloses two or more guide polynucleotides. In some embodiments, each of the two or more guide polynucleotides is enclosed in a separate LNP.

[0021] In one embodiment, the invention is a therapeutic composition for alleviating the symptoms of a genetic disease characterized by autosomal dominant inheritance with a wildtype allele and a mutant allele of a disease-associated gene, the composition comprising a lipid nanoparticle (LNP) containing a therapeutically effective amount of (i) a nucleic acid coding for a CRISPR endonuclease and (ii) a guide polynucleotide comprising a targeting region capable of hybridizing to a target sequence within the mutant allele, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA), wherein the guide polynucleotide promotes cleavage of the mutant allele but not a wildtype allele by the CRISPR endonuclease. In some embodiments, the disease-associated gene is selected from the group consisting of PKD1, PKD2, ACVRL1, ENG, SMAD4, FBN1, ANK1, EPB42, SLC4A1, SPTA1, SPTB, TC0F1, FGFR3, SERPINC1, COL1A1, COL5A1, COL5A2, UGT1A1, CASR, APC, CLCN7, COL 1 Al, PROC, TSC1, TSC2, LDLR mdAPOB.

[0022] In some embodiments, the invention is a method for alleviating the symptoms of a genetic disease characterized by autosomal dominant inheritance with a wild-type allele and a mutant allele of a disease-associated gene, the method comprising administering to a patient exhibiting symptoms of the a genetic disease characterized by autosomal dominant inheritance the CRISPR LNP composition disclosed herein, wherein the administration is selected from the group consisting of intravenous administration, administration to the lung and administration to the gastrointestinal tract.

[0023] In some embodiments, the invention is a method for method for alleviating the symptoms of a genetic disease characterized by autosomal dominant inheritance with a wildtype allele and a mutant allele of a disease-associated gene, the method comprising administering to a patient carrying a wild-type allele and a mutant allele of a disease-associated gene the CRISPR LNP composition disclosed herein, wherein the administration is selected from the group consisting of intravenous administration, administration to the lung and administration to the gastrointestinal tract.

BRIEF DESCRIPTION OF THE FIGURES

[0024] FIGURE 1 is a diagram of CRISPR guide molecules with chemical modifications.

[0025] FIGURE 2 illustrates examples of ionizable cationic lipids used to form lipid nanoparticles (LNPs).

[0026] FIGURE 3 shows expression of luciferase (bioluminescent intensity) in mice transfected with firefly luciferase mRNA using LNPs of various lipid compositions.

[0027] FIGURE 4 shows area under the curve (AUC) of the bioluminescent intensity data from FIGURE 3. [0028] FIGURE 5 shows encapsulation efficiency of the compositions comprising the Casl2a mRNA and each of the three guide RNAs used in this study in the ALC0315 lipid formulation as measured by the RiboGreen assay.

[0029] FIGURE 6 shows the average diameter of LNPs (ALC0315 lipid formulation) enclosing the Casl2a mRNA and each of the three guide molecules.

[0030] FIGURE 7 shows the poly dispersity index (PDI) of LNPs (ALC0315 lipid formulation) enclosing the Casl2a mRNA and each of the three guide molecules.

[0031] FIGURE 8 shows in vivo editing of mouse liver cells at the Ttr locus with crRNA or chRDNAs.

[0032] FIGURE 9 shows changes in plasma TTR levels in mice following in vivo gene editing.

[0033] FIGURE 10 shows dose dependency of in vivo editing of mouse liver cells at the Ttr locus on the amount of Casl2a mRNA and guide molecule during the gene editing step. [0034] FIGURE 11 shows dose dependency of plasma TTR levels on the amount of Casl2a mRNA and guide molecule during the gene editing step.

[0035] FIGURE 12 shows body weight changes following LNP administration.

[0036] FIGURE 13 shows serum levels of TNF-alpha following LNP administration.

[0037] FIGURE 14 shows serum levels of IL-6 following LNP administration.

[0038] FIGURE 15 shows serum levels of the liver enzyme alanine aminotransferase

(ALT) following LNP administration.

[0039] FIGURE 16 shows serum levels of the liver enzyme aspartate aminotransferase (AST) following LNP administration.

[0040] FIGURE 17 shows dose dependency of plasma TTR levels and the rate of gene editing on the amount of Casl2a mRNA and guide molecule during the gene editing step.

[0041] FIGURE 18 shows durability of reduced plasma TTR levels following gene editing with crRNA or chRDNA.

[0042] FIGURE 19 shows in vivo editing of mouse liver cells at the Psck9 locus and the Angptl3 locus with crRNA or chRDNAs.

[0043] FIGURE 20 shows changes in plasma ANGPTL3 levels and plasma cholesterol levels in mice following in vivo gene editing at the Angptl3 locus. [0044] FIGURE 21 shows changes in plasma PCSK9 levels and plasma cholesterol levels in mice following in vivo gene editing at the Pcsk9 locus.

[0045] FIGURE 22 is a diagram of CRISPR guide molecules with chemical modifications used for editing of the human PSCK9 gene.

[0046] FIGURE 23 depicts an exemplary workflow for isolating and transfecting primary mouse hepatocytes (PMH) from transgenic mice.

[0047] FIGURE 24 shows the results of editing or targets in the human APOC3 gene in PMH from APOC3 transgenic mice (crRNA and chRDNA).

[0048] FIGURE 25 shows the results of editing of targets in the human SERPINA1 gene in PMH from SERPINA transgenic mice.

[0049] FIGURE 26 shows the results of editing of targets in the human SERPINA 1 gene in PMH from SERPINA transgenic mice (crRNA and chRDNA).

[0050] FIGURE 27 shows the results of editing of the target tgt21 in the human SERPINA1 gene with chRDNA guides matched and mismatched with the Pi*Z mutation (E366K) in the SERPINA1 gene.

[0051] FIGURE 28 illustrates the design of the multiplex in vivo gene editing experiment.

[0052] FIGURE 29 shows the results of multiplex in vivo gene editing of Ttr and AngptlS.

DETAILED DESCRIPTION OF THE INVENTION

[0053] Definitions

[0054] The following definitions aid in understanding this disclosure.

[0055] As used herein, the terms “guide” and “guide polynucleotide” as used herein refer to one or more polynucleotides that form a nucleoprotein complex with a Cas protein, wherein the nucleoprotein complex preferentially binds a nucleic acid target sequence in a polynucleotide (relative to a polynucleotide that does not comprise the nucleic acid target sequence). Such guides can comprise ribonucleotide bases (e.g, RNA), deoxyribonucleotide bases (e.g., DNA), combinations of ribonucleotide bases and deoxyribonucleotide bases (e.g., RNA/DNA), nucleotide analogs, modified nucleotides, and the like, as well as synthetic, naturally occurring, and non-naturally occurring modified backbone residues or linkages. Many such guides are known, such as but not limited to, single-guide RNA (including miniature and truncated single-guide RNAs), crRNA, dual-guide RNAs, including but not limited to, crRNA/tracrRNA molecules, and the like, the use of which depends on the particular Cas protein.

[0056] As used herein, a “CRISPR polynucleotide” is a polynucleotide sequence comprising at least a portion of a CRISPR guide molecule. In some embodiments, the CRISPR polynucleotide includes a targeting region and/or an activating region.

[0057] With reference to a guide molecule, a “spacer,” “spacer sequence,” “spacer element,” or “targeting region,” as used herein refers to a polynucleotide sequence that can specifically hybridize to a target nucleic acid sequence. The targeting region interacts with the target nucleic acid sequence through hydrogen bonding between complementary base pairs (i. e., paired bases). A targeting region binds to a selected nucleic acid target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell, either in vitro, ex vivo (such as in the generation of CAR-T cells), or in vivo (such as where compositions are administered directly to a subject). The targeting region determines the location of the sitespecific binding and nucleolytic cleavage by the CRISPR endonuclease. Variability of the functional length for a targeting region is known in the art.

[0058] As used herein, the term “capable of hybridizing” refers to a property of complementary nucleic acid strands to form a stable duplex. One of skill in the art would recognize that perfect complementarity over the entire length of the strands is not required for a stable duplex to form. For example, a certain number of mismatches between opposite nucleotides can be tolerated in a stable duplex. Furthermore, not all nucleotides in the duplex need to form Watson-Crick pairs in order for the duplex to remain stable. For example, some nucleotides may comprise non-canonical bases and even abasic (apurinic or apyrimidinic) sites. The number of tolerable mismatches depends on the length of the region of complementarity between the two nucleic acid strands. For example, a stable duplex formed of a longer region of complementarity will tolerate more mismatches than a duplex formed of a shorter region of complementarity. Furthermore, the degree of complementarity required to form a stable duplex varies depending on reaction conditions. For example, a stable duplex present in a high ionic strength solution will tolerate more mismatches than a duplex present in a low ionic strength solution.

[0059] With reference to a guide molecule, the term “activating region” refers to a portion of a polynucleotide capable of associating, or binding with, a CRISPR endonuclease polypeptide, such as for example, a Casl2a polypeptide.

[0060] As used herein, the terms “nucleotide analog,” “non-canonical nucleotide,” and “chemically-modified nucleotide” refer to a compound having structural similarity to a canonical purine or pyrimidine nucleotide occurring in DNA or RNA. The nucleotide analog may contain a modified sugar and/or a modified nucleobase, as compared to a purine or pyrimidine base occurring naturally in DNA or RNA. In some embodiments, the nucleotide analog is inosine or deoxyinosine, such as 2’ -deoxyinosine. In other embodiments, the nucleotide analog is a 2’ -deoxyribonucleotide (in an RNA molecule), or a ribonucleotide (in a DNA molecule). In some embodiments, the nucleotide analog includes a modified base (such as, for example, xanthine, uridine, oxanine (oxanosine), 7-methlguanosine, dihydrouridine, 5- methylcytidine, C3 spacer, 5-hydroxybutynl-2’-deoxyuridine, 5 -nitroindole, 5-methyl isodeoxycytosine, iso deoxyguanosine, other 0-1 purine analogs, N-6-hydroxylaminopurine, nebularine, 7-deaza hypoxanthine, and other 7-deazapurines, a fluoroinosine or a chloroinosine, such as 2-chloroinosine, 6-chloroinosine, 8-chloroinosine, 2-fluoroinosine, 6-fluoroinosine, or 8-fluoroinosine. A nucleotide analog or modified nucleotide may comprise a modified sugar moiety or a modified phosphodiester linkage, e.g., 2’-O-methyl, 2’-O-methoxyethyl, 2’-aza, protein-nucleic acid (PNA), linked nucleic acid (LNA), xeno nucleic acids (XNA), phosphoro- thioate and the like.

[0061] As used herein, the term “CRISPR hybrid RNA/DNA guide” (chRDNA) refers to a polynucleotide guide molecule comprising a targeting region and an activating region, wherein one or both of the targeting region and the activating region comprises one or more deoxyribonucleotides in addition to the ribonucleotides.

[0062] As used herein, the terms “engineered,” “genetically engineered,” “genetically modified,” “recombinant,” “modified,” “non-naturally occurring,” and “non-native” indicate intentional human manipulation of the genome of an organism or a cell. The terms encompass methods of genomic modification that include genomic editing, as defined herein, as well as techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like.

[0063] As used herein, the terms “protospacer adj acent motif’ or “PAM” as used herein refers to double-stranded nucleic acid sequences comprising a CRISPR endonuclease recognition sequence, wherein amino acids of endonuclease protein directly interact with the recognition sequence (e.g., Casl2a protein interacts with the PAM 5’-TTTN-3’ or the PAM 5’- TTTV-3’). PAM sequences are on the non-target strand and can be 5’ or 3’ of a target complement sequence (e.g., in CRISPR-Casl2a systems the PAM 5’-TTTN-3’ or the PAM 5’- TTTV-3’ sequence is on the non-target strand and is 5’ of the target-complement sequence).

[0064] As used herein, the terms “nuclear localization sequence” or “nuclear localization signal” (both abbreviated NLS) refer to a polypeptide sequence within a protein that preferentially increases the subcellular localization of a protein to the nucleus of a cell. NLS sequences are typically positively changed stretches of amino acids located at the terminus of a protein (N-terminus or C-terminus) or internally within the protein sequence. A protein may comprise more than one NLS, a protein (or a combination thereof, i.e., one or more NLS at the N-terminus and one or more NLS at the C-terminus). NLS sequences can be covalently linked to a prokaryotic protein to enable trafficking of the engineered protein to the nucleus of a eukaryotic cell. NLS sequences can be engineered or derived from existing proteins sequences.

[0065] As used herein, the terms “target,” “target sequence,” “nucleic acid target sequence,” “target nucleic acid sequence,” and “on-target sequence” are used interchangeably herein to refer to a nucleic acid sequence that is wholly, or in part, complementary to a nucleic acid target binding sequence of a CRISPR guide polynucleotide (e.g., the targeting region). Typically, the nucleic acid target binding sequence is selected to be 100% complementary to a nucleic acid target sequence to which binding of a CRISPR nucleoprotein complex is being directed; however, to attenuate binding to a nucleic acid target sequence, lower percent complementarity can be used.

[0066] As used herein, the terms “donor polynucleotide,” “donor oligonucleotide,” “donor template,” “non-viral donor,” and “non-viral template” are used interchangeably herein and can be a double-stranded polynucleotide (e.g., DNA), a single-stranded polynucleotide (e.g., DNA or RNA), or a combination thereof. Donor polynucleotides can comprise homology arms flanking the insertion sequence e.g, DSBs in the DNA). The homology arms on each side can vary in length to ensure the desirable level of hybridization at the conditions used.

[0067] As used herein, the term “homology-directed repair” (HDR) refers to the biochemical pathway of DNA repair that takes place in cells, for example, during repair of a DSB in DNA. HDR requires nucleotide sequence homology and uses a donor polynucleotide to repair the sequence wherein the DSB (e.g, within a target DNA sequence) occurred. For example, a donor polynucleotide can be used for repair of the break in the target DNA sequence, wherein the repair results in the transfer of genetic information (e.g, polynucleotide sequences) from the donor polynucleotide at the site or in close proximity of the break in the DNA. Accordingly, new genetic information (e.g., polynucleotide sequences) may be inserted or copied at a target DNA sequence.

[0068] As used herein, the term “homology-independent target integration” (HITI) refers to the biochemical pathway of DNA repair that takes place in a cell, for example, during repair of a DSB in DNA. HITI, unlike HDR, does not require nucleotide sequence homology and uses a donor polynucleotide to repair the sequence wherein the DSB occurred (e.g., within a target DNA sequence). HITI results in the transfer of genetic information from, for example, the donor polynucleotide to the target DNA sequence. Accordingly, new genetic information (e.g., polynucleotide sequences) may be inserted or copied at a target DNA sequence.

[0069] As used herein, the term “genomic region” refers to a segment of a chromosome in the genome of a host cell that is present on either side of the nucleic acid target sequence site or, alternatively, also includes a portion of the nucleic acid target sequence site. The homology arms of the donor polynucleotide have sufficient homology to undergo homologous recombination with the corresponding genomic regions.

[0070] As used herein, the term “non-homologous end joining” (NHEJ) refers to the biochemical pathway of repairing a DSB in DNA by direct ligation of one terminus of the break to the other terminus of the break without a requirement for a donor polynucleotide. NHEJ is a DNA repair pathway available to cells to repair DNA without the use of a repair template. NHEJ in the absence of a donor polynucleotide often results in nucleotides being randomly inserted or deleted at the site of the DSB.

[0071] As used herein, the term “microhomology-mediated end joining” (MMEJ) refers to the biochemical pathway for repairing a DSB in DNA. MMEJ involves deletions flanking a DSB and alignment of microhomologous sequences internal to the break site before joining. MMEJ is genetically defined and requires the activity of, for example, CtIP, Poly(ADP -Ribose) Polymerase 1 (PARP1), DNA polymerase theta (Pol 0), DNA Ligase 1 (Lig 1), or DNA Ligase 3 (Lig 3). Additional genetic components are known in the art. See, e.g., Sfeir et al. Trends in Biochemical Sciences, 2015, 40:701-714).

[0072] As used herein, the term “DNA repair” encompasses any biochemical process whereby cellular machinery repairs damage to a DNA molecule contained in the cell. The damage repaired can include single-strand breaks or double-strand breaks (DSBs). At least three mechanisms exist to repair DSBs: HDR, NHEJ, and MMEJ. “DNA repair” is also used herein to refer to DNA repair resulting from human manipulation, wherein a target locus is modified, e.g., by inserting, deleting, or substituting nucleotides, all of which represent forms of genome editing.

[0073] As used herein, the terms “regulatory sequences,” “regulatory elements,” and “control elements” are interchangeable and refer to polynucleotide sequences that are upstream (5’ non-coding sequences), within, or downstream (3’ non-translated sequences) of a polynucleotide target to be expressed. Regulatory sequences influence, for example, the timing of transcription, amount or level of transcription, RNA processing or stability, and/or translation of the related structural nucleotide sequence. Regulatory sequences may include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, transcription start sites, repressor binding sequences, stem-loop structures, translational initiation sequences, internal ribosome entry sites (IRES), translation leader sequences, transcription termination sequences {e.g., polyadenylation signals and poly-U sequences), translation termination sequences, primer binding sites, and the like.

[0074] As used herein, the term “modulate” refers to a change in the quantity, degree or amount of a function. For example, a CRISPR complex, as disclosed herein, may modulate the activity of a gene sequence by binding to a nucleic acid target sequence. Depending on the action occurring after binding, the CRISPR complex can transiently or permanently induce, enhance, suppress, or inhibit, transcription of a gene, e.g., by cleaving the sequence which is then and imperfectly repaired by cellular DNA repair thereby disrupting the gene sequence. Thus, “modulation” of gene expression includes both gene activation and gene repression, including complete repression of gene transcription.

[0075] As used herein, the term "Significant Mutant Fraction” (SMF) refers to a parameter and a method of calculating gene editing at a particular site in the genome in a population of cells using next-generation sequencing data obtained from a test cell population and a control cell population. SMF is calculated by first determining the "significant" mutations in the matched test and control samples. A mutation is only considered as "significant" if (1) it falls within ±3bp of the predicted cut-site coordinates, and (2) its frequencies in the test and control samples differ significantly as determined by a chi-squared test with p-value threshold of 10'⁴ with Bonferroni multiple-comparison correction. The total frequency of "significant" mutations is calculated for the test and control samples and the control frequency is subtracted from the test frequency to produce the final Significant Mutation Fraction statistic.

[0076] As used herein, the term “lipid nanoparticle” (LNP) refers to a water-in-oil droplet typically between 60 nm and 100 nm in size. One of skill in the art will appreciate that the size of a stable LNP depends on the composition and ionic strength of aqueous solutions within and without the LNP.

[0077] As used herein, the term “encapsulation” refers to successful enclosure of nucleic acids within LNPs. Encapsulation may be expressed as fraction or nucleic acid present in the composition that is enclosed in LNP. For example, 95% encapsulation means that only 5% of nucleic acid is present outside of the LNPs. The non-encapsulated nucleic acid can be reacted with detection reagents or nucleases and thereby quantified. For example, RIBOGREEN® is a fluorescent dye that binds single-stranded nucleic acids including oligonucleotides and can be used to measure encapsulation.

[0078] As used herein, the terms “LNP composition” and “CRISPR LNP composition” refer to an aqueous solution comprising LNPs encapsulating the components of the CRISPR system. Unless otherwise indicated, the LNPs in the LNP composition comprise all of the components of the CRISPR system necessary to produce editing of the targeted locus in the subject organism.

[0079] As used herein, the terms “subject,” “individual,” or “patient” refer to humans and other animals such as laboratory animals. In some embodiments, a cell is derived from a subject. In some embodiments, the subject is a non-human subject.

[0080] As used herein, the term “autosomal dominant disease” refers to a genetic (inherited) disease with autosomal dominant pattern of inheritance, z.e., the disease (mutant) allele exhibits dominance over the normal (non-mutant) allele so that a heterozygous individual with one disease (mutant) allele and one normal (non-mutant) allele exhibits the mutant phenotype including symptoms of the disease.

[0081] As used herein, the terms “effective amount” or “therapeutically effective amount” of a composition or agent refer to a sufficient amount of the composition or agent to provide the desired response. Such responses will depend on the particular disease in question. For example, in a patient being treated for hypercholesteremia using the therapy disclosed herein, a desired response may include reduction in blood LDL levels, or blood triglyceride levels, or reduction or elimination or cardiovascular (CV) adverse events. Preferably, the effective amount also prevents or avoids one or more harmful side effects. The exact effective amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the type of somatic cell being subjected to treatment.

[0082] As used herein, the terms “treatment” or “treating” a particular disease includes preventing, reversing or ameliorating symptoms of the disease. For example, for a patient being treated for hypercholesteremia, treatment may include reducing blood LDL levels, or blood triglyceride levels, or reducing or eliminating any cardiovascular (CV) adverse events.

[0083] As used herein, the terms “pharmaceutically acceptable carrier” and “excipient” refer to aqueous solvents (e.g., water, aqueous solutions of alcohols, saline solutions, sodium chloride, Ringer's solution, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters), as well as dispersion media, coatings, surfactants, gels, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, stabilizers, binders, disintegration agents, lubricants, sweetening agents, flavoring agents, and dyes. The concentration and pH of the various components in a pharmaceutical composition are adjusted according to well-known parameters for each component.

[0084] The present disclosure relies on the ordinary skill in the art as it pertains conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant polynucleotides, as taught, for example, by the following standard publication: Sambrook, Joseph. Molecular Cloning: a Laboratory Manual. 2001, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press; E.A. Greenfield, Antibodies: A Laboratory Manual, 2014, Second edition, Cold Spring Harbor Laboratory Press; R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 2016, 7th Edition, Wiley -Blackwell; J.M. Walker, Methods in Molecular Biology (Series), Humana Press; and Innis, J., Gelfand, D., et al., PCR Protocols: a Guide to Methods and Applications, 1989, Academic Press.

[0085] Then first two approved genome editing therapies involved ex vivo modification of patient’s cells. LYFGENIA, a treatment for sickle cell disease works by adding a functional P-globin gene to patients’ own hematopoietic stem cells (HSCs). Durable production of adult hemoglobin with anti-sickling properties (HbA^T87Q) is possible following successful engraftment of the edited HSCs. Similarly, CASGEVY involves ex vivo genome editing of patient’s HSCs with CRISPR/Cas9 at the erythroid-specific enhancer region of the BCL11A gene. Reduced BCL11A expression leads to increase in fetal hemoglobin (HbF) production in edited HSCs and their progeny.

[0086] In addition to the successful ex vivo gene editing therapies described above, some in vivo gene therapies are currently in development or in clinical trials.

[0087] Hereditary amyloidogenic transthyretin amyloidosis is caused by instability and proteolysis of the mutant transthyretin protein resulting in deposition of amyloid and fatal damage of the peripheral nervous system. One of the approaches to treatment has been gene silencing with small interfering RNA (siRNAs) or anti-sense oligonucleotides (ASO). See Adams, D., et al., (2018) Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis, NEJM 379: 11. While showing promise, RNAi and ASO therapies must be frequently readministered due to the short half-life of RNA molecules. A program using CRISPR/Cas9 to disrupt the TTR gene is ongoing, see Gillmore, J., et al., (2021) CRISPR Cas9 in vivo gene editing for transthyretin amyloidosis, NEJM 385:493. (Phase 1 study completed in 2022).

[0088] One of the major risk factors for cardiovascular disease is abnormal elevation of serum triglycerides and serum cholesterol. One of the factors affecting serum cholesterol levels is the protein PSCK9 that binds to LDL receptors and promotes their internalization and lysosomal degradation thus leaving more LDL in circulation. Depletion of PSCK9 with monoclonal antibodies leads to increased numbers of LDL receptors on the surface of hepatocytes and lowering of serum cholesterol (LDL) levels. Anti-PSCK9 antibody drugs such as REPATHA® and PRALUENT® require monthly or bi-weekly injections in order to maintain the cholesterol-lowering effect.

[0089] Triglycerides in serum are present in triglyceride-rich lipoproteins chylomicrons and VLDL. Lor tissue use triglycerides are liberated from these lipoproteins by lipoprotein lipase (LPL). ANGPTL-family proteins are negative regulators of LPL. Loss-of-function mutations in ANGPTL3 are associated with reduced blood triglycerides in experimental animals. An antisense drug blocking ANGPTL3 expression is in clinical trials and has demonstrated the ability to lower blood triglycerides by 36-47%. Gaudet, D., et al., (2020) Vupanorsen, an N-acetyl galactosamine-conjugated antisense drug to ANGPTL3 mRNA, lowers triglycerides and atherogenic lipoprotein in patients with diabetes, hepatic steatosis, and hypertriglyceridaemia, Eur. Heart J. 41 (40) : 3936. As with PSCK9 inhibitors, weekly or bimonthly injections are required to maintain the physiological effect.

[0090] The instant disclosure describes methods and compositions for successful onetime gene editing of somatic cells in vivo using engineered CRISPR systems with desired long- lasting physiological effect attributable to gene editing.

[0091] The methods disclosed herein utilize a CRISPR endonuclease, i.e., the endonuclease associated with the CRISPR system and encoded by a CRISPR locus. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus is found many prokaryotic genomes and provides resistance to invasion of foreign nucleic acids. Structure, nomenclature, and classification of CRISPR loci are reviewed in Makarova el al., Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology. 2011 June; 9(6): 467-477.

[0092] Briefly, a typical CRISPR locus includes a number of short repeats regularly interspaced with spacers. The CRISPR locus also includes coding sequences for CRISPR- associated (Cas) genes. A spacer-repeat sequence unit encodes a crisprRNA (crRNA). In vivo, a mature crRNAs is processed from a polycistronic transcript referred to as pre-crRNA or pre- crRNA array. The repeats in the pre-crRNA array are recognized by Cas-encoded proteins that bind to and cleave the repeats liberating mature crRNAs. CRISPR systems perform cleavage of a target nucleic acid wherein Cas proteins and crRNA form a CRISPR ribonucleoproteins (crRNP). The crRNA molecule guides the crRNP to the target nucleic acid (e.g., a foreign nucleic acid invading a bacterial cell) and the Cas nuclease proteins cleave the target nucleic acid.

[0093] Class 1, Type I CRISPR systems include means for processing the pre-crRNA array that include a multi-protein complex called CASCADE (CRISPR-associated complex for antiviral defense) comprised of subunits CasA, B, C, D and E. The CASCADE-crRNA complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. The bound nucleoprotein complex recruits the Cas3 helicase/nuclease to facilitate cleavage of target nucleic acid.

[0094] Class 2, Type II CRISPR systems include a trans-activating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to a crRNA repeat in the pre-crRNA array and recruits endogenous RNaselll to cleave the pre-crRNA array. The tracrRNA/crRNA complex can associate with a nuclease, e.g., Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. Hybridization of the crRNA to the target nucleic acid activates the Cas9 nuclease, for target nucleic acid cleavage. [0095] Class 1, Type III CRISPR systems include the RAMP superfamily of endoribonucleases (e.g., Cas6) that cleave the pre-crRNA array with the help of one or more CRISPR polymerase-like proteins. [0096] Class 2, Type V CRISPR systems comprise a different set of Cas-like genes, including Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl4, Casl2g, Casl2h, Casl2i, Casl2j, and Casl2k proteins which are distant homologues of Cas genes in Type I-III CRISPR systems. [0097] CRISPR nucleases do not cleave a fixed sequence but instead are guided by a nucleic acid guide to a target sequence. In addition to the target sequence hybridizing to the nucleic acid guide, the CRISPR endonucleases recognize a sequence termed protospacer adjacent motif (PAM). The CRISPR Class 1 (including CASCADE) endonuclease recognize a PAM sequence selected from 5’-AAG-3’, 5’-AGG-3’, 5’-ATG-3’, 5 ’-GAG-3’, 5’-CAG-3’, 5’- GTG-3’, 5’-TAA-3’, 5’-TGG-3’, 5 ’-AAA-3’, 5’-AAC-3’, 5’-AAT-3’, 5 ’-ATA-3’, 5 ’-TAG-3’, and 5’-TTG-3’. The CRISPR Class 2 endonucleases (including Casl2a) recognize a PAM consisting of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'- NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3', 5’-TTN-3’, 5’-TTTN-3’ and 5’- TTTV-3’.

[0098] The CRISPR guide nucleic acid comprises a targeting region capable of binding the target nucleic acid and an activating region capable of binding the CRISPR endonuclease. In some embodiments, the guide nucleic acid is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, a guide nucleic acid can comprise, in the order of 5' to 3', a spacer extension, a spacer, a minimum CRISPR repeat, a single guide connector, a minimum tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some instances, a nucleic acid-targeting nucleic acid can comprise, a tracrRNA extension, a 3' tracrRNA sequence, a minimum tracrRNA, a single guide connector, a minimum CRISPR repeat, a spacer, and a spacer extension in any order. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. Other chRDNA may be a single guide D(R)NA for use with a Type II CRISPR system comprising a targeting region, and an activating region composed of and a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. For example, the targeting region may comprise DNA or a mixture of DNA and RNA, and an activating region may comprise RNA or a mixture of DNA and RNA. [0099] In some embodiments, the single guide nucleic acid comprises a spacer sequence located 5' of a first duplex which comprises a region of hybridization between a minimum CRISPR repeat and minimum tracrRNA sequence. The first duplex can be interrupted by a bulge. The bulge facilitates recruitment of the endonuclease to the guide nucleic acid. The bulge can be followed by a first stem comprising a linker connecting the minimum CRISPR repeat and the minimum tracrRNA sequence. The last paired nucleotide at the 3' end of the first duplex can be connected to a second linker connecting the first duplex to a mid-tracrRNA. The mid-tracrRNA can comprise one or more additional hairpins.

[00100] In some embodiments, the guide nucleic acid can comprise a dual guide nucleic acid structure. The double guide nucleic acid comprises a spacer extension, a spacer, a minimum CRISPR repeat, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and a tracrRNA extension. The dual guide nucleic acid does not include the single guide connector. Instead, the minimum CRISPR repeat sequence comprises a 3' CRISPR repeat sequence and the minimum tracrRNA sequence comprises a 5' tracrRNA sequence and the dual guide nucleic acids can hybridize via the minimum CRISPR repeat and the minimum tracrRNA sequence.

[00101] In some embodiments, gene editing with CRISPR endonucleases involves disruption of a gene sequence. In some embodiments, disruption of a gene sequence reduces or eliminates transcription of the mRNA from the gene (e.g., by disrupting a promoter or enhancer region). In some embodiments, disruption of a gene sequence reduces or eliminates the amount of the functional protein encoded by the gene (e.g., by disrupting an mRNA splicing site or disrupting the coding sequence with missense or nonsense mutations). Gene disruption with CRISPR endonucleases involves cleavage of the target sequence and subsequent imperfect repair by cellular DNA repair pathways.

[00102] Eukaryotic cells, e.g., mammalian cells possess an innate diversity of DNA repair pathways. The DNA repair pathway involved in repairing double strand breaks (DSB) (such as the ones introduced by the CRISPR-Cas nucleases) includes highly accurate homologous recombination (HR) as well as less accurate pathways of non-homologous end joining (NHEI) and micro-homology-mediated end joining (MMEJ). NHEJ and MMEJ generate a variety of small insertions and deletions at the target site (see Xue and Greene, (2021) DNA repair pathway choices in CRISPR-Cas9 mediated genome editing, Trends Genet. 37:639 ). These error-prone pathways are capable of producing the desired gene disruption through frameshift, nonsense, or missense mutations resulting in reduction or elimination of protein expression.

[00103] Unfortunately, it has been reported that programmable endonucleases such as CRISPR bring about undesirable consequences including large genomic deletions, chromosomal translocations, chromotripsis and other chromosomal abnormalities. A recent review article states “These editing outcomes, while rare, pose safety risks that could negatively impact certain clinical uses of nucleases. These drawbacks of nuclease editing, combined with the fact that nuclease-initiated HDR is inefficient in most therapeutically relevant cell types, have motivated the development of alternative strategies for more precise gene editing [such as primer editing and base editing].” Raguram, A., el al., (2022) Therapeutic in vivo delivery of gene editing agents, Cell 185:P2806. The instant disclosure overcomes these alleged drawbacks of CRISPR endonucleases by presenting compositions for accurate and efficient method of in vivo gene editing followed by a successful therapeutic outcome in an animal model.

[00104] In some embodiments, the CRISPR endonuclease is a Class 2 Type V CRISPR endonuclease. In some embodiments, the CRISPR endonuclease is a Casl2 endonuclease. Casl2 proteins of the present disclosure include wild type Casl2 proteins derived from Type V CRISPR-Cas systems, modified Casl2 proteins, variants of Casl2 proteins, Casl2 orthologs, and combinations thereof. In some embodiments, the Casl2 protein is selected from the group consisting of Casl2a, Casl2b2, Casl2c, Casl2d, Casl2e, Casl2fl, Casl2f2, Casl2f3, Casl2g, Casl2h, Casl2i, Casl2j, and Casl2k. In some embodiments, the Casl2 protein is the Casl2a protein.

[00105] Structure and function of different domains of Casl2 proteins have been disclosed e.g., in the publication WO2022086486 and references cited therein.

[00106] In some embodiments, the Casl2a protein is the Acidaminococcus spp. strain BV316 Casl2a protein. In some embodiments, the Casl2a protein has the sequence of SEQ ID NO: 1.

[00107] In some embodiments, the modified Casl2a protein comprises one or more amino acid changes that can be implemented without a significant effect on the structure or function of the Casl2a protein. In some embodiments, a Casl2 protein can be modified to provide enhanced activity or specificity wherein the modifications occur in regions 226-304, 368-435, 940-956, 978-1158, 1159-1180, and 1181-1298 of SEQ ID NO: 1.

[00108] In some embodiments, the CRISPR endonuclease comprises a nuclear localization sequence (NLS). The NTS sequence can be covalently attached to the endonuclease protein (c. ., Casl2a protein) either directly or via a linker polypeptide. The length of a linker sequence can be optimized depending on the structural characteristics of the particular protein (e.g., solvent accessibility of the termini, the presence of other critical functional peptide sequences at the termini, etc.) to ensure the accessibility of the NLS sequence for cognate importin protein binding and trafficking to the nucleus.

[00109] In some embodiments, a linker sequence contains at least one glycine, serine, and/or threonine residue. In some embodiments, a linker sequence contains at least one glycine residue and at least one serine residue. In some embodiments, a linker sequence contains a plurality of glycine residues and at least one serine residue. In some embodiments, a linker sequence consists of or comprises a GS sequence.

[00110] The NLS sequence may be engineered or derived from another protein. In some embodiments, the NLS sequence is derived from a protein selected from the group consisting of SV40 large T-antigen, Nucleoplasmin, 53BP1, VACM-1/CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrpl, c-Myc, Mouse c-able IV, Mata2 and MINIYO.

[00111] In some embodiments, the NLS sequence is a nucleoplasmin (NPL) NLS sequence connected via a linker (NLS-linker shown as SEQ ID NO: 2).

[00112] In some embodiments, the CRISPR endonuclease is introduced into the cells of the organism as mRNA coding for the endonuclease protein. The CRISPR endonuclease and its mRNA are of prokaryotic origin. In some embodiments, the mRNA is modified for optimal function in a eukaryotic cell within a eukaryotic organism.

[00113] In some embodiments, the mRNA is codon-optimized for translation in eukaryotic cells. Codon optimization is the process of altering the nucleic acid sequence without changing the polypeptide sequence encoded thereby in order to utilize the most prevalent tRNAs present in an organism and increase the efficiency of translation. Codon optimization may be performed manually or with the help of any of the codon optimization software such as GeneArt (ThermoFisher Scientific), GenSmart (Genscript), and Codon Optimization Tool (Integrated DNA Technologies).

[00114] In some embodiments, the mRNA encoding the endonuclease has undergone uridine depletion, i.e., a sequence design process whereby synonymous mRNA codons with no uridines or fewer uridines are substituted for uridine-containing codons. Uridine depletion has been shown to reduce immunogenicity of the mRNA in humans and protect the mRNA from intracellular RNases.

[00115] In some embodiments, the mRNA comprises one or more chemical modifications. In some embodiments, the chemical modifications comprise modified nucleobases or non-canonical nucleobases (i.e., bases other than adenosine, cytosine, guanosine and uridine). In some embodiments, one or more or all of uridines in the nucleic acid are substituted with less immunogenic uridine derivatives in order to further reduce immunogenicity of the nucleic acid. In some embodiments, the uridine derivative is selected from 5-methoxyuridine, 5-methyluridine, 5-carboxymethytl ester uridine, 2-thiouridine and pseudouridine and their various derivatives for which methods of making and methods of use in nucleic acids are disclosed e.g., in the U.S. Patent Nos. 9,428,535 and 9,751,925 and Morais P., et al., (2021) The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines, Front Cell Dev Biol. V. 9 article 789427.

[00116] In some embodiments, the combination of uridine depletion and the use of uridine derivatives results in less immunogenic nucleic acid suitable for in vivo administration as disclosed in Vaidyanathan, S., et al., (2018). Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity w ithout HPLC Purification. Molecular Therapy - Nucleic Acids, 12:530.

[00117] In some embodiments, the nucleic acid is an mRNA containing a 5 ’-cap. In some embodiments, the cap comprises the traditional terminal guanine or “inverted G” structure 3’G(5’)PPP-5’. In some embodiments, the 5’-cap comprises one or more chemical modifications selected from modifications to the guanosine base, the ribose sugar moiety or to the phosphate moiety. In some embodiments, the cap comprises one of more modifications selected from N7-methyl guanosine, 2’-O-methyl ribose, a-thiophosphate, a-methyl phosphate, boranophosphate and selenophosphate.

[00118] In some embodiments, the cap comprises two guanines arranged in the antireverse configuration, e.g., (3’-G(5’)PPP(5')G). As with the single nucleotide cap, the dinucleotide cap may comprise modifications of the guanosine base, ribose and the phosphate group described above. In some embodiments, the guanosine is a N7-(4-chlorophenoxyethyl- G, the N7-(4-chlorophenoxyethyl)-G, the N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G or the N7-(4-bromophenoxyethyl)-m3'-OG(5')ppp(5')G. In some embodiments, the cap is (3’-O- Me-m7G(5’)ppp(5')G). In some embodiments, the cap is m7(3 ’ OMeG)(5 ’ )ppp(5 ’ )m6(2 ’ OMe)pG.

[00119] In some embodiments, the cap is added post-transcriptionally, using enzymes such as the recombinant vaccinia virus capping enzyme and the recombinant 2'-O- methyltransferase enzyme.

[00120] In some embodiments, the mRNA further comprises a poly-A tail, i.e., a structure of about 100-250 adenine ribonucleotides at the 3 ’-end of the mRNA.

[00121] In some embodiments, the mRNA comprises untranslated regions (UTRs). In some embodiments, the mRNA comprises a 5 ’-UTR, or a 3’-UTR or both the 5’-UTR and the 3 ’-UTR.

[00122] In some embodiments, the mRNA comprises a UTR from a mammalian mRNA.

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

[00123] In some embodiments, the mRNA comprises a Kozak sequence that plays a role in translation initiation. In some embodiments, the Kozak sequence includes the AUG start codon of the mRNA. In some embodiment, the Kozak sequence is placed between the 5 ’-UTR and the AUG start codon of the mRNA. [00124] In addition to the CRISPR endonuclease expressed from the mRNA described above, the instant disclosure includes the use of a CRISPR guide molecule capable of interacting with the CRISPR endonuclease. In some embodiments, the guide molecule is a Cast 2 guide molecule capable of forming a ribonucleoprotein complex with its cognate Casl2 protein, such as a Casl2a protein. This Casl2a-guide ribonucleoprotein complex is capable of targeting a target sequence capable of hybridizing to the targeting region of the guide molecule. [00125] In certain embodiments, the activating region is downstream from the targeting region. In certain embodiments, the activating region is upstream from the targeting region. In some embodiments, the activating region is between 10-25 nucleotides in length, e.g., 20 nucleotides in length. In some embodiments, the targeting region is between 10-30 nucleotides in length, e.g., 20 bases in length.

[00126] In some embodiments, the guide molecule is the Acidaminococcus spp. strain BV316 Casl2a guide molecule comprising an activating region (in turn comprising a stem-loop duplex) and a targeting region (in turn comprising a target binding sequence). In some embodiments, the Cast 2a guide molecule further comprises a 3’ extension connected to the 3’- end of the targeting region optionally by via a linker sequence. In some embodiments, the Casl2a guide molecule further comprises a 5’ extension connected to the 5’-end of the targeting region optionally by via a linker sequence.

[00127] In some embodiments, at least one of the activating region and the targeting region of the Casl2 guide molecule comprises DNA or a mixture of DNA and RNA. One or more deoxyribonucleotides may be present at any one or more positions in the targeting region or the activating region. In some embodiments, the activating region and the targeting region each contain one or more deoxyribonucleotides (the remained being ribonucleotides). In some embodiments, the activating region contains one or more deoxyribonucleotides, and the targeting region does not contain any deoxyribonucleotides (e.g., contains only ribonucleotides). In some embodiments, the targeting region contains one or more deoxyribonucleotides, and the activating region does not contain any deoxyribonucleotides (e.g., contains only ribonucleotides). Such hybrid RNA-DNA guides (chRDNAs) are disclosed in the International Patent Application Publication No.: WO2022086486 DNA-containing polynucleotides and guides for CRISPR Type V systems, and methods of making and using the same.

[00128] In some embodiments, at least one of the activating region and the targeting region of the Casl2 guide molecule further comprises chemical modifications selected from a base modification and a backbone modification and including for example, base analogs, modified nucleotides, abasic sites, synthetic, naturally occurring, and non-naturally occurring modified backbone residues or linkages, or combinations thereof. Such modifications may be present at any one or more positions in the targeting region or the activating region. Such guides including chemical modifications (including modified crRNA guides and modified chRDNA guides) are disclosed in the International Patent Application Publication No.: WO2021119006 CRISPR abasic restricted nucleotides and CRISPR accuracy via analogs.

[00129] In some embodiments, the base modification is a base analogs or non-natural bases as defined herein. In some embodiments, the backbone modification of the guide molecule comprises a ribose modification. In some embodiments, the modification is 2’-H (deoxyribose). In some embodiments, the modification is a 2’ -O-m ethoxy ethyl or a 2’-O- methyl. In some embodiments, the modification is a phosphate group modification such as a phosphorothioate or sei enothioate.

[00130] Exemplary placement of deoxyribonucleotides and chemical modifications within the polynucleotide guide is illustrated in FIGURE 1. FIGURE 1 shows a comparison of an Acidaminococcus spp. (Strain BV316) Casl2a editing efficiency when coupled with various guides in the immortalized mouse hepatocyte cell line H2.35. In FIGURE 1, the crRNA is shown as 101. Guides 102-109 include one or more modifications including deoxyribonucleotides. Referring to the numbers above the design, the activating region comprises nucleotides 1-20, and the targeting region comprises nucleotides 21-40. The guides include ribonucleotides (RNA), deoxyribonucleotides (DNA), phosphorothioate nucleotides (P-thioate), and 2’-O-methyl nucleotides (2’-0Me). The editing efficiency of each guide design for the mouse genes Pcsk9, Ttr, and Angptl genes is shown adjacent to the depiction of the design.

[00131] One of the unique advantages of chRDNA guides and guides comprising chemical modifications is increased specificity with little or no sacrifice in efficiency of cleavage by the CRISPR endonuclease. As demonstrated in WO2022086486 and WO2021119006, off-target editing in a mammalian genome can be reduced to undetectable levels (when assessed by NGS), while on-target editing remains close to that of an unmodified crRNA.

[00132] Guide RNA components (such as crRNAs) can be produced by in vitro transcription (e.g., T7 Quick High Yield RNA Synthesis Kit; New England Biolabs, Ipswich, MA) from double-stranded (ds) DNA templates by incorporating a T7 promoter at the 5’ end of the dsDNA template sequences. Guide RNA with modifications including e.g., deoxyribonucleotides and nucleotides with chemical modifications can be synthesized chemically.

[00133] In some embodiments, the Casl2a guide comprises or consists essentially of SEQ ID NO: 141 where “N” represents the nucleotides in the targeting region that are designed to hybridize to the desired target nucleic acid, “r” represents a ribonucleotide, the absence of “r” represents a deoxyribonucleotide, represents phosphorothioate, and “m” represents 2’- O-methylation.

[00134] Examples of sequence species falling within the scope of SEQ ID NO: 141 include e.g., SEQ ID NO: 18 (Pcsk9-targeting chRDNA), SEQ ID NO: 27 (Ttr-targeting chRDNA) and SEQ ID NO: 40 (AngptB -targeting chRDNA). All of SEQ ID NOs: 18, 27 and 40 enable high efficiency of cleavage by Casl2a (FIGURE 8 and FIGURE 19). One of skill in the art would use the well-known rules of Watson-Crick base-pairing (i.e., A-U, C-G, G-C and T-A), to replace each “N” with an appropriate nucleotide thereby designing a Casl2a guide that is a species of SEQ ID NO: 141 and is capable of hybridizing to any chosen target nucleic acid sequence and further, is capable of guiding efficient cleavage by Casl2a.

[00135] In some embodiments, the Casl2a guide has the activating region comprising or consisting essentially of SEQ ID NO: 142 where “r” represents a ribonucleotide, the absence of “r” represents a deoxyribonucleotide, and represents phosphorothioate. [00136] In some embodiments, the delivery of CRISPR components (e.g., mRNA encoding the CRISPR endonuclease and the guide) is achieved by packaging the components into a compartment. The compartment comprising the CRISPR components can be administered in vivo intravenously, intrathecally or into a desired organ. In some embodiments, the compartment is a non-biological compartment selected from nanospheres, liposomes, nanoparticles, microparticles, nanocapsules, vesicles, polyethylene glycol particles, hydrogels, and micelles. In some embodiments, the compartment is a lipid nanoparticle (LNP).

[00137] In some embodiments, the LNP used herein has a diameter of between about 100 nm and about 1 pm, preferably <100 nm. In some embodiments, the LNP includes one or more cationic lipids. The cationic lipids can be selected such that, when combined, the measured value of the pK_a of the combination is no less than 6.1 and no greater than 6.7, e.g., between 6.2 and 6.6; or between 6.3 and 6.5. The cationic lipids can have a head group, one or more hydrophobic tails, and a linker between the head group and the one or more tails. The head group can include an amine which is a site of positive charge. The amine can be a primary, secondary, or tertiary amine, or a quaternary amine. The one or more hydrophobic tails can include two hydrophobic chains, which may be the same or different. The tails can be aliphatic chains, fatty acid chains or other hydrophobic chains. The linker can include, for example, a glyceride linker, an acyclic glyceride analog linker, or a cyclic linker. The linker can include functional groups such as an ether, an ester, a phosphate, a phosphonate, a phosphorothioate, a sulfonate, a disulfide, an acetal, a ketal, an imine, a hydrazone, or an oxime. Cationic lipids include one or more amine group(s) which bear the positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral depending on pH. The ionization of the cationic lipid affects the surface charge of a lipid nanoparticle (LNP) and can influence plasma protein absorption, blood clearance, tissue distribution and the ability to fuse with cellular membranes.

[00138] Methods of making and using lipid nanoparticles for in vivo delivery of nucleic acids are disclosed e.g., in the U.S. Patent Nos. 9,415,109, 9,533,047, and 11,420,931. Briefly, a typical LNP lipid phase comprises an ionizable cationic lipid, cholesterol, a phospholipid, and a polyethylene glycol (PEG) derivative. In some embodiments, the LNP lipid phase comprises an ionizable cationic lipid at about 46-50%, cholesterol at about 38-43%, a phospholipid at about 9-10%, and a PEG derivative at about 1-2%. Polyethylene glycol (PEG) is included in the lipid phase of the LNP as it has an effect of reducing aggregation of LNPs. PEG and PEG derivatives useful for in vivo delivery of nucleic acids are disclosed e.g., in U.S. Patent Application Publication No. US20220047518.

[00139] In some embodiments, the method comprises a step of forming LNPs enclosing (encapsulating) nucleic acids. To form LNPs enclosing nucleic acids, the lipids are diluted in ethanol to a desired concentration, e.g., 25-50 nM and mixed at predetermined molar ratios. The nucleic acid is diluted in a suitable aqueous buffer (e.g., a buffer maintaining stability of the nucleic acid such as sodium citrate buffer) to a desired concentration, e.g., 0.1-10 mg/mL. Formation of lipid nanoparticles can be achieved my mixing the lipid/ethanol solution and the aqueous nucleic acid containing solution at various ratios. The mixing can be effected manually or with the help of pumps or syringes. Ethanol and buffers may be removed via dialysis and additional sterilization steps can be performed according to methods known in the art.

[00140] In some embodiments, the lipid phase of the LNP comprises an ionizable cationic lipid at about 46-50%, cholesterol at about 38-43%, a phospholipid at about 9-10%, and a polyethylene glycol (PEG) derivative at about 1-2%.

[00141] Examples of ionizable cationic lipids ALC-0315, SMI 02 and MC3 are shown in FIGURE 2

[00142] In some embodiments, the lipid phase of the LNP comprises 6-((2- hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l- aminium (ALC-0315), cholesterol, l,2-Distearoyl-sn-glycero-3-PC (1,2-DSPC), and Methoxy - poly ethyleneglycol oxy(2000)-N,N-ditetradecylacetamide (ALC-0159).

[00143] In some embodiments, the lipid phase of the LNP comprises 8-[(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM102), cholesterol, 1,2-DSPC, and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

[00144] In some embodiments, the lipid phase of the LNP comprises 4-(dimethylamino)- butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester (MC3), cholesterol, 1,2-DSPC, and DMG-PEG2000. [00145] In some embodiments, the surface of LNPs is further modified with polymers or lipids e.g., chitosan, cationic polymers, or cationic lipids) or coupled to targeting molecules (antibodies specific for cell-surface receptors or natural ligands of cell surface receptors) to direct the nanoparticle to the appropriate cell type and increase the likelihood of cellular uptake as described e.g., in Jian el al., (2012) Cationic core shell liponanoparticles for ocular gene delivery, Biomaterials 33(30): 7621-30).

[00146] In some embodiments, prior to administration to a patient, LNPs are evaluated to verify that the physical properties are in a suitable range for in vivo administration. In some embodiments, LNPs are evaluated for encapsulation efficiency using a nucleic-acid binding dye that quantifies the amount of non-encapsulated nucleic acid. In some embodiments, the LNPs are used for in vivo administration if encapsulation efficiency is in the range of 70-100%. In some embodiments, LNPs are evaluated for diameter. In some embodiments, the LNPs are used for patient administration if the diameter is in the range of 65-100 nm. In some embodiments, LNPs are evaluated for uniformity. In some embodiments, the LNPs are used for in vivo administration if the polydispersity index (PDI) is at or about 0.25.

[00147] In some embodiments, the method described herein comprises selective cleavage of a target nucleic acid sequence in a cellular genome by a CRISPR endonuclease. The target nucleic acid sequence comprises a region capable of hybridizing to the CRISPR guide polynucleotide and is located adjacent to a protospacer adjacent motif (PAM) recognized by the CRISPR endonuclease. The target nucleic acid comprises a target strand capable of hybridizing to the target-binding sequence of the CRISPR guide polynucleotide. The target nucleic acid further comprises a non-target strand comprising a PAM sequence typically occurring upstream {i.e., in a 5’ direction) relative to the target sequence.

[00148] In some embodiments, the target sequence used herein comprises a 20- nucleotide sequence downstream (in a 3’ direction) of a PAM sequence 5’-TTTV-3’ recognized by Casl2a. In some embodiments, the target sequence is located in a coding region of a gene i.e., an exon). In some embodiments, the target sequence is located in a non-coding region of a gene (i.e., an intron, a promoter or an enhancer region of the gene). [00149] In some embodiments, the target sequence is located in a coding region of a human gene selected from TTR, PCSK9 and ANGPTL3. For example, in mouse homolog genes Ttr, Psck9 or Angptl3 selected from SEQ ID NOs: 5, 7, and 8.

[00150] Table 1. Targeting regions used for in vivo genome editing in animal models.

[00151] One skilled in the art can select an appropriate target within a gene of interest by the following method: select sequences (about 20 nucleotides-long) downstream (in a 3’ direction) of a 5’- TTTV PAM motif (or the appropriate PAM motif for a CRISPR endonuclease other than Casl2a) in the coding regions of the genes or any regulatory region of the gene that is to be edited. The list of sequences selected may be narrowed down by taking into account one or more of the following: homology to other regions in the genome; GC content; melting temperature; and presence of homopolymers within the spacer (guide nucleic acid binding sequence).

[00152] Exemplary target sequences selected by the method set forth above for the human PSCK9 gene (SEQ ID NOs: 63-82) are shown in Example 7 section B.

[00153] Other genes can be targeted in order to address the disease or condition caused by aberrant expression of a gene as listed in Table 2.

[00154] Table 2. Diseases and conditions and genes that can be targeted in vivo.

[00155] In some embodiments, the target gene is selected from human genes HSD17B 13, DGAT2, PNPLA3, HNF1, HNF4, SERPINA1, TTR, LPA, ANGPTL3, PCSK9, AGT, APOA, APOB, APOC3, TM6SF2, HMGCR, TERT-hTR, BMPR2, ALAS1, GSDla, HAO1, LDHA, XDH, SERPINC1, F7, F8, F9, F10, F12, F13, IDS, C5, C3, CFB, ALDH2, KLKB1, F12, SERPING1, USH2A, OT, HFE, IDUA, CEP290, RHO and an HBV gene. In some embodiments, the guide polynucleotide targeting the gene from the above list comprises a targeting region (spacer) selected from Table 14 (SEQ ID NOs: 63-82) or from SEQ ID NOs: 143-434 (genes listed in Table 23). In some embodiments, the guide polynucleotide comprises or consists of a sequence from Table 3 (SEQ ID NOs: 10-36) or Table 15 (SEQ ID NOs: 83- 140).

[00156] In some embodiments, genome editing by CRISPR systems produces a genome modification resulting in reduced expression or elimination of expression of one or more genes listed in Table 2 in the modified cell and this brings about alleviation of the symptoms of the disease or condition. In some embodiments, the gene expression is aberrant {i.e., is or becomes abnormally high) and reducing or eliminating the excessive gene transcript results is in alleviation of the symptoms of the disease or condition. In some embodiment, the gene contains a mutation and expression of the mutant protein results in the symptoms of the disease so that eliminating expression of the mutant protein results is in alleviation of the symptoms of the disease or condition.

[00157] In some embodiments, genome editing by CRISPR systems allows insertion of a nucleic acid sequence that results in expression of one or more genes listed in Table 2 in the modified cell and this brings about alleviation of the symptoms of the disease or condition. In some embodiment, the gene contains a mutation and expression of the gene is either abolished or produced a non-functional protein. In some embodiments, the gene expression is or becomes aberrant i.e., abnormally low or absent) and inserting a functional copy of the gene results in restoring gene expression and alleviation of the symptoms of the disease or condition. [00158] In some embodiments, the target nucleic acid comprises a gene that is expressed (or aberrantly expressed) in the liver, e.g., in hepatocytes or sinusoidal endothelial cells of the liver. In some embodiments the gene is expressed in hematopoietic cells throughout the body. In such embodiments, the LNP comprising the CRISPR system is administered systemically, i.e., intravenously.

[00159] In some embodiments, the target nucleic acid comprises a gene that is expressed (or aberrantly expressed) in the cells of the eye. In some embodiments, the LNPs comprising the CRISPR system are delivered into the eye (intraocular delivery). I some embodiments, the delivery is intravitreal. In some embodiments, the delivery is directly to the retina to reach the retinal pigment epithelium.

[00160] In some embodiments, more than one target nucleic acid is cleaved by the CRISPR system. In some embodiments, the additional target nucleic acids are located in the same gene. In some embodiments, the first target nucleic acid is located in the first gene and the additional (i.e., second, third, etc.) target nucleic acids are located in a different (i.e., second, third, etc.) gene. In some embodiments, the first gene and the second, third, etc., genes are active in the same pathway, e.g., lipid metabolism pathway, uric acid/oxalate metabolism, coagulation and the like.

[00161] In some embodiments, the invention is a therapeutic composition comprising the LNP described above (“the LNP composition”) suitable for administration to humans. In some embodiments, the LNP composition comprise a therapeutically effective amount of the CRISPR system components. In some embodiments, the therapeutically effective amount comprises between 0.5 mg/kg and 2 mg/kg of total nucleic acid (including the Casl2a mRNA and the CRISPR guide), hi some embodiments, the therapeutically effective amount comprises between 1 mg/kg of total nucleic acid. In some embodiments, the therapeutically effective amount comprises between 30 mg and 80 mg of total nucleic acid. In some embodiments, the therapeutically effective amount comprises between 50 mg and 80 mg of total nucleic acid.

[00162] In some embodiments, the amount of total nucleic acid has been shown to achieve sufficient genome editing to produce the desired physiological response. In some embodiments, the sufficient genome editing is less than 100% in cells of a target organ. In some embodiments, the sufficient genome editing is at least 65% in cells of a target organ.

[00163] In some embodiments, the LNP composition comprises Casl2a mRNA and two or more different guides targeting two or more target nucleic acids. In some embodiments, the additional guides target one or more target nucleic acids located in the same gene. In some embodiments, the additional guides target one or more target nucleic acids in different genes. In some embodiments, the different genes are active in the same pathway, e.g., lipid metabolism pathway, uric acid/oxalate metabolism pathway, coagulation pathway and the like.

[00164] In some embodiments, the LNP composition comprising two or more different guides is designed for being administered simultaneously. In some embodiments, the composition comprises two or more different guides and the Casl2a mRNA in the same LNP (single LNP composition). In some embodiments, the composition comprises two or more different LNPs, each with the Casl2a mRNA and one of the guides. Such composition comprises a mixture of two or more LNP compositions.

[00165] In some embodiments, the LNP composition comprising two or more different guides is designed for being administered sequentially (two or more LNP compositions for administration at different times). In some embodiments, only the first LNP composition comprises the Casl2a mRNA along with a guide (z.e., the first guide for the first target nucleic acid), while the subsequent compositions include only the guide (z.e., the second guide for the second target nucleic acid) and no Casl2a mRNA. In some embodiments, the first LNP composition and all subsequent LNP compositions comprise a guide (i.e., the first guide or the second guide) and the Casl2a mRNA.

[00166] In some embodiments, the LNP composition undergoes validation studies and safety studies prior to being administered to a human patient. In some embodiments, validation is in vitro validation. In some embodiments, validation is in vivo validation in experimental animals. In some embodiments, validation is a combination of in vitro validation and in vivo validation in experimental animals.

[00167] In some embodiments, prior to administration to a patient, the LNP composition comprising the CRISPR system targeting a particular gene is tested in vitro to assess genome editing properties. Preferably, the system is tested on the cell type that is to be edited in vivo, e.g., hepatocyte cell line or primary hepatocytes for liver editing, or retinal cell lines (retinal pigment epithelium cell lines) for editing cells of the retina.

[00168] In some embodiments, for use in in vitro testing gene editing capabilities of the CRISPR system, the CRISPR endonuclease is recombinantly expressed in . coli and purified using chromatographic methods.

[00169] In some embodiments, for in vitro gene editing, a nucleoprotein complex is formed between the CRISPR endonuclease and the guide. To form an endonuclease-guide nucleoprotein complex, the endonuclease and the guide are mixed at a desired ratio (e.g., 1 :3 or 80 pmol:240 pmol proteimguide and incubated in a suitable buffer (e g., guide, 60mM TRIS- acetate, 150 mM potassium acetate, 30 mM magnesium acetate, at pH 7.9) heated and allowed to equilibrate to room temperature.

[00170] In some embodiments, for in vitro testing gene editing capabilities of the CRISPR system, endonuclease-guide nucleoprotein complexes are transfected into the appropriate cell type by nucleofection, e.g., using the Nucleofector™ 96-well Shuttle System (Lonza, Allendale, N.J.). In some embodiments, after nucleofection, the cells are allowed to incubate, e.g., for 48 hours prior to assessing gene editing.

[00171] In some embodiments, to assess genome editing, genomic DNA from cells is isolated and the site of desired editing is assessed by DNA sequencing. In some embodiments, DNA sequencing is next-generation sequencing (NGS).

[00172] In some embodiments, the sequencing step utilizes an adaptor added at least one end of a nucleic acid or nucleic acid strand. The adaptor can be double-stranded or partially double-stranded and comprises a double-stranded portion that can be ligated to the double stranded nucleic acid to be sequenced. Adaptors of various shapes and functions are known in the art, see e.g., U.S. Patent Nos. 8,822,150 (Y-shaped adaptor); 8,455,193 (stem-loop/hairpin adaptor); and 11,085,084 (various shapes of partially double-stranded adaptors). In some embodiments, the function of an adaptor is to introduce certain useful elements into a nucleic acid, such as barcodes, amplification primer binding sites, sequencing primer binding sites, enzyme recognition sites, and ligation-enabling sites. In some embodiments, the adaptor molecules are in vitro synthesized artificial sequences. In other embodiments, the adaptor molecules are in vitro synthesized naturally occurring sequences. In yet other embodiments, the adaptor molecules are isolated naturally occurring molecules or isolated non-naturally occurring molecules.

[00173] Adaptor ligation can be performed according to methods widely known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, 4^th Ed. Cold Spring Harbor Lab Press (2012). A suitable ligase enzyme catalyzes the formation of phosphodiester linkages between the strands of two nucleic acids strand, e.g., a single-strand DNA ligase such as CircLigase™ ssDNA ligase (Epicentre Biotechnologies, Madison, Wise., or Lucigen, Middleton, Wise.), or a double-strand DNA ligase selected from T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, or E. coli DNA ligase. In some embodiments, the ligation step is preceded by addition of the 5' phosphate e.g., with a polynucleotide kinase such as T4 polynucleotide kinase. In some embodiments, the ligation step is preceded by addition of the 3’-dA (“dA-tailing”) e.g., with a DNA polymerase capable of template-independent addition of a nucleotide such as Taq DNA polymerase.

[00174] In some embodiments, the sequencing step utilizes barcodes. Analyzing individual nucleic acid molecules by massively parallel sequencing typically requires a separate level of barcoding for sample identification and for error correction.

[00175] The use of unique molecular barcodes is described e.g., in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368. A unique molecular identifying barcode (abbreviated UMI or UID) is added to each molecule to be sequenced to mark the molecule and its progeny (e.g., amplicons generated by PCR). In some embodiments, a UMI is present in the 5 ’-portion of an amplification primer. In some embodiments, a UMI is present in an adaptor ligated to the nucleic acid.

[00176] A UMI has multiple uses including counting the number of original target molecules in the sample and error correction (Newman, A., et al., (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nature Medicine doi: 10.1038/nm.3519). Briefly, the entire progeny of a single target molecule is marked with the same UMI barcode and thus forms a barcoded family. A variation in the sequence not shared by all (or the majority) of the members of the barcoded family is discarded as an artefact. UMI barcodes can also be used for positional deduplication and target quantification, as the entire family represents a single molecule in the original sample (Newman, A., et al., (2016) Integrated digital error suppression for improved detection of circulating tumor DNA, Nature Biotechnology 34:547).

[00177] A sample identifying barcode is used for multiplex sequencing. A multiplex sample ID barcode (abbreviated MID or SID) is used to identify the source of the nucleic acid where two or more samples are mixed prior to the sequencing step (e.g., application to a flowcell of a sequencing instrument).

[00178] In some embodiments, the nucleic acid molecule to be sequenced includes a UMI and an MID. In some embodiments, a single barcode is used as both UMI and MID. In some embodiments, a barcode is composed of several parts. For example, the unique identifying information is comprised of a barcode sequence and a nucleic acid end sequence. In some embodiments, a barcode is comprised of several subcodes as described in the U.S. Patent Application Pub. No. 20200109397 “Modular Nucleic Acid Adaptors ”

[00179] In some embodiments, each barcode comprises a predefined sequence. In other embodiments, the barcode comprises a random sequence. The barcodes are about 4-20 bases long, so that between 96 and 384 different adaptors, each with a different pair of identical barcodes can be added to a human genomic sample. In some embodiments, the number of UMIs in the reaction can be in excess of the number of molecules to be labelled. A person of ordinary skill in the relevant art would recognize that the number of barcodes depends on the complexity of the sample (i.e., expected number of unique target molecules) and would be able to design a suitable number of barcodes of suitable lengths for each sequencing run.

[00180] In some embodiments, the method includes sequencing the nucleic acid adapted by the methods described herein. Any of a number of sequencing technologies or sequencing assays can be utilized. The term "Next Generation Sequencing (NGS)" as used herein refers to sequencing methods that allow for massively parallel sequencing of single molecules or clonally amplified single molecules.

[00181] Non-limiting examples of sequence assays that are suitable for use with the methods disclosed herein include nanopore sequencing (U.S. Pat. Publ. Nos. 2013/0244340, 2013/0264207, 2014/0134616, 2015/0119259 and 2015/0337366), Sanger sequencing, capillary array sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman etal., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nature Biotech., 16:381-384 (1998)), sequencing by hybridization (Drmanac et al., Nature Biotech., 16:54-58 (1998), and NGS methods, including but not limited to sequencing by synthesis (e.g., HiSeq™, MiSeq™, or Genome Analyzer, each available from Illumina), sequencing by ligation (e.g., SOLiD™, Life Technologies), ion semiconductor sequencing (e.g., Ion Torrent™, Life Technologies), and SMRT® sequencing (e.g., Pacific Biosciences).

[00182] Commercially available sequencing technologies include sequencing-by- hybridization platforms from Affymetrix Inc. (Sunnyvale, Calif.), sequencing-by-synthesis platforms from Illumina/Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.). Other sequencing technologies include, but are not limited to, the Ion Torrent technology (ThermoFisher Scientific, Waltham, Mass.), Single Molecule Real-Time (SMRT®) sequencing (Pacific Biosciences, Menlo Park, Calif.) and Oxford Nanopore Technologies (Oxford, UK).

[00183] In some embodiments, the sequencing step involves sequence aligning and determining a consensus sequence. In some embodiments, a consensus sequence is determined from a plurality of sequences all having an identical UMI. The sequenced having an identical UMI are presumed to derive from the same original molecule through amplification. In other embodiments, UMI is used to eliminate artifacts, i.e., variations existing in the progeny of a single molecule resulting from PCR errors or sequencing errors.

[00184] In some embodiments, the number or representation of each sequence in a sample can be quantified by quantifying relative numbers of sequences with each UMI among the population having the same multiplex sample ID (MID). A person skilled in the relevant art will be able to determine the number of sequence reads per UMI (“sequence depth”) necessary to determine a consensus sequence with a desired degree of confidence. In some embodiments, the desired depth is 5-50 reads per UMI.

[00185] In some embodiments, the computational script was designed to executes the following tasks: align reads to the mouse genome (e.g., the latest build, currently mm 10) using any suitable software; compare aligned reads to the expected wild type genomic locus sequence; discard reads not aligning to any part of the wild type locus; tally reads matching the wild type sequence; categorize reads with indels (insertion or deletion of bases) by indel type and tally; and determine the proportion (e.g., percentage) of mutant (edited) reads by dividing the tally of indel reads by the sum of wild type reads and indel reads.

[00186] In some embodiments, the invention comprises an amplification step preceding the sequencing step. The amplification step can involve linear or exponential amplification, e.g., PCR. Amplification may be isothermal or involve thermocycling. In some embodiments, the amplification is exponential and involves PCR or any of its variations including real-time PCR, digital droplet PCR (ddPCR), emulsion PCR and the like. In some embodiments, a universal amplification primer is used, i.e., a primer that hybridizes to a universal primer binding site present in the adaptor ligated to all nucleic acids in the sample. The number of amplification cycles where universal primers are used can be low, but also can be 10, 20 or as high as 30 or more cycles, depending on the amount of amplification product needed for the subsequent steps. Because amplification with universal primers has reduced sequence bias, the number of amplification cycles need not be limited out of concern for amplification bias.

[00187] In some embodiments, the LNP composition comprising the CRISPR system is used for patient administration if editing efficiency is at least 65%. The inventors discovered that 100% editing efficiency in the liver is not required to achieve the desired physiological effect. As is seen from FIGURE 8, FIGURE 9, and FIGURE 17, 65% editing results in reduction of Ttr gene expression to undetectable levels. Similarly, less than 100% editing of the Psck9 and AngptlS loci results in reduction of the corresponding gene expression and reduction in serum cholesterol levels FIGURE 19, FIGURE 20, and FIGURE 21.

[00188] One of skill in the relevant art would be able to experimentally determine the minimal editing efficiency required to achieve physiological effect for each target gene in each target organ. The therapeutically effective amount of the LNP composition would include the sufficient amount of CRISPR system components to effect at least the minimal editing efficiency. [00189] In some embodiments, prior to patient administration, the LNP composition comprising the CRISPR system is assessed for causing chromosomal translocations. It has been reported that genome editing involving double-strand breaks (e.g., editing with CRISPR endonucleases) occasionally results in balanced chromosomal translocations between two cleavage sites located on different chromosomes. A detection assay can be designed for the predicted most likely translocation e.g., between a target cleavage site and the predicted most likely off-target cleavage site. One example of such a detection assay is disclosed in the U.S. Provisional Application Ser. No. 63/515,762 In vitro validation methods for CD19-targeting cell therapies, filed on July 26, 2023. Briefly, a series of amplification primers can be designed adjacent to each known or predicted cleavage site involved in the translocation to be detected. In some embodiments, the LNP composition comprising the CRISPR system is administered to a patient if no translocations are detected during the testing or if the rate of translocations falls below a predetermined safety threshold.

[00190] In some embodiments, prior to patient administration, the LNP composition comprising the CRISPR system is assessed for causing off-target editing.

[00191] In some embodiments, potential sites for off-target editing in a given genome are found empirically, e.g., by performing whole-genome sequencing of edited cells and identifying and rating any genome changes not present in unedited genomes. In some embodiments, potential off-target sites are located using algorithms developed for that purpose, e.g., DeWierdt, P., et al. (2021) Optimization ofAsCas!2a for combinatorial genetic screens in human cells, Nat. Biotech. 39(1):94), SITE-Seq, Cas-OFFinder, CRISPRme, and GUIDE-Seq. In some embodiments, the most likely off-target sites are selected for testing, e.g., off-target sites with no more than 4 mismatches with the target site, or no more than 6 mismatches with the target site.

[00192] In some embodiments, amplification primers are designed to amplify and sequence each of the selected potential off-target sites in edited cells to assess genome editing. In some embodiments, the editing rates are determined as Significant Mutant Fraction (SMF) calculated by first determining the "significant" mutations in the matched test and control samples. A mutation (edit) is only considered "significant" if (1) it falls within ±3bp of the predicted cut-site coordinates, and (2) its frequencies in the test and control samples differ significantly as determined by a chi-squared test with p-value threshold of 10'⁴ with Bonferroni multiple-comparison correction. The total frequency of "significant" mutations (edits) is calculated for the test and control samples and the control frequency is subtracted from the test frequency to produce the final Significant Mutation Fraction statistic. In some embodiments, subtraction of the control mutant rate results in a negative number (mutation frequency less than in the control sample). In some embodiments, the negative numbers are recorded as zero.

[00193] In some embodiments, the CRISPR system is used for patient administration if off-target editing is no greater than 0.02% across all off-sites tested.

[00194] With respect to the precision of genomic editing, the inventors devised methods and compositions for in vivo gene editing that substantially improve upon the state of the art. Gillmore et al., report that in the case of in vivo TTR gene editing with CRSIRP-Cas9, up to 7% of off-target editing has occurred. (Gillmore, J. etal., (2021) CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis, NEJM 385:493, appendix, Table SI). In contrast, in the instant disclosure, examples show that off-target editing was below the level of detection (which was 0.03%). An extensive sequencing exploration of off-target sites found no experimental animal with an editing rate exceeding the editing rate in negative control animals (Example 4, section E, Table 11). The methods of selecting and assessing off-target sites were essentially the same as in Gillmore.

[00195] In some embodiments, the LNP composition comprising the CRISPR system is administered to a patient if no off-target editing is detected or if the rate of off-target editing falls below a predetermined safety threshold.

[00196] In some embodiments, prior to administration to a patient, the LNP composition comprising the CRISPR system is tested in vivo in experimental animals to extra-organ editing e.g., extrahepatic editing in case of targeting the liver). In some embodiments, the LNP composition comprising the CRISPR system is used for patient administration if extra-organ editing is no greater than 0.5% across all (or relevant) extra organs tested.

[00197] In some embodiments, prior to administration to a patient, the LNP composition comprising the CRISPR system is tested in vivo for tolerability in experimental animals. In some embodiments, the tolerability is assessed by measuring immune response following the treatment, e.g., 6 hours following the treatment. In some embodiments, immune response is assessed by measuring serum levels of cytokines selected from IL-1, IL-2, IL-2-receptor-a, IL- 6, IL-8, IL-10, IFNy, TNFa, MCP-1 and GM-CSF. In some embodiments cytokine levels are compared before and after the treatment. In some embodiments, cytokine levels are remeasured, e.g„ after 1 or 2 days. In some embodiments, cytokine levels are measured by ELISA.

[00198] In some embodiments, the LNP composition comprising the CRISPR system is used for patient administration if no excessive cytokine levels are observed in experimental animals after 6 hours and cytokine levels return to baseline after one or two days.

[00199] In some embodiments, the composition also includes one or more pharmaceutically acceptable excipients. Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[00200] In some embodiments, the composition further comprises an antimicrobial agent for preventing or deterring microbial growth. In some embodiments, the antimicrobial agent is selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof. [00201] In some embodiments, the composition further comprises an antioxidant added to prevent the deterioration of the lymphocytes. In some embodiments, the antioxidant is selected from ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[00202] In some embodiments, the composition further comprises a surfactant. In some embodiments, the surfactant is selected from polysorbates, sorbitan esters, lipids, such as phospholipids (lecithin and other phosphatidylcholines), phosphatidylethanolamines, fatty acids and fatty esters; steroids, such as cholesterol.

[00203] In some embodiments, the composition further comprises a freezing agent such as 3% to 12% dimethylsulfoxide (DMSO) or 1% to 5% human albumin.

[00204] In some embodiments, the invention is a method of treating a patient by administering to the patient in need thereof an amount of the LNP composition comprises between 0.7 mg/kg and 80 mg of total nucleic acid (including the Casl2a mRNA and the guide and a suitable excipient and optionally, also one or more of a suitable antimicrobial agent, an antioxidant, a surfactant, and a freezing agent.

[00205] In some embodiments, the invention is a method of treating a patient by administering to the patient in need thereof an amount of the LNP composition comprises between 30 mg and 2 mg/kg of total nucleic acid (including the Casl2a mRNA and the guide and a suitable excipient and optionally, also one or more of a suitable antimicrobial agent, an antioxidant, a surfactant, and a freezing agent.

[00206] In some embodiments, the method includes multiplex targeting of genes in the patient. In some embodiments, the method comprises administering to the patient in need thereof an amount of the LNP composition comprising the Casl2a mRNA and two or more guides targeting two or more target nucleic acids. In some embodiments, the two or more guides target two or more target nucleic acids located in the same gene. In some embodiments, the two or more guides target two or more target nucleic acids in different genes. In some embodiments, the different genes are active in the same pathway, e.g., lipid metabolism pathway, uric acid/oxalate metabolism pathway, coagulation pathway and the like. [00207] In some embodiments, the method comprises steps where the LNP comprising the first guide and the LNP comprising additional guides are administered simultaneously. In some embodiments, simultaneous administration involves encapsulation of the two or more guides along with the Casl2a mRNA into the same LNP (single LNP composition). In some embodiments, simultaneous administration involves encapsulation of the two or more guides along with the Casl2a mRNA into different LNPs (a mixture of two or more LNP compositions).

[00208] In some embodiments, the method comprises steps where the first guide and the additional guides are administered sequentially. In some embodiments, only the first LNP composition comprises the Casl2a mRNA along with the guide (i.e., the first guide for the first target nucleic acid). The subsequent compositions include only the guide (i.e., the second guide for the second target nucleic acid) and no Casl2a mRNA. In some embodiments, the first LNP composition and all subsequent LNP compositions comprise the Casl2a mRNA along with the guide (i.e., the first guide or the second guide).

[00209] In some embodiments, the method comprises sequential administrations that are spaced by a period of 24, 48, 72 or 96 hours. The inventors have discovered that in some embodiments, a single administration of Casl2a mRNA supports editing with the coadministered first guide as well as with a second guide administered 24, 48, 72 and even 96 hours later. Without wishing to be bound by a particular theory, the inventors propose that 24- 96 hour delay between multiple edits results in better outcomes because the delay allows repair of the first cleavage by the CRISPR endonuclease before the second cleavage is introduced. The repair prevents chromosomal translocations sometimes associated with multiple double strand breaks occurring in the cell at the same time.

[00210] In some embodiments, the method of treating a patient further involved steps of monitoring the patient for safety and effectiveness of the treatment.

[00211] In some embodiments, the patient is monitored for tolerability of the treatment with LNPs containing the CRISPR system components described herein. In some embodiments, the tolerability is assessed by measuring immune response following the treatment, e.g., 6 hours following the treatment. In some embodiments, immune response is assessed by measuring serum levels of one or more cytokines selected from IL-1, IL-2, IL-2-receptor-a, IL-6, IL-8, IL-10, IFNy, INF a, MCP-1 and GM-CSF. In some embodiments cytokine levels are compared before and after the treatment. In some embodiments, cytokine levels are remeasured, e.g., after 1 or 2 days. In some embodiments, cytokine levels are measured by ELISA.

[00212] In some embodiments, in cases of liver editing, the tolerability is assessed by measuring the amount of liver enzymes. In some embodiments, liver enzymes are aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In some embodiments, the amount of liver enzymes is assessed in vivo in experimental animals. In some embodiments, the LNP composition comprising the CRISPR system is used for patient administration if no significant change in the amount of liver enzymes is observed.

[00213] In some embodiments, the patient is periodically assessed for durability of response to treatment with LNPs containing the CRISPR system components described herein. In some embodiments, the patient is assessed for the presence of the protein whose gene has been edited or for physiological response. In some embodiments, e.g., when the gene is TTR, PSCK9 or ANGPTL3, the patient is assessed for serum level of the respective protein. In some embodiments, e.g., when the gene is PSCK9, the patient is assessed for serum cholesterol level, e.g., LDL cholesterol level or total cholesterol level. In some embodiments, e.g., when the gene is ANGPTL3, the patient is assessed for serum triglyceride level, e.g., VLDL or chylomicron level or total triglyceride level. In some embodiments, the patient is assessed monthly or biweekly.

[00214] In some embodiments, the invention is a method of treating an autosomal dominant disease, i.e., an inherited disease where a single mutant allele (despite the presence of the normal allele on the other chromosome) brings about the disease phenotype. The inventors have surprisingly discovered that the CRISPR LNP composition disclosed herein is able to discriminate between an allele perfectly matched to the targeting region of the guide polynucleotide and an allele differing from the targeting region of the guide polynucleotide by a single nucleotide (FIGURE 27). The inventors utilized this newly discovered property of the CRISPR LNP composition to devise a method of treating an autosomal dominant disease by administering the CRISPR LNP composition in order to selectively cleave the mutant (disease- causing) allele and spare the wild-type allele thereby inactivating the disease-causing allele while preserving the function of the normal allele.

[00215] In some embodiments, the invention is a method of treating or alleviating an autosomal dominant disease or condition characterized by a mutant allele dominant over a wildtype allele, the alleles differing by at least one nucleotide change in a gene sequence, the method comprising administering to a patient the CRISPR LNP composition disclosed herein wherein the guide polynucleotide of the composition is capable of promoting cleavage of the mutant allele but not the wild-type allele.

[00216] In some embodiments, the mutant gene associated with an autosomal dominant disease is selected from Table 24.

[00217] Table 24. Diseases with autosomal dominant inheritance and associated genes

[00218] In some embodiments, the mutant gene associated with an autosomal dominant disease is selected from Table 24.

[00219] Many of the genes listed in Table 24 exert their function through expression in the liver. In some embodiments, the invention comprises administering the CRISPR LNP composition intravenously thereby inactivating the disease allele expressed in the liver. Some of the genes listed in Table 24 exert their function through expression in the lung or the gastrointestinal (GI) tract. In some embodiments, the invention comprises administering the CRISPR LNP composition directly to the lung or the GI tract thereby inactivating the disease allele expressed in the cells of those organs.

[00220] In some embodiments, the method comprises administering the CRISPR LNP composition to a patient exhibiting symptoms of an autosomal dominant disease (having the disease phenotype). In some diseases, the symptoms involve irreversible degenerative changes to tissues and organs. To prevent the irreversible damage, in some embodiments the method comprises administering the CRISPR LNP composition to an asymptomatic or presymptomatic patient having the heterozygous genotype associated with autosomal dominant disease.

[00221] In some embodiments, the mutant allele and a wild-type allele differ by a single nucleotide (SNP). In some embodiments, the targeting region of the guide polynucleotide is perfectly complementary to the mutant allele and has at least one mismatch with the wild-type allele at the mutant (SNP) site. In some embodiments, the entire length of the targeting region of the guide polynucleotide is perfectly complementary to the mutant allele. In some embodiments, the entire length of the targeting region of the guide polynucleotide is not perfectly complementary to the mutant allele but is sufficiently complementary to the mutant allele to enable cleavage of the mutant allele by the CRISPR endonuclease and to not enable cleavage of the wild-type allele by the CRISPR endonuclease.

[00222] In some embodiments, multiple mutations in the disease-associated gene are known to be associated with the autosomal dominant disease. In some embodiments, a patient exhibiting the disease phenotype is first tested to identify the genotype, i.e., the mutation carried by the patient and the CRISPR LNP composition comprising the guide polynucleotide targeting the patient’s mutation is administered to the patient. In some embodiments, the method comprises administering to a patient exhibiting the disease phenotype a mixture of several LNPs each LNP enclosing a CRISPR system comprising a guide polynucleotide targeting a different mutation so that the entire mixture of LNPs contains a guide polynucleotide for each of the known mutations.

[00223] EXAMPLES

[00224] Example 1. In vitro gene editing in a mouse hepatocyte cell line Casl2a-guide nucleoprotein complexes^

[00225] This example describes a method for cloning, expressing, and purifying Casl2a, methods of producing Casl2a guide components forming Casl2a-guide nucleoprotein complexes, and nucleofection of the complexes into mouse hepatocytes.

[00226] A. Cloning of the Casl2 protein

[00227] The Acidaminococcus spp. (strain BV3L6) catalytically active Casl2a protein sequence was codon optimized for expression in E. coli cells. SEQ ID NO; 1 is the AsCasl2a protein with a nuclear localization sequence (NLS) connected to the C-end of the protein via a linker. NLS with the linker is shown separately as SEQ ID NO: 2. Oligonucleotide sequences coding for the Casl2a-NLS protein (referred to as the AsCasl2a and Casl2a protein in the following Examples) were provided to commercial manufacturers for synthesis. DNA sequences were then cloned into suitable bacterial expression vectors using standard cloning methods.

[00228] B. Expression and purification of the Casl2a protein

[00229] The AsCasl2a protein was expressed in E. coli using an expression vector and purified using affinity chromatography, ion exchange, and size exclusion chromatography, essentially as described in, for example, Swarts et al. (Molecular Cell, 2017, 66:221-233).

[00230] C. Designing Casl2a guides [00231] Casl2a guides were designed to target the following mouse genes proprotein convertase subtilisin/kexin type 9 Pcsk9 transthyretin (Hr). and angiopoietin-like 3 (Angpt3

[00232] A collection of 20-nucleotide sequences downstream (in a 3’ direction) of a 5’- TTTV PAM motif in the coding regions of the genes encoding mouse Pcsk9, Ttr, and Angptl3 were selected for targeting (Pcsk9'. SEQ ID NO: 5, Ttr'. SEQ ID No: 7, and Angptl3'. SEQ ID No: 9). Target selection criteria included, but were not limited to, homology to other regions in the genome; percent G-C content; melting temperature; and presence of homopolymer within the spacer.

[00233] To generate the all-RNA (“crRNA”) control guides, the identified 20-nucleotide sequences were appended downstream (in a 3’ direction) to the AsCasl2a activating region sequence (SEQ ID NO: 3). For the chemically modified (“chem-mod”) the activating region and the targeting region of each guide was modified with phosphorothioate, 2'-O-methylation, and deoxyribonucleotides at various positions (FIGURE 1, Table 3). Sequences were provided to commercial manufacturers for synthesis.

[00234] Table 3. crRNA and chemically modified Casl2a guides.

“r”: RNA, phosphorothioate, “m”: 2’-O-methylation

[00235] D. Production of Casl2a guide components

[00236] Casl2a guides were produced by linking a targeting region to a particular Casl2a guide activating region. A targeting region, or spacer, preferably comprised a 20- nucleotide target binding sequence. The target binding sequence was complementary to a target sequence that occurred downstream (in a 3’ direction) of a 5’- TTTV or 5’ - TTTN PAM. An exemplary Casl2a guide activating region for the Acidaminococcus spp Cas l 2a species is SEQ ID NO: 3. An exemplary crRNA with the targeting region is SEQ ID NO: 4.

[00237] Casl2a guide sequences (such as crRNAs and chRDNA) were provided to a commercial manufacturer for synthesis.

[00238] E. Assembly of a Casl2a-guide nucleoprotein complex

[00239] Acidaminococcus spp. Casl2a (AsCasl2a) tagged with a C-terminal nuclear localization sequence (NLS) was recombinantly expressed in E. coli and purified using chromatographic methods. Nucleoprotein complexes were formed at a concentration of 80 pmol Casl2a protein:240 pmol guide, unless otherwise stated. Prior to assembly with Casl2a protein, each of the guide components (e.g., crRNA or chRDNA) was adjusted to the desired total concentration (240 pmol) in a final volume of 1 pl, incubated for 2 minutes at 95°C, removed from a thermocycler, and allowed to equilibrate to room temperature. The Casl2a protein was diluted to an appropriate concentration in binding buffer (60mM TRIS-acetate, 150 mM potassium acetate, 30 mM magnesium acetate, at pH 7.9) to a final volume of 1.5 pl and mixed with the 1 pl of the guide components, followed by incubation at 37°C for 10 minutes.

[00240] F. Nucleofection of H2.35 hepatocytes with Casl2a-guide nucleoprotein complexes.

[00241] The Casl2a-guide nucleoprotein complexes of were transfected into H2.35 cells using the Nucleofector™ 96-well Shuttle System (Lonza, Allendale, N.J.). The Casl2a-guide nucleoprotein complex was dispensed in a 2.5 pl final volume into individual wells of a 96- well plate. The adherent H2.35 cells were washed with lOmL of calcium and magnesium -free phosphate-buffered saline (PBS) and then PBS aspirated, followed by the addition of 5mL of ACCUTASE (Innovative Cell Technologies, Inc., San Diego, Cal.) and incubated for 5-10 minutes at room temperature. Flask was rocked side to side and tapped against the palm of the hand to detach cells. H2.35 cell suspension was pelleted by centrifugation for 5 minutes at 200 x g, washed with calcium and PBS, and the cell pellet was resuspended in 10 ml of calcium and magnesium-free PBS. The cells were counted using the Countess® II Automated Cell Counter (Life Technologies; Grand Island, N.Y.).

[00242] 10⁷ cells were transferred to a 15 ml conical tube and pelleted. The PBS was aspirated, and the cells resuspended in CTS Xenon Electroporation Buffer (ThermoFisher Scientific, Wilmington, Del.) solution to a density of 10⁵ - 2.5 x 10⁵ cells/ml per sample. 18 pl of the cell suspension was then added to each well containing 2.5 pl of the Casl2a-guide nucleoprotein complexes, and the entire volume from each well was transferred to a well of a 96-well Nucleocuvette™ Plate (Lonza). The plate was loaded onto the Nucleofector™ 96-well Shuttle and cells nucleofected using the EH-110 Nucleofector™ program (Lonza). Post- nucleofection, 77.5 pl of DMEM (4g/L glucose) medium supplemented with lOOnM dexamethasone and 4% fetal bovine serum was added to each well, and the entire volume of transfected cell suspension was transferred to a 96-well cell culture plate containing 100 pl pre- warmed DMEM complete medium. The plate was transferred to a tissue culture incubator and maintained at 33°C in 10% CO2 for 48 hours before downstream analysis.

[00243] G. Determining genome editing efficiency

[00244] Genomic DNA (gDNA) was isolated from the nucleofected H2.35 cells 48 hours after transfection with 50 pL QuickExtract™ DNA extraction solution (Epicentre, Madison, Wise.) per well, followed by incubation at 37°C for 10 minutes, 65°C for 30 minutes, and 95°C for 3 minutes to stop the reaction. The isolated gDNA was diluted with 50 pL sterile water and samples were stored at -80°C.

[00245] Using the isolated gDNA, a first PCR was performed using Q5 Hot Start High- Fidelity 2X Master Mix (New England Biolabs, Ipswich, Mass.) at lx concentration, primers designed to amplify the region around the Casl2a target were used at 0.5 pM each, and 3.75 pL of gDNA was used in a final volume of 10 pL. Amplification was conducted by an initial cycle at 98°C for 1 minute, 35 cycles of 10s at 98°C, and 20 seconds at 60°C, 30 seconds at 72°C; and a final extension at 72° C for 2 minutes. The PCR reactions were diluted 1 : 100 in water.

[00246] PCR with barcoded primers was performed using a reaction mix comprising Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) at lx concentration, primers at 0.5 pM each, and 1 pL of 1: 100 diluted first PCR in a final volume of 10 pL. The reaction mixtures were amplified as follows: 98°C for 1 minute; followed by 12 cycles of 10s at 98°C, 20 seconds at 60°C, and 30 seconds at 72°C; with a final extension reaction at 72°C for 2 minutes.

[00247] The PCR reactions were pooled and transferred into a single microfuge tube for SPRIselect (Beckman Coulter, Pasadena, Cal.) bead-based cleanup of amplicons for sequencing.

[00248] To the amplicon, 0.9x volumes of SPRIselect beads were added, mixed, and incubated at room temperature for 10 minutes. The microfuge tube was placed on a magnetic tube stand until the solution cleared. Supernatant was removed and discarded, the residual beads were washed with 1 volume of 85% ethanol, and the beads were incubated at room temperature for 30 seconds. After incubation, ethanol was aspirated, and the beads were air-dried at room temperature for 10 minutes. The microfuge tube was removed from the magnetic stand and 0.25x volumes of Qiagen EB buffer (Qiagen, Valencia, Cal.) was added to the beads, mixed vigorously, and incubated for 2 minutes at room temperature. The microfuge tube was returned to the magnet, incubated until the solution had cleared, and supernatant containing the purified amplicons was dispensed into a clean microfuge tube. The purified amplicons were quantified using the Nanodrop™ 2000 System (ThermoFisher Scientific, Wilmington, Del.) and library quality analyzed using the Fragment Analyzer™ System and the DNF-910 dsDNA Reagent Kit (Advanced Analytical Technologies, Ames, Iowa).

[00249] The pooled amplicons were normalized to a 4 nM concentration as calculated from the Nanodrop™ 2000 System values and the average size of the amplicons. The library was analyzed on a MiSeq Sequencer with MiSeq Reagent Kit v2 (Illumina, San Diego, Cal.) for 300 cycles with two 151-cycle paired-end runs and two 8-cycle index reads.

[00250] The identities of products in the sequencing data were determined based on the index barcode sequences adapted onto the amplicons in the barcoding PCR. A computational script was used to process the MiSeq data that executes, for example, the following tasks: a. Reads were aligned to the mouse genome (build mm 10) using Bowtie software; b. Aligned reads were compared to the expected wild type genomic locus sequence, and reads not aligning to any part of the wild type locus discarded; c. Reads matching wild type sequence were tallied; d. Reads with indels (insertion or deletion of bases) were categorized by indel type and tallied; and e. Total indel reads were divided by the sum of wild type reads and indel reads to give percent-mutated reads.

[00251] Through the identification of indel sequences at regions targeted by the Cas 12a- guide nucleoprotein complexes, the resulting genome editing efficiency was determined.

[00252] The editing efficiency of three replicates from the in-cell editing experiment are shown in Table 4 below.

[00253] Table 4 Editing efficiency of crRNA and chemically modified Casl2a guides.

[00254] Example 2. In vivo luciferase gene delivery via lipid nanoparticles (LNP)

[00255] In this example, various LNP formulations were used to establish effective in vivo delivery of luciferase mRNA into mice. Firefly luciferase (ffLuc) mRNA (“CleanCap FLuc mRNA”) was obtained from TriLink Biotechnologies (San Diego, Cal.).

[00256] A. LNP preparation

[00257] The lipid mixtures were prepared in ethanol as shown in Table 5. 1 mL of each lipid mixture was loaded into a syringe for the encapsulation step. A commercially available lipid mix for LNP composition Gen-Voy™ (Precision Nanosystems, Vancouver, B.C.) was used for comparison. Structures of the lipid components is shown in FIGURE 2.

[00258] Table 5 Lipid mixtures.

[00259] RNA was prepared in 50mM sodium acetate. RNA concentration was confirmed with Nano-drop (ThermoFisher Scientific), and 3 mL containing 200 ug (or 300 ug) of RNA in sodium citrate was loaded into a syringe for the encapsulation step.

[00260] For LNP formation (encapsulation), a syringe with 3 mL of an RNA mixture and a syringe with 1 mL of a lipid mixture was inserted into the NanoAssemblr® Ignite™ (Precision Nanosystems, Vancouver, BC). The NanoAssemblr® Ignite™ was pre-loaded with a NanoAssemblr Ignite Cartridges (NxGen) to form water-in-oil droplets using rapid mixing. Encapsulation efficiency was assessed using the Quant-iT™ RiboGreen Assay Kit (ThermoFisher Scientific). LNP size and polydispersity was measured with an UNCLE instrument (Unchained Labs, Pleasanton, Cal.) using Dynamic Light Scattering (DLS).

[00261] B. LNP delivery

[00262] Seven groups of 3 NGS mice per group received injections into the tail vein of 200 uL (or 300 uL) as listed in Table 6 to deliver 1 mg/kg of RNA.

[00263] Table 6. In vivo delivery of luciferase

[00264] In vivo imaging to measure bioluminescence was performed with the IVIS® Spectrum in vivo imaging system on days 0, 2, 3, 6, and 8. Results for days 0-8 are shown in FIGURE 3 (bioluminescence intensity) and FIGURE 4 (area under the curve (AUC) of bioluminescence intensity).

[00265] Example 3. Encapsulating CRISPR components in LNP for in vivo delivery.

[00266] This example describes forming LNPs containing Casl2a mRNA and guides (crRNA or chRDNA) targeting Pcsk9, Ttr and Angptl3. [00267] Lipid mixtures “ALC-0315” were prepared essentially as described in Example 2 for that lipid formulation. A combination of 109.5 ug Casl2a mRNA and 109.5 ug of each of the guides SEQ ID NOs: 19 (crRNA) and SEQ ID NO: 27 (chRDNA) was mixed with the lipid mixture as described in Example 2 to form LNPs.

[00268] LNPs were evaluated for encapsulation efficiency (FIGURE 5) using the Quant-iT™ RiboGreen Assay Kit (ThermoFisher Scientific, Wilmington, Del.) to confirm that the LNPs fall into the range of 70-100% suitable for in vivo delivery. LNPs were further evaluated for diameter (FIGURE 6) with an UNCLE Instrument (Unchained Labs, Pleasanton, Cal.) using Dynamic Light Scattering (DLS) to confirm that the LNPs fall into the range of 65- 100 nm range that does not cause immune response and is optimal for receptor-mediated cellular uptake. Lastly, LNPs were evaluated for uniformity (FIGURE 7) evaluated as poly dispersity index (PDI) measured with an UNCLE instrument using Dynamic Light Scattering to confirm that the LNPs are uniform as indicated by PDI below 0.25.

[00269] Example 4. In vivo liver Ttr gene disruption

[00270] In this example, an LNP composition containing CRISPR components targeting the mouse Ttr gene were delivered, genome editing and physiological effects on the mice were observed.

[00271] A. Administering CRISPR components via LNP

[00272] Seven-week-old BALB/c mice (4 mice per treatment group, average weight of 20g/mouse) were injected with an LNP composition containing ~40ug of Cast 2a mRNA and one of the guides of SEQ ID NOs: 19 (crRNA) and SEQ ID NO: 27 (chRDNA) (Example 3). Each mouse received three IV injections of 2mg/kg of total RNA with a 48-hour spacing between injections. The control group received saline injections on the same schedule. The mice were taken down after 12 days to assess editing of the Ttr gene and plasma levels of TTR.

[00273] B. Measuring Ttr gene editing

[00274] To assess genome editing, genomic DNA was isolated and subjected to nextgeneration sequencing to assess degree of genome editing. Genomic DNA was extracted from mouse liver punchouts using a DNeasy Blood & Tissue Kit (Qiagen). Three technical replicates were subjected to next-generation sequencing for each mouse. Target amplification and sequencing were performed on the Illumina MiSeq instrument according to manufacturer’s instructions. The amplification primers for the Ttr locus are shown in Table 7.

[00275] Table 7. Primers for the Ttr locus.

[00276] Comparable levels of genome editing (-65%) were observed for crRNA anc chRDNA (FIGURE 8). TTR protein was deleted in serum by ELISA on days zero and 16 post- LNP injection using a commercial ELISA kit (Aviva Systems Biology, San Diego, Cal.) #OKIA00111. Briefly, 5ul of plasma were diluted to a final dilution of 1/20,000 following manufacturer’s recommendations. TTR was readily detected in the control sample but was undetectable in both the crRNA mice and the chRDNA mice (FIGURE 9).

[00277] C. Dose-dependent reduction of plasma TTR levels

[00278] The treatment protocol described in A. was performed with three doses of crRNA (SEQ ID NO: 19): 0.3 mg/kg, 1 mg/kg and 3 mg/kg. Gene editing and serum TTR were assessed as in B. A dose-dependent level of genome editing was observed on day 25 (FIGURE 10). A dose-dependent reduction of serum TTR was observed on day 19 (FIGURE 11).

[00279] D. Tolerability of the LNP treatment

[00280] Weight measurements were taken of mice in C. between days 0 and 20. Minimal body weight changes where observed (FIGURE 12). Immunogenicity was assessed 6 hours and 12 days after the injection. Serum levels of TNF-alpha and IL-6 were assessed by ELISA using commercially available kits (MTA00B-1 andM6000B-l respectively from R&D systems (Minneapolis, Minn.). After the initial increase, cytokines levels became negligible by day 12 (FIGURE 13 and FIGURE 14). Liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assessed 24 hours post-LNP injection. No significant increase compared to the saline control was observed for ALT (FIGURE 15) or for AST (FIGURE 16).

[00281] E. Off-target editing and extrahepatic editing [00282] In this experiment, genomic DNA samples were taken from each mouse (Example 4) to detect gene alterations at the Ttr target site and at the off-target sites in the liver and extrahepatic organs.

[00283] To select potential off-target sites for Casl2a editing, the algorithm described in DeWierdt (see DeWierdt, P., et al. (2021) Optimization ofAsCas!2a for combinatorial genetic screens in human cells, Nat. Biotech. 39( 1 ) : 94) was applied to the mouse genome (Mus musculus genome build mm 10). The algorithm yields a “CFD score” indicating a likelihood that the off- target site will be edited by the Casl2a endonuclease. For the initial screen, the sites with 3 and 4 mismatches relative to the Ttr target were selected, and additional sites with 5 and 6 mismatches were selected by descending CFD score. The initial screen yielded 2000+ hits. For sequencing, top 10 off-target sites were selected (Table 8) The selected sites included 3-4 mismatches, and any sites with mismatches in the seed region of the spacer were excluded.

[00284] Table 8. Off-target sites tested by sequencing.

[00285] Amplification primers (Table 9) were designed for the selected sites.

[00286] Table 9. Amplification primers for off-target sites.

[00287] Table 10. Amplicon length.

[00288] To assess off-target editing in the liver, genomic DNA was extracted from mouse liver punchouts. Three technical replicates were subjected to next-generation sequencing for each mouse. Target amplification and sequencing were performed on the Illumina MiSeq instrument according to manufacturer’s instructions. [00289] The results are shown in Table 11. The editing values were determined as Significant Mutant Fraction (SMF) defined in the Definitions section. Each result is the maximum value of editing among the four mice in each category (see Example 4). For each mouse, the value of editing was determined as the average of three replicates. Negative values (fewer mutations than the control sample) are represented as 0.

[00290] Table 11. Off-target editing for mice in different treatment categories.

[00291] To assess extrahepatic editing of the Ttr target site, genomic DNA was extracted in three replicates from each organ and amplification and sequencing performed on MiSeq according to the manufacturer’s instructions. The forward and reverse primers for the Ttr locus are shown in Table 12.

[00292] Table 12. Primers for the Ttr locus. [00293] The average editing rate or three replicates for each mouse is shown in Table

13.

[00294] Table 13. Editing of the Ttr locus in the liver and other organs. [00295] F. Dose escalation study

[00296] In this example, seven-week-old BALB/c mice (4 mice per treatment group, average weight of 20g/mouse) received a single injection of the LNP composition (Example 3) with increasing amounts of Cast 2a mRNA and of the /'//'-targeting crRNA (SEQ ID NO: 19) (for final doses of 0.125 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, and 2 mg/kg). The control group received a saline injection. Plasma levels of TTR and Ttr gene editing were assessed on day 12 as described in Example 4 sections B and C respectively. Results are shown in FIGURE 17.

[00297] G. Durability of response

[00298] In this example, a long-term study was undertaken to assess the durability of reduction in plasma Ttr following a single administration of LNPs. 4 mice in each group received injections of the LNP composition (Example 3) with either ~20 or ~40ug of Casl2a mRNA and the ///'-targeting guides, either the crRNA (SEQ ID NO: 19) or a chRDNA (SEQ ID NO: 27) at either 1 mg/kg or 2 mg/kg. The control group received a saline injection. Blood samples were taken monthly to assess plasma TTR levels (Example 4, section B). Results are shown in FIGURE 18.

[00299] Example 5. In vivo disruption ofPsck9 andAngptl3 and effect on serum cholesterol

[00300] In this example, the LNP-based method was applied to mouse genes Psck9 and Angptl3.

[00301] For Pcsk9, 8 mice in each group received a single injection of the LNP composition (Example 3) with ~40ug of the Casl2a mRNA and one of the guides for Psck9, crRNA (SEQ ID NO: 10) and chRDNA (SEQ ID NO: 18) (2 mg/kg). For Angptl3, each mouse of the group (4 mice in each group) received three IV injections of 2mg/kg of total RNA with a 48-hour spacing (3x2 mg/kg). LNP composition (Example 3) was ~40ug of the Casl2a mRNA and one of the guides for Angptl3: crRNA (SEQ ID NO: 39) and chRDNA (SEQ ID NO: 40). Gene editing at the Psck9 and Angptl3 loci was assessed per Example 4, section B. Results are shown in FIGURE 19. Plasma protein levels for PCSK9 and ANGPTL3 were assessed by ELISA, following manufacturer’s recommendations Angptl3: R&D Systems, #MNL30; Pcsk9: R&D Systems, #MPC900). Serum cholesterol levels were measured from blood plasma by IDEXX BioAnalytics Inc. (Columbia, Mo.) Results are shown in FIGURE 20 (Psck9) and FIGURE 21 (AngptlS).

[00302] Example 6: Preparation of human cytotoxic T cells (CD4+ and CD8+) from PBMCs and culture of primary cells

[00303] This Example illustrates the preparation of CD4+ and CD8+ T cells from donor peripheral blood mononuclear cells (PBMCs).

[00304] CD4+ and CD8+ T cells were prepared from donor PBMCs essentially as follows. T cells were isolated from peripheral blood mononuclear cells (PBMCs) using RoboSep-S (STEMCELL Technologies, Cambridge, Mass) and EasySep™ Human T cell Isolation Kit (STEMCELL Technologies) and activated for 3 days in the presence of anti- CD3/CD28 beads (Dynabeads™; ThermoFisher Scientific, Gibco brand) in ImmunoCult-XF complete medium (ImmunoCult-XF T Cell Expansion Medium (STEMCELL Technologies), CTS Immune Cell SR (Gibco A2596102), Antibiotics-Antimycotics (100X, Coming 30-004- Cl)) supplemented with recombinant human (rh) IL-2 (100 units/mL). After 3 days, beads were removed via magnetic separation and cells were expanded for 1 day in ImmunoCult-XF complete medium supplemented with IL-2 (100 units/mL).

[00305] Table 2

[00306] : Nucleofection of T Cells (CD4+ and CD8+) with Casl2a-guide nucleoprotein complexes

[00307] This example describes the nucleofection of activated T cells with a Casl2a- guide nucleoprotein complex.

[00308] The Casl2a-guide nucleoprotein complexes of Example 2 were transfected into primary activated T cells (CD4+ and CD8+) (prepared as described in Example 6) using the Nucleofector™ 96-well Shuttle System (Lonza, Allendale, N.J.). The Casl2a-guide nucleoprotein complex was dispensed in a 2.5 pl final volume into individual wells of a 96- well plate. The suspended T cells were pelleted by centrifugation for 10 minutes at 200 x g, washed with calcium and magnesium -free phosphate buffered saline (PBS), and the cell pellet was resuspended in 10 ml of calcium and magnesium-free PBS. The cells were counted using the Countess® II Automated Cell Counter (Life Technologies; Grand Island, N.Y.). [00309] 2.2 x 10⁷ cells were transferred to a 15 ml conical tube and pelleted. The PBS was aspirated, and the cells resuspended in Nucleofector™ P4 or P3 (Lonza, Allendale, N.J.) solution to a density of 2 x 10⁵- 10⁶ cells/ml per sample. 20 pl of the cell suspension was then added to each well containing 2.5 pl of the Casl2a-guide nucleoprotein complexes, and the entire volume from each well was transferred to a well of a 96-well Nucleocuvette™ Plate (Lonza). The plate was loaded onto the Nucleofector™ 96-well Shuttle (Lonza) and cells nucleofected using the CA137 Nucleofector™ program (Lonza). Post-nucleofection, 77.5 pl of ImmunoCult-XF complete medium supplemented with IL-2 (100 units/mL) was added to each well, and the entire volume of transfected cell suspension was transferred to a 96-well cell culture plate containing 100 pl pre-warmed ImmunoCult-XF complete medium supplemented with IL-2 (100 units/mL). The plate was transferred to a tissue culture incubator and maintained at 37°C in 5% CCL for 48 hours before downstream analysis.

[00310] Example 8: Tiling of human PCSK9 gene with Casl2a

[00311] This Example illustrates the targeting of human CD4+ and CD8+ T cells from donor peripheral blood mononuclear cells (PBMCs) with Casl2a guides targeting the /TIS' gene.

[00312] A. Designing the AsCas 12a crRNA guides

[00313] All 20-nucleotide sequences downstream (in a 3’ direction) of a 5’ - TTTV PAM motif in the coding regions of the genes encoding human PCSK9 were selected for targeting (SEQ ID NOs: 63-82). Target selection criteria included, but were not limited to, homology to other regions in the genome; percent G-C content; melting temperature; and presence of homopolymer within the spacer.

[00314] The identified 20-nucleotide sequences were appended downstream (in a 3’ direction) to the AsCasl2a activating region sequence (SEQ ID NO: 3)

[00315] Sequences were provided to commercial manufacturers for synthesis. Then, individual Casl2a-guide nucleoprotein complexes were prepared as described in Example 6 and transfected into primary T cells as described in Example 7.

[00316] B. Determining genome editing efficiency

[00317] For deep sequencing, genomic DNA was isolated from the nucleofected primary T cells 48 hours after transfection using the Casl2a guide/nucleoprotein complexes and 50 pL QuickExtract™ DNA extraction solution (Epicentre, Madison, Wise.) per well, followed by incubation at 37°C for 10 minutes, 65°C for 30 minutes, and 95°C for 3 minutes to stop the reaction. Editing efficiency of the isolated gDNA was determined in a manner similar to Example 1 section D, and editing efficiency of three technical replicate (n=3) is presented in Table 14:

[00318] Data presented in Table 14 demonstrates the targeting of the human PCSK9 gene with Casl2a. Other human genes can be targeted as described herein.

[00319] Example 9: Chemically modified Casl2a guides targeting human PCSK9

[00320] The following Example describes the engineering of AsCasl2a guide molecules to comprise DNA bases or both chemically modified and DNA bases in the activating region and target binding sequence.

[00321] A. In silica Casl2a chRDNA guide design

[00322] Two AsCasl2a guide targeting human PSCK9 (SEQ ID NO: 79 and SEQ ID NO: 71) were selected for engineering of chemically modified “chemmod” guides. Phosphorothioate, 2’-O-methylation, deoxyribonucleotides, or phosphorothioate deoxyribonucleotides were engineered in the activating region and target binding sequence of the AsCasl2a crRNA molecules targeting PCSK9-tgtl7 (SEQ ID NO: 83) and PCSK9-tgt9 (SEQ ID NO: 112). Engineered Casl2a guides are shown in Table 15 below (ribonucleotides are shown with “r” (the absence of “r” indicates deoxyribonucleotides); phosphorothioate nucleotides are shown with a after the nucleotides, 2’-O-methylation is shown with a “m”). Control crRNAs (SEQ ID NOs: 83 and 112) are also shown.

[00323] The guides are also illustrated in FIGURE 22. In FIGURE 22, the left-hand nucleotides 1-20 represent the activating region, and the right-hand nucleotides 1-20 represent the targeting region. Each group of 29 guides (SEQ ID NOs: 83-111 for PSCK9 target 17 and SEQ ID NOs.: 112-140 for PSCK9 target 9) is represented by the diagram. The top guide in the diagram represents the unmodified crRNA for each of the two groups (SEQ ID NO: 83 for PSCK9 target 17 and SEQ ID NO.: 112 for PSCK9 target 9).

[00324] Sequences presented in Table 15 were provided to a commercial manufacturer for synthesis.

[00325] Individual Casl2a-guide nucleoprotein complexes for screening were prepared essentially as described in Example 1. The nucleoprotein complexes were transfected into primary T cells as described in Example 7, and the resulting genome editing efficiency of the Casl2a-guide nucleoprotein complexes was determined as described in Example 8. The results of the in-cell editing experiment are shown in Table 16 below.

[00326] The editing results in Table 16 above demonstrate that Casl2a chRDNA and Casl2a chemically modified chRDNAs are capable of editing at a rate comparable to the crRNA across multiple targets.

[00327] Example 9. Primary hepatocytes from transgenic mice carrying human liveractive genes.

[00328] Transgenic mice carrying human genes were obtained from The Jackson Laboratory.

[00329] The human APOC3 transgenic mice B6;CBA-Tg(APOC3)3707Bres/J, strain # 006907 are described in Reaven G.M., el al., (1994) Elypertriglyceridemic mice transgenic for the human apolipoprotein C-III gene are neither insulin resistant nor hyperinsulinemic, J Lipid Res 35(5): 820-4. According to the description provided by the supplier, to produce the strain, transgenic construct containing the human apolipoprotein C-III gene (including 2.5 kb 5' and 1.1 kb 3' flanking sequences) was injected into the male pronucleus of fertilized eggs (both from (C57BL/6J x CBA/J)F1 mice). The resulting animals were bred to (C57BL/6J x CBA/J)F1 to establish a founder line. Given the high expression of the transgene in the mice, multiple copies are likely.

[00330] The human SERPINA1 transgenic mice C57BL/6J- Tg(SERPINAl*E366K)lMlb/J, strain # 037670 are described in Lu Y., et al., (2022) The unfolded protein response to PI*Z alpha-1 antitrypsin in human hepatocellular and murine models, Hepatol Commun 6(9)2354-2367. The PI*Z mice express a mutant human SERPINA 1 gene which carries the mutation E366K. According to the description provided by the supplier, to produce the strain, the linearized transgenic construct containing the entire mutant human E366K SERPINA1 gene under its endogenous promoters (both hepatocyte and macrophage), plus 5 kB of the 5' and 3 kb of the 3' flanking genomic DNA sequences was microinjected into fertilized C57BL/6J oocytes. Founder 1 carrying 5-6 copies of the transgene was maintained on the C57BL/6J background.

[00331] Primary hepatocytes from transgenic mice (PMH) were isolated with a Liver Perfusion Kit (Miltenyi Biotec, 130-128-030) following the manufacturer’s protocol. The isolated cells were plated in 96-well plate at 4xl0⁴ cells/well and transfected within 2-4 hours after plating using Casl2a mRNA and guides essentially as described in Example 2. The guides targeted the gene regions listed in Table 17. The guides consisted of the activating region of SEQ ID NO: 3 linked to one of the spacers selected from Table 17.

[00332] Table 17. Targets in human genes for use in PMH of transgenic mice

[00333] Editing efficiency was assessed using next generation sequencing essentially as described in Example 1G. Results are shown in FIGURES 24, 25, 26 and 27.

[00334] For the APOC3 experiment, the all-RNA guides (crRNA) were compared with chRDNA guides having the same sequence and the chemical modification pattern mps_D_24 (FIGURE 1, chemical modification pattern 109). Results are shown in Table 18 and FIGURE 24. In Table 18, each number (% edited) is a technical replicate with three replicates per condition.

[00335] Table 18. Editing human APOC3 in transgenic PMH

[00336] For the SERPINA1 experiment, editing was performed with all-RNA guides (crRNA) Results are shown in Table 19 and FIGURE 25. In Table 19, each number (% edited) is a technical replicate with three replicates per condition.

[00337] Table 19. Editing human SERPINA1 in transgenic PMH

[00338] For SERPINA1 targets 7, 9 and 26, the all-RNA guides (crRNA) were compared with chRDNA guides having the same sequence and the chemical modification pattern mps_D_24 (FIGURE 1, 109). Results are shown in Table 20 and FIGURE 26. In Table 20, each number (% edited) is a technical replicate with three replicates per condition.

[00339] Table 20. SERPINA1 edits with crRNA and chRDNA

[00340] The SERPINA1 target 21 contained the Pi*Z single nucleotide polymorphism (SNP) leading to the amino acid change E366K. The two chRDNA guides with chemical modifications mps_D_24 (FIGURE 1, 109) were designed to match the wild-type sequence or the Pi*Z SNP. Results are shown in Table 21 and FIGURE 27. In Table 21, each number (% edited) is a technical replicate with three replicates per condition

[00341] Table 21. Single nucleotide discrimination in SERPINA1 gene

[00342] Example 10. In vivo multiplex editing

[00343] In this example, the Ttr and AngptlA genes were sequentially targeted in vivo (FIGURE 28). The LNP with Casl2a mRNA and Ttr crRNA was prepared with the LNP composition and the protocol described in Example 4(A). The LNP with Angptl3 crRNA was prepared with the LNP composition and the protocol described in Example 4(A) but without the addition of any Casl2a mRNA. For Ttr, the crRNA had the activating region SEQ ID NO: 3 and the targeting region SEQ ID NO: 7. For Angptl3, the crRNA had the activating region SEQ ID NO: 3 and the targeting region SEQ ID NO: 8.

[00344] BALB/c mice (4 mice per treatment group) were injected intravenously (IV) with the first LNP composition containing Casl2a mRNA and Ttr crRNA at 1 mg/kg. After a period selected from 24, 48, 72 or 96 hours, the mice were IV injected with the second LNP composition containing only Angptl3 crRNA at 0.5 mg/kg. A control group received no LNP (saline injection), another control group received the Casl2a-Ttr LNP but no Angptl3 LNP, and the last control group received no Casl2a-Ttr LNP but only the Angptl3 LNP. Gene editing was assessed by NGS after 7 days from the first injection as described in Example 1(B). Results are shown in Table 22 and FIGURE 29. In Table 22, each number represents an average of three sequencing replicates from the same mouse sample.

[00345] Table 22. Multiplex editing

[00346] Example 11. Targeting additional genes in human cells

[00347] Hep G2 cells (hepatocellular carcinoma, ATCC HB-8065) were transfected with Casl2a-guide nucleoprotein complexes as described in Example 7 with the following modifications. Hep G2 were cultured in Eagle's Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Coming) and plated on poly-D-ly sine- coated plates (Thermo Fisher Scientific). For targeting each gene, 10⁵ Hep G2 cells were resuspended in SF buffer (Lonza) and transfected with an ALT-R™ crRNA guide (Integrated DNA Technologies) along with Casl2a. Each guide consisted of the activating region of SEQ ID NO: 3 linked to one of the spacers selected from Table 23. Genome editing was assessed by NGS as described in Example 8(B). Results are shown in Table 23.

[00348] Table 23. Gene editing in human cells

ND = not detected

[00349] While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus, the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

Claims

We claim:

1. A method of modifying a sequence of a target nucleic acid in a somatic cell in a living organism, the method comprising: contacting the organism with (i) a nucleic acid coding for a CRISPR endonuclease and (ii) a guide polynucleotide comprising a targeting region capable of hybridizing to a target sequence within the target nucleic acid, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA), and wherein the CRISPR endonuclease binds the guide polynucleotide and cleaves the target nucleic acid and the sequence of the target nucleic acid is modified within the somatic cell.

2. The method of claim 1, wherein both the targeting region and the activating region of the guide polynucleotide comprise DNA and ribonucleic acid (RNA).

3. The method of claim 1, wherein at least one of the targeting region and the activating region further comprises a chemical modification.

4. The method of claim 2, wherein the chemical modification is selected from a deoxyribonucleotide, a phosphorothioate ribonucleotide, a phosphorothioate deoxyribonucleotide, and a 2’ -O-m ethyl nucleotide.

5. The method of claim 1, wherein the guide polynucleotide comprises SEQ ID NOs:

141.

6. The method of claim 1, wherein the guide polynucleotide comprises SEQ ID NOs:

142.

7. The method of claim 1, wherein the guide polynucleotide comprises a targeting region selected from the group consisting of SEQ ID NOs: 63-82 and SEQ ID NOs: 143-434.

8. The method of claim 1, wherein the guide polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 10-36 and 83-140.

9. The method of claim 1, wherein the CRISPR endonuclease is a CRISPR Class 2 endonuclease.

10. The method of claim 9, wherein the CRISPR Class 2 endonuclease is Casl2a.

11. The method of claim 1, wherein the CRISPR endonuclease comprises a nuclear localization signal (NLS).

12. The method of claim 11, wherein the NLS is selected from the group consisting of SV40 large T-antigen, nucleoplasmin, 53BP1, VACM-1/CUL5, CXCR4, VP1, ING4, IER5, ERK5, UL79, EWS, Hrpl, c-Myc, Mouse c-able IV, Mata2 and MINIYO NLS.

13. The method of claim 11, wherein the NLS is the nucleoplasmin NLS conjugated to a linker to form SEQ ID NO: 2.

14. The method of claim 11, wherein the Casl2a comprises the amino acid sequence of SEQ ID NO: 1

15. The method of claim 1, wherein the nucleic acid coding for the CRISPR endonuclease is an mRNA.

16. The method of claim 15, wherein the mRNA comprises codon optimization for optimizing mRNA expression in mammalian cells.

17. The method of claim 15, wherein the mRNA comprises modifications minimizing immunogenicity in mammalian cells.

18. The method of claim 17, wherein the modification minimizing immunogenicity is a uridine modification or a cytidine modification.

19. The method of claim 11, wherein the uridine modification is selected from the group consisting of 5-methoxyuridine, 5-methyluridine, 5-carboxymethytl ester uridine, 2- thiouridine and pseudouridine and derivatives thereof.

20. The method of claim 17, wherein the cytidine modification is 2-methoxycytidine or its derivatives.

21. The method of claim 15, wherein the mRNA comprises a 5’-cap having a formula selected from the group consisting of 3’G(5’)PPP-5’, N7-(4-chlorophenoxyethyl)-m3'- OG(5 ')ppp(5 ')G, N7-(4-bromophenoxyethyl)-m3 '-OG(5 ')ppp(5 ')G, 3 ’ -O-Me- m7G(5’)ppp(5')G). and m7(3’OMeG)(5’)ppp(5’)m6(2’OMe)pG.

22. The method of claim 21, wherein the 5 ’-cap comprises a chemical modification selected from a modification to the guanosine base, a modification to the ribose sugar moiety, and a modification or to the phosphate moiety.

23. The method of claim 21, wherein the 5 ’-cap comprises one of more modifications selected from the group consisting of N7-methyl guanosine, 2’-O-methyl ribose, a- thiophosphate, a-methyl phosphate, boranophosphate and selenophosphate.

24. The method of claim 15, wherein the mRNA further comprises a 5 ’-untranslated region (UTR) and a 3’-UTR comprising eukaryotic sequences.

25. The method of claim 1, wherein the target sequence is in a gene selected from human genes HSD17B 13, DGAT2, PNPLA3, HNF1, HNF4, SERPINA1, TTR, LPA, ANGPTL3, PCSK9, AGP, APOC3, APOA, APOB, TM6SF2, HMGCR, TERT-hTR, BMPR2, ALAS1, GSDla, HA01, LDHA, XDH, SERPINC1, F7, F8, F9, F10, F12, F13, IDS, C5, C3, CFB, ALDH2, KLKB1, Fl 2, SERPING1, USH2A, OT, HFE, IDUA, CEP290, RHO and an HBV gene.

26. The method of claim 1, wherein the nucleic acid coding for the CRISPR endonuclease and the guide polynucleotide are present in a lipid nanoparticle (LNP).

27. The method of claim 26, wherein the LNP comprises a lipid phase comprising an ionizable cationic lipid at about 46-50%, cholesterol at about 38-43%, a phospholipid at about 9-10%, and a polyethylene glycol (PEG) derivative at about 1-2%.

28. The method of claim 27, wherein the lipid phase comprises 6-((2-hexyldecanoyl)oxy)- N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l-aminium (ALC-0315), cholesterol, l,2-Distearoyl-sn-glycero-3-PC (1,2-DSPC), and Methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (ALC-0159).

29. The method of claim 27, wherein the lipid phase comprises ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about 1-2%.

30. The method of claim 27, wherein the lipid phase comprises 8-[(2-hydroxyethyl)[6- oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM102), cholesterol, 1,2-DSPC, and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

31. The method of claim 27, wherein the lipid phase comprises SMI 02 at about 50%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and DMG-PEG2000 at about 1-2%.

32. The method of claim 27, wherein the lipid phase comprises 4-(dimethylamino)- butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester (MC3), cholesterol, 1,2-DSPC, and DMG-PEG2000.

33. The method of claim 27, wherein the lipid phase comprises MC3 at about 50%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and DMG-PEG2000 at about 1-2%.

34. The method of claim 26, wherein the LNP are characterized by encapsulation efficiency of 70-100%.

35. The method of claim 26, wherein the LNP are characterized by poly dispersity index of 0-0.25.

36. The method of claim 26, wherein the LNP are characterized by diameter of 65-100 nanometers.

37. The method of claim 1, wherein contacting the organism is via systemic administration.

38. The method of claim 1, wherein the rate of sequence modification at a selected genomic locus of at least 65% in vitro or in vivo in experimental animals.

39. The method of claim 1, wherein upon sequence modification, the rate of chromosomal translocations is undetectable by sequencing.

40. The method of claim 1, wherein upon sequence modification, the rate of off-target sequence modification is undetectable by sequencing.

41. The method of claim 1, wherein upon sequence modification, the rate of extra-organ sequence modification is no greater than 0.6%

42. The method of claim 1, wherein sequence modification results in reduced expression of a gene.

43. The method of claim 1, further comprising the step of assessing the reduction in the expression of the gene.

44. The method of claim 43, wherein the gene is TTR and the assessing comprises assessing levels of TTR protein in the blood plasma.

45. The method of claim 43, wherein the gene is PSCK9 and the assessing comprises assessing levels of PCSK9 protein or levels of LDL cholesterol in the blood plasma.

46. The method of claim 43, wherein the gene is ANGPTL3 and the assessing comprises assessing levels of ANGPTL3 protein or levels of triglycerides or LDL in the blood plasma.

47. The method of claim 1, further comprising monitoring the patient for excessive immune response.

48. The method of claim 1, further comprising monitoring the patient for change in the function of the target organ.

49. The method of claim 48, where the target organ is the liver and the change in the function is the change in the amount of liver-secreted enzymes.

50. A therapeutic composition for modifying a sequence of a target nucleic acid in a somatic cell in a living organism the composition comprising a lipid nanoparticle (LNP) wherein the lipid phase comprises ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2- DSPC at about 9-10%, and ALC-0159 at about 1-2%, and the LNP contains a therapeutically effective amount of (i) a nucleic acid coding for a CRISPR endonuclease and (ii) a guide polynucleotide comprising a targeting region capable of hybridizing to a target sequence within the target nucleic acid, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA).

51. The therapeutic composition of claim 50, wherein the therapeutically effective amount is between 0.7 mg/kg and 2 mg/kg of total nucleic acid.

52. The therapeutic composition of claim 50, wherein the therapeutically effective amount is between 1 mg/kg and 2 mg/kg of total nucleic acid.

53. The therapeutic composition of claim 50, wherein the therapeutically effective amount is between 30 mg and 80 mg of total nucleic acid.

54. The therapeutic composition of claim 50, wherein the therapeutically effective amount is between 55 mg and 80 mg of total nucleic acid.

55. The therapeutic composition of claim 50, further comprising one or more of excipient, antimicrobial agent, an antioxidant, a surfactant, and a freezing agent.

56. The therapeutic composition of claim 50, wherein the LNP are characterized by encapsulation efficiency of 70-100%.

57. The therapeutic composition of claim 50, wherein the LNP are characterized by poly dispersity index of 0-0.25.

58. The therapeutic composition of claim 50, wherein the LNP are characterized by diameter of 65-100 nanometers.

59. The therapeutic composition of claim 50, wherein the therapeutically effective amount is capable of achieving the rate of sequence modification at a selected genomic locus of at least 65% in vitro or in vivo in experimental animals.

60. The therapeutic composition of claim 50, wherein upon sequence modification, the rate of chromosomal translocations undetectable by sequencing.

61. The therapeutic composition of claim 50, wherein upon sequence modification, the rate of off-target sequence modification is undetectable by sequencing.

62. The therapeutic composition of claim 53, wherein upon sequence modification, the rate of extra-organ sequence modifications is no greater than 0.6%.

63. The therapeutic composition of claim 50, wherein the targeting region is selected from SEQ ID NOs: 63-82 and 143-434.

64. A method of treating a disease or condition comprising a step of systemic administration to a patient of the composition of claim 50, wherein the somatic cell is a hepatocyte.

65. The method of claim 64, wherein the target nucleic acid is within the PSCK9 gene, and the guide polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 10-36 and 83-140.

66. The method of claim 64, wherein the target nucleic acid is within the TTR gene, and wherein the level of the TTR protein in the patient’s plasma is reduced.

67. The method of claim 64, wherein the target nucleic acid is within the PCSK9 gene, and wherein the level of the PCSK9 protein or the level of LDL cholesterol in the patient’s plasma is reduced.

68. The method of claim 64, wherein the target nucleic acid is within the ANGPTL3 gene, and wherein the level of the ANGPTL3 protein or the level of triglycerides in the patient’s plasma is reduced.

69. A method of making the therapeutic composition of claim 50, the method comprising combining the lipid phase comprising ALC-0315 at about 46%, cholesterol at about 38-43%, 1,2-DSPC at about 9-10%, and ALC-0159 at about 1-2%, and a therapeutically effective amount of (i) a nucleic acid coding for a CRISPR endonuclease and (ii) a guide polynucleotide comprising a targeting region capable of hybridizing to a target sequence within the target nucleic acid, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA).

70. The method of claim 69, wherein the therapeutically effective amount is between 0.7 mg/kg and 2 mg/kg of total nucleic acid.

71. The method of claim 69, wherein the therapeutically effective amount is between 1 mg/kg and 2 mg/kg of total nucleic acid.

72. The method of claim 69, wherein the therapeutically effective amount is between 30 mg and 80 mg of total nucleic acid.

73. The method of claim 69, wherein the therapeutically effective amount is between 55 mg and 80 mg of total nucleic acid.

74. The method of claim 69, further comprising adding one or more of excipient, antimicrobial agent, an antioxidant, a surfactant, and a freezing agent.

75. The method of claim 69, wherein the resulting LNP are characterized by encapsulation efficiency of 70-100%.

76. The method of claim 69, wherein the resulting LNP are characterized by poly dispersity index of 0-0.25.

77. The method of claim 69, wherein the resulting LNP are characterized by diameter of 65-100 nanometers.

78. A method of modifying a sequence of two or more target nucleic acids in a somatic cell in a living organism, the method comprising: contacting the organism with lipid nanoparticles (LNP) enclosing: (i) a nucleic acid coding for a CRISPR endonuclease and (ii) two or more guide polynucleotides, each polynucleotide comprising a targeting region capable of hybridizing to a target sequence within one of the two or more target nucleic acids, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA), and wherein the CRISPR endonuclease binds the guide polynucleotide and cleaves each target nucleic acid and the sequence of each target nucleic acid is modified within the somatic cell.

79. The method of claim 78, wherein the two or more guide polynucleotides target two or more target sequences located in the same gene.

80. The method of claim 78, wherein the two or more guide polynucleotides target two or more target sequences located in two or more genes.

81. The method of claim 78, wherein the same LNP encloses two or more guide polynucleotides.

82. The method of claim 78, wherein each of the two or more guide polynucleotides is enclosed in a separate LNP and the method comprises administering a first LNP enclosing a first guide polynucleotide and a second LNP enclosing a second guide polynucleotide.

83. The method of claim 82, wherein the first LNP and the second LNP are administered sequentially after an interval selected from 24, 48, 72 and 96 hours.

84. A composition for modifying a sequence of two or more target nucleic acids in a somatic cell in a living organism, the composition comprising lipid nanoparticles (LNP) enclosing: (i) a nucleic acid coding for a CRISPR endonuclease and (ii) two or more guide polynucleotides, each polynucleotide comprising a targeting region capable of hybridizing to a target sequence within one of the two or more target nucleic acids, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA), and wherein the CRISPR endonuclease is capable of binds the guide polynucleotide and cleaving each target nucleic acid.

85. The method of claim 84, wherein the two or more guide polynucleotides target two or more target sequences located in the same gene.

86. The method of claim 84, wherein the two or more guide polynucleotides target two or more target sequences located in two or more genes.

87. The method of claim 84, wherein the same LNP encloses two or more guide polynucleotides.

88. The method of claim 84, wherein each of the two or more guide polynucleotides is enclosed in a separate LNP.

89. A therapeutic composition for alleviating the symptoms of a genetic disease characterized by autosomal dominant inheritance with a wild-type allele and a mutant allele of a disease-associated gene, the composition comprising a lipid nanoparticle (LNP) containing a therapeutically effective amount of (i) a nucleic acid coding for a CRISPR endonuclease and (ii) a guide polynucleotide comprising a targeting region capable of hybridizing to a target sequence within the mutant allele, and an activating region adjacent to the targeting region and capable of binding to the CRISPR endonuclease, wherein at least one of the targeting region and the activating region comprises deoxyribose nucleic acid (DNA), wherein the guide polynucleotide promotes cleavage of the mutant allele but not a wild-type allele by the CRISPR endonuclease.

90. The composition of claim 89, wherein the disease-associated gene is selected from the group consisting of PKD1, PKD2, ACVRL1, ENG, SMAD4, FBN1, ANK1, EPB42, SLC4A1, SPTA1, SPTB, TCOF1, FGFR3, SERPINC1, COL1A1, COL5A1, COL5A2, UGT1A1, CASR, APC, CLCN7, COL1A1, PROC, TSC1, TSC2, LDLR and APOB.

91. A method for alleviating the symptoms of a genetic disease characterized by autosomal dominant inheritance with a wild-type allele and a mutant allele of a disease- associated gene, the method comprising administering to a patient exhibiting symptoms of the a genetic disease characterized by autosomal dominant inheritance the composition of claim 89, wherein the administration is selected from the group consisting of intravenous administration, administration to the lung and administration to the gastrointestinal tract.

92. A method for alleviating the symptoms of a genetic disease characterized by autosomal dominant inheritance with a wild-type allele and a mutant allele of a disease- associated gene, the method comprising administering to a patient carrying a wild-type allele and a mutant allele of a disease-associated gene the composition of claim 89, wherein the administration is selected from the group consisting of intravenous administration, administration to the lung and administration to the gastrointestinal tract.