Attorney Docket No.: 063586-522001WO GENE EDITING SYSTEMS TARGETING PCSK9 AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing dates of U.S. Provisional Application No.63/510,253, filed June 26, 2023, U.S. Provisional Application No.63/618,064, filed January 5, 2024, U.S. Provisional Application No.63/625,522, filed January 26, 2024, and U.S. Provisional Application No.63/575,038, filed April 5, 2024. The entire contents of each of the priority applications is incorporated by reference herein. SEQUENCE LISTING The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 26, 2024, is named “063586-522001WO_Sequence Listing-v2.xml” and is 283,455 bytes in size. BACKGROUND OF THE INVENTION PCSK9 gene encodes the 9
th member of the protein convertase family, Proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 protein is an enzyme that binds to and degrades the low-density lipoprotein particle receptor (LDLR), which is located on the membrane of liver cells and other types of cells. LDLR is involved in transporting low-density lipoprotein particles (LDL), including cholesterol, from extracellular fluid into cells. The LDL-LDLR complex normally would be targeted to lysosomes for destruction in the presence of PCSK9. When activity of PCSK9 is blocked, the LDL-LDLR complex separates during trafficking with LDL digested in lysosomes and LDLRs recycled back to the cell surface to remove additional LDL particles from the extracellular fluid. Thus, blocking PCSK9 can lower blood LDL concentrations, thereby benefiting conditions associated with LDL. SUMMARY OF THE INVENTION The present disclosure is based, at least in part, on the development of PCSK9- targeting gene editing systems involving the use of a CRISPR nuclease and a guide RNA (gRNA), also known as an RNA guide, targeting a genomic site in the PCSK9 gene. A number of genomic sites (e.g., within exon 1, exon 4, exon 8, or exon 11 of the PCSK9 gene) has been identified as target sites by the gene editing system to achieve high levels of Indel
Attorney Docket No.: 063586-522001WO and high levels of PCSK protein knockdown. Accordingly, provided herein, in some aspects, is a gene editing system for genetic editing of a proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, the gene editing system comprising: (i) a CRISPR nuclease or a first nucleic acid encoding the CRISPR nuclease; and (ii) an RNA guide or a second nucleic acid encoding the RNA guide. The RNA guide comprises a spacer sequence specific to a target sequence within an PCSK9 gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) recognizable by the CRISPR nuclease. In some instances, the PAM comprises the motif of 5’-NTTR-3’, in which N is any of A, T, G, and C; and R is A or G. The PAM motif is located 5’ to the target sequence. In some embodiments, the CRISPR nuclease comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1. In some examples, the CRISPR nuclease comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some examples, the CRISPR nuclease comprises an amino acid sequence at least 98% identical to SEQ ID NO: 1. In some examples, the CRISPR nuclease comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1. In some embodiments, the CRISPR nuclease comprises an amino acid sequence at least 90% (e.g., at least 95%, 98%, or above) identical to SEQ ID NO:1 and comprise one or more mutations relative to SEQ ID NO: 1. In some examples, the one or more mutations are amino acid substitutions, for example, at positions P14 (e.g., P14R), D32 (e.g., D32R), I61 (e.g., I61R), E311 (e.g., E311R), T338 (e.g., T338G), and/or E736 (e.g., E736G) in SEQ ID NO:1. In specific examples, the mutations include P14R, D32R, I61R, and E311R. In other specific examples, the mutations include P14R, D32R, I61R, E311R, T338G, and E736G. In some specific examples, the CRISPR nuclease comprises the amino acid sequence of SEQ ID NO: 2. In other specific examples, the CRISPR nuclease comprises the amino acid sequence of SEQ ID NO: 3. Any of the CRISPR nucleases disclosed herein may further comprise one or more functional motifs, which are heterologous to the nuclease moiety. Such functional motifs may comprise one or more nuclear localization signals (NLS) and/or one or more peptide linkers such as those provided in Examples below. In some embodiments, the gene editing system provided herein comprises the first nucleic acid encoding the CRISPR nuclease. In some examples, the first nucleic acid is a messenger RNA (mRNA). Alternatively, the first nucleic acid is located in a viral vector, which optionally is an adeno-associated viral (AAV) vector.
Attorney Docket No.: 063586-522001WO In some embodiments, the target sequence is within exon 1 of the PCSK9 gene. In some examples, the target sequence comprises 5’- GCGCAGCGGTGGAAGGTGGC-3’ (SEQ ID NO: 15). In some examples, the spacer sequences specific to such a target sequence may comprise 5’- GCGCAGCGGUGGAAGGUGGC-3’ (SEQ ID NO: 74). In some embodiments, the target sequence is within exon 2 of the PCSK9 gene. In some examples, the target sequence comprises 5’- CATGGGGCCAGGATCCGTGG-3’ (SEQ ID NO: 17). In some examples, the spacer sequences specific to such a target sequence may comprise 5’- CAUGGGGCCAGGAUCCGUGG-3’ (SEQ ID NO: 76). In some embodiments, the target sequence is within exon 3 of the PCSK9 gene. In some examples, the target sequence comprises 5’- AAGTTGCCCCATGTCGACTA-3’ (SEQ ID NO: 19); 5’- CCCCATGTCGACTACATCGA-3’ (SEQ ID NO: 20); 5’- CCCAGAGCATCCCGTGGAAC-3’ (SEQ ID NO: 21); 5’- CCCCTCCACGGTACCGGGCG -3’ (SEQ ID NO: 22); or 5’- AGCAGAGTCCCCCGGCCTCT-3’ (SEQ ID NO: 24). In some examples, the spacer sequences specific to such target sequences may comprise 5’- AAGUUGCCCCAUGUCGACUA-3’ (SEQ ID NO: 78); 5’- CCCCAUGUCGACUACAUCGA-3’ (SEQ ID NO: 79); 5’- CCCAGAGCAUCCCGUGGAAC-3’ (SEQ ID NO: 80); 5’- CCCCUCCACGGUACCGGGCG-3’ (SEQ ID NO: 81); 5’- AGCAGAGUCCCCCGGCCUCU-3’ (SEQ ID NO: 83). In some embodiments, the target sequence is within exon 4 of the PCSK9 gene. In some examples, the target sequence comprises 5’- GAAAGACGGAGGCAGCCTGG-3’ (SEQ ID NO: 26). In some examples, the spacer sequences specific to such a target sequence may comprise 5’- GAAAGACGGAGGCAGCCUGG-3’ (SEQ ID NO: 85). In some embodiments, the target sequence is within exon 5 of the PCSK9 gene. In some examples, the target sequence comprises 5’- TGTTCGTCGAGCAGGCCAGC-3’ (SEQ ID NO: 27). In some examples, the spacer sequences specific to such a target sequence may comprise 5’- UGUUCGUCGAGCAGGCCAGC-3’ (SEQ ID NO: 86). In some embodiments, the target sequence is within exon 7 of the PCSK9 gene. In some examples, the target sequence comprises 5’- CCCCAGGGGAGGACATCATT-3’ (SEQ ID NO: 43); or 5’- GGCTCCTTTCTCTGCCACCC-3’ (SEQ ID NO: 47). In some examples, the spacer sequences specific to such target sequences may comprise 5’- CCCCAGGGGAGGACAUCAUU-3’ (SEQ ID NO: 102) or 5’- GGCUCCUUUCUCUGCCACCC-3’ (SEQ ID NO: 106). In some embodiments, the target sequence is within exon 8 of the PCSK9 gene. In
Attorney Docket No.: 063586-522001WO some examples, the target sequence comprises 5’- ATGACATCTTTGGCAGAGAA-3’ (SEQ ID NO: 51); or 5’- GCAGAGAAGTGGATCAGTCT-3’ (SEQ ID NO: 52). In some examples, the spacer sequences specific to such target sequences may comprise 5’- AUGACAUCUUUGGCAGAGAA-3’ (SEQ ID NO: 110) or 5’- GCAGAGAAGUGGAUCAGUCU-3’ (SEQ ID NO: 111). In some embodiments, the target sequence is within exon 10 of the PCSK9 gene. In some examples, the target sequence comprises 5’- GCCTGGGGTAGCAGGCAGCA-3’ (SEQ ID NO: 60); or 5’- ACTCTAAGGCCCAAGGGGGC-3’ (SEQ ID NO: 64). In some examples, the spacer sequences specific to such target sequences may comprise 5’- GCCUGGGGUAGCAGGCAGCA-3’ (SEQ ID NO: 119) or 5’- ACUCUAAGGCCCAAGGGGGC-3’ (SEQ ID NO: 123). In some embodiments, the target sequence is within exon 11 of the PCSK9 gene. In some examples, the target sequence comprises 5’- GCACCCACAAGCCGCCTGTG-3’ (SEQ ID NO: 65); 5’- ACTTTGCATTCCAGACCTGG-3’ (SEQ ID NO: 66); 5’- CATTCCAGACCTGGGGCATG-3’ (SEQ ID NO: 67); 5’- GGCTGACCTCGTGGCCTCAG-3’ (SEQ ID NO: 68); or 5’- TGGGTGCCAAGGTCCTCCAC-3’ (SEQ ID NO: 69). In some examples, the spacer sequences specific to such target sequences may comprise 5’- GCACCCACAAGCCGCCUGUG-3’ (SEQ ID NO: 124); 5’- ACUUUGCAUUCCAGACCUGG-3’ (SEQ ID NO: 125); 5’- CAUUCCAGACCUGGGGCAUG-3’ (SEQ ID NO: 126); 5’- GGCUGACCUCGUGGCCUCAG-3’ (SEQ ID NO: 127); or 5’- UGGGUGCCAAGGUCCUCCAC-3’ (SEQ ID NO: 128). In some examples, the spacer sequence is 20-30-nucleotide in length. In specific examples, the spacer is 20-nucleotide in length. In other examples, the spacer sequence is 15- 19-nucleotide in length. In specific examples, the spacer is 16-nucleotide in length. As reported herein, shorter spacers exhibited better gene editing efficacy and decreased off-target editing. In some instances, the shorter spacers have a 3’ end truncation (e.g., up to 5- nucleotides, such as 4-nt, 3-nt, 2-nt or 1nt) relative to the counterpart spacers that have the same length as the target sequence (e.g., 20-nt in length). Such truncated versions of any of the specific spacer sequences disclosed herein are within the scope of the present disclosure. In specific examples, the spacer sequence may be set forth as SEQ ID NO: 217, 218, 220, or 221. In some instances, any of the RNA guides disclosed herein may comprise a spacer as disclosed herein and a direct repeat sequence. In some examples, the direct repeat sequence is
Attorney Docket No.: 063586-522001WO 23-36-nucleotide in length. In some examples, the direct repeat sequence is at least 90% identical to SEQ ID NO: 2 or a fragment thereof, which optionally is at least 23-nucleotide in length. For example, the direct repeat sequence is SEQ ID NO: 2, or a fragment thereof, which optionally is at least 23-nucleotide in length. In one specific example, the direct repeat sequence is 5’- CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAAC-3’ (SEQ ID NO: 4). In some embodiments, the RNA guide may comprise one or more modifications. In some examples, the RNA guide comprises one or more phosphorothioate (PS) bonds and/or 2’-O-methylated bases, optionally at the 5’ and 3’ end thereof. In some examples, the RNA guide does not contain 2’-O-methylation at the 5’ end. In specific examples, the RNA guide may have the modification pattern shown in Table 5, for example, mod2, mod4, or mod6 (e.g., mod2 or mod4). Exemplary RNA guides for use in the gene editing system provided herein include E1T3, E2T6, E3T2, E3T3, E3T5, E3T6, E3T10, E5T1, E7T7, E7T18, E8T8, E8T10, E10T2, E10T8, E11T3, E11T5, E11T8, E11T10, and E11T11. In some examples, the RNA guide is E1T3, E4T3, E10T8, or E11T3. See Table 3 below. In some examples, the RNA guides may have a 3’-truncation (e.g., up to 5 nucleotides) in the spacer sequence of any of the exemplary RNA guides as disclosed herein. In some embodiments, the gene editing system comprises the second nucleic acid encoding the RNA guide. In some instances, the nucleic acid encoding the RNA guide is located in a viral vector. In some examples, the gene editing system comprises a viral vector that comprises both the first nucleic acid encoding the CRISPR nuclease and the second nucleic acid encoding the RNA guide. In some examples, the system comprises the first nucleic acid encoding the CRISPR nuclease, which is located in a first vector, and the second nucleic acid encoding the RNA guide, which is located in a second vector. In some instances, the first and/or the second vector is a viral vector. In other instances, the first vector and second vector are the same vector. In other examples, the system comprises one or more lipid nanoparticles (LNPs), to which the CRISPR nuclease or the encoding nucleic acid and/or the RNA guide or the encoding nucleic acid are associated. In other aspects, the present disclosure provides a pharmaceutical composition comprising any of the gene editing systems disclosed herein. In addition, the present disclosure provides a kit comprising the CRISPR nuclease or its encoding nucleic acid and the RNA guide or its encoding nucleic acid of any of the gene editing systems disclosed herein.
Attorney Docket No.: 063586-522001WO In yet other aspects, the present disclosure features a method for editing a PCSK9 gene in a cell, the method comprising contacting a host cell with the gene editing system provided herein to genetically edit the PCSK9 gene in the host cell. In some embodiments, the host cell is cultured in vitro. In other embodiments, the contacting step is performed by administering the system for editing the PCSK9 gene to a subject comprising the host cell (e.g., liver cell). Any modified cells comprising a disrupted PCSK9 gene produced by contacting a host cell with the gene editing system is also within the scope of the present disclosure. Further, the present disclosure features a method for treating a disease associated with PCSK9 in a subject, comprising administering to a subject in need thereof any of the gene editing systems provided herein or the modified cells generated thereby. In some instances, the subject is a human patient having or suspected of having the disease associated with PCSK9, which optionally is a cardiovascular disease. Also within the scope of the present disclosure are any of the gene editing systems disclosed herein for use in treating a disease associated with PCSK9 gene via genetically editing the PCSK9 gene in target cells such as in liver cells of a subject in need thereof (e.g., a human patient). Further provided herein is the use of the gene editing system for manufacturing a medicament for use in treating the target disease. In addition, the present disclosure features an RNA guide, comprising (i) a spacer sequence that is specific to a target sequence in a PCSK9 gene; and (ii) a direct repeat sequence (e.g., those disclosed herein), which may be recognizable by any of the CRISPR nucleases provided herein. The target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5’-NTTR-3’, which is located 5’ to the target sequence. N is any one of A, T, G, and C and R is A or G. In some instances, the spacer may be 20-30-nucleotide in length. In specific examples, the spacer is 20-nucleotide in length. Alternatively, the spacer sequence is 15-19-nucleotide in length. In specific examples, the spacer is 16-nucleotide in length. Alternatively or in addition, the direct repeat sequence is 23-36-nucleotide in length. In specific examples, the direct repeat is 23-nucleotide in length. In some instances, the direct repeat sequence is any one of those listed in Table 2 below or a fragment thereof having at least 23 nucleotides. In some embodiments, the RNA guide may comprise one or more modifications. In some examples, the RNA guide comprises one or more phosphorothioate (PS) bonds and/or 2’-O-methylated bases, optionally at the 5’ and 3’ end thereof. In some examples, the RNA guide does not contain 2’-O-methylation at the 5’ end. In specific examples, the RNA guide may have the modification pattern shown in Table 5, for example, mod2, mod4, or mod6
Attorney Docket No.: 063586-522001WO (e.g., mod2 or mod4). In some embodiments, the RNA guide may be specific to a target sequence as those disclosed above. In some examples, the RNA guide may comprise a spacer sequence specific to the target sequence such as those provided above. Exemplary RNA guides for use in the gene editing system provided herein include E1T3, E2T6, E3T2, E3T3, E3T5, E3T6, E3T10, E5T1, E7T7, E7T18, E8T8, E8T10, E10T2, E10T8, E11T3, E11T5, E11T8, E11T10, and E11T11. In some examples, the RNA guide is E1T3, E4T3, E10T8, or E11T3. See Table 3 below. In some examples, the RNA guides may have a 3’-truncation (e.g., up to 5-nucleotides) in the spacer sequence of any of the exemplary RNA guides as disclosed herein. See Table 6 below. In specific examples, the RNA guide can be of SEQ ID NO: 195, 222, or 230. In other specific examples, the RNA guide can be SEQ ID NO: 196, 223, or 229. Additional embodiments provided herein include: A gene editing system, comprising (i) a CRISPR nuclease or a first nucleic acid encoding the CRISPR nuclease, wherein the CRISPR nuclease comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1; and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within a target gene and a direct repeat sequence recognizable by the CRISPR nuclease, the target sequence being adjacent to a protospacer adjacent motif (PAM), wherein the PAM comprises the motif of 5’-NTTR-3’, which is located 5’ to the target sequence; wherein N is A, T, G, or C, and R is A or G. The RNA guide comprises modifications of phosphorothioate (PS) bonds and 2’-O-methylated bases at the 5’ and/or 3’ end. In some instances, the modifications are set forth in mod2, mod4, or mod6 in Table 5. In one example, the modifications have the pattern set forth in mod2. In another example, the modifications have the pattern set forth in mod4. A gene editing system, comprising (i) a CRISPR nuclease or a first nucleic acid encoding the CRISPR nuclease, wherein the CRISPR nuclease comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1; and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within a target gene and a direct repeat sequence recognizable by the CRISPR nuclease, the target sequence being adjacent to a protospacer adjacent motif (PAM), wherein the PAM comprises the motif of 5’-NTTR-3’, which is located 5’ to the target sequence; wherein N is A, T, G, or C, and R is A or G. The spacer sequence in the RNA guide is 15-19-nucleotide in length. In one example, the spacer is 16-nucleotide in length. The details of one or more embodiments of the invention are set forth in the
Attorney Docket No.: 063586-522001WO description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein. FIG.1 is a diagram showing PCSK9 indels in HEK293T and HepG2 cells following RNP transfection with the variant CRISPR nuclease and RNA guides of Table 3. Each circle represents the average of three technical replicates across one biological replicate. Use of an additional 64 RNA guides targeting PCSK9 exon sequences with 5’-NTTY-3’ (Y is C or T) PAM sequences did not result in indels or resulted in indels of less than 10%. FIG.2 is a diagram showing PCSK9 indels in a PHH donor (HUM190171) following RNP transfection the variant CRISPR nuclease and RNA guides of Table 2.19 RNA guides resulted in indels of at least 40% at the highest concentration tested. Expression of PCSK9 was then measured for the RNA guides resulting in the highest percent indels. FIG.3 is a diagram showing PCSK9 expression (measured by MSD) relative to control expression in a PHH donor (HU2021) following RNP transfection with the highest performing RNA guides identified in FIG.2. Many of the RNA guides reduced PCSK9 expression to under 10% at the highest RNP concentration tested. E4T3, E11T3, E8T10, and E1T3 also substantially decreased PCSK9 expression at the lower RNP concentrations tested. FIG.4 are diagrams showing indels at PCSK9 target site in a PHH donor (HU8403, Thermofisher) following electroporation of mRNA effector with modified RNA guides. Each bar represents the average of three technical replicates across one biological replicate. Error bars represent standard deviation. The data shows high on-target editing activity for mod2, mod4, and mod6 across both targets tested. FIGS.5A-5B are diagrams showing indels at PCSK9 target site in a PHH donor (HU2186, Thermofisher) following electroporation of mRNA effector with RNA guides containing either 16 nt, 18 nt, or 20 nt spacers for PCSK9-E1T3 (FIG.5A) or PCSK9-E10T8 (FIG.5B). Each dot represents the average of three technical replicates across one biological replicate. Error bars represent standard deviation. The data shows higher on-target activity for 18 nt compared to 16 nt and 20 nt spacers.
Attorney Docket No.: 063586-522001WO FIGS.6A-6E are diagrams showing indels at in silico predicted off-target sites in a PHH donor (HU2186, Thermofisher) following electroporation of mRNA effector with RNA guides containing either 16 nt, 18 nt, or 20 nt spacers for PCSK9-E1T3 (FIGS.6A-6B) or PCSK9-E10T8 (FIGS.6C-6E). Each dot represents the average of three technical replicates across one biological replicate. Error bars represent standard deviation. The data shows higher off-target activity for 18 nt compared to 16 nt and 20 nt spacers, and elimination of off-target activity at sites tested for the 16 nt spacer length guides. FIG.7 is a diagram showing interim PCSK9 levels in genetically edited mice. DETAILED DESCRIPTION OF THE INVENTION LDL cholesterol constitutes the majority of cholesterol in human bodies. High levels of LDL cholesterol can raise risks for heart disease and stroke. PCSK9 regulates the amount of LDL cholesterol in the bloodstream. As such, PCSK9 plays a role in lipoprotein homeostasis. Agents that block PCSK9 can lower the concentration of LDL cholesterol, thereby reducing the risk of diseases associated with LDL cholesterol, such as cardiovascular conditions. The PCSK9 gene resides on chromosome 1 at the band 1p32.3 and includes 15 exons. medlineplus.gov/genetics/gene/pcsk9/Gene; and NCBI.15 May 2023. Genomic context. Retrieved 20 May 2023, the relevant disclosures of each of which are incorporated by reference herein. This gene produces two isoforms through alternative splicing. The present disclosure reports the development of CRISPR nuclease-based gene editing systems for effective disruption of the PCSK9 gene in, for example, liver cells. Also reported herein is the identification of genetic sites in the PCSK9 gene where gene editing can occur at high efficiency, e.g., showing high indels in edited PCSK9 genes and high protein knockdown in liver cells. The gene editing systems disclosed herein can effectively disrupt the PCSK9 in target cells (e.g., in liver cells), thereby benefiting treatment of diseases associated with LDL cholesterol such as cardiovascular diseases. Accordingly, provided herein are gene editing systems targeting the PCSK9 gene, methods for editing the PCSK9 gene in suitable target cells such as in liver cells using such a gene editing system, and methods for treating target diseases such as cardiovascular diseases using the gene editing system or edited cells produced thereby. Also provided herein are guide RNAs showing high editing efficiencies of the PCSK9 gene. I. CRISPR Nuclease-Containing Gene Editing Systems Targeting PCSK9 The gene editing system provided herein for genetic editing of a proprotein convertase
Attorney Docket No.: 063586-522001WO subtilisin/kexin type 9 (PCSK9) gene may comprise (i) a CRISPR nuclease or a first nucleic acid encoding the CRISPR nuclease; and (ii) an RNA guide targeting a genetic site within the PCSK9 gene or a second nucleic acid encoding the RNA guide. A. CRISPR Nucleases CRISPR nuclease refers to an RNA-guided endonuclease in a CRISPR editing system that is capable of cleaving DNA at specific sites guided by an RNA guide. In some embodiments, the CRISPR nuclease used in the gene editing system disclosed herein comprises the amino acid sequence of SEQ ID NO: 1. See Table 1 below. In other embodiments, the CRISPR nuclease may comprise an amino acid sequence that is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. The “percent identity” (a.k.a., sequence identity) of two nucleic acids or of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol.215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. In some instances, the CRISPR nuclease of the present disclosure has enzymatic activity, e.g., nuclease or endonuclease activity similar to SEQ ID NO:1, and comprising an amino acid sequence, which differs from the amino acid sequences of SEQ ID NO: 1 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods. In some instances, the CRISPR nuclease may comprise one or more conservative amino acid substitutions as compared with SEQ ID NO: 1. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative
Attorney Docket No.: 063586-522001WO charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In some examples, the CRISPR nuclease comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or above) identical to SEQ ID NO:1 and one or more mutations relative to SEQ ID NO: 1. In some examples, the mutations may be at position P14, D32, I61, E311, T338, E736, or a combination thereof in SEQ ID NO:1. In some instances, the mutations are amino acid residue substitutions, for example, at the above noted position(s). In some examples, the amino acid residue substitution is P14R, D32R, I61R, E311R, T338G, E736G, or a combination thereof. In specific examples, the mutations are at positions P14R, D32R, I61R, and E311R in SEQ ID NO: 1, for example, P14R, D32R, I61R, and E311R. In other examples, the mutations are at positions P14R, D32R, I61R, E311, T338, and E736, for example, P14R, D32R, I61R, E311R, T338G, and E736G. Table 1 below provided the amino acid sequences of exemplary CRISPR nuclease. Table 1. Exemplary CRISPR Nucleases Description Sequence SEQ ID NO
Attorney Docket No.: 063586-522001WO (P14R, EDIEMQVMYEIIKYSLNKKSDWDNFISYIENVENPNIDNINRYKLLRECF D32R, CENENMIKNKLELLSVEQLKKFGGCIMKPHINSMTINIQDFKIEEKENSL I 1R GFILHLPLNKKQYQIELLGNRQIKKGTKEIHETLVDITNTHGENIVFTIK
dd t ona C S nuc eases or use n t e gene ed t ng system d sc osed ere n can be found, e.g., in WO2021050534, WO2022192391, and WO2024020567, the relevant disclosures of each of which are incorporated by reference herein for the subject matter and purpose referenced herein. B. RNA Guides In some embodiments, the gene editing system described herein comprises an RNA guide targeting a PCSK9 gene, for example, targeting exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or exon 12. In some embodiments, the RNA guide may target exon 1, exon 2, exon 3, exon 5, exon 7, exon 8, exon 10, or exon 11 of the PCSK9 gene. In one specific example, the RNA guide may target exon 1 of the PCSK9 gene. In another specific example, example, the RNA guide may target exon 4 of the PCSK9 gene. In yet another specific example, example, the RNA guide may target exon 8 of the PCSK9 gene. In another specific example, the RNA guide may target exon 11 of the PCSK9 gene. In some instances, the gene editing system described herein may comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting PCSK9. The RNA guide may direct the CRISPR nuclease contained in the gene editing system as described herein to a PCSK9 target sequence. Two or more RNA guides may direct two or
Attorney Docket No.: 063586-522001WO more separate CRISPR nucleases as described herein to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) PCSK9 target sequences. Those skilled in the art reading the below examples of particular kinds of RNA guides will understand that, in some embodiments, an RNA guide is PCSK9 target-specific. That is, the RNA guide binds specifically to one or more PCSK9 target sequences (e.g., within a cell such as a liver cell) and not to non-targeted sequences (e.g., non-specific DNA or random sequences within the same cell). In some embodiments, the RNA guide comprises a spacer sequence followed by a direct repeat sequence, referring to the sequences in the 5’ to 3’ direction. In some examples, the RNA guide comprises a first direct repeat sequence followed by a spacer sequence and a second direct repeat sequence, referring to the sequences in the 5’ to 3’ direction. In some examples, the first and second direct repeats of such an RNA guide are identical. In other examples, the first and second direct repeats of such an RNA guide are different. In some instances, the RNA guide disclosed herein may further comprise a linker sequence, a 5’ end and/or 3’ end protection fragment (see disclosures herein), or a combination thereof. In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present within the same RNA molecule. For example, the spacer and direct repeat sequences are linked directly to one another. Alternatively, a short linker is present between the spacer and direct repeat sequences, e.g., an RNA linker of 1, 2, or 3 nucleotides in length. In other examples, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present in separate molecules, which are joined to one another by base pairing interactions. The RNA guide provided herein may comprise a spacer sequence specific to a target sequence within an PCSK9 gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5’-NTTR-3’ (N being any of A, T, G, and C; and R being A or G), which is located 5’ to the target sequence. As used herein, the term “protospacer adjacent motif” or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., an PCSK9 target sequence) to which a complex comprising an RNA guide (e.g., an PCSK9-targeting RNA guide) and a CRISPR nuclease binds. In a double-stranded DNA molecule, the strand containing the PAM motif is called the “PAM-strand” and the complementary strand is called the “non-PAM strand.” The RNA guide binds to a site in the non-PAM strand that is complementary to a target sequence disclosed herein. As used herein, the term “target sequence” refers to a DNA fragment adjacent to a
Attorney Docket No.: 063586-522001WO PAM motif (on the PAM strand). The complementary region of the target sequence is on the non-PAM strand. A target sequence may be immediately adjacent to the PAM motif. Alternatively, the target sequence and the PAM may be separately by a small sequence segment (e.g., up to 5 nucleotides, for example, up to 4, 3, 2, or 1 nucleotide). A target sequence may be located at the 3’ end of the PAM motif or at the 5’ end of the PAM motif, depending upon the CRISPR nuclease that recognizes the PAM motif, which is known in the art. For example, a target sequence is located at the 3’ end of a PAM motif for a CRISPR nuclease such as those disclosed herein. In some embodiments, the target sequence is a sequence within a PCSK9 gene. As used herein, the term “adjacent to” refers to a nucleotide or amino acid sequence in close proximity to another nucleotide or amino acid sequence. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (i.e., immediately adjacent). In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides separate the two sequences (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by up to 2 nucleotides, up to 5 nucleotides, up to 8 nucleotides, up to 10 nucleotides, up to 12 nucleotides, or up to 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by 2-5 nucleotides, 4-6 nucleotides, 4-8 nucleotides, 4-10 nucleotides, 6-8 nucleotides, 6-10 nucleotides, 6-12 nucleotides, 8-10 nucleotides, 8-12 nucleotides, 10-12 nucleotides, 10-15 nucleotides, or 12- 15 nucleotides. As used herein, the term “spacer” or “spacer sequence” is a portion in an RNA guide that is the RNA equivalent of a target sequence (a DNA sequence) in the gene for editing (here PCSK9). The spacer contains a sequence capable of binding to the non-PAM strand via base-pairing at the site complementary to the target sequence (in the PAM strand). Such a spacer is also known as specific to the target sequence. In some instances, the spacer may be at least 75% identical to the target sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%), except for the RNA-DNA sequence difference. In some instances, the spacer may be 100% identical to the target sequence except for the RNA- DNA sequence difference.
Attorney Docket No.: 063586-522001WO (i). Spacer Sequence In some embodiments, the spacer sequence of the RNA guide provided herein may have a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary to a non-PAM strand sequence. In some embodiments, the spacer sequence is designed to be complementary to a specific DNA strand, e.g., of a genomic locus. As used herein, the term “complementary” refers to a first polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a second polynucleotide (e.g., the complementary sequence of a target sequence) such that the first and second polynucleotides can form a double-stranded complex via base-pairing to permit an effector polypeptide that is complexed with the first polynucleotide to act on (e.g., cleave) the second polynucleotide. In some embodiments, the first polynucleotide may be substantially complementary to the second polynucleotide, i.e., having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second polynucleotide. In some embodiments, the first polynucleotide is completely complementary to the second polynucleotide, i.e., having 100% complementarity to the second polynucleotide. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. In some embodiments, the PAM strand is a coding (e.g., sense) strand. In other embodiments, the PAM strand is a non-coding (e.g., antisense strand). Since an RNA guide binds the non-PAM strand via base-pairing, the non-PAM strand is also known as the target strand, while the PAM strand is also known as the non-target strand. In some embodiments, the spacer sequence can be any of the spacer sequences provided in Table 3 below. In some examples, the spacer sequence may be specific to a target sequence within exon 1, exon 2, exon 3, exon 5, exon 7, exon 8, exon 10, or exon 11 of the PCSK9 gene. In one example, the spacer sequence may be specific to exon 1 of the PCSK9 gene. In another example, example, the spacer sequence may be specific to exon 4 of the PCSK9 gene. In yet another example, example, the spacer sequence may be specific to exon 8 of the PCSK9 gene. In another specific example, the spacer sequence may be specific to exon 11 of the PCSK9 gene. In some instances, the gene editing system described herein may comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting PCSK9. Exemplary spacer sequences are provided herein. In one example, the spacer sequence
Attorney Docket No.: 063586-522001WO is specific to a target sequence of 5’- GCGCAGCGGTGGAAGGTGGC-3’ (SEQ ID NO: 15); Such a spacer sequence may comprise the nucleotide sequence of 5’- GCGCAGCGGUGGAAGGUGGC-3’ (SEQ ID NO: 74). In another example, the spacer sequence is specific to a target sequence of GAAAGACGGAGGCAGCCTGG (SEQ ID NO: 26). In yet another example, the spacer sequence is specific to a target sequence of 5’- GCAGAGAAGTGGATCAGTCT-3’ (SEQ ID NO: 52); Such a spacer sequence may comprise the nucleotide sequence of 5’- GCAGAGAAGUGGAUCAGUCU-3’ (SEQ ID NO: 111). In still another example, the spacer sequence is specific to a target sequence of 5’- GCACCCACAAGCCGCCTGTG-3’ (SEQ ID NO: 65); Such a spacer sequence may comprise the nucleotide sequence of 5’- GCACCCACAAGCCGCCUGUG-3’(SEQ ID NO: 124). In some instances, the gRNA for the CRISPR nucleases disclosed herein may have a shortened spacer sequence, for example, 15-19-nt long. In specific examples, the spacer sequence may be 16-nt in length. It is reported that gRNAs for the CRISPR nucleases disclosed herein that have a shortened spacer sequence exhibit higher gene editing efficiencies and lower off-target effects relative to the counterpart gRNAs having a longer spacer sequence, regardless of the target gene intended for editing. In some instances, the shortened spacer sequence may have 3’ end truncations relative to the counterpart spacer sequence that matches the target sequence (i.e., having the same length as the target sequence, which may have 20-nt in length). The 3’ end truncation may include a deletion of up to 5 nucleotides, for example, up to 4 nucleotides, up to 3 nucleotides, or up to 2 nucleotides. Such shortened spacer sequences derived from any of the exemplar guide RNAs provided herein (e.g., those listed in Table 3) are within the scope of the present disclosure. Examples of such shortened spacer sequences are provided in Table 6 below and elsewhere in the present disclosure, all of which are within the scope of the present disclosure. In some embodiments, a spacer sequence provided herein may comprise only RNA bases. In some embodiments, the spacer sequence may comprise a DNA base (e.g., the spacer comprises at least one thymine). In some embodiments, the spacer sequence may comprise RNA bases and DNA bases (e.g., the spacer sequence comprises at least one thymine and at least one uracil). (ii). Direct Repeat In some embodiments, the RNA guide comprises a direct repeat sequence. In some embodiments, the direct repeat sequence of the RNA guide has a length of between 12-100,
Attorney Docket No.: 063586-522001WO 13-75, 14-50, or 15-40 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In some embodiments, the direct repeat sequence is a sequence provided in Table 2 or a portion thereof. Table 2. CRISPR Nuclease Direct Repeat Sequences SEQ ID NO Direct Repeat Sequence 4 CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAAC
n some ns ances, e rec repea sequence s any one o Q s: - provided in Table 2 above. In one example, the direct repeat sequence comprises (e.g., consists of) SEQ ID NO: 4. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2. In some examples, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to SEQ ID NO: 4. Additional direct repeat sequence for use in constructing the RNA guides provided herein can be found, e.g., in WO2021050534 or WO2022192391, the relevant disclosures of each of which are incorporated by reference herein for the subject matter and purpose referenced herein. (iii). Exemplary RNA Guides The present disclosure provides RNA guides that comprise any and all combinations of the direct repeats and spacers described herein (e.g., as set forth in Table 3 below). In some embodiments, the sequence of an RNA guide has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a gRNA sequence provided in Table 3. In some embodiments, an RNA guide has a sequence of any of those provided in Table 3. Exemplary RNA guides include E1T3, E2T6, E3T2, E3T3, E3T5, E3T6, E3T10,
Attorney Docket No.: 063586-522001WO E4T3, E5T1, E7T7, E7T18, E8T8, E8T10, E10T2, E10T8, E11T3, E11T5, E11T8, E11T10, and E11T11 provided in Table 3. In one example, the RNA guide is E1T3. In another example, the RNA guide is E4T3. In yet another example, the RNA guide is E8T10. In still another example, the RNA guide is E11T3. In another example, the RNA guide is E10T8. The above-listed exemplary guides all have a spacer sequence of 20-nt in length. In some instances, the RNA guides for use in the gene editing system provided herein can be a variant of any of the above listed exemplary guides (reference guides), the variant having a shortened spacer sequence relative to the reference guide. The shortened spacer sequence may have 15-19-nt in length, for example, 16-18-nt in length. In some examples, the variant guide has a shortened spacer, which, relative to the spacer sequence in the reference guide, has a 3’ truncation of up to 5 nucleotides, for example, up to 4 nucleotides, up to 3 nucleotide, or up to 2 nucleotides. The shortened spacer can be a 3’ truncated version relative to the spacer in the reference guide with 16-nt, 17-nt, 18-nt, or 19 nt. In one example, the variant guide has a 3’ truncated 16-nt spacer sequence. In another example, the variant guide has a 3’ truncated 18-nt spacer sequence. In some specific examples, the RNA guide used in the gene editing systems provided herein is E1T3 or a variant thereof with a shortened spacer sequence. Examples are provided below: PCSK9_E1T3_20mer: 5’-CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACGCGCAGCGGU GGAAGGUGGC-3’ (SEQ ID NO: 132) PCSK9_E1T3_18mer: 5’-CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACGCGCAGCGGU GGAAGGUG-3’ (SEQ ID NO: 194) PCSK9_E1T3_16mer_unmodifed: 5’-CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACGCGCAGCGGU GGAAGG-3’ (SEQ ID NO: 224) In other specific examples, the RNA guide used in the gene editing systems provided herein is E10T8 or a variant thereof with a shortened spacer sequence. Examples are provided below: PCSK9_E10T8_20mer: 5’-CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACACUCUAAGGC CCAAGGGGGC-3’ (SEQ ID NO: 181)
Attorney Docket No.: 063586-522001WO PCSK9_E10T8_18mer: 5’-CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACACUCUAAGGC CCAAGGGG-3’ (SEQ ID NO: 231) PCSK9_E10T8_16mer_ 5’-CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACACUCUAAGGC CCAAGG-3’ (SEQ ID NO: 232) (iv). Modifications The RNA guides provided herein may include one or more covalent modifications with respect to a reference sequence, in particular the parent polyribonucleotide, which are included within the scope of the present disclosure. Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below. The RNA guide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein. In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to RNA guide- protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210. Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not
Attorney Docket No.: 063586-522001WO substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). In some embodiments, sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its internucleoside backbone. Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged. The modified nucleotides, which may be incorporated into the sequence, can be
Attorney Docket No.: 063586-522001WO modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). The α-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5’-O-(1-thiophosphate)-adenosine, 5’-O-(1-thiophosphate)-cytidine (a-thio-cytidine), 5’- O-(1-thiophosphate)-guanosine, 5’-O-(1-thiophosphate)-uridine, or 5’-O-(1-thiophosphate)- pseudouridine). Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein. In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4’-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2’-deoxy-2’-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1-beta-D- arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1- (2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5’-
Attorney Docket No.: 063586-522001WO elaidic acid ester). In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc). The one or more post- transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl- pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio- pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4- acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1- methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6-diaminopurine, 7-deaza- adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7- deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-
Attorney Docket No.: 063586-522001WO dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl- guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7- methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res.25, 1283-1284, which is incorporated by reference in its entirety. In some embodiments, one or more of the nucleotides of an RNA guide comprises a 2’-O-methyl phosphorothioate modification. In some embodiments, each of the first three nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification. In some embodiments, each of the last four nucleotides of the RNA guide comprises a 2’-O- methyl phosphorothioate modification. In some embodiments, each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified. In some embodiments, each of the first three nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification, and each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification. When a gene editing system disclosed herein comprises nucleic acids encoding the CRISPR nuclease disclosed herein, e.g., mRNA molecules, such nucleic acid molecules may contain any of the modifications disclosed herein, where applicable. In specific examples, the gRNAs in association with the CRISPR nucleases disclosed herein (regardless of the target gene) may contain 1-3 (e.g., 1, 2, or 3) 2’-O-methylated bases
Attorney Docket No.: 063586-522001WO and 1-3 (e.g., 1, 2, or 3) PS bonds at the 5’ end, at the 3’ end, or both. In some instances, the gRNA may have no 2’-O-methylated base at the 5’ end. Exemplary modification patterns are provided in Table 5 below. In one example, the modification pattern is mod2. In another example, the modification pattern is mod4. In yet another example, the modification pattern is mod6. The present disclosure shows that gRNAs for the CRISPR nucleases disclosed herein that have such modification patterns would exhibit higher gene editing efficiencies relative to the unmodified counterparts or gRNAs having the same sequence but different modifications, regardless of the target gene intended for editing. Any of the RNA guides provided in the present disclosure, either in modified form or unmodified form, is within the scope of the present disclosure. In some specific examples, the RNA guide used in the gene editing systems provided herein is E1T3 or a variant thereof with a shortened spacer sequence and a modification patter of mod2 or mod4. Examples are provided below: PCSK9_E1T3_20mer_mod2: 5’-C*U*U*GUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACGCGCAGC GGUGGAAGGU*mG*mG*mC-3’ (SEQ ID NO: 195) PCSK9_E1T3_18mer_mod2: 5’-C*U*U*GUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACGCGCAGC GGUGGAAG*mG*mU*mG*-3’ (SEQ ID NO: 222) PCSK9_E1T3_16mer_mod2: 5’-C*U*U*GUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACGCGCAGCG GUGGA*mA*mG*mG’3’ (SEQ ID NO: 230) PCSK9_E1T3_20mer_mod4: 5’-C*UUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACGCGCAGCGG UGGAAGGU*mG*mG*mC-3’ (SEQ ID NO: 196) PCSK9_E1T3_18mer_mod4: 5’-C*UUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACGCGCAGCGG UGGAAG*mG*mU*mG-3’ (SEQ ID NO: 223) PCSK9_E1T3_16mer_mod4: 5’-C*UUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACGCGCAGCGGU GGA*mA*mG*mG-3’ (SEQ ID NO: 229) In some specific examples, the RNA guide used in the gene editing systems provided herein is E10T8 or a variant thereof with a shortened spacer sequence and a modification
Attorney Docket No.: 063586-522001WO patter of mod2 or mod4. Examples are provided below: PCSK9_E10T8_20mer_mod2: 5’-C*U*U*GUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACACUCUAAG GCCCAAGGG*mG*mG*mC-3’ (SEQ ID NO: 233) PCSK9_E10T8_18mer_mod2: 5’-C*U*U*GUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACACUCUAAG GCCCAAG*mG*mG*mG-3’ (SEQ ID NO: 234) PCSK9_E10T8_16mer_mod2: 5’-C*U*U*GUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACACUCUAAG GCCCA*mA*mG*mG*-3’ (SEQ ID NO: 235) PCSK9_E10T8_20mer_mod4: 5’-C*UUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACACUCUAAGG CCCAAGGG*mG*mG*mC-3’ (SEQ ID NO: 225) PCSK9_E10T8_18mer_mod4: 5’-C*UUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACACUCUAAGGC CCAAG*mG*mG*mG-3’ (SEQ ID NO: 226) PCSK9_E10T8_16mer_mod4: 5’-C*UUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAACACUCUAAGGC CCA*mA*mG*mG-3’ (SEQ ID NO: 227) “*” represents phosphorothioate bonds and mN represents 2’-O-methyl modified base Any of the PCSK9-targeting gRNAs, as well as nucleic acids encoding such, is also within the scope of the present disclosure. II. Preparation of Gene Editing System Components The present disclosure provides methods for production of components of the gene editing systems disclosed herein, e.g., the RNA guide and the CRISPR nuclease, as well as methods for complexing the RNA guide and the CRISPR nuclease. A. RNA Guide In some embodiments, the RNA guide can be made by in vitro transcription of a DNA template. Thus, for example, in some embodiments, the RNA guide is generated by in vitro transcription of a DNA template encoding the RNA guide using an upstream promoter
Attorney Docket No.: 063586-522001WO sequence (e.g., a T7 polymerase promoter sequence). In some embodiments, the DNA template encodes multiple RNA guides or the in vitro transcription reaction includes multiple different DNA templates, each encoding a different RNA guide. In some embodiments, the RNA guide is made using chemical synthetic methods. In some embodiments, the RNA guide is made by expressing the RNA guide sequence in cells transfected with a plasmid including sequences that encode the RNA guide. In some embodiments, the plasmid encodes multiple different RNA guides. In some embodiments, multiple different plasmids, each encoding a different RNA guide, are transfected into the cells. In some embodiments, the RNA guide is expressed from a plasmid that encodes the RNA guide and also encodes the CRISPR nuclease. In some embodiments, the RNA guide is expressed from a plasmid that expresses the RNA guide but not the CRISPR nuclease. In some embodiments, the RNA guide is purchased from a commercial vendor. In some embodiments, the RNA guide is synthesized using one or more modified nucleotide, e.g., as described above. B. CRISPR nuclease In some embodiments, the CRISPR nuclease disclosed in the present disclosure can be prepared by the conventional recombinant technology. For example, nucleic acids encoding the CRISPR nuclease can be cloned into one expression vector, the coding sequence being in operable linkage to a suitable promoter. The expression vector can be introduced into suitable host cells, which can be cultured under suitable conditions allowing for the production of the polypeptide, which can be isolated and purified from the cell culture. Optionally, the CRISPR nuclease can be complexed with an RNA guide to form a complex. Suitable host cells are not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells). The method for transferring the expression vector described above into host cells, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used. After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production of the CRISPR nuclease. After expression of the CRISPR nuclease, the host cells can be collected and CRISPR nuclease purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel
Attorney Docket No.: 063586-522001WO filtration chromatography, ion exchange chromatography, etc.). A variety of methods can be used to determine the level of production of the CRISPR nuclease in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the CRISPR nuclease or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med.158:1211 [1983]). The present disclosure provides methods of in vivo expression of the CRISPR nuclease in a cell, comprising providing a polyribonucleotide encoding the CRISPR nuclease to a host cell wherein the polyribonucleotide encodes the CRISPR nuclease, expressing the CRISPR nuclease in the cell, and obtaining the CRISPR nuclease from the cell. The present disclosure further provides methods of in vivo expression of the CRISPR nuclease in a cell, comprising providing a polyribonucleotide (e.g., an mRNA molecule) encoding the CRISPR nuclease to a host cell located in a subject such as a human patient. In some examples, the polyribonucleotide encoding the CRISPR nuclease can be delivered to the cell with an RNA guide and, once expressed in the cell, the CRISPR nuclease and the RNA guide form a complex. C. CRISPR Nuclease-gRNA Complexes In some embodiments, an RNA guide targeting PCSK9 can be complexed with the CRISPR nuclease disclosed herein to form a ribonucleoprotein (RNP) complex. In some embodiments, complexation of the RNA guide and CRISPR nuclease occurs at a temperature lower than about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, or 55°C. In some embodiments, the RNA guide does not dissociate from the CRISPR nuclease at about 37°C over an incubation period of at least about any one of 10mins, 15mins, 20mins, 25mins, 30mins, 35mins, 40mins, 45mins, 50mins, 55mins, 1hr, 2hr, 3hr, 4hr, or more hours. In some embodiments, the RNA guide and CRISPR nuclease are complexed in a complexation buffer. In some embodiments, the CRISPR nuclease is stored in a buffer that is replaced with a complexation buffer to form a complex with the RNA guide. In some embodiments, the CRISPR nuclease is stored in a complexation buffer. In some embodiments, the complexation buffer has a pH in a range of about 7.3 to 8.6.
Attorney Docket No.: 063586-522001WO In one embodiment, the pH of the complexation buffer is about 7.3. In one embodiment, the pH of the complexation buffer is about 7.4. In one embodiment, the pH of the complexation buffer is about 7.5. In one embodiment, the pH of the complexation buffer is about 7.6. In one embodiment, the pH of the complexation buffer is about 7.7. In one embodiment, the pH of the complexation buffer is about 7.8. In one embodiment, the pH of the complexation buffer is about 7.9. In one embodiment, the pH of the complexation buffer is about 8.0. In one embodiment, the pH of the complexation buffer is about 8.1. In one embodiment, the pH of the complexation buffer is about 8.2. In one embodiment, the pH of the complexation buffer is about 8.3. In one embodiment, the pH of the complexation buffer is about 8.4. In one embodiment, the pH of the complexation buffer is about 8.5. In one embodiment, the pH of the complexation buffer is about 8.6. In some embodiments, the CRISPR nuclease can be overexpressed and complexed with the RNA guide in a host cell prior to purification as described herein. In other embodiments, mRNA or DNA encoding the CRISPR nuclease can be introduced into a cell so that the CRISPR nuclease is expressed in the cell. In some embodiments, the RNA guide is also introduced into the cell, whether simultaneously, separately, or sequentially from a single mRNA or DNA construct, such that the ribonucleoprotein complex is formed in the cell. In some instances, the cell can be a target cell (e.g., a liver cell) in which editing of the PCSK9 gene is intended. III. Methods for Genetic Editing of PCSK9 Gene The disclosure also provides methods of genetically editing the PCSK9 gene in target cells such as liver cells to disrupt expression of PCSK9. In some instances, the methods comprise introducing any of the gene editing systems disclosed herein into cultured target cells (in vitro editing). For example, an PCSK9-targeting RNA guide and the CRISPR nuclease provided herein can be introduced into a population of cultured cells. Alternatively, the methods comprise introducing the gene editing system into a subject such as a human patient to achieve editing of PCSK9 in vivo. The PCSK9-targeting RNA guide and CRISPR nuclease can form a ribonucleoprotein complex, which can be introduced into target cells. Alternatively, the PCSK9-targeting RNA guide and CRISPR nuclease can be encoded by a nucleic acid vector(s), which can be introduced into the target cells. In another example, an mRNA molecule encoding the CRISPR nuclease can be introduced into target cells. Alternatively or in addition, the RNA guide can be introduced directly into the target cells.
Attorney Docket No.: 063586-522001WO Any of the gene editing systems disclosed herein may be used to genetically engineered a PCSK9 gene in target cells of interest (e.g., liver cells). The gene editing system may comprise an RNA guide and the CRISPR nuclease such as those provided herein, e.g., in Table 1 above. The RNA guide comprises a spacer sequence specific to a target sequence in the PCSK9 gene, e.g., specific to a region in exon 1, exon 4, exon 8, or exon 11 of the PCSK9 gene. A. Delivery of Gene Editing System Components of any of the gene editing systems disclosed herein may be formulated, for example, including a carrier, such as a carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to target cells (e.g., a prokaryotic, eukaryotic, plant, mammalian, etc.). In some examples, the target cells are liver cells. Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)), microinjection, microprojectile bombardment (“gene gun”), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome- mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the CRISPR nuclease disclosed herein, one or more RNA guides also disclosed herein, etc.), one or more transcripts thereof, and/or a pre-formed RNA guide/CRISPR nuclease complex to target cells. In some instances, an RNA guide and an RNA (e.g., mRNA) encoding the CRISPR nuclease are delivered together in a single composition. Alternatively, an RNA guide and an RNA (e.g., mRNA) encoding the CRISPR nuclease are delivered in separate compositions. For example, the RNA guide and the RNA encoding the CRISPR nuclease delivered in separate compositions are delivered using the same delivery technology. In other examples, the RNA guide and the RNA encoding the CRISPR nuclease delivered in separate compositions are delivered using different delivery technologies. Exemplary intracellular delivery methods, include, but are not limited to: viruses, such as AAV, or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, lipid nanoparticles, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast
Attorney Docket No.: 063586-522001WO fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, a lipid nanoparticle comprises an mRNA encoding the CRISPR nuclease (e.g., those provided herein such as the one in Table 1 above), an RNA guide, or an mRNA encoding the CRISPR nuclease and the RNA guide can be used for delivering the gene editing components to target cells such as to liver cells. Any suitable LNP components that have been used for delivering nucleic acids to cells can also be used in the instant disclosure, for example, the LNP particles used for delivering the COVID-19 mRNA vaccines as reported in Schoenmaker et al., International J. Pharmaceutics, 601 (2021) 120586, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. Effectiveness of PCSK9 editing in the target cells can be examined via conventional technology and/or those provided in Examples below. B. Genetically Modified Cells Any of the gene editing systems disclosed herein can be delivered to a variety of cells to genetically edit the PCSK9 gene, which can lead to substantially reduced or no expression of the PCSK9 gene. In some embodiments, the target cells can be cultured cells or a co-culture of two or more types of cells. In some examples, the target cells can be obtained from a living organism (e.g., a mammal such as a human subject) and maintained in a cell culture. In some embodiments, the cell can be a single-cellular organism. For example, the cell can be a prokaryotic cell. In some examples, the cell is a bacterial cell or derived from a bacterial cell. In other examples, the cell is an archaeal cell or derived from an archaeal cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a plant cell or derived from a plant cell. In some embodiments, the cell is a fungal cell or derived from a fungal cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebra fish cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to,
Attorney Docket No.: 063586-522001WO 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell. For example, in some embodiments, the differentiated cell is a liver cell (e.g., a hepatocyte), a biliary cell (e.g., a cholangiocyte), a stellate cell, a Kupffer cell, a liver sinusoidal endothelial cell, a muscle cell (e.g., a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a fibroblast, or a sex cell. In some embodiments, the cell is a terminally differentiated cell. For example, in some embodiments, the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiomyocyte, a skeletal muscle cell, an epidermal cell, or a gut cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a Natural Killer (NK) cell. In some embodiments, the immune cell is a Tumor Infiltrating Lymphocyte (TIL). In some embodiments, the cell is a mammalian cell, e.g., a human cell or a murine cell. In some embodiments, the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease-specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism. Any of the genetically modified cells produced using any of the gene editing system disclosed herein is also within the scope of the present disclosure. Such modified cells may comprise a disrupted PCSK9 gene. In some instances, the disrupted PCSK9 gene may comprise one or more mutations (e.g., deletion, insertion, and/or nucleotide substitution) in exon 2. In other instances, the disrupted PCSK9 gene may comprise one or more mutations (e.g., deletion, insertion, and/or nucleotide substitution) in exon 11. In some instances, the modified cells carrying a disrupted PCSK9 gene show substantially reduced expression of PCSK9 as relative to a wild-type counterpart. For example, the level of PCSK9 protein in the modified cells may be reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or over. In
Attorney Docket No.: 063586-522001WO specific examples, the level of PCSK9 protein or the level of PCSK9 activity is not detectable in the modified cells by a conventional approach. C. Therapeutic Applications Any of the gene editing systems or modified cells (e.g., modified liver cells) generated using such a gene editing system as disclosed herein may be used for treating a disease associated with LDL cholesterol, such as cardiovascular conditions (e.g., heart diseases and stroke). High cholesterol levels can lead to development of fatty deposits in blood vessels, which can make it difficult for enough blood to flow through arteries and/or form a clot, causing a heart attack or stroke. PCSK9 modulates the level of LDL cholesterol in the blood stream. Blocking the level and/or activity of PCSK9 can lower blood levels of LDL cholesterol, thereby benefiting diseases associated with PCSK9 (e.g., diseases associated with high levels of LDL cholesterol). In some embodiments, provided herein is a method for treating a target disease associated with PCSK9 and/or LDL cholesterol such as cardiovascular conditions (e.g., coronary artery disease, peripheral arterial disease), heart attack, stroke, hypercholesterolemia as disclosed herein. The method may comprise administering to a subject (e.g., a human patient) in need of the treatment any of the gene editing systems disclosed herein. The gene editing system may be delivered to a specific tissue (e.g., liver) or specific type of cells (e.g., liver cells) where the gene edit is needed. The gene editing system may comprise LNPs encompassing one or more of the components, one or more vectors (e.g., viral vectors) encoding one or more of the components, or a combination thereof. Components of the gene editing system may be formulated to form a pharmaceutical composition, which may further comprise one or more pharmaceutically acceptable carriers. In some embodiments, provided herein is a composition comprising the gene editing system disclosed herein or components thereof. Such a composition can be a pharmaceutical composition. A pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation for a suitable delivery route, e.g., parenteral, intra-lesional, intra-organ or another route of administration. A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a
Attorney Docket No.: 063586-522001WO dosage. A formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which that are useful include those which may comprise the cells in a packaged form, in a liposomal preparation, or as a component of a biodegradable polymer system. Some compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. IV. Kits for Genetic Editing of PCSK9 Gene and Alleviating Target Diseases The present disclosure also provides kits that can be used, for example, to carry out a gene editing method described herein for genetical editing of the PCSK9 gene as disclosed herein. In some embodiments, the kits include an RNA guide and the CRISPR nuclease (e.g., those provided herein such as the one provided in Table 1). In some embodiments, the kits include an RNA guide and the CRISPR nuclease. In other embodiments, the kits include a polynucleotide that encodes such the CRISPR nuclease, and optionally the polynucleotide is comprised within a vector, e.g., as described herein. In some embodiments, the kits include a
Attorney Docket No.: 063586-522001WO polynucleotide that encodes an RNA guide disclosed herein. The CRISPR nuclease (or polynucleotide encoding the CRISPR nuclease) and the RNA guide (e.g., as a ribonucleoprotein) can be packaged within the same or other vessel within a kit or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The CRISPR nuclease and the RNA guide can be packaged within the same or other vessel within a kit or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The kits can additionally include, optionally, a buffer and/or instructions for use of the RNA guide and CRISPR nuclease. General techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed.1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed.1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds.1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (lRL Press, (1986»; and B. Perbal, A practical Guide
Attorney Docket No.: 063586-522001WO To Molecular Cloning (1984); F.M. Ausubel et al. (eds.). Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. Example 1 - CRISPR Nuclease-Mediated Editing of PCSK9 Target Sequences in HEK293T and HepG2 cells This Example describes the genomic editing of the PCSK9 gene using a variant CRISPR nuclease introduced into cells by RNP in the cell lines HEK293T and HepG2. RNA guides were designed and ordered from Integrated DNA Technologies (IDT). For initial RNA guide screening in HEK293T and HepG2 cells, RNA guides were designed to be specific to target sequences within each of the coding exons of PCSK9 with 5’-NTTR-3’ PAM sequences (the PAM sequence is on the 5’ end of the target sequence, N is any of A, G, C, and T and R is A or G). RNA guides were not designed for any target sequences having an identical sequence elsewhere in the genome were not used. See RNA guide sequences in Table 3. Table 3. Target and RNA Guide Sequences for PCSK9 Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID O 1 2 3 4 5
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID NO NO NO 6 7 8 9 0 1 2 3 4 5 6 7 8
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID NO NO NO 9 0 1 2 3 4 5 6 7 8 9 0
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID NO NO NO 1 2 3 4 5 6 7 8 9 0 1 2 3
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID NO NO NO 4 5 6 7 8 9 0 1 2 3 4 5
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID NO NO NO 6 7 8
CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAAC (SEQ ID NO: 72). The DNA counterpart of this DR sequence is CTTGTTGTATATGTCCTTTTATAGGTATTAAACAAC (SEQ ID NO: 191). RNP complexation reactions were made by mixing purified CRISPR nuclease (SEQ ID NO: 3) (100 µM in 20 mM HEPES pH 7.5, 300 mM NaCl, 10 % Glycerol, 0.5 mM TCEP) with RNA guide (250 µM in 250 mM NaCl) at a 1:1 (CRISPR nuclease:RNA guide) volume ratio (2.5:1 RNA guide:CRISPR nuclease molar ratio). Complexations were incubated on ice for 45-60 min. RNP electroporation in HEK293T cells During RNP complexation incubation, HEK293T cells were harvested using TrypLE
TM (ThermoFisher Scientific) and counted. Cells were washed once with PBS and resuspended in SF buffer + supplement (Lonza #V4SC-2096). Resuspended cells were dispensed at 3e5 cells/reaction into Lonza 96-well electroporation plates. Complexed RNP was added to each reaction at a final concentration of 20 µM, and transfection enhancer oligos were then added at a final concentration of 4 µM. Non-targeting RNA guides were used as negative controls. The plates were electroporated using an electroporation device (program CM-130, Lonza 4D- nucleofector). Immediately following electroporation, pre-warmed DMEM + 10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, 30,000 cells of diluted nucleofected cells were plated into pre-warmed 96-well plate with wells containing DMEM + 10% FBS. Editing plates were incubated for 3 days at 37
oC with 5% CO2.
Attorney Docket No.: 063586-522001WO After 3 days, wells were harvested using TrypLE
TM (ThermoFisher Scientific) and transferred to 96-well twin.tec
® PCR plates (Eppendorf). Media was flicked off, and cells were resuspended in DNA extraction buffer (QuickExtract
TM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at - 20°C, and subsequently analyzed for PCSK9 indels using Next Generation Sequencing (NGS), as described below. RNP electroporation in HepG2 cells During RNP complexation incubation, HepG2 cells were harvested using TrypLE
TM (ThermoFisher Scientific) and counted. Cells were washed once with PBS and resuspended in SF buffer + supplement (Lonza #V4SC-2096). Resuspended cells were dispensed at 2.5e5 cells/reaction into Lonza 96-well electroporation plates. Complexed RNP was added to each reaction at a final concentration of 20 µM, with no transfection enhancer oligo. Non-targeting RNA guides were used as negative controls. The plates were electroporated using an electroporation device (program DJ-100, Lonza 4D-nucleofector). Immediately following electroporation, pre-warmed EMEM (ATCC) + 10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 25,000 cells of diluted nucleofected cells were plated into pre-warmed 96-well plate with wells containing EMEM (ATCC) + 10% FBS. Editing plates were incubated for 3 days at 37
oC with 5% CO2. After 3 days, media was collected, and PCSK9 protein expression in the media was measured using Meso Scale Discovery (MSD) immunoassays, as described below. In parallel, cells in the wells were harvested using TrypLE
TM (ThermoFisher Scientific) and transferred to 96-well twin.tec
® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in DNA extraction buffer (QuickExtract
TM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at -20°C, and subsequently analyzed for PCSK9 indels using Next Generation Sequencing (NGS), as described below. NGS library preparation and analysis Samples for Next Generation Sequencing (NGS) were prepared by rounds of PCR. The first round (PCR I) was used to amplify the genomic regions flanking the target site and add NGS adapters. The second round (PCR II) was used to add NGS indexes. Reactions were then pooled, purified by column purification, and quantified on a fluorometer (Qubit). Sequencing
Attorney Docket No.: 063586-522001WO runs were done using a 150 cycle NGS instrument (NextSeq v2.5) mid or high output kit and run on an NGS instrument (NextSeq 550). For NGS analysis, the indel mapping function used a sample’s fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well. Up to 50,000 reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion. The % indels was calculated as the number of indel-containing reads divided by the number of reads analyzed (reads passing filters up to 50,000). The QC standard for the minimum number of reads passing filters was 10,000. As shown in FIG.1, PCSK9 indels in HEK293T and HepG2 cells following RNP transfection with the variant CRISPR nuclease and RNA guides listed in Table 3. Each circle represents the average of three technical replicates across one biological replicate. Use of an additional 64 RNA guides targeting PCSK9 exon sequences with 5’-NTTY-3’ (Y is C or T) PAM sequences did not result in indels or resulted in indels of less than 10%. 25 RNA guides resulted in indel percentages of at least 80% in HEK293T and at least 50% in HepG2 cells. See FIG.1. These RNA guides were then selected to be tested in primary human hepatocytes. Example 2 - CRISPR Nuclease-Mediated Editing of PCSK9 Target Sequences in Primary Human Hepatocyte (PHH) Donor Cells This Example describes genomic editing of the PCSK9 gene using a variant CRISPR nuclease introduced into cells by RNP in primary human hepatocyte (PHH) donors. The RNA guides that produced greater than 80% indels in HEK293T cell and greater than 50% indels in HepG2 cells as observed in Example 1 were selected for use in this study, including E1T3, E2T6, E3T2, E3T3, E3T5, E3T10, E4T3, E5T1, E6T8, E6T10, E7T7, E7T18, E8T3, E8T8, E8T10, E9T5, E10T2, E10T8, E11T3, E11T5, E11T8, E11T10, E11T11, and E12T8. Structural information of these RNA guides can be found in Table 3 above. RNP electroporation in primary human hepatocyte (PHH) donor cells During RNP complexation incubation, PHH cells from human donors (HUM190171
Attorney Docket No.: 063586-522001WO from Lonza) were thawed from liquid nitrogen quickly in a 37
oC water bath. The cells were added to pre-warmed hepatocyte recovery media (Thermofisher, CM7000) and centrifuged for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams’ Medium E, Thermofisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum-containing), Thermofisher CM3000). The cells were subjected to a trypan blue viability count using the Vi-CELL BLU cell counter. The desired number of viable cells were then washed in PBS and resuspended in P3 buffer + supplement (Lonza, V4SP-3096) and transfection enhancer oligo (final concentration of 4 µM). Resuspended cells were dispensed at 125,000 cells/reaction into Lonza 96-well electroporation plates. Complexed RNP was added to each reaction at a final concentration 5 µM, 1.25 µM, or 0.625 µM. Non-targeting RNA guides were used as negative controls. The strips were electroporated using an electroporation device (program DS-150, Lonza 4D-nucleofector).5 minutes following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, 125,000 cells of diluted nucleofected cells were plated into a pre-warmed collagen-coated 96-well plate (Thermofisher) with wells containing Hepatocyte plating medium. The cells were then incubated in a 37
oC incubator. The media was changed to hepatocyte maintenance media (Williams’ Medium E, Thermofisher A1217601 supplemented with William’s E medium Cell Maintenance Cocktail, Thermofisher CM 4000) after the cells attached after 4 hours. Fresh hepatocyte maintenance media was replaced after 3 days. After 7 days post RNP electroporation, media was collected and PCSK9 protein expression in the media was measured using Meso Scale Discovery (MSD) immunoassays, as described below. In parallel, cells in the wells were harvested using Accutase and transferred to 96-well twin.tec
® PCR plates (Eppendorf) and centrifuged. Media was flicked off and cells were resuspended in DNA extraction buffer (QuickExtract
TM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at -20°C, and subsequently analyzed for PCSK9 indels using Next Generation Sequencing (NGS) as described in Example 1. FIG.2 shows PCSK9 indels in a PHH donor (HUM190171) following RNP transfection of the variant CRISPR nuclease and the selected RNA guides.19 RNA guides resulted in indels of at least 40% at the highest concentration tested. Expression of PCSK9 was then measured for the RNA guides resulting in the highest percent indels.
Attorney Docket No.: 063586-522001WO Meso Scale Discovery (MSD) immunoassays Samples for MSD immunoassays were collected from media on PHH cells electroporated with RNP, 3 days post electroporation. First, U-Plex linkages were incubated with either PCSK9 or fibronectin (for normalization) capture antibodies, then added to MSD plates for antibody coating. Calibrator curves were prepared, and samples were added to coated plates, using a 5x dilution of spent PHH media in Diluent 101. Following 1 hour shaking incubation, plates were washed, detecting antibody was added, and plates were shaken and incubated for 1 hour. Following subsequent washes, MSD Gold Read Buffer B was added to each well, and plates were read for detection of PCSK9 and fibronectin expression. FIG.3 shows PCSK9 expression (measured by MSD) relative to control expression in a PHH donor (HU2021) following RNP transfection with the highest performing RNA guides identified in FIG.2. Many of the RNA guides reduced PCSK9 expression to under 10% at the highest RNP concentration tested. E4T3, E11T3, E8T10, and E1T3 also substantially decreased PCSK9 expression at the lower RNP concentrations tested. Example 3 – CRISPR Nuclease-Mediated Editing of PCSK9 Target Sequences in Primary Human Hepatocyte (PHH) Donor Cells Using Various RNA Guide Modifications This Example describes genomic editing of the PCSK9 gene using a variant CRISPR nuclease introduced into cells by mRNA with synthetic RNA guides containing various modifications in primary human hepatocytes (PHH) donors. On-target indels were determined and compared between cells transfected with mRNA effector and RNA guides containing different modification configurations. Structural information of these RNA guides can be found in Table 4 and modification nomenclature can be found in Table 5. RNA guides were designed with various chemical modifications (modification formats shown in Table 5) and ordered from Integrated DNA Technologies (IDT). Guides contained phosphorothioate (PS) bonds and/or 2’-O-methylated bases at various positions at the 5’ and 3’ end of the guide (see Table 4 for specific sequence information of guides). Table 4. Exemplary Target and RNA Guide Sequences for PCSK9 Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ D O 2
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID O 5 6 7 8 9 0 1
y All of the guides listed in Table 4 above have a DR sequence of SEQ ID NO: 72.
Attorney Docket No.: 063586-522001WO Table 5. RNA Guide Modification Nomenclature Modification name Modification information Generic guide sequence (N = SEQ ID base) NO 2 3 4 5 6 7 8 5
= - - e y o e ase Spacer sequence represented by the “N” residues uses the 20-nt length as an example. The length of the spacer can be 16-nt, 17-nt, 18-nt, 19-nt, or 20-nt.
Attorney Docket No.: 063586-522001WO mRNA electroporation in primary human hepatocyte (PHH) donor cells A CRISPR nuclease sequence encoding the nuclease variant of SEQ ID NO: 3 and a nuclear localization signal (NLS) at both the N-terminus and C-terminus, which is connected to the nuclease variant via a flexible peptide linker, was cloned into an in vitro transcription (IVT) backbone and reverse transcribed into mRNA using an in vitro transcription kit. The full amino acid sequence of the CRISPR nuclease is listed below (SEQ ID NO: 228). A working solution of mRNA for expression was prepared in water at a concentration of 1 mg/mL. MKRPAATKKAGQAKKKKSAGGGGSGGGGSGGGGSGMIKSIQLKVKGECRITKDVINEYKEYYNNCSRW IKNNLTSITIGEMAKFLQSLSDKEVAYRSMGLSDEWKDKPLYHLFTKKYHTKNADNLLYYYIKEKNLD GYKGNTLNISNTSFRQFGYFKLVVSNYRTKIRTLNCKIKRKKIDADSTSEDIEMQVMYEIIKYSLNKK SDWDNFISYIENVENPNIDNINRYKLLRECFCENENMIKNKLELLSVEQLKKFGGCIMKPHINSMTIN IQDFKIEEKENSLGFILHLPLNKKQYQIELLGNRQIKKGTKEIHETLVDITNTHGENIVFTIKNDNLY IVFSYRSEFEKEEVNFAKTVGLDVNFKHAFFVGSEKDNCHLDGYINLYKYLLEHDEFTNLLTEDERKD YEELSKVVTFCPFENQLLFARYNKMSKFCKKEQVLSKLLYALQKKLKDENRTKEYIYVSCVNKLRAKY VSYFILKEKYYEKQKEYDIEMGFVDDSTESKESMDKRRTEYPFRNTPVANELLSKLNNVQQDINGCLK NIINYIYKIFEQNGYKVVALENLENSNFEKKQVLPTIKSLLKYHKLENQNVNDIKASDKVKEYIENGY YELMTNENNEIVDAKYTEKGAMKVKNANFFNLMMKSLHFASVKDEFVLLSNNGKTQIALVPSEFTSQM DSTDHCLYMKKNDKGKLVKADKKEVRTKQERHINGLNADFNAANNIKYIVENEVWRGIFCTRPKKTEY NVPSLDTTKKGPSAILNMLKKIGAIKVLETEKSAGGGGSGGGGSGGGGSGKRPAATKKAGQAKKKK (SEQ ID NO: 228) PHH cells from human donors (from Thermofisher or Lonza) were thawed from liquid nitrogen quickly in a 37
oC water bath. The cells were added to pre-warmed hepatocyte recovery media (Thermofisher, CM7000) and centrifuged for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams’ Medium E, Thermofisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum- containing), Thermofisher CM3000). The cells were subjected to a trypan blue viability count using the Vi-CELL BLU cell counter. The desired number of viable cells were then washed in PBS and resuspended in P3 buffer + supplement (Lonza, V4SP-3096) and transfection enhancer oligo (final concentration of 4 µM). Resuspended cells were dispensed at 125,000 cells/reaction into Lonza 96-well electroporation plates. mRNA effector (1 mg/mL in water) was mixed with synthetic RNA guides (1 mM in water) at a 1:1 volume ratio. mRNA/guide RNA mixtures were added to each reaction at a final mRNA concentration of 25 nM, 6.25 nM, 1.56 nM, or 0.39 nM. The strips were electroporated using an electroporation device (program DS-150, Lonza 4D-nucleofector).5 minutes following electroporation, pre-warmed Hepatocyte plating
Attorney Docket No.: 063586-522001WO medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, 125,000 cells of diluted nucleofected cells were plated into a pre-warmed collagen-coated 96-well plate (Thermofisher) with wells containing Hepatocyte plating medium. The cells were then incubated in a 37
oC incubator. The media was changed to hepatocyte maintenance media (Williams’ Medium E, Thermofisher A1217601 supplemented with William’s E medium Cell Maintenance Cocktail, Thermofisher CM 4000) after the cells attached after 4 hours. After 3 days post electroporation, cells in the wells were harvested using Accutase and transferred to 96-well twin.tec
® PCR plates (Eppendorf) and centrifuged. Media was flicked off and cells were resuspended in DNA extraction buffer (QuickExtract
TM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at -20°C, and subsequently analyzed for PCSK9 indels using Next Generation Sequencing (NGS) as described in Example 1. FIG.4 shows PCSK9 indels in a PHH donor following electroporation of the variant CRISPR nuclease and the selected modified RNA guides. All three modifications (mod2, mod4, mod6) showed high on-target activity for guides tested, with unmodified guides demonstrating reduced activity when delivered with an mRNA expressing the nuclease. Additional guide modification formats were tested (Table 5, e.g.: mod1, mod3 and mod5) but showed significantly lower on-target indel activity compared to mod2, mod4, and mod6. Example 4 – CRISPR Nuclease-Mediated Editing of PCSK9 Target Sequences in Primary Human Hepatocyte (PHH) Donor Cells Using Various RNA Guide Spacer Nucleotide Lengths This Example describes genomic editing of the PCSK9 gene using a variant CRISPR nuclease introduced into cells by mRNA with synthetic RNA guides of various lengths in primary human hepatocytes (PHH) donors. The synthetic RNA guides used with the CRISPR nuclease contained spacer lengths ranging from 16 nucleotides (nt) to 20 nt. On-target indels and/or off-target indels were determined and compared between cells transfected with RNA guides of different spacer lengths. Structural information of these RNA guides can be found in Table 6. RNA guides were designed and ordered from Integrated DNA Technologies (IDT). Guides contained one phosphorothioate (PS) bond at the 5’ end and three PS bonds + three 2’- O-methylated bases at the 3’ end of the guide (see Table 6 for specific sequence information
Attorney Docket No.: 063586-522001WO of guides). Spacer lengths were either 16 nt, 18 nt, or 20 nt, with truncations occurring at the 3’ end. Table 6. Exemplary Target and RNA Guide Sequences for PCSK9 Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID O 6 3 9 5 6 7
All guides listed above have the DR sequence of SEQ ID NO: 72. mRNA electroporation in primary human hepatocyte (PHH) donor cells A CRISPR nuclease sequence encoding the nuclease variant of SEQ ID NO: 3 and a nuclear localization signal (NLS) at both the N-terminus and C-terminus, which is connected to the nuclease variant via a flexible peptide linker was cloned into an in vitro transcription (IVT) backbone and reverse transcribed into mRNA using an in vitro transcription kit. A working solution of mRNA for expression was prepared in water at a concentration of 1 mg/mL. PHH cells from human donors (from Thermofisher or Lonza) were thawed from liquid
Attorney Docket No.: 063586-522001WO nitrogen quickly in a 37
oC water bath. The cells were added to pre-warmed hepatocyte recovery media (Thermofisher, CM7000) and centrifuged for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams’ Medium E, Thermofisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum- containing), Thermofisher CM3000). The cells were subjected to a trypan blue viability count using the Vi-CELL BLU cell counter. The desired number of viable cells were then washed in PBS and resuspended in P3 buffer + supplement (Lonza, V4SP-3096) and transfection enhancer oligo (final concentration of 4 µM). Resuspended cells were dispensed at 125,000 cells/reaction into Lonza 96-well electroporation plates. mRNA effector (1 mg/mL in water) was mixed with synthetic RNA guides (1 mM in water) at a 1:1 volume ratio. mRNA/guide RNA mixtures were added to each reaction at a final mRNA concentration of 25 nM, 6.25 nM, 1.56 nM, 0.78 nM, 0.39 nM, 0.19 nM, 0.09 nM, or 0.05 nM. The strips were electroporated using an electroporation device (program DS-150, Lonza 4D-nucleofector).5 minutes following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, 125,000 cells of diluted nucleofected cells were plated into a pre-warmed collagen-coated 96-well plate (Thermofisher) with wells containing Hepatocyte plating medium. The cells were then incubated in a 37
oC incubator. The media was changed to hepatocyte maintenance media (Williams’ Medium E, Thermofisher A1217601 supplemented with William’s E medium Cell Maintenance Cocktail, Thermofisher CM 4000) after the cells attached after 4 hours. After 3 days post electroporation, cells in the wells were harvested using Accutase and transferred to 96-well twin.tec
® PCR plates (Eppendorf) and centrifuged. Media was flicked off and cells were resuspended in DNA extraction buffer (QuickExtract
TM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at -20°C, and subsequently analyzed for PCSK9 indels and indels of in silico predicted off-target sites using Next Generation Sequencing (NGS) as described in Example 1. FIG.5A shows PCSK9 indels in a PHH donor following electroporation of the variant CRISPR nuclease and the selected modified RNA guides. The 18 nt spacer length guides showed significantly higher on-target activity at PCSK9, while 16 nt and 20 nt spacer length guides showed equivalent editing. FIG.5B shows indels at in silico predicted off-target sites for PCSK9 targets. The 18 nt spacer length guides showed significantly higher off-target
Attorney Docket No.: 063586-522001WO activity at sites tested.20 nt spacer length guides showed significantly lower, but detectable levels of editing at off-targets, while 16 nt spacer length guides showed no off-target activity at sites tested. Guides with 18 nt spacers showed higher on-target and higher off-target activity for the PCSK9-targeting guides tested. Guides with 16 nt spacers showed equivalent on-target activity compared to 20 nt spacers, but significantly lower off-target activity across several predicted off-target sites. Off-target analysis of PCSK9-E1T3 guides with various spacer lengths in PHH To assess off-target activity at a larger scale, rhAmpSeq
TM (Integrated DNA Technologies) was performed in PHH cells, targeting in silico predicted off-targets for a 20 nt spacer PCSK9-E1T3 guide. Briefly, gDNA was extracted from primary human hepatocytes using the Zymo Quick- DNA Miniprep Plus kit, quantified by high sensitivity DNA Qubit, and was then normalized to 8ng/uL with IDTE pH 8.0. rhAmp primer pools for the first round of PCR (PCR1) were resuspended in IDTE pH7.5 to variable 10X concentrations based on the number of primers in each pool. If the panel contained ≥ 500 primers, the 10X rhAmp primer pool was 50uM total. If the panel contained between 100 and 500 primers, the 10X rhAmp Primer Pool was 100nM of each primer. If the panel contained ≤ 100 primers, the 10X rhAmp Primer Pool was 250nM of each primer. rhAmp index primers for the second round of PCR (PCR2) were resuspended in IDTE pH8.0 to a working concentration of 5uM. Next Generation Sequencing (NGS) samples were prepared by two rounds of PCR. Two technical replicates were analyzed per sample or negative. PCR1 was used to amplify genomic regions. Reactions then underwent a 1.5X solid-phase reversible immobilization (SPRI) to purify the product, removing unused primers and primer dimer. PCR2 was performed to add Illumina adapters and indices. Reactions then underwent a 1X SPRI to purify the product. Diluted product was then run on a 2% agarose gel for QC, checking the size of the product against the design provided by IDT. Reactions were then pooled by scaling the volume pooled to the number of amplicons in each product so that each amplicon was equally represented in the library pool. Library pools were then run on a high sensitivity D1000 Tapestation kit to obtain an average fragment length, quantified by high sensitivity DNA Qubit, and normalized to 0.8nM. The number of required reads was determined by multiplying the total number of amplicons in a library pool, the estimated cell equivalents of the input gDNA (given an estimation of 6pg gDNA per cell), and a coverage multiplier of ten.
Attorney Docket No.: 063586-522001WO Libraries were then sequenced on a 500 Cycle NovaSeq 6000 SP Reagent Kit v1.5. For NGS analysis, the indel mapping function used a sample’s fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well. All reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion. The QC standard for the minimum number of reads passing filters was 1,000. For each amplicon, indel ratios, referring to the percentage of NGS reads comprising indels, were calculated for each sample and its cognate non-targeting control (negative). Amplicons with potential off-target activity were flagged by comparing the sample to its negative and observing a difference in one or more of the following representations of the NGS data: 1. Indel ratio 2. Heatmap of the fraction of reads with a given indel length 3. Figure with the indel ratio per position on the y-axis and amplicon position on the x-axis Additionally, a background-corrected indel ratio was calculated by removing all reads from the sample which contained indels that also appeared in its negative and by removing all reads from the sample which contained indels that did not overlap with a window 20bp upstream and 20bp downstream from the putative cut site. Amplicons with background- corrected indel ratios >0.05% were flagged. For each flagged amplicon, the list of the unique reads (hashmap) from a sample was compared to its negative to determine if there was true off-target activity or it the flag originated from sequencing noise, amplification errors, primer dimerization, or mispriming. Similar to what was observed using NGS at select targeted sites (FIGS.6A-6E), rhAmpSeq
TM in PHH demonstrated higher off-target activity in samples treated with 18 nt spacer length guides compared to 16 nt and 20 nt lengths (Table 7). The gRNA having the 16- nt spacer showed higher on-target editing efficacy as compared with the gRNAs having the 18-nt or 20-nt spacer. A further reduction in off-target activity was observed at the 16 nt length compared to 20 nt (Table 7).
Attorney Docket No.: 063586-522001WO Table 7. Off-target Indel Activity Measured by rhAmpSeq for PCSK9-E1T3 Target ID 16 nt spacer 18 nt spacer 20 nt spacer On-target 73.63% 69.88% 53.68%
Humanized PCSK9 Transgenic Mice This example provides a method for evaluating efficacy of a gene editing system comprising an mRNA encoding a CRISPR nuclease (e.g., a CRISPR nuclease polypeptide comprising the nuclease of SEQ ID NO: 2 or 3, for example, the CRISPR nuclease polypeptide comprising SEQ ID NO: 228) and a PCSK9 guide RNA, targeting a genomic site within the PCSK9 gene (e.g., those provided herein) with varying spacer length and modifications. mRNAs expressing a CRISPR nuclease and gRNA targeting PCSK9 are loaded into lipid nanoparticles and administered to mice expressing human PCSK9 and lacking expression of mouse PCSK9 (Essalmani et al, Biol Chem.2018, 399(12):1363-1374). Treatment groups include two dose levels with short- and long-term endpoints as described in Table 8. Table 8. Mice Treatment Groups Group mRNA Guide Deliv Animal Dose Admin Duration- # RNA # (m /k ) R E d i
Attorney Docket No.: 063586-522001WO 3 Encoding PCSK9 LNP 5 1.5 Tail 1-2 weeks the gRNA Vein or
examined in vivo. Treatment groups are described in Table 9. Table 9. Mice Treatment Groups Group # mRNA Guide Delivery Animal Dose Admin Duration/ RNA # (mg/kg) Route Endpoint
The following exemplary guides are to be investigated: PCSK9_E1T3_20mer_mod4 (SEQ ID NO: 196) PCSK9_E1T3_16mer_mod4 (SEQ ID NO: 229) PCSK9_E1T3_20mer_mod2 (SEQ ID NO: 195)
Attorney Docket No.: 063586-522001WO PCSK9_E1T3_16mer_mod2 (SEQ ID NO: 230) PCSK9_E1T3_20mer_unmodified (SEQ ID NO: 132) PCSK9_E1T3_16mer_unmodifed (SEQ ID NO: 224) Interim readouts for PCSK9 levels, LDL-C levels, and Triglycerides will be done weekly or biweekly for the first month and then monthly thereafter. At study endpoint, terminal blood is collected for assessment of PCSK9, LDL-C, and triglycerides. Liver tissue are also collected at necropsy for the assessment of editing by NGS. These studies enable the assessment and optimization of a PCSK9 gene editing system including the assessment of dose dependency on editing levels, guide RNA modification approach, spacer length, and the physiological benefit of editing as it relates the therapeutically relevant biomarkers, and the long-term durability. Example 6 – Method for Determining Efficacy of a PCSK9 Gene Editing System with LNP-Based Delivery Approach on Humanized PCSK Transgenic Mice This example investigates the efficacy of a gene editing system comprising an mRNA encoding the CRISPR nuclease polypeptide of SEQ ID NO: 228 and a PCSK9 guide RNA, with varying spacer lengths (16-nt or 20-nt) with mod4 modifications. The mRNAs expressing the CRISPR nuclease polypeptide and the gRNA were loaded into lipid nanoparticles and administered to mice expressing human PCSK9 and lacking expression of mouse PCSK9 (Essalmani et al, Biol. Chem, 399(12):1363-1374, 2018). The lipid nanoparticles contain 46.3% cationic lipid 6-((2-hexyldecanoyl)oxy)-N-(6- ((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium, 9.4% phospholipid 1,2- Distearoyl-sn-glycerol-3-phosphocholine (DSPC), 42.7% cholesterol, and 1.6% PEG lipid 2- [(polyethylene glycol)-2000]-N,N ditetradecylacetamide, and was formulated with a Molar N/P ratio of ~6. The LNPs was prepared according to the general procedures described in (Schoenmaker, IJPharm, 601:120586, 2021), the relevant disclosures of which are incorporated by reference herein for the subject matter and purpose referenced herein. The nitrogen:phosphate (N/P) ratio is calculated from the results of HPLC (for determination of aminolipid mol) and ribogreen (for determination of mol of total RNAs), as indicated by the formula below: ^^^ ^^^^^^^^^^ (^^^^) ^^^ ^^^^^ ^^^ (^^^^^^^^^)
Attorney Docket No.: 063586-522001WO Mice of two treatment groups were dosed at the two dose levels with short- and long- term endpoints as described in Table 10. The impact of guide RNA spacer length is examined in vivo. Table 10. Mice Treatment Groups Grou mRNA Guide Deliver Animal Dose Admin Duration p # RNA y # (mg/kg) Route Endpoint ar ar ar ar
The guide RNA sequences and modifications are provided in Example 5 above. Interim readouts for PCSK9 levels were done biweekly for the first month and then monthly thereafter (FIG.7). The interim results for this experiment demonstrate that modification 4 (mod4) is able to achieve PCSK9 lowering in vivo when used with guides of a 20mer and a 16mer spacer in a dose-dependent manner. At study endpoint, terminal blood will be collected for endpoint assessment of PCSK9, LDL-C, and triglycerides. Liver tissue will be collected at necropsy for the assessment of editing by NGS. Results from these studies enable the assessment and optimization of a PCSK9 gene editing system as provided herein, including the assessment of dose dependency on editing levels, guide RNA modification approach, spacer length, and the physiological benefit of editing as it relates the therapeutically relevant biomarkers, and the long term durability.
Attorney Docket No.: 063586-522001WO OTHER EMBODIMENTS All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. EQUIVALENTS While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
Attorney Docket No.: 063586-522001WO All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or
Attorney Docket No.: 063586-522001WO unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID NO NO NO 9 0 1 2 3 4 5 6 7 8 9 0
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID NO NO NO 1 2 3 4 5 6 7 8 9 0 1 2 3
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID NO NO NO 4 5 6 7 8 9 0 1 2 3 4 5
Attorney Docket No.: 063586-522001WO Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ Sequence ID Homology Sequence ID ID NO NO NO 6 7 8
CUUGUUGUAUAUGUCCUUUUAUAGGUAUUAAACAAC (SEQ ID NO: 72). The DNA counterpart of this DR sequence is CTTGTTGTATATGTCCTTTTATAGGTATTAAACAAC (SEQ ID NO: 191). RNP complexation reactions were made by mixing purified CRISPR nuclease (SEQ ID NO: 3) (100 µM in 20 mM HEPES pH 7.5, 300 mM NaCl, 10 % Glycerol, 0.5 mM TCEP) with RNA guide (250 µM in 250 mM NaCl) at a 1:1 (CRISPR nuclease:RNA guide) volume ratio (2.5:1 RNA guide:CRISPR nuclease molar ratio). Complexations were incubated on ice for 45-60 min. RNP electroporation in HEK293T cells During RNP complexation incubation, HEK293T cells were harvested using TrypLETM (ThermoFisher Scientific) and counted. Cells were washed once with PBS and resuspended in SF buffer + supplement (Lonza #V4SC-2096). Resuspended cells were dispensed at 3e5 cells/reaction into Lonza 96-well electroporation plates. Complexed RNP was added to each reaction at a final concentration of 20 µM, and transfection enhancer oligos were then added at a final concentration of 4 µM. Non-targeting RNA guides were used as negative controls. The plates were electroporated using an electroporation device (program CM-130, Lonza 4D- nucleofector). Immediately following electroporation, pre-warmed DMEM + 10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, 30,000 cells of diluted nucleofected cells were plated into pre-warmed 96-well plate with wells containing DMEM + 10% FBS. Editing plates were incubated for 3 days at 37oC with 5% CO2.
Attorney Docket No.: 063586-522001WO After 3 days, wells were harvested using TrypLETM (ThermoFisher Scientific) and transferred to 96-well twin.tec® PCR plates (Eppendorf). Media was flicked off, and cells were resuspended in DNA extraction buffer (QuickExtractTM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at - 20°C, and subsequently analyzed for PCSK9 indels using Next Generation Sequencing (NGS), as described below. RNP electroporation in HepG2 cells During RNP complexation incubation, HepG2 cells were harvested using TrypLETM (ThermoFisher Scientific) and counted. Cells were washed once with PBS and resuspended in SF buffer + supplement (Lonza #V4SC-2096). Resuspended cells were dispensed at 2.5e5 cells/reaction into Lonza 96-well electroporation plates. Complexed RNP was added to each reaction at a final concentration of 20 µM, with no transfection enhancer oligo. Non-targeting RNA guides were used as negative controls. The plates were electroporated using an electroporation device (program DJ-100, Lonza 4D-nucleofector). Immediately following electroporation, pre-warmed EMEM (ATCC) + 10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 25,000 cells of diluted nucleofected cells were plated into pre-warmed 96-well plate with wells containing EMEM (ATCC) + 10% FBS. Editing plates were incubated for 3 days at 37oC with 5% CO2. After 3 days, media was collected, and PCSK9 protein expression in the media was measured using Meso Scale Discovery (MSD) immunoassays, as described below. In parallel, cells in the wells were harvested using TrypLETM (ThermoFisher Scientific) and transferred to 96-well twin.tec® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in DNA extraction buffer (QuickExtractTM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at -20°C, and subsequently analyzed for PCSK9 indels using Next Generation Sequencing (NGS), as described below. NGS library preparation and analysis Samples for Next Generation Sequencing (NGS) were prepared by rounds of PCR. The first round (PCR I) was used to amplify the genomic regions flanking the target site and add NGS adapters. The second round (PCR II) was used to add NGS indexes. Reactions were then pooled, purified by column purification, and quantified on a fluorometer (Qubit). Sequencing
Attorney Docket No.: 063586-522001WO runs were done using a 150 cycle NGS instrument (NextSeq v2.5) mid or high output kit and run on an NGS instrument (NextSeq 550). For NGS analysis, the indel mapping function used a sample’s fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well. Up to 50,000 reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion. The % indels was calculated as the number of indel-containing reads divided by the number of reads analyzed (reads passing filters up to 50,000). The QC standard for the minimum number of reads passing filters was 10,000. As shown in FIG.1, PCSK9 indels in HEK293T and HepG2 cells following RNP transfection with the variant CRISPR nuclease and RNA guides listed in Table 3. Each circle represents the average of three technical replicates across one biological replicate. Use of an additional 64 RNA guides targeting PCSK9 exon sequences with 5’-NTTY-3’ (Y is C or T) PAM sequences did not result in indels or resulted in indels of less than 10%. 25 RNA guides resulted in indel percentages of at least 80% in HEK293T and at least 50% in HepG2 cells. See FIG.1. These RNA guides were then selected to be tested in primary human hepatocytes. Example 2 - CRISPR Nuclease-Mediated Editing of PCSK9 Target Sequences in Primary Human Hepatocyte (PHH) Donor Cells This Example describes genomic editing of the PCSK9 gene using a variant CRISPR nuclease introduced into cells by RNP in primary human hepatocyte (PHH) donors. The RNA guides that produced greater than 80% indels in HEK293T cell and greater than 50% indels in HepG2 cells as observed in Example 1 were selected for use in this study, including E1T3, E2T6, E3T2, E3T3, E3T5, E3T10, E4T3, E5T1, E6T8, E6T10, E7T7, E7T18, E8T3, E8T8, E8T10, E9T5, E10T2, E10T8, E11T3, E11T5, E11T8, E11T10, E11T11, and E12T8. Structural information of these RNA guides can be found in Table 3 above. RNP electroporation in primary human hepatocyte (PHH) donor cells During RNP complexation incubation, PHH cells from human donors (HUM190171
Attorney Docket No.: 063586-522001WO from Lonza) were thawed from liquid nitrogen quickly in a 37oC water bath. The cells were added to pre-warmed hepatocyte recovery media (Thermofisher, CM7000) and centrifuged for 10 minutes. The cell pellet was resuspended in appropriate volume of hepatocyte plating Medium (Williams’ Medium E, Thermofisher A1217601 supplemented with Hepatocyte Plating Supplement Pack (serum-containing), Thermofisher CM3000). The cells were subjected to a trypan blue viability count using the Vi-CELL BLU cell counter. The desired number of viable cells were then washed in PBS and resuspended in P3 buffer + supplement (Lonza, V4SP-3096) and transfection enhancer oligo (final concentration of 4 µM). Resuspended cells were dispensed at 125,000 cells/reaction into Lonza 96-well electroporation plates. Complexed RNP was added to each reaction at a final concentration 5 µM, 1.25 µM, or 0.625 µM. Non-targeting RNA guides were used as negative controls. The strips were electroporated using an electroporation device (program DS-150, Lonza 4D-nucleofector).5 minutes following electroporation, pre-warmed Hepatocyte plating medium was added to each well and mixed very gently by pipetting. For each technical replicate plate, 125,000 cells of diluted nucleofected cells were plated into a pre-warmed collagen-coated 96-well plate (Thermofisher) with wells containing Hepatocyte plating medium. The cells were then incubated in a 37oC incubator. The media was changed to hepatocyte maintenance media (Williams’ Medium E, Thermofisher A1217601 supplemented with William’s E medium Cell Maintenance Cocktail, Thermofisher CM 4000) after the cells attached after 4 hours. Fresh hepatocyte maintenance media was replaced after 3 days. After 7 days post RNP electroporation, media was collected and PCSK9 protein expression in the media was measured using Meso Scale Discovery (MSD) immunoassays, as described below. In parallel, cells in the wells were harvested using Accutase and transferred to 96-well twin.tec® PCR plates (Eppendorf) and centrifuged. Media was flicked off and cells were resuspended in DNA extraction buffer (QuickExtractTM). Samples were then cycled in a PCR machine at 65°C for 15 min, 68°C for 15 min, 98°C for 10 min. Samples were then frozen at -20°C, and subsequently analyzed for PCSK9 indels using Next Generation Sequencing (NGS) as described in Example 1. FIG.2 shows PCSK9 indels in a PHH donor (HUM190171) following RNP transfection of the variant CRISPR nuclease and the selected RNA guides.19 RNA guides resulted in indels of at least 40% at the highest concentration tested. Expression of PCSK9 was then measured for the RNA guides resulting in the highest percent indels.
Attorney Docket No.: 063586-522001WO Meso Scale Discovery (MSD) immunoassays Samples for MSD immunoassays were collected from media on PHH cells electroporated with RNP, 3 days post electroporation. First, U-Plex linkages were incubated with either PCSK9 or fibronectin (for normalization) capture antibodies, then added to MSD plates for antibody coating. Calibrator curves were prepared, and samples were added to coated plates, using a 5x dilution of spent PHH media in Diluent 101. Following 1 hour shaking incubation, plates were washed, detecting antibody was added, and plates were shaken and incubated for 1 hour. Following subsequent washes, MSD Gold Read Buffer B was added to each well, and plates were read for detection of PCSK9 and fibronectin expression. FIG.3 shows PCSK9 expression (measured by MSD) relative to control expression in a PHH donor (HU2021) following RNP transfection with the highest performing RNA guides identified in FIG.2. Many of the RNA guides reduced PCSK9 expression to under 10% at the highest RNP concentration tested. E4T3, E11T3, E8T10, and E1T3 also substantially decreased PCSK9 expression at the lower RNP concentrations tested. Example 3 – CRISPR Nuclease-Mediated Editing of PCSK9 Target Sequences in Primary Human Hepatocyte (PHH) Donor Cells Using Various RNA Guide Modifications This Example describes genomic editing of the PCSK9 gene using a variant CRISPR nuclease introduced into cells by mRNA with synthetic RNA guides containing various modifications in primary human hepatocytes (PHH) donors. On-target indels were determined and compared between cells transfected with mRNA effector and RNA guides containing different modification configurations. Structural information of these RNA guides can be found in Table 4 and modification nomenclature can be found in Table 5. RNA guides were designed with various chemical modifications (modification formats shown in Table 5) and ordered from Integrated DNA Technologies (IDT). Guides contained phosphorothioate (PS) bonds and/or 2’-O-methylated bases at various positions at the 5’ and 3’ end of the guide (see Table 4 for specific sequence information of guides). Table 4. Exemplary Target and RNA Guide Sequences for PCSK9 Guide PAM Target SEQ NHP Spacer SEQ RNA Guide Sequence SEQ D O 2
y All of the guides listed in Table 4 above have a DR sequence of SEQ ID NO: 72.
Attorney Docket No.: 063586-522001WO Table 5. RNA Guide Modification Nomenclature Modification name Modification information Generic guide sequence (N = SEQ ID base) NO 2 3 4 5 6 7 8 5
Attorney Docket No.: 063586-522001WO 3 Encoding PCSK9 LNP 5 1.5 Tail 1-2 weeks the gRNA Vein or
examined in vivo. Treatment groups are described in Table 9. Table 9. Mice Treatment Groups Group # mRNA Guide Delivery Animal Dose Admin Duration/ RNA # (mg/kg) Route Endpoint