Guide nucleic acids targeting VEGFA and uses thereof

Abstract

Systems and methods for modifying VEGFA expression are provided.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of priority and filing dates of (related to) PCT / CN2023 / 087915, filed on April 12, 2023, and PCT / CN2023 / 089908, filed on April 21, 2023, the entire contents of which (including any drawings and sequence listing) are incorporated herein by reference.

[0003] References to electronic sequence listings

[0004] This disclosure contains a sequence listing XML file that has been electronically submitted in XML format, and the sequence listing is hereby incorporated by reference in its entirety. The XML copy, created on April 12, 2024 by the software "WIPO Sequence" in accordance with WIPO Standard ST.26, is named HGP024PCT.xml and is 54,485 bytes in size.

[0005] According to WIPO Standard ST.26, the symbol "t" is used to represent both T in DNA and U in RNA. Therefore, in this sequence listing prepared according to ST.26, in any case where the sequence is RNA, T in the sequence should be regarded as U. Background Art

[0006] The VEGFA gene (also known as VPF, VEGF or MVCD1) encodes vascular endothelial growth factor A (VEGFA), which plays a vital role in the physiological development of vascular structures and pathological angiogenesis. VEGFA is reportedly associated with diseases such as neovascular age-related macular degeneration (nAMD) (also known as wet AMD) and diabetic macular edema (DME). AMD is the main cause of visual dysfunction in adults over 50 years old, with nAMD accounting for 90% of AMD blindness. Choroidal neovascularization (CNV) is a hallmark of nAMD. Overexpression of VEGFA promotes CNV. WO 2022 / 188797 (PCT / CN2022 / 079890), which is incorporated herein by reference in its entirety, discloses a hfCas13X.1 (also known as hfCas13e.1) system delivered by AAV9 to knock down VEGFA mRNA. It is expected to develop tools and methods for knocking down VEGFA mRNA and / or treating VEGFA-related diseases with improved efficiency and / or safety.

[0007] Citation or identification of any document in this disclosure is not an admission that such document is available as prior art to the present disclosure. Each of the references mentioned or cited in this disclosure is incorporated by reference in its entirety. Summary of the Invention

[0008] In some aspects, the present disclosure provides tools and methods for downregulating VEGFA expression with improved efficiency and / or safety. Figure 12 Representative high-fidelity Cas13Y (hfCas13Y) endonuclease-based therapeutic systems provided in some aspects of the present disclosure are presented. Figure 12 A shows a schematic diagram of a representative therapeutic system HG202 based on the high-fidelity Cas13Y endonuclease developed by the applicant for specific and efficient knockdown of target RNA.

[0009] Figure 12 B shows the mechanism of the therapeutic system HG202, in which HG202 downregulates VEGFA expression through hfCas13Y-based VEGFA mRNA knockdown with improved efficiency and / or safety.

[0010] In one aspect, the present disclosure provides a system comprising:

[0011] (1) a guide nucleic acid comprising

[0012] a) a scaffold sequence capable of forming a complex with the Cas13 endonuclease, and

[0013] b) a guide sequence capable of hybridizing to a target sequence on a transcript of the VEGFA gene, thereby directing the complex to the transcript, or a polynucleotide encoding the guide nucleic acid; and

[0014] (2) the Cas13 endonuclease or a polynucleotide encoding the Cas13 endonuclease;

[0015] wherein the Cas13 endonuclease comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 16 or 32, comprises substitutions D160A, D642A, Y666A, and Y677A relative to a wild-type version of the Cas13 endonuclease of SEQ ID NO: 16, and has a guide sequence-specific RNA endonuclease activity substantially the same (e.g., at least about 80%, 90%, 95%, 99% or more) as the Cas13 endonuclease of SEQ ID NO: 16;

[0016] wherein the guide sequence comprises (1) the sequence of SEQ ID NO: 1 or 2, or a 5' or 3' truncation thereof which is truncated by 1, 2, 3, 4, 5 or 6 nucleotides at the 5' or 3' end; or (2) a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 1 or 2, or a 5' or 3' truncation thereof which is truncated by 1, 2, 3, 4, 5 or 6 nucleotides at the 5' or 3' end; or (3) a sequence having at most 1, 2, 3, 4, 5 or 6 nucleotides that differ from SEQ ID NO: 1 or 2, whether contiguous or not.

[0017] In some embodiments, the scaffold sequence has a secondary structure that is substantially identical to the secondary structure of the sequence of SEQ ID NO:5; or wherein the scaffold sequence comprises (1) the sequence of SEQ ID NO:5 or a 5' or 3' truncation thereof that is truncated by 1, 2, 3, 4, 5, or 6 nucleotides at the 5' or 3' end; or (2) a sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:5 or a 5' or 3' truncation thereof that is truncated by 1, 2, 3, 4, 5, or 6 nucleotides at the 5' or 3' end; or (3) a sequence that differs from SEQ ID NO:5 by at most 1, 2, 3, 4, 5, or 6 nucleotides, whether contiguous or not.

[0018] In some embodiments, the guide nucleic acid comprises, from 5' to 3', one scaffold sequence, one guide sequence, and one scaffold sequence, wherein the scaffold sequences are the same or different.

[0019] In some embodiments, the guide nucleic acid comprises, from 5' to 3', one said scaffold sequence, one said guide sequence, one said scaffold sequence, one said guide sequence, and one said scaffold sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0020] In some embodiments, the guide nucleic acid comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of any one of SEQ ID NOs: 17 and 30-31; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of any one of SEQ ID NOs: 17 and 30-31.

[0021] In some embodiments, the transcript of the VEGFA gene is VEGFA pre-mRNA or VEGFA mRNA.

[0022] In some embodiments, the VEGFA gene is a human VEGFA gene.

[0023] In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector genome comprising:

[0024] (1) a first polynucleotide sequence comprising a sequence containing or encoding a guide nucleic acid of the present disclosure; and

[0025] (2) a second polynucleotide sequence comprising a sequence encoding a Cas13 endonuclease of the present disclosure,

[0026] The rAAV vector genome is suitable for encapsulation into rAAV particles.

[0027] In some embodiments, the first polynucleotide sequence comprises a U6 promoter (e.g., SEQ ID NO: 4 or 22) operably linked to a sequence comprising or encoding the guide nucleic acid.

[0028] In some embodiments, the second polynucleotide sequence comprises a Cbh promoter (e.g., SEQ ID NO: 6) operably linked to a sequence encoding the Cas13 endonuclease.

[0029] In some embodiments, the second polynucleotide comprises a first sequence encoding a first NLS at the 5' end of the sequence encoding the Cas13 endonuclease and / or a second sequence encoding a second NLS at the 3' end of the sequence encoding the Cas13 endonuclease.

[0030] In some embodiments, the first NLS and / or the second NLS is an SV40 NLS (eg, SEQ ID NO: 26), a bpSV40 NLS (eg, SEQ ID NO: 15), or a nucleoplasmin NLS (npNLS).

[0031] In some embodiments, the second polynucleotide sequence comprises a WPRE sequence downstream of a sequence encoding the Cas13 endonuclease; optionally wherein the WPRE sequence is selected from the group consisting of a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), WPRE3 (e.g., SEQ ID NO: 11), and functional variants thereof (e.g., functional truncations).

[0032] In some embodiments, the second polynucleotide sequence comprises a sequence encoding a bGH polyA signal downstream of a sequence encoding the Cas13 endonuclease (e.g., SEQ ID NO: 12).

[0033] In some embodiments, the second polynucleotide sequence comprises, from 5' to 3', a promoter of SEQ ID NO: 6, a Kozak sequence of gccacc, an in-frame start codon ATG, a first sequence encoding a first NLS of SEQ ID NO: 15, a sequence encoding a Cas13 endonuclease of SEQ ID NO: 16 or 32, a second sequence encoding a second NLS of SEQ ID NO: 15, an in-frame stop codon, a WPRE sequence of SEQ ID NO: 11, and a sequence encoding a polyA signal of SEQ ID NO: 12.

[0034] In some embodiments, the rAAV vector genome comprises the first polynucleotide sequence and the second polynucleotide sequence from 5' to 3'; or comprises the second polynucleotide sequence and the first polynucleotide sequence from 5' to 3'.

[0035] In some embodiments, the rAAV vector genome comprises, from 5' to 3':

[0036] (1) 5' ITR of SEQ ID NO: 3;

[0037] (2) the promoter of SEQ ID NO: 4;

[0038] (3) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0039] (4) a sequence encoding the guide sequence of SEQ ID NO: 1;

[0040] (5) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0041] (6) a sequence encoding the guide sequence of SEQ ID NO: 2;

[0042] (7) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0043] (8) the promoter of SEQ ID NO: 6;

[0044] (9) Kozak sequence of gccacc;

[0045] (10) in-frame start codon ATG;

[0046] (11) a first sequence of SEQ ID NO: 8 encoding the first NLS of SEQ ID NO: 15;

[0047] (12) a sequence of SEQ ID NO: 33 encoding the Cas13 endonuclease of SEQ ID NO: 32;

[0048] (13) a second sequence SEQ ID NO: 10 encoding a second NLS of SEQ ID NO: 15;

[0049] (14) in-frame stop codon;

[0050] (15) WPRE sequence of SEQ ID NO: 11;

[0051] (16) a sequence encoding a polyA signal of SEQ ID NO: 12; and

[0052] (17) 3' ITR of SEQ ID NO: 13;

[0053] The rAAV vector genome comprises an optional linker between any two adjacent components among components (1) to (17).

[0054] In some embodiments, the rAAV vector genome comprises, consists essentially of, or consists of a polynucleotide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to the sequence of SEQ ID NO: 14.

[0055] In some embodiments, the rAAV vector genome comprises, from 5' to 3':

[0056] (1) 5' ITR of SEQ ID NO: 3;

[0057] (2) the promoter of SEQ ID NO: 4;

[0058] (3) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0059] (4) a sequence encoding the guide sequence of SEQ ID NO: 1;

[0060] (5) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0061] (6) a sequence encoding the guide sequence of SEQ ID NO: 2;

[0062] (7) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0063] (8) the promoter of SEQ ID NO: 6;

[0064] (9) Kozak sequence of gccacc;

[0065] (10) in-frame start codon ATG;

[0066] (11) a first sequence of SEQ ID NO: 8 encoding the first NLS of SEQ ID NO: 15;

[0067] (12) a sequence of SEQ ID NO: 33 encoding the Cas13 endonuclease of SEQ ID NO: 32;

[0068] (13) a second sequence SEQ ID NO: 10 encoding a second NLS of SEQ ID NO: 15;

[0069] (14) in-frame stop codon;

[0070] (15) WPRE sequence of SEQ ID NO: 11;

[0071] (16) a sequence encoding a polyA signal of SEQ ID NO: 12; and

[0072] (17) 3' ITR of SEQ ID NO: 13;

[0073] wherein the rAAV vector genome comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to the sequence of SEQ ID NO: 14;

[0074] wherein the rAAV vector genome does not comprise any nucleotide difference in any of the components (1) to (17) compared to the sequence of SEQ ID NO: 14;

[0075] wherein the rAAV vector genome comprises one or more nucleotide differences compared to the sequence of SEQ ID NO: 14 or does not comprise any nucleotide differences compared to the sequence of SEQ ID NO: 14 between any two adjacent components among the components (1) to (17).

[0076] In some embodiments, the rAAV vector genome comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 14.

[0077] In yet another aspect, the present disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the present disclosure.

[0078] In some embodiments, the rAAV particle comprises a capsid of serotype AAV8.

[0079] In some embodiments, the AAV8 is wild-type AAV8 or a functional variant thereof.

[0080] In yet another aspect, the present disclosure provides a method for producing rAAV particles, comprising culturing a transgenic plasmid comprising the rAAV vector genome of the present disclosure in a host cell, thereby encapsulating the rAAV vector genome into a capsid of serotype AAV8.

[0081] In yet another aspect, the present disclosure provides a cell comprising a transgenic plasmid comprising the rAAV vector genome of the present disclosure.

[0082] In yet another aspect, the present disclosure provides use of a cell comprising a transgenic plasmid comprising the rAAV vector genome of the present disclosure for producing rAAV particles.

[0083] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising (1) a system of the present disclosure or a rAAV particle of the present disclosure and (2) a pharmaceutically acceptable excipient.

[0084] In yet another aspect, the present disclosure provides a method of downregulating the expression of a VEGFA gene, comprising contacting a transcript of the VEGFA gene with a system of the present disclosure, an rAAV particle of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0085] In yet another aspect, the present disclosure provides a cell or progeny thereof comprising the system of the present disclosure or the rAAV particle of the present disclosure.

[0086] In yet another aspect, the disclosure provides a cell or progeny thereof comprising a VEGFA transcript modified by a system of the disclosure, an rAAV particle of the disclosure, or a method of the disclosure.

[0087] In another aspect, the present disclosure provides a method for preventing, diagnosing and / or treating a VEGFA-related disease in a subject in need thereof, comprising administering to the subject a system of the present disclosure, an rAAV particle of the present disclosure or a pharmaceutical composition of the present disclosure, wherein the Cas13 nuclease modifies the transcript of the VEGFA gene, thereby resulting in downregulation of expression of the VEGFA gene, thereby treating the VEGFA-related disease.

[0088] In some embodiments, the VEGFA-related disease is neovascular age-related macular degeneration (nAMD), diabetic macular edema (DME), or cancer.

[0089] In some embodiments, the subject is unresponsive to anti-VEGF therapy, such as an anti-VEGF antibody.

[0090] The details of one or more embodiments of the present disclosure are set forth in the following description. Other features or advantages of the present disclosure should be clear from the following drawings and detailed description of several embodiments and from the appended claims. It should be understood that, unless otherwise indicated, any aspect or embodiment of the present disclosure may be combined with any other aspect or embodiments of the present disclosure (including aspects or embodiments described only in a subsection, only in the examples, or only in the claims) to constitute another embodiment disclosed herein, explicitly or implicitly. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] An understanding of certain features and advantages of the present disclosure will be gained by referring to the following detailed description, which sets forth illustrative embodiments in which the principles of the present disclosure may be utilized, and to the accompanying drawings, in which:

[0092] Figure 1 is a schematic diagram showing an exemplary target dsDNA, an exemplary guide RNA, and an exemplary napDNA bp.

[0093] Figure 2 is another schematic diagram showing an exemplary target dsDNA, an exemplary guide RNA, and an exemplary napDNAbp.

[0094] Figure 3 is a schematic diagram showing an exemplary target dsDNA, an exemplary transcript transcribed from the target dsDNA (target RNA), an exemplary guide RNA, and an exemplary napRNAbp.

[0095] Figure 4is another schematic diagram showing an exemplary target dsDNA, an exemplary transcript transcribed from the target dsDNA (target RNA), an exemplary guide RNA, and an exemplary napRNAbp, wherein the guide sequence contains a mismatch with the target sequence.

[0096] Figure 5 Shown is a comparison of the in vivo VEGFA knockdown efficiency of HG202 and HG203.

[0097] Figure 6 Shown are the results of in vitro gRNA screening targeting VEGFA.

[0098] Figure 7 In vitro expression of hfCas13Y and gRNA (SEQ ID NO: 17) and VEGFA knockdown are shown.

[0099] Figure 8 In vitro off-target analysis of hfCas13Y and gRNA (SEQ ID NO: 17) is shown.

[0100] Figure 9 In vivo VEGFA knockdown of HG202 is shown.

[0101] Figure 10 HG202 is shown to reduce CNV in vivo.

[0102] Figure 11 This shows the safety of HG202.

[0103] Figure 12 Representative high-fidelity Cas13Y (hfCas13Y) endonuclease-based therapeutic systems are shown.

[0104] Figure 13 Schematic diagram of animal modeling and treatment is shown.

[0105] The drawings herein are for illustration purposes only and are not necessarily drawn to scale.

[0106] definition

[0107] The present disclosure will be described with respect to specific embodiments, but the present disclosure is not limited thereto in any respect. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Unless otherwise indicated, the terms set forth below are generally understood in their simple and ordinary meanings or common sense.

[0108] Overview

[0109] Nucleic acid programmable binding protein (napBP), e.g., nucleic acid programmable DNA binding protein (napDNAbp), such as Cas9, Cas12, IscB, nucleic acid programmable RNA binding protein (napRNAbp), such as Cas13, can bind to a target nucleic acid (e.g., dsDNA, mRNA) under the guidance of a guide nucleic acid (e.g., guide RNA) comprising a guide sequence targeting the target nucleic acid. In some embodiments, the target nucleic acid is a eukaryotic nucleic acid.

[0110] Without wishing to be bound by theory, in some embodiments, a guide nucleic acid comprises a scaffold sequence responsible for forming a complex with the napBP, and a guide sequence intentionally designed to hybridize to a target sequence of a target nucleic acid, thereby directing the complex comprising the napBP and the guide nucleic acid to the target nucleic acid.

[0111] refer to Figure 1 , an exemplary target dsDNA (eg, VEGFA gene) is depicted as comprising a 5' to 3' single DNA strand and a 3' to 5' single DNA strand.

[0112] Exemplary guide nucleic acids (e.g., guide RNAs) are depicted as comprising a guide sequence and a scaffold sequence. The guide sequence is designed to hybridize with a portion of a 3' to 5' single DNA strand, and the guide sequence is therefore "targeted" to this portion. Thus, the 3' to 5' single DNA strand is referred to as the "target strand (TS)" of the target dsDNA, while the opposite 5' to 3' single DNA strand is referred to as the "non-target strand (NTS)" of the target dsDNA. This portion of the target strand based on which the guide sequence is designed and to which the guide sequence can hybridize is referred to as the "target sequence," and the opposite portion corresponding to this portion on the non-target strand is referred to as a "protospacer sequence," which is 100% (completely) reverse complementary to the target sequence and is referred to in the present disclosure as "corresponding" to the target sequence.

[0113] refer to Figure 3 , an exemplary target dsDNA (e.g., VEGFA gene) is depicted as comprising a 5' to 3' single DNA strand and a 3' to 5' single DNA strand. According to conventional transcription processes, an exemplary target RNA (transcript, e.g., pre-mRNA) can be transcribed using the 3' to 5' single DNA strand as a synthesis template, so the 3' to 5' single DNA strand is referred to as the "template strand" or "antisense strand." The transcript so transcribed has the same primary sequence as the 5' to 3' single DNA strand (except that T is replaced by U), so the 5' to 3' single DNA strand is referred to as the "coding strand" or "sense strand."

[0114] Exemplary guide nucleic acids (e.g., guide RNAs) are depicted as comprising a guide sequence and a scaffold sequence. The guide sequence is designed to hybridize with a portion of the transcript (target RNA), and the guide sequence is therefore "targeted" to that portion. Thus, the portion of the target RNA upon which the guide sequence is designed and to which the guide sequence can hybridize is referred to as the "target sequence." In some embodiments, the guide sequence is 100% (perfectly) reverse complementary to the target sequence. In some other embodiments, the guide sequence is reverse complementary to the target sequence and contains mismatches (e.g., Figure 4 exemplified in ).

[0115] Typically, nucleic acid sequences (eg, DNA sequences, RNA sequences) are written in a 5' to 3' direction / orientation, unless specifically indicated otherwise, as is conventional in the art.

[0116] For example, the DNA sequence of ATGC is generally understood to be 5'-ATGC-3' unless otherwise indicated. The reverse sequence is 5'-CGTA-3'. The perfect complementary sequence is 5'-TACG-3'. The perfect reverse complementary sequence is 5'-GCAT-3'. Note that a perfect complementary sequence generally does not have the ability to base pair / hybridize with the original sequence.

[0117] Generally, unless otherwise indicated, the double-stranded sequence of a dsDNA can be represented by the sequence of its 5' to 3' single DNA strand, conventionally written in a 5' to 3' direction / orientation.

[0118] For example, for dsDNA having a 5' to 3' single DNA strand of 5'-ATGC-3' and a 3' to 5' single DNA strand of 3'-TACG-5', the dsDNA can be simply expressed as 5'-ATGC-3'.

[0119] 5'-----ATGC-----3'

[0120] 3'-----TACG-----5'

[0121] It should be noted that the 5' to 3' single DNA strand or the 3' to 5' single DNA strand of the dsDNA can be a non-target strand from which the protospacer sequence is selected.

[0122] Typically, for a gene that is dsDNA, the 5' to 3' single DNA strand is the sense strand of the gene, and the 3' to 5' single DNA strand is the antisense strand of the gene. Note that the sense strand or antisense strand of a gene may be a non-target strand from which the protospacer sequence is selected.

[0123] Normally, the transcript (target RNA) transcribed from dsDNA then has a (target) sequence of 5'-AUGC-3'.

[0124] To hybridize to the target dsDNA, in one embodiment, the guide sequence of the guide nucleic acid is designed to have a sequence of 5'-AUGC-3' that is completely reverse complementary to the 3' to 5' strand of the target dsRNA, which sequence will be listed as ATGC in the electronic sequence listing, but labeled as an RNA sequence; and in another embodiment, the guide sequence of the guide nucleic acid is designed to have a sequence of 5'-GCAU-3' that is completely reverse complementary to the 5' to 3' strand of the target dsRNA, which sequence will be listed as GCAT in the electronic sequence listing, but labeled as an RNA sequence.

[0125] In the case where the guide sequence of a guide nucleic acid is completely reverse complementary to the target sequence and the target sequence is completely reverse complementary to the protospacer sequence, the guide sequence is identical to the protospacer sequence except for the U in the guide sequence (due to its RNA nature) and the T in the corresponding protospacer sequence (due to its DNA nature). According to WIPO standard ST.26, the symbol "t" is used to represent both T in DNA and U in RNA (see "Table 1: List of nucleotide symbols", the definition of symbol "t" is "thymine in DNA / uracil in RNA (t / u)"). Therefore, in the electronic sequence listing of the present disclosure prepared according to ST.26, such a guide sequence can be listed in the same order as the corresponding protospacer sequence. For convenience, a single SEQ ID NO in the electronic sequence listing can be used to represent both such a guide sequence and the protospacer sequence, regardless of whether such a single SEQ ID NO is labeled as DNA or RNA in the electronic sequence listing. When reference is made to such a SEQ ID NO showing a protospacer sequence / guide sequence, it refers to the protospacer sequence as a DNA sequence or the guide sequence as an RNA sequence depending on the context, regardless of whether it is labeled as DNA or RNA in the electronic sequence listing.

[0126] In order to hybridize to the target RNA, in one embodiment, the guide sequence of the guide nucleic acid is designed to have a 5'-GCAU-3' sequence that is completely reverse complementary to the (target) sequence of the target RNA, which sequence will be listed in GCAT in the electronic sequence listing but labeled as an RNA sequence.

[0127] the term

[0128] As used herein, if a DNA sequence, e.g., 5'-ATGC-3', is transcribed into an RNA sequence (e.g., 5'-AUGC-3') in which each dT (deoxythymidine, or simply "T") in the primary sequence is replaced with U (uridine), and the other dA (deoxyadenosine, or simply "A"), dG (deoxyguanosine, or simply "G"), and dC (deoxycytidine, or simply "C") are replaced with A (adenosine), G (guanosine), and C (cytidine), respectively, it is said in the present disclosure that the DNA sequence "encodes" the RNA sequence.

[0129] As used herein, the term "activity" refers to biological activity. In some embodiments, the activity includes enzymatic activity, such as the catalytic ability of the effector. For example, the activity can include nuclease activity, such as dsDNA endonuclease activity, RNA endonuclease activity.

[0130] As used herein, the term "nucleic acid programmable binding protein (napBP)" can be used interchangeably with "nucleic acid programmable binding domain (napBD)" to refer to a protein that can associate with (e.g., bind to) a programmable nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid (e.g., gRNA), which is capable of guiding a protein to a specific sequence of a target nucleic acid via an interaction (e.g., hybridization) between a programmable nucleic acid (e.g., a guide sequence of a programmable nucleic acid) and a target nucleic acid (e.g., a target sequence of a target nucleic acid). The napBP can be indirectly associated with (e.g., bind to) a target nucleic acid via an interaction (e.g., binding) between the napBP and the programmable nucleic acid (e.g., a scaffold sequence of a programmable nucleic acid) and an interaction (e.g., hybridization) between the programmable nucleic acid (e.g., a guide sequence of a programmable nucleic acid) and the target nucleic acid (e.g., a target sequence of a target nucleic acid). In some embodiments, the napBP is a nucleic acid programmable DNA binding protein (napDNAbp). In some embodiments, the napBP is a nucleic acid programmable RNA binding protein (napRNAbp).

[0131] As used herein, the term "complex" refers to a grouping of two or more molecules. In some embodiments, the complex comprises a polypeptide and a nucleic acid that interact (e.g., bind, contact, adhere) with each other. As used herein, the term "complex" may refer to a grouping of a guide nucleic acid and a polypeptide (e.g., napBP). As used herein, the term "complex" may refer to a grouping of a guide nucleic acid, a polypeptide (e.g., napBP), and a target nucleic acid.

[0132] As used herein, the term "protospacer adjacent motif" or "PAM" refers to a short DNA sequence (or DNA motif) adjacent to the protospacer sequence on the non-target strand of dsDNA. As used herein, the term "adjacent" includes situations where there are no nucleotides between the protospacer sequence and the PAM, and situations where there are a small amount (e.g., 1, 2, 3, 4, or 5) of nucleotides between the protospacer sequence and the PAM. As used herein, A is "immediately adjacent" to B (immediately adjacent to B), A is "immediately adjacent" to the 5' of B, and A is "immediately adjacent" to the 3' of B, meaning that there are no nucleotides between A and B. In some embodiments, the PAM is adjacent to the 5' of the protospacer sequence. In some embodiments, the PAM is adjacent to the 3' of the protospacer sequence.

[0133] As used herein, the term "guide nucleic acid" refers to any nucleic acid that helps target napBP to a target nucleic acid. For this purpose, the guide nucleic acid can be designed to include a guide sequence that can hybridize to a specific sequence of the target nucleic acid, and the guide nucleic acid can also include a scaffold sequence that helps guide the napBP to the target nucleic acid. In some embodiments, the guide nucleic acid is a guide RNA. In some embodiments, the guide nucleic acid is a nucleic acid that encodes a guide RNA.

[0134] As used herein, the terms "nucleic acid," "polynucleotide," and "nucleotide sequence" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs or modifications thereof.

[0135] As used in the context of CRISPR-Cas technology (e.g., CRISPR-Cas12 technology), the term "guide RNA" is used interchangeably with the term "CRISPR RNA (crRNA)", "single guide RNA (sgRNA)" or "RNA guide", the term "guide sequence" is used interchangeably with the term "spacer sequence", and the term "scaffold sequence" is used interchangeably with the term "direct repeat sequence".

[0136] As described herein, guide sequences are designed to be able to hybridize with target sequences. As used herein, the term "hybridize, hybridizing or hybridization" refers to a reaction in which one or more polynucleotide sequences react to form a complex that is stabilized via hydrogen bonds between the bases of the polynucleotide sequences. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. A polynucleotide sequence that can hybridize with a given polynucleotide sequence is referred to as the "complementary sequence" of a given polynucleotide sequence. As used herein, the hybridization of a guide sequence and a target sequence is so stable as to allow an effector polypeptide (e.g., napBP) complexed with a nucleic acid comprising a guide sequence or a functional domain associated with (e.g., fused to) the effector polypeptide to act (e.g., cleave, deaminize) on the target sequence or its complementary sequence or a nearby sequence.

[0137] For the purpose of hybridization, in some embodiments, the guide sequence is reverse complementary to the target sequence. As used herein, the term "reverse complement" refers to the ability of the nucleobases of a first polynucleotide sequence (such as a guide sequence) to base pair with the nucleobases of a second polynucleotide sequence (such as a target sequence) by traditional Watson-Crick base pairing. Two reverse complementary polynucleotide sequences are able to non-covalently bind under appropriate temperature and solution ionic strength conditions. In some embodiments, the first polynucleotide sequence (e.g., a guide sequence) has 100% (complete) reverse complementarity with the second nucleic acid (e.g., a target sequence). In some embodiments, a first polynucleotide sequence is complementary to a second nucleic acid if the first polynucleotide sequence (e.g., a guide sequence) is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to a second polynucleotide sequence (e.g., a target sequence) (i.e., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides of the first polynucleotide sequence can base pair with nucleotides of the second polynucleotide sequence). As used herein, the term "substantially complementary" means that a first polynucleotide sequence (e.g., a guide sequence) has a certain level of complementarity with a second polynucleotide sequence (e.g., a target sequence) (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides of the first polynucleotide sequence can be base paired with nucleotides of the second polynucleotide sequence, or a maximum of 1, 2, 3, 4, or 5 consecutive or non-contiguous nucleotides of the first polynucleotide sequence are mismatched with nucleotides of the second polynucleotide sequence). In some embodiments, the level of complementarity is such that a first polynucleotide sequence (e.g., a guide sequence) is capable of hybridizing with sufficient affinity to a second polynucleotide sequence (e.g., a target sequence) to allow an effector polypeptide (e.g., a napBP) complexed with a nucleic acid comprising the first polynucleotide sequence, or a functional domain associated with (e.g., fused to) the effector polypeptide, to act (e.g., cleave, deaminize) the target sequence, its complement, or a nearby sequence. In some embodiments, a guide sequence that is substantially complementary to a target sequence has less than 100% complementarity with the target sequence.In some embodiments, a guide sequence that is substantially complementary to a target sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence and / or has a maximum of 1, 2, 3, 4, or 5 contiguous or non-contiguous nucleotide mismatches compared to the target sequence.

[0138] As used herein, the term "sequence identity" is related to sequence homology. Homology comparisons can be performed by eye, or more generally by means of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage sequence identity (%) between two or more sequences (polypeptide or polynucleotide sequences). Sequence homology can be generated by any of a variety of computer programs known in the art (such as BLAST, FASTA). The appropriate computer program for performing such comparisons is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research [nucleic acid research] 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, BLAST packages (see Ausubel et al., 1999 ibid-Chapter 18), FASTA (Atschul et al., 1990, J.Mol.Biol. [Molecular Biology Journal], 403-410) and GENEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searching (see Ausubel et al., 1999 supra, pp. 7-58 to pp. 7-60). Common online tools for calculating the percent sequence identity between two or more sequences (polypeptide or polynucleotide sequences) are available on the website of the European Bioinformatics Institute at EMBL (www dot ebi dot ac dot uk slashjdispatcher slash), which allow for rapid online calculation of percent sequence identity by global or local alignment.

[0139] As used herein, the terms "polypeptide" and "peptide" are used interchangeably herein and refer to polymers of amino acids of any length. A protein may have one or more polypeptides. Amino acid polymers may also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0140] As used herein, "variant" is interpreted to mean a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide, respectively, but retains essential properties (e.g., the binding properties of napBP). A typical variant of a polynucleotide differs from another reference polynucleotide in nucleic acid sequence. Changes in the nucleic acid sequence of a polynucleotide variant may or may not change the amino acid sequence of a polypeptide encoded by a reference polynucleotide. Changes in the nucleic acid sequence of a polynucleotide variant may result in amino acid substitutions, additions, and / or deletions in a polypeptide encoded by a reference polynucleotide. A typical variant of a polypeptide differs from another reference polypeptide in amino acid sequence. Typically, the differences are limited, so that the sequences of the reference polypeptide and the polypeptide variant are generally very similar and identical in many regions. A polypeptide variant and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and / or deletions in any combination. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant known not to be naturally occurring. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0141] As used herein, the terms "upstream" and "downstream" refer to the relative positions of two or more elements in a nucleic acid in the 5' to 3' direction. When the 3' end of the first sequence is present to the left of the 5' end of the second sequence, the first sequence is upstream of the second sequence. When the 5' end of the first sequence is present to the right of the 3' end of the second sequence, the first sequence is downstream of the second sequence. In some embodiments, the PAM is upstream of the indel induced by napBP, and the indel induced by napBP is downstream of the PAM. In some embodiments, the PAM is downstream of the indel induced by napBP, and the indel induced by napBP is upstream of the PAM.

[0142] As used herein, the term "wild type" has the meaning commonly understood by those skilled in the art, i.e., refers to the typical form of an organism, strain, gene, or trait as it occurs in nature, distinguishing it from mutants or variants. It can be isolated from a source in nature without being intentionally modified.

[0143] As used herein, the terms "non-naturally occurring" and "engineered" are used interchangeably and refer to the presence of human effort. When these terms are used to describe a nucleic acid or polypeptide, it means that the nucleic acid or polypeptide is at least substantially free from at least one other component with which it is found in nature or with which it is associated in nature.

[0144] As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of nucleotide sequences in many types of host cells, as well as those that direct expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Regulatory elements can also direct expression in a time-dependent manner, for example, in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue- or cell-type specific.

[0145] As used herein, the term "cell" is understood to refer not only to a particular individual cell, but also to the progeny or potential progeny of that cell. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term.

[0146] As used herein, the term "in vivo" refers to the body of an organism, while the term "ex vivo" or "in vitro" refers to outside of an organism.

[0147] As used herein, the term "treatment" refers to a method for obtaining beneficial or desired results (including clinical results). For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, weakening the degree of the disease, stabilizing the disease (e.g., delaying the worsening of the disease), delaying the spread of the disease (e.g., metastasis), delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing down the progress of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dosage of one or more other drugs required for treating the disease, delaying disease progression, increasing quality of life and / or prolonging survival." treatment" also encompasses the pathological results that reduce diseases (such as cancer). Any one or more of these therapeutic aspects of the method of this disclosure are contemplated for treatment.

[0148] As used herein, the term "disease" includes the terms "disorder" and "condition" and is not limited to medically well-defined terms.

[0149] As used herein, reference to a value or parameter that is "not" generally means and describes a value or parameter that is "different from." For example, a method is not for treating cancer type X, which means that the method can be used to treat cancers other than type X.

[0150] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0151] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" in phrases such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0152] As used herein, when the term "about" precedes a series of numbers (e.g., about 1, 2, 3), it is understood that each of the numbers in the series is modified by the term "about" (i.e., about 1, about 2, about 3). As used herein, the term "about XY" or "about X to Y" has the same meaning as "about X to about Y."

[0153] It should be understood that the embodiments of the present disclosure described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0154] It is also noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as an antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. DETAILED DESCRIPTION

[0155] Overview

[0156] The VEGFA gene (also known as VPF, VEGF or MVCD1) encodes vascular endothelial growth factor A (VEGFA), which plays a vital role in the physiological development of vascular structures and pathological angiogenesis. VEGFA is reportedly associated with diseases such as neovascular age-related macular degeneration (nAMD) (also known as wet AMD) and diabetic macular edema (DME). AMD is the main cause of visual dysfunction in adults over 50 years old, with nAMD accounting for 90% of AMD blindness. Choroidal neovascularization (CNV) is a hallmark of nAMD. Overexpression of VEGFA promotes CNV. WO 2022 / 188797 (PCT / CN2022 / 079890), which is incorporated herein by reference in its entirety, discloses a hfCas13X.1 (also known as hfCas13e.1) system delivered by AAV9 to knock down VEGFA mRNA. It is expected to develop tools and methods for knocking down VEGFA mRNA and / or treating VEGFA-related diseases with improved efficiency and / or safety.

[0157] In some aspects, the present disclosure provides tools and methods for treating VEGFA-related diseases, such as nAMD and DME.

[0158] As used herein, the term "VEGFA-related disease" includes diseases associated with the VEGFA gene, its transcription (including transcripts; for example, increased or decreased transcription, non-functional or dysfunctional transcripts) and / or its expression (including expression and expression products; for example, increased or decreased expression, non-functional or dysfunctional expression products), including diseases directly or indirectly caused by the transcription of the VEGFA gene (including transcription and transcripts, for example, VEGFA mRNA), and diseases directly or indirectly caused by the expression of the VEGFA gene (including expression products, for example, vascular endothelial growth factor A). In some embodiments, the term "VEGFA-related disease" refers to a disease caused by abnormal expression (e.g., overexpression or underexpression) of the VEGFA gene. In some embodiments, the term "VEGFA-related disease" refers to a disease caused by a lack of a functional expression product (e.g., vascular endothelial growth factor A) of the VEGFA gene.

[0159] As used herein, the term "transcript" includes any transcription product transcribed from a gene, including mRNA, non-coding RNA, and any variants, derivatives, or ancestors thereof, such as pre-mRNA, as well as any transcript or isoform produced from a gene or pre-mRNA by, for example, alternative promoter usage, alternative splicing, alternative initiation, and any naturally occurring variants or processed products thereof. The transcript of the VEGFA gene is also referred to in this disclosure as a VEGFA transcript.

[0160] In some embodiments, the VEGFA gene is a human VEGFA gene. The wild-type human VEGFA gene has NCBI Gene ID: 7422. In some embodiments, the transcript is human VEGFA mRNA. Human VEGFA mRNA has NCBI Accession Number: NM_001025366. In some embodiments, the expression product is human vascular endothelial growth factor A. Human vascular endothelial growth factor A has NCBI Accession Number: NP_001020537.

[0161] In some embodiments, the present disclosure provides tools for reducing / downregulating the expression of the VEGFA gene by cutting (e.g., relying on the function of a Cas13 endonuclease) VEGFA transcripts (e.g., VEGFA mRNA). These tools can be delivered to a subject in need, for example, via an rAAV vector. In some embodiments, these tools include a guide nucleic acid comprising a guide sequence that is designed to interact with a VEGFA gene.

[0162] VEGFA mRNA hybridization, thereby guiding the complex comprising the guide nucleic acid and napRNAn to the VEGFA mRNA, and enabling napRNAn to modify (specifically cut) VEGFA mRNA, thereby leading to the degradation of VEGFA mRNA. When VEGFA expression is appropriately downregulated, it is reasonable to expect that syndromes caused by abnormal VEGFA expression (e.g., syndromes of nAMD) can be alleviated.

[0163] Guide nucleic acid

[0164] In one aspect, the present disclosure provides a guide nucleic acid comprising a guide sequence capable of hybridizing to a target sequence on a target strand of a VEGFA gene or a target sequence on a transcript of a VEGFA gene.

[0165] In some embodiments, the guide nucleic acid comprises a scaffold sequence capable of forming a complex with a nucleic acid programmable binding protein (napBP), and wherein hybridization of the guide sequence to the target sequence directs the complex to the VEGFA gene or transcript.

[0166] Components of guide nucleic acids are described in more detail in other subsections herein.

[0167] system

[0168] In another aspect, the present disclosure provides a system comprising:

[0169] (1) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid (e.g., DNA, RNA, or a DNA / RNA mixture), the guide nucleic acid comprising:

[0170] (a) a scaffold sequence capable of forming a complex with a nucleic acid programmable binding protein (napBP), and

[0171] (b) a guide sequence capable of hybridizing to a target sequence on the target strand of the VEGFA gene or a target sequence on a transcript of the VEGFA gene, thereby directing the complex to the VEGFA gene or transcript; and

[0172] (2) napBP or a polynucleotide encoding napBP.

[0173] In some embodiments, the system is a complex comprising napBP complexed with a guide nucleic acid. In some embodiments, the complex further comprises a VEGFA gene or transcript thereof hybridized to the guide sequence.

[0174] In some embodiments, the system is a composition comprising component (1) and component (2).

[0175] The components of the system are described in more detail in other sections of this document.

[0176] rAAV vector genome

[0177] The present disclosure provides various delivery of the system of the present disclosure, such as delivery via rAAV vectors. In some embodiments, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector genome encoding or comprising the system of the present disclosure. In some embodiments, the rAAV vector genome is DNA (e.g., ssDNA, dsDNA) or RNA.

[0178] Thus, in yet another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector genome (e.g., a DNA rAAV vector genome, an RNA rAAV vector genome) comprising:

[0179] (1) a first polynucleotide sequence comprising a sequence encoding a guide nucleic acid comprising:

[0180] (a) a scaffold sequence capable of forming a complex with a nucleic acid programmable binding protein (napBP), and

[0181] (b) a guide sequence capable of hybridizing to a target sequence on the target strand of the VEGFA gene or a target sequence on a transcript of the VEGFA gene, thereby directing the complex to the VEGFA gene or transcript; and

[0182] (2) a second polynucleotide sequence comprising a sequence encoding napBP,

[0183] The rAAV vector genome is suitable for encapsulation into rAAV particles (eg, DNA-encapsulated rAAV particles, RNA-encapsulated rAAV particles).

[0184] The components of the rAAV vector genome are described in more detail in other sections herein.

[0185] Modification method

[0186] In yet another aspect, the present disclosure provides a method of modifying (e.g., downregulating) expression of a VEGFA gene, comprising contacting the VEGFA gene or its transcript with a system comprising:

[0187] (1) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid (e.g., DNA, RNA, or a DNA / RNA mixture), the guide nucleic acid comprising:

[0188] (a) a scaffold sequence capable of forming a complex with a nucleic acid programmable binding protein (napBP), and

[0189] (b) a guide sequence capable of hybridizing to a target sequence on the target strand of the VEGFA gene or a target sequence on the transcript of the VEGFA gene, thereby directing the complex to the VEGFA gene or transcript and modifying the expression of the VEGFA gene; and

[0190] (2) napBP or a polynucleotide encoding napBP.

[0191] In some embodiments, modification of expression of the VEGFA gene treats, detects, or diagnoses a VEGFA-related disease. In some embodiments, modification of expression of the VEGFA gene detects or diagnoses the presence, progression, or development of a VEGFA-related disease.

[0192] The components of the method are described in more detail in other subsections of this article.

[0193] mechanism

[0194] The present disclosure provides various mechanisms for modifying the expression of the VEGFA gene for various purposes, including but not limited to treating or diagnosing VEGFA-related diseases. The modification can be at the gene level, transcription level, transcript level, or translation level. In some embodiments, the modification results in downregulating the expression of the VEGFA gene to treat

[0195] VEGFA-related diseases.

[0196] As used herein, the phrase "modifying the expression of the VEGFA gene" includes, but is not limited to, modifying any one or more stages of the transcription and translation process initiated from the VEGFA gene to the expression product of the VEGFA gene, e.g., vascular endothelial growth factor A. Non-limiting examples of modifications include:

[0197] (1) modifying the VEGFA gene, such as cleaving the VEGFA gene (and thereby introducing an insertion / deletion mutation into the VEGFA gene), changing (e.g., adding, deleting, or substituting) any one or more nucleotides of the VEGFA gene by, for example, base editing or prime editing;

[0198] (2) modifying the transcription of the VEGFA gene, such as increasing or decreasing transcription by, for example, methylating or demethylating the VEGFA gene via epigenetic modification;

[0199] (3) modifying a transcript of a VEGFA gene, such as VEGFA mRNA, e.g., cleaving (and typically degrading) the transcript, changing (e.g., adding, deleting, substituting) any one or more nucleotides of the transcript by, e.g., base editing; and

[0200] (4) Modifying the translation of a transcript of the VEGFA gene (eg, VEGFA mRNA), such as increasing or decreasing translation of the transcript by, for example, epigenetic modification.

[0201] DNA modification

[0202] The present disclosure provides a variety of ways to modify the expression of the VEGFA gene at the DNA level. In some embodiments, the VEGFA gene comprises a mutation. In some embodiments, the VEGFA gene is a human VEGFA gene comprising a mutation. Various VEGFA mutations that cause VEGFA-related diseases are known in the art. In some embodiments, napBP is a nucleic acid programmable DNA binding protein (napDNAbp).

[0203] DNA cleavage

[0204] Provided in this disclosure are tools and methods for modifying the expression of the VEGFA gene using DNA cleavage.

[0205] In some embodiments, the napDNAbp is a nucleic acid programmable dsDNA endonuclease (napDNAn).

[0206] In some embodiments, directing the complex to the VEGFA gene enables napDNAn to specifically cleave the VEGFA gene in a guide sequence-specific manner.

[0207] In some embodiments, specific cleavage of the VEGFA gene results in the incorporation of insertion and / or deletion (indel) mutations into the VEGFA gene.

[0208] In some embodiments, specific cutting or insertion and / or deletion mutation of VEGFA gene generates in-frame stop codon in VEGFA gene.As well-known, the existence of in-frame stop codon in gene and / or from the transcript of gene transcription usually stops protein from the translation of transcript.Usually, stop codon is TAG, TAA or TGA in DNA, is UAG, UAA or UGA in RNA.

[0209] In some embodiments, specific cleavage, or insertion and / or deletion mutation of the VEGFA gene generates a 3n+1 frameshift mutation, a 3n+2 frameshift mutation, a 3n-1 frameshift mutation, or a 3n-2 frameshift mutation in the VEGFA gene, where n is 0 or a positive integer (e.g., 1, 2, 3).

[0210] In some embodiments, the frameshift mutation reduces or eliminates transcription of the VEGFA gene and / or translation of a transcript (e.g., mRNA) of the VEGFA gene. In some embodiments, the frameshift mutation generates an in-frame stop codon in the VEGFA gene.

[0211] In some embodiments, the frameshift mutation results in expression of a mutant of vascular endothelial growth factor A. In some embodiments, the mutant is functional. In some embodiments, the mutant is non-functional.

[0212] In some embodiments, the frameshift mutation results in the expression of a functional mutant of vascular endothelial growth factor A. As used herein, the term "functional mutant" of a protein refers to a mutant of the protein that substantially has one or more functions (e.g., all known functions) of the protein, for example, a mutant that has at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the function of the protein or about 100% of the function of the protein.

[0213] In some embodiments, the frameshift mutation results in the expression of a non-functional mutant of vascular endothelial growth factor A. As used herein, the term "non-functional mutant" of a protein refers to a mutant of a protein that substantially lacks one or more functions (e.g., all known functions) of the protein, for example, a mutant that has at most about 35%, 30%, 20%, 15%, 10%, 5% or 1% of the function of the protein or has about 0% of the function of the protein.

[0214] In some embodiments, the frameshift mutation results in the expression of a non-pathogenic mutant of vascular endothelial growth factor A. As used herein, the term "non-pathogenic mutant" of a protein refers to a mutant of a protein that is non-pathogenic or has not been reported to cause any disease.

[0215] In some embodiments, the frameshift mutation does not result in the expression of a pathogenic mutant of vascular endothelial growth factor A. As used herein, the term "pathogenic mutant" of a protein refers to a mutant of a protein that is pathogenic or has been reported to cause any disease.

[0216] In some embodiments, the system of the present disclosure is combined with a supplement to deliver the wild-type expression product of the VEGFA gene. For example, in some embodiments, a cDNA encoding the wild-type expression product of the VEGFA gene is delivered in the same or separate rAAV particles in combination with the system of the present disclosure delivered in rAAV particles.

[0217] DNA base editing

[0218] Also provided in this disclosure are tools and methods for modifying the expression of the VEGFA gene using DNA base editing.

[0219] In some embodiments, napDNAbp is a nickase and further comprises a base editing domain. As used herein, the term "nickase" means that it substantially lacks dsDNA endonuclease activity (i.e., it is substantially unable to cut two chains of dsDNA) and is substantially capable of cutting one chain of dsDNA. In some embodiments, one chain is the target chain of dsDNA. In some embodiments, one chain is the sense chain or antisense chain of dsDNA.

[0220] In some embodiments, the napDNAbp is an endonuclease-deficient napDNAbp and further comprises a base editing domain. As used herein, the term "endonuclease-deficient napDNAbp" is used interchangeably with "endonuclease-inactivated napDNAbp" or "dead napDNAbp," meaning that it substantially lacks dsDNA endonuclease activity (i.e., it substantially cannot cut the double strand of dsDNA) and that it substantially lacks nickase activity (i.e., it substantially cannot cut either strand of dsDNA).

[0221] In some embodiments, directing the complex to the VEGFA gene enables the napDNAbp, which acts as a nickase or dead napDNAbp and further comprises a base editing domain, to specifically base edit the VEGFA gene in a guide sequence-specific manner.

[0222] As used herein, the term "base editing" means editing the base of a nucleotide (e.g., ribonucleotide, deoxyribonucleotide), including but not limited to insertion (addition), deletion (excision), and substitution of bases.

[0223] In some embodiments, the base editing of the VEGFA gene replaces a nucleotide of the VEGFA gene with a different nucleotide. In some embodiments, the base editing of the VEGFA gene replaces one nucleotide of the VEGFA gene with a different nucleotide, which is also referred to as single base editing. In some embodiments, the base editing of the VEGFA gene replaces two or three or more nucleotides (whether or not consecutive) of the VEGFA gene with two or three or more different nucleotides, which is also referred to as multiple or multiplexed base editing.

[0224] As well known in the art and as shown in the table below, codon includes 61 non-terminator codons and three stop codons (that is, TAA, TAG, TGA) of encoding 22 amino acids. Each codon is made up of three nucleotides. The nucleotide changes in the non-terminator codon can convert the non-terminator codon into another non-terminator codon of encoding the same or different amino acids, or be converted into a stop codon. The nucleotide changes in the stop codon can convert a stop codon or another stop codon into a non-terminator codon. Unless otherwise indicated, the codons in the present disclosure (for example, non-terminator codon, stop codon, start codon) are in-frame codons.

[0225] Nucleotide position in the codon

[0226]

[0227]

[0228] From a process perspective, in some embodiments, base editing replaces an A in a codon in the VEGFA gene with a T, C, or G. In some embodiments, base editing replaces a T in a codon in the VEGFA gene with an A, C, or G. In some embodiments, base editing replaces a C in a codon in the VEGFA gene with an A, T, or G. In some embodiments, base editing replaces a G in a codon in the VEGFA gene with an A, T, or C.

[0229] From the results, in some embodiments, the base editor replaces the stop codon of the VEGFA gene with a non-stop codon. In some embodiments, the base editor replaces the non-stop codon of the VEGFA gene with a different non-stop codon. In some embodiments, the base editor replaces the non-stop codon of the VEGFA gene with a stop codon.

[0230] The VEGFA gene can contain a pathogenic mutation, which can be a pathogenic codon substitution, wherein the wild-type codon of the VEGFA gene is replaced by a pathogenic codon. In some embodiments, the wild-type codon is a stop codon, and the pathogenic codon is a non-stop codon. The pathogenic codon substitution of a wild-type stop codon to a non-stop codon can result in the expression of a mutant of the expression product of the VEGFA gene containing an additional C-terminal amino acid, and the mutant may be pathogenic (e.g., non-functional). In some embodiments, the wild-type codon is a non-stop codon, and the pathogenic codon is a stop codon or a non-stop codon different from the wild-type non-stop codon. The pathogenic codon substitution of a wild-type non-stop codon with a stop codon can result in premature termination (partial) translation of the transcript of the VEGFA gene, which may generate an N-terminal truncation of the expression product of the VEGFA gene or no expression product. The N-terminal truncation of the expression product may be pathogenic (e.g., non-functional) and may be stable or unstable (e.g., rapidly degraded by cellular enzymes). Substitution of a pathogenic codon for a wild-type non-stop codon with a non-stop codon that is different from the wild-type non-stop codon can result in the expression of a mutant of the expression product of the VEGFA gene that may be pathogenic (e.g., non-functional) and that may be stable or unstable (e.g., rapidly degraded by cellular enzymes).

[0231] In some embodiments, base editing of the VEGFA gene reduces or eliminates transcription of the VEGFA gene and / or translation of the transcript (e.g., mRNA) of the VEGFA gene. For example, base editing of the VEGFA gene eliminates the complete expression of the VEGFA gene by replacing a non-stop codon in the VEGFA gene with a stop codon, thereby causing the transcript of the VEGFA gene to not be translated or to terminate (partial) translation prematurely.

[0232] In some embodiments, base editing of the VEGFA gene generates an in-frame stop codon in the VEGFA gene.

[0233] In some embodiments, base editing of the VEGFA gene results in expression of a functional expression product of the VEGFA gene (e.g., a functional mutant of vascular endothelial growth factor A).

[0234] In some embodiments, base editing of the VEGFA gene results in expression of a mutant of vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon. In some embodiments, the mutant of vascular endothelial growth factor A is functional. In some embodiments, the mutant of vascular endothelial growth factor A is non-functional.

[0235] In some embodiments, base editing of the VEGFA gene results in the expression of a non-functional mutant of vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon, and the resulting expression product of the base-edited VEGFA gene is a non-functional mutant of vascular endothelial growth factor A.

[0236] In some embodiments, base editing of the VEGFA gene results in the expression of wild-type vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon or a non-wild-type non-stop codon with a wild-type non-stop codon, and the resulting expression product of the base-edited VEGFA gene is wild-type vascular endothelial growth factor A.

[0237] In some embodiments, base editing of the VEGFA gene results in the expression of a functional mutant of vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon, and the resulting expression product of the base-edited VEGFA gene is a functional mutant of vascular endothelial growth factor A.

[0238] In some embodiments, base editing of the VEGFA gene results in the expression of a non-pathogenic mutant of vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon, and the resulting expression product of the base-edited VEGFA gene is non-pathogenic.

[0239] In some embodiments, base editing of the VEGFA gene does not result in expression of a pathogenic mutant of vascular endothelial growth factor A.

[0240] DNA prime editing

[0241] Also provided in this disclosure are tools and methods for modifying the expression of the VEGFA gene using DNA prime editing.

[0242] In some embodiments, napDNAbp is a nickase and further comprises a reverse transcriptase domain, and the guide nucleic acid is a lead editing guide nucleic acid (e.g., pegRNA). As used herein, the term "nickase" means that it substantially lacks dsDNA endonuclease activity (i.e., it is substantially unable to cut two chains of dsDNA) and is substantially capable of cutting a chain of dsDNA. In some embodiments, a chain is a non-target chain of dsDNA. In some embodiments, a chain is a sense strand or an antisense strand of dsDNA.

[0243] In some embodiments, directing the complex to the VEGFA gene enables napDNAbp, which acts as a nickase and further comprises a reverse transcriptase domain, to specifically prime the VEGFA gene in a guide sequence-specific manner.

[0244] For a general introduction to prime editing technology, see “Prime Editing: Adding Precision and Flexibility to CRISPR Editing” (blogdot addgene dot org slash prime-editing-crisp-cas-reverse-transcriptase).

[0245] Prime editing can allow for the insertion (addition), deletion, or substitution of one or more nucleotides of the VEGFA gene.

[0246] In some embodiments, lead editing of the VEGFA gene reduces or eliminates transcription of the VEGFA gene and / or translation of a transcript (eg, mRNA) of the VEGFA gene.

[0247] In some embodiments, prime editing of the VEGFA gene results in expression of a functional expression product of the VEGFA gene (eg, a functional mutant of vascular endothelial growth factor A).

[0248] In some embodiments, the lead editing of the VEGFA gene generates an in-frame stop codon in the VEGFA gene, such as by inserting a stop codon, by deleting one or more nucleotides to generate a frameshift mutation that further generates a stop codon, or by replacing a non-stop codon with a stop codon.

[0249] In some embodiments, the prime editing of the VEGFA gene results in expression of a mutant of vascular endothelial growth factor A, such as by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon. In some embodiments, the mutant of vascular endothelial growth factor A is functional. In some embodiments, the mutant of vascular endothelial growth factor A is non-functional.

[0250] In some embodiments, the lead editing of the VEGFA gene results in the expression of a non-functional mutant of vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon, and the resulting expression product of the lead-edited VEGFA gene is a non-functional mutant of vascular endothelial growth factor A.

[0251] In some embodiments, the lead editing of the VEGFA gene results in the expression of wild-type vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon or a non-wild-type non-stop codon with a wild-type non-stop codon, and the resulting expression product of the lead-edited VEGFA gene is wild-type vascular endothelial growth factor A.

[0252] In some embodiments, the lead editing of the VEGFA gene results in the expression of a functional mutant of vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon, and the resulting expression product of the lead-edited VEGFA gene is a functional mutant of vascular endothelial growth factor A.

[0253] In some embodiments, lead editing of the VEGFA gene results in expression of a non-pathogenic mutant of vascular endothelial growth factor A, such as by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon, and the resulting expression product of the lead-edited VEGFA gene is non-pathogenic.

[0254] In some embodiments, the prime editing of the VEGFA gene does not result in the expression of a pathogenic mutant of vascular endothelial growth factor A.

[0255] DNA epigenome modifications

[0256] In some embodiments, the napDNAbp is an endonuclease-deficient napDNAbp and further comprises an epigenomic modification domain, such as a methylation domain, a demethylation domain. As used herein, the term "endonuclease-deficient napDNAbp" is used interchangeably with "endonuclease-inactivated napDNAbp" or "dead napDNAbp" to mean that it substantially lacks dsDNA endonuclease activity (i.e., it substantially cannot cut the double strand of dsDNA) and that it substantially lacks nickase activity (i.e., it substantially cannot cut either strand of dsDNA).

[0257] In some embodiments, directing the complex to the VEGFA gene enables the napDNAbp, which is an endonuclease-deficient napDNAbp and further comprises an epigenomic modification domain (e.g., a methylation domain, a demethylation domain), to specifically perform epigenetic modification on the VEGFA gene in a sequence-specific manner.

[0258] In some embodiments, the epigenomic modification of the VEGFA gene reduces or eliminates transcription of the VEGFA gene and / or translation of a transcript (eg, mRNA) of the VEGFA gene.

[0259] In some embodiments, the epigenomic modification of the VEGFA gene increases transcription of the VEGFA gene and / or translation of a transcript (eg, mRNA) of the VEGFA gene.

[0260] RNA modification

[0261] The present disclosure provides a variety of ways to modify the expression of the VEGFA gene at the RNA level. In some embodiments, the transcript of the VEGFA gene is VEGFA pre-mRNA or VEGFA mRNA. In some embodiments, the transcript comprises a mutation. In some embodiments, the transcript is a transcript of a human VEGFA gene comprising a mutation. In some embodiments, napBP is a nucleic acid programmable RNA binding protein (napRNAbp).

[0262] RNA cleavage

[0263] Provided in this disclosure are tools and methods for modifying the expression of the VEGFA gene using RNA cleavage.

[0264] In some embodiments, the napRNAbp is a nucleic acid programmable RNA endonuclease (napRNAn).

[0265] In some embodiments, directing the complex to the transcript of the VEGFA gene enables napRNAn to specifically cleave the transcript of the VEGFA gene in a guide sequence-specific manner. Typically, cleavage of a transcript by an RNA endonuclease results in degradation of the transcript.

[0266] RNA base editing

[0267] Also provided in this disclosure are tools and methods for modifying the expression of the VEGFA gene using RNA base editing.

[0268] In some embodiments, the napRNAbp is an endonuclease-deficient napRNAbp and further comprises a base editing domain. As used herein, the term "endonuclease-deficient napRNAbp" is used interchangeably with "endonuclease-inactive napRNAbp" or "dead napRNAbp," meaning that it substantially lacks RNA endonuclease activity (i.e., it substantially cannot cleave RNA).

[0269] In some embodiments, directing the complex to the transcript of the VEGFA gene enables the napRNAbp, which is an endonuclease-deficient napRNAbp and further comprises a base editing domain, to specifically base edit the transcript in a guide sequence-specific manner.

[0270] In some embodiments, base editing of a transcript replaces a nucleotide of the transcript with a different nucleotide. In some embodiments, base editing of a transcript replaces one nucleotide of the transcript with one different nucleotide, which is also referred to as single base editing. In some embodiments, base editing of a transcript replaces two or three or more nucleotides of the transcript (whether or not consecutive) with two or three or more different nucleotides, which is also referred to as multiple or multiplexed base editing.

[0271] From a process perspective, in some embodiments, base editing replaces A in a transcript codon with U, C, or G; replaces U in a transcript codon with A, C, or G; replaces C in a transcript codon with A, U, or G; or replaces G in a transcript codon with A, U, or C.

[0272] From the results, in some embodiments, the base editor replaces the stop codon of the transcript with a non-stop codon; replaces the non-stop codon of the transcript with a different non-stop codon; or replaces the non-stop codon of the transcript with a stop codon.

[0273] The transcript may contain a pathogenic mutation, which may be a pathogenic codon replacement, wherein the wild-type codon of the transcript is replaced by a pathogenic codon. In some embodiments, the wild-type codon is a stop codon, and the pathogenic codon is a non-stop codon. The substitution of a wild-type stop codon to a pathogenic codon of a non-stop codon may result in the translation of a mutant of the expression product of the VEGFA gene containing an additional C-terminal amino acid, which may be pathogenic (e.g., non-functional). In some embodiments, the wild-type codon is a non-stop codon, and the pathogenic codon is a stop codon or a non-stop codon that is different from the wild-type non-stop codon. The substitution of a pathogenic codon with a stop codon for a wild-type non-stop codon may result in the premature termination (partial) translation of the transcript, which may generate an N-terminal truncation of the expression product of the VEGFA gene or may not generate an expression product. The N-terminal truncation of the expression product may be pathogenic (e.g., non-functional) and may be stable or unstable (e.g., rapidly degraded by cellular enzymes). Substitution of a pathogenic codon for a wild-type non-stop codon with a non-stop codon that is different from the wild-type non-stop codon can result in translation of a mutant of the expression product of the VEGFA gene that may be pathogenic (e.g., non-functional) and that may be stable or unstable (e.g., rapidly degraded by cellular enzymes).

[0274] In some embodiments, base editing of a transcript reduces or eliminates translation of the transcript. For example, base editing of a transcript eliminates complete translation of the transcript by replacing a non-stop codon in the transcript with a stop codon, thereby causing the transcript to be untranslated or prematurely terminate (partial) translation.

[0275] In some embodiments, base editing of a transcript generates an in-frame stop codon in the transcript.

[0276] In some embodiments, base editing of a transcript results in translation of a functional expression product of the transcript (e.g., a functional mutant of vascular endothelial growth factor A).

[0277] In some embodiments, base editing of the transcript results in expression of a mutant of vascular endothelial growth factor A, such as by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon. In some embodiments, the mutant of vascular endothelial growth factor A is functional. In some embodiments, the mutant of vascular endothelial growth factor A is non-functional.

[0278] In some embodiments, base editing of the transcript results in expression of a non-functional mutant of vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon, and the resulting expression product of the transcript is a non-functional mutant of vascular endothelial growth factor A.

[0279] In some embodiments, base editing of the transcript results in expression of wild-type vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon or a non-wild-type non-stop codon with a wild-type non-stop codon, and the resulting expression product of the transcript is wild-type vascular endothelial growth factor A.

[0280] In some embodiments, base editing of the transcript results in expression of a functional mutant of vascular endothelial growth factor A, for example, by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon, and the resulting expression product of the transcript is a functional mutant of vascular endothelial growth factor A.

[0281] In some embodiments, base editing of a transcript results in expression of a non-pathogenic mutant of vascular endothelial growth factor A, such as by replacing a non-wild-type stop codon with a non-wild-type non-stop codon or replacing a non-wild-type non-stop codon with a different non-wild-type non-stop codon, and the resulting expression product of the transcript is non-pathogenic.

[0282] In some embodiments, base editing of the transcript does not result in expression of a pathogenic mutant of vascular endothelial growth factor A.

[0283] napBP

[0284] For the purposes of the present disclosure, napBP is capable of forming a complex with the guide nucleic acid of the present disclosure by compounding with the scaffold sequence of the guide nucleic acid, and thereby being guided to the VEGFA gene or its transcript via hybridization of the guide sequence of the guide nucleic acid with the target sequence of the VEGFA gene or transcript. When napBP is guided to the VEGFA gene or transcript, the activity of napBP (or a functional domain associated with (e.g., bound to) napBP) acts to modify the VEGFA gene or its transcript, thereby resulting in modification of the expression of the VEGFA gene. Generally, any such napBP can be used with the guide nucleic acid of the present disclosure, and when selecting napBP, a scaffold sequence compatible with napBP for compounding with napBP can also be selected accordingly. Scaffold sequences are generally conserved.

[0285] In some embodiments, the napBP (e.g., napDNAbp) is capable of recognizing a protospacer adjacent motif (PAM) on the non-target strand of the VEGFA gene, wherein the PAM is immediately 5' or 3' to the protospacer sequence on the non-target strand of the VEGFA gene, and wherein the protospacer sequence is completely reverse complementary to the target sequence.

[0286] In some embodiments, the PAM comprises the sequence 5'-NN-3', 5'-NNN-3', 5'-NNNN-3', 5'-NNNNN-3', or 5'-NNNNNN-3', wherein N is A, T, G, or C.

[0287] Non-limiting examples of napBPs include CRISPR-associated (Cas) proteins, IscB, TAL nucleases, meganucleases, and zinc finger nucleases. Non-limiting examples of CRISPR-associated (Cas) proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12f / Cas14, Cas12g, Cas12h, Cas12i, and Cas12k. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12f / Cas14, Cas12g, Cas12h, Cas12i, Cas12k, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csn1, Csn2, Csn3, Csn4, Csn5e, Csn1, Csn2, Csn3, Csn4, Csn5e, Csn1, Csn2, Csn3, Csn4, Csn5e, Csn1 sc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, C mr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Cs x11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, IscB, homologs thereof, or modified or engineered versions thereof. Other napDNA bps are also within the scope of the present disclosure, such as IscB, IsrB, although they may not be specifically listed in the present disclosure.See, e.g., Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct;1:325-336. doi:10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems." Science. 2019 Jan 4;363(6422):88-91. doi:10.1126 / science.aav7271, the entire contents of each of which are hereby incorporated by reference.

[0288] In some embodiments, the Cas protein is an endonuclease, a nickase, or a death Cas.

[0289] In some embodiments, the PAM comprises the sequence 5'-NTN-3', wherein N is A, T, G or C, and wherein the PAM is immediately adjacent to the 5' portion of the protospacer sequence. In some embodiments, the PAM comprises the sequence 5'-TTN-3', wherein N is A, T, G or C. For example, in some embodiments, the napBP is a Class 2 V-type CRISPR-associated protein (Cas12). In some embodiments, Cas12 is Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14), Cas12i or Cas12k (C2c10, C2C7), such as Cas12i1, Cas12i1, Cas12i3, Cas12i4, xCas12i (SiCas12i), Cas12Max, hfCas12Max or a mutant thereof.

[0290] In some embodiments, the PAM comprises the sequence 5'-NGG-3', wherein N is A, T, G, or C, and wherein the PAM is immediately 3' to the protospacer sequence. For example, in some embodiments, the napBP is a Class 2 type II CRISPR-associated protein (Cas9), such as SaCas9, SpCas9, or a mutant thereof.

[0291] In some embodiments, the PAM comprises the sequence 5'-NNNGAN-3', wherein N is A, T, G, or C, and wherein the PAM is immediately 3' to the protospacer sequence. For example, in some embodiments, the napBP is an IscB protein, such as OgeuIscB or a mutant thereof.

[0292] In some cases, the ability of napBP to recognize a target nucleic acid may not be limited to any particular PAM, meaning that napBP can recognize any PAM such that the PAM does not substantially restrict the choice of protospacer sequence or target sequence. Such napBPs are referred to as "PAM-free," e.g., a PAM-free SpCas9 mutant. In some embodiments, napBP is PAM-free.

[0293] In some embodiments, napBP is a 2-type VI CRISPR-associated protein (Cas13). Cas13 can target RNA by guiding nucleic acids. Cas13 is particularly useful because when Cas13 is used as a napBP, there is no PAM restriction on eukaryotic transcripts. In some embodiments, Cas13 is Cas13a (C2c2), Cas13b (such as Cas13b1, Cas13b2), Cas13c, Cas13d, Cas13e (Cas13X), Cas13f (Cas13Y) or a mutant thereof. In some embodiments. In PCT / CN2022 / 122833, napBP is hfCas13Y (hfCas13f / hfCas13f.1). As used herein, the terms "Cas13Y" and "Cas13f" are used interchangeably, and the terms "hfCas13Y" and "hfCas13f" and "hfCas13f.1" are used interchangeably.

[0294] Typically, Cas proteins (e.g., Cas9, Cas12, Cas13) can be associated with CRISPR RNA (crRNA) comprising a spacer sequence that guides the Cas protein to a specific sequence that is reverse complementary to the crRNA spacer sequence and capable of hybridizing therewith. The crRNA also comprises a scaffold sequence that can be complexed with the Cas protein.

[0295] In some embodiments, the napBP comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 16, or an N-terminal truncation thereof without the first N-terminal methionine (SEQ ID NO: 32). In some embodiments, the napBP retains at least 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the guide sequence-specific DNA endonuclease activity of the sequence of SEQ ID NO: 16 or 32.

[0296] In some embodiments, the napBP comprises the sequence of SEQ ID NO: 16 or 32.

[0297] In some embodiments, the sequence encoding the napBP comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 9 or a 5' truncation thereof without the first 5' ATG codon (SEQ ID NO: 33). In some embodiments, the napBP encoded by the sequence retains at least 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the guide sequence-specific DNA endonuclease activity of the sequence of SEQ ID NO: 9 or 33.

[0298] In some embodiments, the sequence encoding napBP comprises the sequence of SEQ ID NO: 9 or 33.

[0299] Base editing

[0300] In some embodiments, the napBP further comprises a base editing domain.

[0301] In some embodiments, a base editing domain is capable of replacing a base of a nucleotide with a different base.

[0302] In some embodiments, the base editing domain is capable of deaminating a base of a nucleotide.

[0303] In some embodiments, the base editing domain comprises a deaminase domain capable of deaminating a base of a nucleotide (e.g., adenine, guanine, cytosine, thymine, uracil). In some embodiments, the deaminase domain is capable of deaminating adenine (A) to hypoxanthine (I). In some embodiments, adenine is deaminated to hypoxanthine and adenosine (A) or deoxyadenosine (dA) containing adenine is converted to guanosine (G) or deoxyguanosine (dG). In some embodiments, the deaminase domain is capable of deaminating cytosine (C) to uracil (U). In some embodiments, cytosine is deaminated to uracil and cytidine (C) or deoxycytidine (dC) containing cytosine is converted to uridine (U) or deoxythymidine (dT).

[0304] In some embodiments, a base editing domain is capable of excising a nucleotide base (e.g., adenine, guanine, cytosine, thymine, uracil).

[0305] In some embodiments, the base editing domain comprises a base excision domain capable of excising a base of a nucleotide.

[0306] In some embodiments, the base editing domain comprises a deaminase domain and a base excision domain.

[0307] In some embodiments, the deaminase domain is a double-stranded RNA-specific adenosine deaminase (ADAR), such as ADAR1, ADAR2 (ADARB1), ADAR3 (ADARB2), or a deaminase domain thereof, or a mutant thereof.

[0308] In some embodiments, the ADAR is a human ADAR or an ADAR from a non-human species. In some embodiments, the ADAR is an octopus ADAR.

[0309] In some embodiments, the deaminase domain is the human ADAR2 deaminase domain (hADAR2DD).

[0310] In some embodiments, the deaminase domain is a mutant of hADAR2DD, such as hADAR2DD-E488Q, hADAR2DD-E488Q+T375G, hADAR2DD-E488Q+V351G+S486A+T375S+S370C+P462A+N597I+L332I+I398V+K350I+M383L+D6 19G+S582T+V440I+S495N+K418E+S661T, hADAR2DD-E488Q+V351G+S486A+T375A+S370C +P462A+N597I+L332I+I398V+K350I+M383L+D619G+S582T+V440I+S495N+K418E+S661T.

[0311] In some embodiments, the deaminase domain is an apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC), such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, or a deaminase domain thereof, or a mutant thereof.

[0312] In some embodiments, the deaminase domain is activation-induced cytidine deaminase (AID, AICDA, single-stranded DNA cytosine deaminase), or a deaminase domain thereof, or a mutant thereof.

[0313] In some embodiments, the deaminase domain is tRNA adenosine deaminase (TadA), or a deaminase domain thereof, or a mutant thereof.

[0314] Epigenomic modifications

[0315] In some embodiments, the napBP further comprises a transcriptional repression domain (eg, a KRAB domain or a SID domain). In some embodiments, the napBP further comprises a KRAB domain.

[0316] In some embodiments, the napBP further comprises a DNA methyltransferase, such as DNMT31, DNMT3a.

[0317] In some embodiments, the napBP further comprises a DNMT31 domain and a DNMT3a domain.

[0318] In some embodiments, the napBP further comprises a DNMT31 domain, a DNMT3a domain, and a KRAB domain.

[0319] In some embodiments, the napBP, KRAB domain, DNMT31 domain, and DNMT3a domain are arranged from N-terminus to C-terminus.

[0320] Design of protospacer sequence / target sequence; target site

[0321] For the purposes of the present disclosure, in some embodiments, the protospacer sequence or target sequence is positioned such that the VEGFA gene or its transcript can be specifically modified by a napBP or a functional domain associated with a napBP.

[0322] In order to facilitate the evaluation of the selected protospacer sequence or target sequence and the designed guide sequence in a mouse model, in some embodiments, the protospacer sequence or target sequence is positioned so that the mouse VEGFA gene or its transcript can be specifically modified by napBP or a functional domain associated with napBP. In some embodiments, the protospacer sequence or target sequence is positioned so that both the human VEGFA gene or its transcript and the mouse VEGFA gene or its transcript can be specifically modified by napBP or a functional domain associated with napBP. That is, the protospacer sequence or target sequence is selected to have cross-reactivity with both human and mouse species.

[0323] In some embodiments, the protospacer sequence is a stretch of contiguous nucleotides identified from the non-target strand of the VEGFA gene by identifying a stretch of contiguous nucleotides immediately 5' or 3' (and optionally 3') to the PAM on the non-target strand.

[0324] In some embodiments, the protospacer sequence is a stretch of about, at least about, or at most about 14, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more consecutive nucleotides on the non-target strand of the VEGFA gene, or a range of consecutive nucleotides on the non-target strand of the VEGFA gene. In some embodiments, the protospacer sequence is a stretch of about 20, 30, or 50 contiguous nucleotides on the non-target strand of the VEGFA gene.

[0325] In some embodiments, the protospacer sequence on the non-target strand of the VEGFA gene corresponding to the target sequence is immediately 3' to the PAM of 5'-NTN-3', wherein N is A, T, G, or C.

[0326] In some embodiments, the protospacer sequence on the non-target strand of the VEGFA gene corresponding to the target sequence is immediately 5' to the PAM of 5'-NGG-3', where N is A, T, G, or C.

[0327] In some embodiments, the protospacer sequence on the non-target strand of the VEGFA gene corresponding to the target sequence is immediately 5' to the PAM of 5'-NNNGAN-3', where N is A, T, G, or C.

[0328] In some embodiments, the protospacer sequence is a stretch of about 20, 30, or 50 consecutive nucleotides of the non-target strand of the VEGFA gene immediately 3' to the PAM of 5'-NTN-3', wherein N is A, T, G, or C.

[0329] In some embodiments, the protospacer sequence is a stretch of about 20, 30, or 50 consecutive nucleotides of the non-target strand of the VEGFA gene immediately 5' to the PAM of 5'-NGG-3', wherein N is A, T, G, or C.

[0330] In some embodiments, the protospacer sequence is a stretch of about 20, 30, or 50 consecutive nucleotides of the non-target strand of the VEGFA gene immediately 5' to the PAM of 5'-NNNGAN-3', wherein N is A, T, G, or C.

[0331] In some embodiments, the target sequence is a stretch of contiguous nucleotides identified from the target strand of the VEGFA gene or from a transcript thereof.

[0332] 65, 66, 67, 68, 69, 70, or more consecutive nucleotides on the target strand of a VEGFA gene or a transcript thereof, or a range of consecutive nucleotides on the target strand of a VEGFA gene or a transcript thereof, for example, 16 to about 50 consecutive nucleotides. In some embodiments, the target sequence is a stretch of about 20, 30, or 50 consecutive nucleotides on the target strand of the VEGFA gene or its transcript.

[0333] In some embodiments, the non-target strand is the sense strand of the VEGFA gene.

[0334] In some embodiments, the non-target strand is the antisense strand of the VEGFA gene.

[0335] In some embodiments, the target strand is the sense strand of the VEGFA gene.

[0336] In some embodiments, the target strand is the antisense strand of the VEGFA gene.

[0337] In some embodiments, the protospacer sequence corresponding to the target sequence on the non-target strand of the VEGFA gene, or the target sequence on the target strand of the VEGFA gene is located at or within an exon of the VEGFA gene or its transcript, or at or within a splice donor or splice acceptor of an exon.

[0338] In some embodiments, the exon is selected from exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8.

[0339] In some embodiments, the VEGFA gene is a human VEGFA gene, a non-human primate VEGFA gene, or a mouse VEGFA gene. In some embodiments, the VEGFA mRNA is a human VEGFA mRNA, a non-human primate VEGFA mRNA, or a mouse VEGFA mRNA.

[0340] In some embodiments, the VEGFA gene or its transcript is in a eukaryotic cell, such as a human cell, a non-human primate cell, or a mouse cell. In some embodiments, the VEGFA gene or its transcript is in a retinal cell.

[0341] Design guide sequence based on protospacer / target sequence

[0342] 65, 66, 67, 68, 69, 70, or more nucleotides in length, or a nucleoside length ranges between any two of the foregoing values, such as about 16 to about 50 nucleotides in length. In some embodiments, the guide sequence is about 20, 30, or 50 nucleotides in length.

[0343] In some embodiments, (1) the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (perfect) reverse complementary to the target sequence, optionally about 100% (perfect) reverse complementary; (2) the guide sequence contains no more than 5, 4, 3, 2, or 1 mismatches to the target sequence or no mismatches; or (3) the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (perfect) reverse complementary to the target sequence; (4) the guide sequence contains no more than 5, 4, 3, 2, or 1 mismatches to the target sequence; or (5) the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (perfect) reverse complementary to the target sequence; 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides.

[0344] In some embodiments, the guide sequence contains one mismatch with the target sequence. In some embodiments, the guide sequence is about 98% reverse complementary to the target sequence. In some embodiments, one mismatch in the guide sequence is at a position corresponding to a nucleotide of the target sequence that is intended to be replaced.

[0345] Choice of protospacer sequence / target sequence / guide sequence; role of the system

[0346] In some embodiments, the protospacer sequence, target sequence or guide sequence is selected based on the knockdown efficiency of napRNAbp directed to the VEGFA transcript by the guide sequence. The knockdown efficiency can be represented by the fold change of VEGFA transcript or protein measured, for example, by RT-qPCR or Western blot. The higher the knockdown efficiency, the more suitable the protospacer sequence, target sequence or guide sequence.

[0347] In some embodiments, the protospacer sequence, target sequence or guide sequence is selected based on the transcription level of the VEGFA gene and / or the translation level of the transcript (e.g., mRNA) of the VEGFA gene in an in vivo animal model (the expression level of the VEGFA gene). The lower the level, the more suitable the protospacer sequence, target sequence or guide sequence.

[0348] In some embodiments, after administration of the system of the present disclosure to a cell model (e.g., a HEK293T cell model) or an animal model (e.g., a mouse model, a non-human primate model), the level of the transcript (e.g., mRNA) of the VEGFA gene in the cell model or animal model is reduced by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more compared to the level of the transcript (e.g., mRNA) of the VEGFA gene in the same cell model or animal model that did not receive the administration.

[0349] In some embodiments, following administration of a system of the present disclosure to a cell model (e.g., a HEK293T cell model) or an animal model (e.g., a mouse model, a non-human primate model), the level of the expression product of the VEGFA gene (e.g., vascular endothelial growth factor A) in the cell or animal model is reduced by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more compared to the level of the expression product of the VEGFA gene (e.g., vascular endothelial growth factor A) in the same cell model or animal model that did not receive the administration.

[0350] The overall structure of the guide nucleic acid

[0351] In some embodiments, the guide nucleic acid comprises a scaffold sequence 5' to the guide sequence. In some embodiments, the guide nucleic acid comprises a scaffold sequence 3' to the guide sequence.

[0352] In some embodiments, a guide nucleic acid comprises a scaffold sequence and a guide sequence.

[0353] In some embodiments, a guide nucleic acid comprises a scaffold sequence 5' of a guide sequence. In some embodiments, a guide nucleic acid comprises a scaffold sequence 3' of a guide sequence.

[0354] In some embodiments, a guide nucleic acid comprises one or more scaffold sequences and / or one or more guide sequences, with the proviso that a guide nucleic acid does not comprise a scaffold sequence and a guide sequence.

[0355] In some embodiments, a guide nucleic acid comprises, from 5' to 3', a scaffold sequence, a guide sequence, and a scaffold sequence, wherein the scaffold sequences are the same or different.

[0356] In some embodiments, a guide nucleic acid comprises, from 5' to 3', a guide sequence, a scaffold sequence, and a guide sequence, wherein the guide sequences are the same or different.

[0357] In some embodiments, the guide nucleic acid comprises, from 5' to 3', one scaffold sequence, one guide sequence, one scaffold sequence, and one guide sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0358] In some embodiments, the guide nucleic acid comprises, from 5' to 3', one guide sequence, one scaffold sequence, one guide sequence and one scaffold sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0359] In some embodiments, a guide nucleic acid comprises, from 5' to 3', one scaffold sequence, one guide sequence, one scaffold sequence, one guide sequence, and one scaffold sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0360] In some embodiments, the guide nucleic acid comprises, from 5' to 3', one guide sequence, one scaffold sequence, one guide sequence, one scaffold sequence, and one guide sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0361] In some embodiments, a guide nucleic acid comprises, from 5' to 3', one scaffold sequence, one guide sequence, one scaffold sequence, one guide sequence, one scaffold sequence, and one guide sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0362] In some embodiments, the guide nucleic acid comprises, from 5' to 3', one guide sequence, one scaffold sequence, one guide sequence, one scaffold sequence, one guide sequence, and one scaffold sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0363] In some embodiments, the guide nucleic acid comprises a linker or does not comprise a linker between any adjacent scaffold sequence and the guide sequence. In some embodiments, the guide nucleic acid does not comprise a linker between any adjacent scaffold sequence and the guide sequence.

[0364] Multiple guide nucleic acids

[0365] The systems or rAAV vector genomes of the present disclosure can comprise or encode one guide nucleic acid, or can comprise or encode multiple (e.g., 2, 3, 4, or more) guide nucleic acids, e.g., for the purpose of improving the editing efficiency of the system.

[0366] In some embodiments, the system further comprises one or more additional guide nucleic acids, or the first polynucleotide sequence further comprises one or more additional sequences encoding one or more additional guide nucleic acids, each of the one or more additional guide nucleic acids comprising:

[0367] (1) an additional scaffold sequence capable of forming a complex with napBP, and

[0368] (2) an additional guide sequence capable of hybridizing to an additional target sequence on the target strand of the VEGFA gene or an additional target sequence on its transcript, thereby directing the complex to the VEGFA gene or transcript.

[0369] In some embodiments, the additional protospacer sequence is on the same strand as the protospacer sequence.

[0370] In some embodiments, the additional protospacer sequence is on a different strand than the protospacer sequence.

[0371] In some embodiments, the additional protospacer sequence is the same as or different from the protospacer sequence.

[0372] In some embodiments, the additional target sequence is the same as or different from the target sequence.

[0373] In some embodiments, the additional guide sequence is the same as or different from the guide sequence.

[0374] In some embodiments, the additional scaffold sequence is the same or different from the scaffold sequence. In some embodiments in which the system comprises the same napBP and multiple guide nucleic acids, the scaffold sequences of the multiple guide nucleic acids can be the same or different (e.g., differing by no more than 5, 4, 3, 2, or 1 nucleotides) so as to be compatible with the same napBP. In some embodiments in which the system comprises different napBPs (e.g., Cas12i and Cas9) and multiple guide nucleic acids, the scaffold sequences of the multiple guide nucleic acids can be different so as to be compatible with different napBPs, respectively.

[0375] In some embodiments, the additional guide nucleic acid and the guide nucleic acid are operably linked to or under the regulation of the same regulatory element (e.g., promoter) or separate regulatory elements (e.g., separate promoters).

[0376] Directing the properties and modification of nucleic acids

[0377] In some embodiments, the guide nucleic acid (e.g., guide nucleic acid, additional guide nucleic acid) is RNA, i.e., guide RNA (gRNA). In some embodiments, the guide nucleic acid is an unmodified guide RNA. In some embodiments, the guide nucleic acid is a modified guide RNA. In some embodiments, the guide nucleic acid comprises a modification. In some embodiments, the guide nucleic acid is a modified RNA containing modified ribonucleotides. In some embodiments, the guide nucleic acid is a modified RNA containing deoxyribonucleotides. In some embodiments, the guide nucleic acid is a modified RNA containing modified deoxyribonucleotides. In some embodiments, the guide nucleic acid comprises modified or unmodified deoxyribonucleotides and modified or unmodified ribonucleotides.

[0378] Specific target sequence and guide sequence

[0379] In some embodiments, the guide sequence or additional guide sequence comprises (1) the sequence of SEQ ID NO: 1 or 2, or a 5' or 3' truncation thereof by 1, 2, 3, 4, 5, or 6 nucleotides at the 5' or 3' end; or (2) a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 1 or 2, or a 5' or 3' truncation thereof by 1, 2, 3, 4, 5, or 6 nucleotides at the 5' or 3' end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotides that differ from SEQ ID NO: 1 or 2, whether contiguous or not.

[0380] In some embodiments, the guide sequence or additional guide sequence comprises the sequence of SEQ ID NO: 1 or 2.

[0381] In some embodiments, the target sequence or additional target sequence comprises (1) the sequence of SEQ ID NO: 18 or 19, or a 5' or 3' truncation thereof at the 5' or 3' end thereof by 1, 2, 3, 4, 5, or 6 nucleotides; or (2) a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 18 or 19, or a 5' or 3' truncation thereof at the 5' or 3' end thereof by 1, 2, 3, 4, 5, or 6 nucleotides; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences from SEQ ID NO: 18 or 19, whether contiguous or not.

[0382] Specific scaffold sequence

[0383] For purposes of the present disclosure, support sequence is compatible with napBP of the present disclosure, and can be compounded with napBP. Support sequence can be the naturally occurring support sequence identified together with napBP or its maintenance variant of the ability compounded with napBP. Usually, as long as the secondary structure of variant is substantially the same as the secondary structure of naturally occurring support sequence, just can maintain the ability compounded with napBP. Nucleotide deletion, insertion or replacement in the primary sequence of support sequence may not necessarily change the secondary structure of support sequence (for example, the relative position and / or size of the stem, projection and ring of support sequence can not significantly deviate from the relative position and / or size of original stem, projection and ring). For example, Nucleotide deletion, insertion or replacement can be in the projection or loop region of support sequence, so that projection and therefore the overall symmetry of secondary structure roughly keep identical. Nucleotide deletion, insertion or replacement can also be in the stem of support sequence, so that the length of stem does not significantly deviate from the length of original stem (for example, in each of two stems, add or lack a base pair corresponding to 4 base changes altogether).

[0384] In some embodiments, the scaffold sequence or additional scaffold sequence has a secondary structure that is substantially identical to the secondary structure of the sequence of SEQ ID NO:5.

[0385] In some embodiments, the scaffold sequence or additional scaffold sequence comprises (1) the sequence of SEQ ID NO:5 or a 5' or 3' truncation thereof at the 5' or 3' end truncated by 1, 2, 3, 4, 5, or 6 nucleotides; or (2) a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:5 or a 5' or 3' truncation thereof at the 5' or 3' end truncated by 1, 2, 3, 4, 5, or 6 nucleotides; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences from SEQ ID NO:5, whether contiguous or not.

[0386] In some embodiments, the scaffold sequence or additional scaffold sequence comprises the sequence of SEQ ID NO:5.

[0387] Specific guide nucleic acid sequence

[0388] In some embodiments, the guide nucleic acid comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of any one of SEQ ID NOs: 17 and 30-31; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of any one of SEQ ID NOs: 17 and 30-31.

[0389] In some embodiments, the guide nucleic acid comprises the sequence of any one of SEQ ID NOs: 17 and 30-31.

[0390] Directing the regulation of nucleic acids

[0391] Also provided in the present disclosure are polynucleotides comprising or encoding guide nucleic acids.

[0392] In some embodiments, the polynucleotide comprising or encoding a guide nucleic acid is DNA, RNA, or a DNA / RNA mixture. A "DNA / RNA mixture" refers to a nucleic acid comprising one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether or not consecutive. However, "DNA" or "RNA" may also refer to a DNA comprising one or more modified or unmodified ribonucleotides, whether or not consecutive, or an RNA comprising one or more modified or unmodified deoxyribonucleotides, whether or not consecutive.

[0393] In some embodiments, the guide nucleic acid is operably linked to or under the regulation of a promoter.

[0394] In some embodiments, the first polynucleotide sequence comprises a promoter operably linked to a sequence encoding a guide nucleic acid.

[0395] In some embodiments, the promoter is a broad spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter.

[0396] Suitable promoters are known in the art and include, for example, Cbh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retroviral Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1 alpha short (EFS) promoter, beta glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (le) enhancer and / or promoter, chicken β-actin (CBA) promoter or derivatives thereof such as CAG promoter, CB promoter, (human) elongation factor 1 alpha-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, synapsin (Syn) promoter, synapsin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, and myelin basic protein (MBP) promoter.

[0397] In some embodiments, the promoter is a U6 promoter.

[0398] In some embodiments, the promoter comprises a sequence that has at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 4 or 22; or a sequence that differs from the sequence of SEQ ID NO: 4 or 22 in at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, whether contiguous or not.

[0399] In some embodiments, the first polynucleotide sequence comprises, from 5′ to 3′, a promoter of SEQ ID NO: 4 and a first sequence encoding a guide nucleic acid of any one of SEQ ID NOs: 17 and 30-31.

[0400] Regulation of napBP

[0401] In some embodiments, the polynucleotide encoding napBP is DNA, RNA, or a DNA / RNA mixture. A "DNA / RNA mixture" refers to a nucleic acid comprising one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether or not consecutive. However, "DNA" or "RNA" may also refer to a DNA comprising one or more modified or unmodified ribonucleotides, whether or not consecutive, or an RNA comprising one or more modified or unmodified deoxyribonucleotides, whether or not consecutive.

[0402] In some embodiments, the polynucleotide encoding the napBP is operably linked to or under the regulation of a promoter.

[0403] In some embodiments, the second polynucleotide sequence comprises a promoter operably linked to a sequence encoding napBP.

[0404] In some embodiments, the promoter is a broad spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter.

[0405] Suitable promoters are known in the art and include, for example, the Cbh promoter, the Cba promoter, the pol I promoter, the pol II promoter, the pol III promoter, T7 promoter, U6 promoter, H1 promoter, retroviral Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1 alpha short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or derivatives thereof such as CAG promoter, CB promoter, (human) elongation factor 1 alpha-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, synaptophysin (Syn) promoter, human synapsin (hSyn) promoter, synapsin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, myelin basic protein (MBP) promoter, VEGFA promoter, GRK1 promoter, CRX promoter, NRL promoter, MECP2 promoter, mMECP2 promoter, hMECP2 promoter, APP promoter, and RCVRN promoter.

[0406] In some embodiments, the promoter is an eye- or retina-specific promoter.

[0407] In some embodiments, the promoter is the Cbh promoter.

[0408] In some embodiments, the second polynucleotide sequence comprises a Kozak sequence (gccacc) 5' to the sequence encoding napBP.

[0409] In some embodiments, the second polynucleotide sequence comprises a sequence encoding a nuclear localization signal (NLS) 5' and / or 3' to the sequence encoding napBP.

[0410] In some embodiments, the second polynucleotide sequence comprises a sequence encoding a nuclear export signal (NES) 5' and / or 3' to the sequence encoding napBP.

[0411] In some embodiments, the second polynucleotide sequence comprises a sequence encoding a first NLS 5' to the sequence encoding napBP and a second sequence encoding a second NLS 3' to the sequence encoding napBP.

[0412] In some embodiments, the NLS, the first NLS, and / or the second NLS is an SV40 NLS (SEQ ID NO: 26), a bpSV40 NLS (SEQ ID NO: 15), or a nucleoplasmin NLS (npNLS).

[0413] In some embodiments, the napBP comprises a bpSV40 NLS at the N-terminus of the napBP and a bpSV40 NLS at the C-terminus of the napBP.

[0414] In some embodiments, the napBP comprises an NLS inserted between the N-terminal Met and the remaining amino acid sequence of the napBP.

[0415] In some embodiments, the second polynucleotide comprises a sequence encoding a first NLS 5' to the sequence encoding the Cas13 endonuclease.

[0416] In some embodiments, the second polynucleotide comprises a sequence encoding a first NLS inserted between the 5' start codon ATG of the sequence encoding the Cas13 endonuclease and the remaining polynucleotide sequence.

[0417] In some embodiments, the second polynucleotide comprises a sequence encoding a second NLS 3' to the sequence encoding the Cas13 endonuclease.

[0418] In some embodiments, the second polynucleotide sequence comprises a WPRE sequence downstream of the sequence encoding napBP.

[0419] In some embodiments, the WPRE sequence is selected from the group consisting of a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), WPRE3 (shortened WPRE), and functional variants thereof (eg, functional truncations).

[0420] In some embodiments, the second polynucleotide sequence comprises a sequence encoding a polyadenylation (polyA) signal downstream of the sequence encoding napBP.

[0421] In some embodiments, the second polynucleotide sequence comprises a WPRE sequence downstream of a sequence encoding napBP, followed by a sequence encoding a polyadenylation (polyA) signal.

[0422] In some embodiments, the polyA signal is selected from the group consisting of a bovine growth hormone polyadenylation (bGH polyA) signal, a small polyA (SPA) signal, a human growth hormone polyadenylation (hGH polyA) signal, an SV40 polyA (SV40polyA) signal, a rabbit beta globin polyA (rBG polyA) signal, an SV40 late polyadenylation signal upstream element, and a combination of SV40 late polyadenylation signals, and functional variants thereof (e.g., functional truncations).

[0423] In some embodiments, the polyA signal is a bGH polyA signal.

[0424] In some embodiments, the second polynucleotide sequence comprises, from 5′ to 3′, a promoter, a Kozak sequence, an in-frame start codon ATG, a first sequence encoding a first NLS, a sequence encoding napBP, a second sequence encoding a second NLS, an in-frame stop codon, a WPRE sequence, and a sequence encoding a polyA signal.

[0425] In some embodiments, the promoter comprises a sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 6; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, from the sequence of SEQ ID NO: 6.

[0426] In some embodiments, the Kozak sequence is gccacc.

[0427] In some embodiments, the NLS, the first NLS, and / or the second NLS comprises a sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 15; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences, whether contiguous or not, compared to the sequence of SEQ ID NO: 15.

[0428] In some embodiments, the sequence encoding an NLS, the first sequence encoding a first NLS, and / or the second sequence encoding a second NLS comprises a sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 8 or 10; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO: 8 or 10.

[0429] In some embodiments, the WPRE sequence comprises a sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 11; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that differ from the sequence of SEQ ID NO: 11, whether contiguous or not.

[0430] In some embodiments, the sequence encoding the polyA signal comprises a sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 12; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that differ from the sequence of SEQ ID NO: 12, whether contiguous or not.

[0431] In some embodiments, the second polynucleotide sequence comprises, from 5′ to 3′, a promoter of SEQ ID NO: 6, a Kozak sequence of gccacc, an in-frame start codon ATG, a first sequence encoding a first NLS of SEQ ID NO: 15, a sequence encoding napBP of SEQ ID NO: 16 or 32, a second sequence encoding a second NLS of SEQ ID NO: 15, an in-frame stop codon, a WPRE sequence of SEQ ID NO: 11, and a sequence encoding a polyA signal of SEQ ID NO: 12.

[0432] In some embodiments, the second polynucleotide sequence comprises, from 5' to 3', a promoter of SEQ ID NO: 6, a Kozak sequence of gccacc, an in-frame start codon ATG, a first sequence of SEQ ID NO: 8 or 10 encoding a first NLS, a sequence of SEQ ID NO: 9 or 33 encoding napBP, a second sequence of SEQ ID NO: 8 or 10 encoding a second NLS, an in-frame stop codon, a WPRE sequence of SEQ ID NO: 11, and a sequence encoding a polyA signal of SEQ ID NO: 12.

[0433] Full-length rAAV vector genome

[0434] In some embodiments, the rAAV vector genome comprises a 5' inverted terminal repeat (ITR) sequence and a 3' ITR sequence.

[0435] In some embodiments, the 5' ITR sequence and the 3' ITR sequence are both wild-type ITR sequences from the following: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, or a member of the evolutionary branch to which any one of AAV1-AAV13 belongs, or a functional variant (e.g., a functional truncation) thereof.

[0436] In some embodiments, the 5' ITR sequence comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 3 or 28.

[0437] In some embodiments, the 3' ITR sequence comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 13 or 29.

[0438] In some embodiments, the rAAV vector genome comprises, from 5' to 3', a first polynucleotide sequence and a second polynucleotide sequence.

[0439] In some embodiments, the rAAV vector genome comprises, from 5' to 3', the second polynucleotide sequence and the first polynucleotide sequence.

[0440] In some embodiments, the rAAV vector genome comprises, from 5' to 3':

[0441] (1) a 5' ITR as described in the present disclosure;

[0442] (2) a promoter as described in the present disclosure operably linked to a sequence encoding a guide nucleic acid;

[0443] (3) a sequence encoding a guide nucleic acid as described in the present disclosure;

[0444] (4) a promoter as described in the present disclosure operably linked to a sequence encoding napBP;

[0445] (5) optionally, a Kozak sequence as described in the present disclosure;

[0446] (6) in-frame start codon ATG;

[0447] (7) optionally, a first sequence encoding a first NLS as described in the present disclosure;

[0448] (8) a sequence encoding napBP as described in the present disclosure;

[0449] (9) optionally, a second sequence encoding a second NLS as described in the present disclosure;

[0450] (10) In-frame stop codon,

[0451] (11) optionally, a WPRE sequence as described in the present disclosure;

[0452] (12) a sequence encoding a polyA signal as described in the present disclosure; and

[0453] (13) A 3' ITR as described in the present disclosure.

[0454] In some embodiments, the rAAV vector genome comprises, from 5' to 3':

[0455] (1) 5' ITR of SEQ ID NO: 3;

[0456] (2) the promoter of SEQ ID NO: 4;

[0457] (3) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0458] (4) a sequence encoding the guide sequence of SEQ ID NO: 1;

[0459] (5) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0460] (6) a sequence encoding the guide sequence of SEQ ID NO: 2;

[0461] (7) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0462] (8) the promoter of SEQ ID NO: 6;

[0463] (9) Kozak sequence of gccacc;

[0464] (10) in-frame start codon ATG;

[0465] (11) a first sequence of SEQ ID NO: 8 encoding the first NLS of SEQ ID NO: 15;

[0466] (12) a sequence of SEQ ID NO: 33 encoding the Cas13 endonuclease of SEQ ID NO: 32;

[0467] (13) a second sequence SEQ ID NO: 10 encoding a second NLS of SEQ ID NO: 15;

[0468] (14) in-frame stop codon;

[0469] (15) WPRE sequence of SEQ ID NO: 11;

[0470] (16) a sequence encoding a polyA signal of SEQ ID NO: 12; and

[0471] (17) 3' ITR of SEQ ID NO: 13;

[0472] The rAAV vector genome comprises an optional linker between any two adjacent components among components (1) to (17).

[0473] In some embodiments, the rAAV vector genome comprises, consists essentially of, or consists of a polynucleotide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to the sequence of SEQ ID NO: 14. In some embodiments, the polynucleotide encodes the napRNA of SEQ ID NO: 32 and the targeting

[0474] Guide RNA for VEGFA mRNA.

[0475] In some embodiments, the rAAV vector genome comprises, from 5' to 3':

[0476] (1) 5' ITR of SEQ ID NO: 3;

[0477] (2) the promoter of SEQ ID NO: 4;

[0478] (3) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0479] (4) a sequence encoding the guide sequence of SEQ ID NO: 1;

[0480] (5) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0481] (6) a sequence encoding the guide sequence of SEQ ID NO: 2;

[0482] (7) a sequence encoding the scaffold sequence of SEQ ID NO: 5;

[0483] (8) the promoter of SEQ ID NO: 6;

[0484] (9) Kozak sequence of gccacc;

[0485] (10) in-frame start codon ATG;

[0486] (11) a first sequence of SEQ ID NO: 8 encoding the first NLS of SEQ ID NO: 15;

[0487] (12) a sequence of SEQ ID NO: 33 encoding the napRNAn of SEQ ID NO: 32;

[0488] (13) a second sequence SEQ ID NO: 10 encoding a second NLS of SEQ ID NO: 15;

[0489] (14) in-frame stop codon;

[0490] (15) WPRE sequence of SEQ ID NO: 11;

[0491] (16) a sequence encoding a polyA signal of SEQ ID NO: 12; and

[0492] (17) 3' ITR of SEQ ID NO: 13;

[0493] wherein the rAAV vector genome comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to the sequence of SEQ ID NO: 14;

[0494] wherein the rAAV vector genome does not comprise any nucleotide difference in any of the components (1) to (17) compared to the sequence of SEQ ID NO: 14;

[0495] wherein the rAAV vector genome comprises one or more nucleotide differences compared to the sequence of SEQ ID NO: 14 or does not comprise any nucleotide differences compared to the sequence of SEQ ID NO: 14 between any two adjacent components among the components (1) to (17).

[0496] In some embodiments, the rAAV vector genome comprises the sequence of SEQ ID NO:14.

[0497] In some embodiments, the rAAV vector genome of the present disclosure is a DNA rAAV vector genome or an RNA rAAV vector genome. "DNA rAAV vector genome" means that the rAAV vector genome is DNA that can be encapsulated into rAAV particles. "RNA rAAV vector genome" means that the rAAV vector genome is RNA that can be encapsulated into rAAV particles.

[0498] rAAV particles

[0499] In another aspect, the present disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the present disclosure. A brief introduction to AAV for delivery can be found in the "Adeno-associated virus (AAV) Guide"

[0500] (addgene.org / guides / aav / ).

[0501] Adeno-associated virus (AAV), when engineered for delivery of, for example, a protein coding sequence of interest, may be referred to as a (r)AAV vector, (r)AAV vector particle, or (r)AAV particle, where "r" stands for "recombinant." Furthermore, the genome packaged in an AAV vector for delivery may be referred to as a (r)AAV vector genome, vector genome, or simply vg, while viral genome may refer to the original viral genome of native AAV.

[0502] The serotype of the rAAV particle capsid can be matched to the type of target cell. For example, Table 2 of WO 2018002719 A1 lists exemplary cell types that can be transduced by the indicated AAV serotypes (incorporated herein by reference).

[0503] In some embodiments, the rAAV particles comprise a capsid having a serotype suitable for delivery to retinal cells. In some embodiments, the rAAV particles comprise a capsid of a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV.PHP.eB, AAV1-AAV13 to which any one belongs, or a functional variant thereof (e.g., a functional truncation), which encapsulates the rAAV vector genome. In some embodiments, the serotype of the capsid is a wild-type serotype or a functional variant thereof. In some embodiments, the serotype of the capsid is a wild-type AAV8 or a functional variant thereof.

[0504] The general principles of rAAV particle production are known in the art. In some embodiments, rAAV particles can be produced using the triple transfection method (described in detail in US Pat. No. 6,001,650).

[0505] Vector titer is usually expressed as vector genomes / ml (vg / ml). In some embodiments, the vector titer is greater than 1×10 9 , higher than 5×10 10 , higher than 1×10 11 , higher than 5×10 11 , higher than 1×10 12 , higher than 5×10 12 or higher than 1×10 13 vg / ml.

[0506] As an alternative to packaging single-stranded (ss) DNA sequences as vector genomes for rAAV particles, systems and methods for packaging RNA sequences as vector genomes into rAAV particles have recently been developed and can be applied herein. See PCT / CN2022 / 075366, which is incorporated herein by reference in its entirety.

[0507] When the vector genome is RNA, such as in PCT / CN2022 / 075366, for simplicity of description and declaration, the sequence elements described herein for the DNA vector genome, when present in the RNA vector genome, should generally be considered to be applicable to the RNA vector genome, except that the deoxyribonucleotides in the DNA sequence are the corresponding ribonucleotides in the RNA sequence (e.g., dT is equivalent to U, and dA is equivalent to A) and / or the elements in the DNA sequence are replaced with corresponding elements having corresponding functions in the RNA sequence, or are omitted because their functions are not required in the RNA sequence, and / or additional elements necessary for the RNA vector genome are introduced.

[0508] As used herein, a coding sequence (e.g., as a sequence element of an rAAV vector genome herein) is interpreted, understood, and considered to cover and encompass both DNA coding sequences and RNA coding sequences. When it is a DNA coding sequence, an RNA sequence can be transcribed from the DNA coding sequence, and optionally a protein can be further translated from the transcribed RNA sequence as needed. When it is an RNA coding sequence, the RNA coding sequence itself can be a functional RNA sequence for use, or an RNA sequence can be generated from the RNA coding sequence (e.g., by RNA processing), or a protein can be translated from the RNA coding sequence.

[0509] For example, a Cas13 coding sequence encoding a Cas13 polypeptide encompasses a Cas13 DNA coding sequence from which the Cas13 polypeptide is expressed (indirectly via transcription and translation) or a Cas13 RNA coding sequence from which the Cas13 polypeptide is translated (directly).

[0510] For example, a gRNA coding sequence encoding a gRNA encompasses a gRNA DNA coding sequence from which the gRNA is transcribed or a gRNA RNA coding sequence that (1) is itself a functional gRNA for use or (2) from which a gRNA is produced (e.g., by RNA processing).

[0511] In some embodiments of the rAAV RNA vector genome, the 5'-ITR and / or 3'-ITR may not be necessary as DNA packaging signals and may be at least partially omitted, but RNA packaging signals may be introduced.

[0512] In some embodiments of the rAAV RNA vector genome, the promoter driving transcription of the DNA sequence may not be necessary and may be at least partially omitted.

[0513] In some embodiments of the rAAV RNA vector genome, the sequence encoding the polyA signal may be unnecessary and may be at least partially omitted, but a polyA tail may be introduced.

[0514] Similarly, other DNA elements of the rAAV DNA vector genome can be omitted or replaced with corresponding RNA elements and / or additional RNA elements can be introduced to adapt the strategy of delivering the RNA vector genome by rAAV particles.

[0515] In another aspect, the present disclosure provides a method for producing rAAV particles, comprising culturing a transgenic plasmid comprising the rAAV vector genome of the present disclosure in a host cell, thereby encapsulating the rAAV vector genome into a capsid of serotype AAV8. In another aspect, the present disclosure provides a cell comprising a transgenic plasmid comprising the rAAV vector genome of the present disclosure. In another aspect, the present disclosure provides the use of a cell comprising a transgenic plasmid comprising the rAAV vector genome of the present disclosure for producing rAAV particles.

[0516] Pharmaceutical composition

[0517] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising (1) a system of the present disclosure or a rAAV particle of the present disclosure and (2) a pharmaceutically acceptable excipient.

[0518] In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration selected from about 1×10 10 vg / mL, 2×10 10 vg / mL, 3×10 10 vg / mL, 4×10 10 vg / mL, 5×10 10 vg / mL, 6×10 10 vg / mL, 7×10 10 vg / mL, 8×10 10 vg / mL, 9×10 10 vg / mL, 1×10 11 vg / mL, 2×10 11 vg / mL, 3×10 11 vg / mL, 4×10 11 vg / mL, 5×10 11 vg / mL, 6×10 11 vg / mL, 7×10 11 vg / mL, 8×10 11 vg / mL, 9×10 11 vg / mL, 1×10 12 vg / mL, 2×10 12 vg / mL, 3×10 12 vg / mL, 4×10 12vg / mL, 5×10 12 vg / mL, 6×10 12 vg / mL, 7×10 12 vg / mL, 8×10 12 vg / mL, 9×10 12 vg / mL, 1×10 13 vg / mL, or a concentration in a numerical range between any two of the aforementioned values, such as about 9×10 10 vg / mL to about 8×10 11 The concentration of vg / mL.

[0519] In some embodiments, the pharmaceutical composition is an injectable formulation, such as a subretinal injection formulation.

[0520] In some embodiments, the volume of the injection is selected from about 1 microliter, 10 microliters, 50 microliters, 100 microliters, 150 microliters, 200 microliters, 250 microliters, 300 microliters, 350 microliters, 400 microliters, 450 microliters, 500 microliters, 550 microliters, 600 microliters, 650 microliters, 700 microliters, 750 microliters, 800 microliters, 850 microliters, 900 microliters, 950 microliters, 1000 microliters, and volumes in a range of values ​​between any two of the foregoing values, for example, a concentration of about 10 microliters to about 750 microliters.

[0521] cell

[0522] In yet another aspect, the present disclosure provides a cell or progeny thereof comprising a guide nucleic acid of the present disclosure, a system of the present disclosure, or an rAAV particle of the present disclosure. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human retinal cell.

[0523] In yet another aspect, the present disclosure provides a cell or progeny thereof comprising a VEGFA gene or transcript thereof modified by a system of the present disclosure, an rAAV particle of the present disclosure, or a method of the present disclosure. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human retinal cell.

[0524] In some embodiments, the cell is not in the body of an organism (such as a human or animal). In some embodiments, the cell is not a human embryonic stem cell. In some embodiments, the cell is not a human germ cell.

[0525] Treatment

[0526] In another aspect, the present disclosure provides a method for preventing, diagnosing and / or treating a VEGFA-related disease in a subject in need thereof, comprising administering to the subject a system of the present disclosure, an rAAV particle of the present disclosure, or a pharmaceutical composition of the present disclosure, wherein napBP modifies the VEGFA gene or its transcript, and wherein modification of the VEGFA gene or its transcript treats the disease. In some embodiments, napBP modifies the transcript of the VEGFA gene, thereby resulting in downregulation of expression of the VEGFA gene, thereby treating the VEGFA-related disease.

[0527] In some embodiments, the VEGFA-related disease is neovascular age-related macular degeneration (nAMD) (also known as wet AMD), diabetic macular edema (DME), or a cancer such as non-squamous non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), glioblastoma multiforme, ovarian cancer, and cervical cancer. In some embodiments, the cancer is a carcinoma, sarcoma, myeloma, leukemia, lymphoma, and mixed tumors. Non-limiting examples of cancers that can be treated by the methods and compositions described herein include cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. Furthermore, the cancer may specifically be of the following histological types, but is not limited thereto: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; hepatocellular carcinoma with cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma with adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic cell carcinoma; oncocytic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; carcinoma of the skin appendages; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; stromal tumor of the ovary, malignant; thecoma, malignant; granulosa cell tumor, malignant; and fibroblastoma, malignant; Sertoli cell carcinoma; Leydig cell carcinoma cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant;Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; goiter of the ovary, malignant; choriocarcinoma; mesonephroblastoma, malignant; angiosarcoma; angioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; neuro Ganglioneuroma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative enteropathy; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; plasmacytoma, colorectal cancer, rectal cancer, and hairy cell leukemia.

[0528] In some embodiments, the VEGFA gene or its transcript is in a eukaryotic cell, eg, a human cell, a non-human primate cell, or a mouse cell, such as a human retinal cell.

[0529] In some embodiments, administering comprises local administration or systemic administration.

[0530] In some embodiments, administration comprises subretinal administration, intrathecal administration, intramuscular administration, intravenous administration, transdermal administration, intranasal administration, oral administration, mucosal administration, intraperitoneal administration, intracranial administration, intracerebroventricular administration, or stereotactic administration.

[0531] In some embodiments, administration is injection or infusion, such as subretinal injection.

[0532] In some embodiments, the subject is a human, a non-human primate, or a mouse.

[0533] In some embodiments, the subject is unresponsive to anti-VEGF therapy, such as an anti-VEGF antibody.

[0534] In some embodiments, the level of the transcript (e.g., mRNA) of the VEGFA gene in the subject is reduced by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more compared to the level of the transcript (e.g., mRNA) of the VEGFA gene in the subject before administration.

[0535] In some embodiments, the level of the expression product of the VEGFA gene (e.g., vascular endothelial growth factor A) in the subject is reduced by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more compared to the level of the expression product of the VEGFA gene (e.g., vascular endothelial growth factor A) in the subject before administration.

[0536] In some embodiments, choroidal neovascularization (CNV) formation is inhibited.

[0537] In some embodiments, the median survival of subjects who have the disease but receive the administration is 5 days, 10 days, 20 days, 30 days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more longer than the median survival of a subject or population of subjects who have the disease and do not receive the administration.

[0538] The dosage of rAAV particles for treating VEGFA-related diseases can be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage can vary greatly depending on a variety of factors, such as vector selection, target cells, organisms, tissues, the general condition of the subject to be treated, the degree of transformation / modification sought, the route of administration, the mode of administration, the type of transformation / modification sought, etc.

[0539] In some embodiments, the rAAV particles are administered at a therapeutically effective dose. For example, a therapeutically effective dose of rAAV particles can be about 1.0E+7 (1.0×10 7), 2.0E+7, 3.0E+7, 4.0E+7, 6.0E+7, 8.0E+7, 1.0E+8, 2.0E+8, 3.0E+8, 4. 0E+8, 6.0E+8, 8.0E+8, 1.0E+9, 2.0E+9, 3.0E+9, 4.0E+9, 6.0E+9, 8.0E+9, 1.0E+10, 2.0E+10, 3.0E+10, 4.0E+10, 6.0E+10, 8.0E+10, 1.0E+11, 2.0E +11, 3.0E+11, 4.0E+11, 6.0E+11, 8.0E+11, 1.0E+12, 2.0E+12, 3.0E+12, 4 +15, 2.0E+15, 3.0E+15, 4.0E+15, 6.0E+15, 8.0E+15, 1.0E+16, 2.0E+16, 3.0E+16, 4.0E+16, 6.0E+16, 8.0E+16, or 1.0E+17 vg, or within the range of any two of these point values. vg represents the number of vector genomes per rAAV particle to be administered. In some embodiments, the therapeutically effective dose of rAAV particles is expressed in terms of vg per eye.

[0540] Reagent test kit

[0541] In yet another aspect, the present disclosure provides a kit comprising a system of the present disclosure, an rAAV particle of the present disclosure, or a pharmaceutical composition of the present disclosure, or any one, two, or all components thereof.

[0542] In some embodiments, the kit further includes instructions for using one or more components contained therein, and / or for combining with one or more additional components that may be obtained or required elsewhere.

[0543] In some embodiments, the kit further comprises one or more buffers that can be used to dissolve any of the components contained therein and / or provide suitable reaction conditions for one or more of the components. Such buffers can include one or more of PBS, HEPES, Tris, MOPS, Na2CO3, NaHCO3, NaB, or a combination thereof. In some embodiments, the reaction conditions include a suitable pH, such as an alkaline pH. In some embodiments, the pH is between 7-10.

[0544] In some embodiments, any one or more of the kit components can be stored in a suitable container or at a suitable temperature, such as 4 degrees Celsius.

[0545] Further embodiments are described in the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0546] Example

[0547] The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the invention; by their exemplary nature it will be understood that other procedures, methods or techniques known to those skilled in the art may be used instead.

[0548] Example 1. Knockdown of VEGFA mRNA in vitro by the napRNAn system

[0549] This example demonstrates the in vitro knockdown efficiency of the disclosed napRNAn system comprising a guide sequence targeting VEGFA mRNA.

[0550] The napRNAn system disclosed herein is composed of napRNAn and guide RNA (gRNA), which includes a scaffold sequence capable of forming a complex with napRNAn (compatible with napRNAn) and a guide sequence designed to hybridize with human VEGFA mRNA. napRNAn is hfCas13Y (containing four mutations: D160A, D642A, Y666A, and Y677A; see WO 2023 / 051734 (PCT / CN2022 / 122833), which is incorporated herein by reference in its entirety). The amino acid sequence of napRNAn is listed in SEQ ID NO: 16, and the polynucleotide encoding napRNAn is listed in SEQ ID NO: 9. The guide sequence is listed in SEQ ID NO: 1 or 2 (g1-g2, respectively), and the corresponding target sequence of VEGFA mRNA is listed in SEQ ID NO: 18 or 19 (T1-T2, respectively). The scaffold sequence is listed in SEQ ID NO: 5. The gRNAs are listed in SEQ ID NO: 30 or 31 (corresponding to g1-g2, respectively) in the Scaffold-g1-Scaffold or Scaffold-g2-Scaffold (SGS) (DR-Spacer-DR) configuration.

[0551] HEK293T cells were transfected with expression plasmids encoding a gRNA targeting VEGFA driven by the U6 promoter and a napRNAn driven by the CBH promoter. An NLS was inserted between the N-terminal Met and the remaining amino acid sequence of the napRNAn. In other words, the NLS coding sequence was inserted between the 5' in-frame start codon ATG of the napRNAn coding sequence and the remaining polynucleotide sequence. The VEGFA knockdown efficiency of the napRNAn system was measured as the fold change in VEGFA mRNA and protein in transfected HEK293T cells.

[0552] hfCas13Y-mediated RNA knockdown of VEGFA mRNA in vitro

[0553] It was observed that the designed gRNA together with hfCas13Y showed high expression and significantly reduced VEGFA expression in vitro.

[0554] Figure 6 and Figure 7 Shown are the results of in vitro gRNA screening targeting VEGFA. Figure 6 A shows a schematic diagram of the designed gRNA (with guide sequences g1-g6) targeting VEGFAmRNA. Figure 6 B shows the knockdown efficiency of hfCas13Y guided by various single gRNAs containing a guide sequence targeting VEGFA (one of g1-g6) in HEK293T cells, wherein gRNA-1 (SEQ ID NO: 30; having a guide sequence g1 of SEQ ID NO: 1) and gRNA-2 (SEQ ID NO: 31; having a guide sequence g2 of SEQ ID NO: 2) exhibited high VEGFA mRNA knockdown efficiency. NT (non-targeting) means that a non-targeting guide sequence that cannot hybridize with VEGFA mRNA is used instead of a VEGFA-targeting guide sequence as a negative control (NC). ***P<0.001.

[0555] The two guide sequences g1 and g2 were then combined to generate a gRNA carrying both g1 and g2 listed in SEQ ID NO: 17, which was in a scaffold-g1-scaffold-g2-scaffold (SGSGS) (DR-spacer-DR-spacer-DR) configuration. The results showed that hfCas13Y together with gRNA (SEQ ID NO: 17) exhibited approximately 100% VEGFA mRNA knockdown efficiency in HEK293T cells ( Figure 6 C). hfCas13Y and gRNA (SEQ ID NO: 17) are both highly expressed in HEK293T cells ( Figure 7A). Compared with the negative control in HEK293T cells, VEGFA mRNA ( Figure 7 B) and protein ( Figure 7 C) Levels were significantly reduced by 86% and 52%, respectively. **P<0.01. ****P<0.0001.

[0556] In vitro off-target analysis

[0557] It was further observed that the combination of hfCas13Y and gRNA (SEQ ID NO: 17) (denoted as "HG202" in the accompanying drawings) showed highly specific targeting and only downregulated VEGFA in vitro, which demonstrated its high safety.

[0558] Figure 8 Off-target analysis is shown. RNA-seq results showed that the HG202 combination specifically targeted and downregulated only VEGFA RNA ( Figure 8 A). Figure 8 B shows indel efficiency at target VEGFA and potential DNA off-target sites. Figure 8 C shows knockdown efficiency at VEGFA and potential RNA off-target sites. NC = negative control. ****P < 0.0001. Each circle or triangle represents one sample.

[0559] Example 2. In vivo knockdown of VEGFA mRNA by the napRNAn system delivered via AAV

[0560] This example demonstrates the in vivo knockdown efficiency of the disclosed AAV-delivered napRNAn system comprising a guide sequence targeting VEGFA mRNA.

[0561] The napRNAn system disclosed herein is composed of napRNAn and gRNA, which includes a scaffold sequence capable of forming a complex with napRNAn (compatible with napRNAn) and two different guide sequences designed to hybridize with human VEGFA mRNA. napRNAn is hfCas13Y (containing four mutations: D160A, D642A, Y666A, and Y677A; see WO 2023 / 051734 (PCT / CN2022 / 122833), which is incorporated herein by reference in its entirety). The amino acid sequence of napRNAn is listed in SEQ ID NO: 16, and the polynucleotide encoding napRNAn is listed in SEQ ID NO: 9. Two different guide sequences are listed in SEQ ID NO: 1-2 (g1-g2), and the corresponding target sequences of VEGFA mRNA are listed in SEQ ID NO: 18-19 (T1-T2). The scaffold sequence is listed in SEQ ID NO: 5. The gRNA is listed in SEQ ID NO: 17 in the configuration of Scaffold-g1-Scaffold-g2-Scaffold (SGSGS)(DR-Spacer-DR-Spacer-DR).

[0562] The control napRNAn system according to PCT / CN2022 / 079890 is used for comparison with the napRNAn system of the present disclosure. The control napRNAn system is composed of napRNAn and gRNA, which includes a scaffold sequence capable of forming a complex with napRNAn (compatible with napRNAn) and two different guide sequences designed to hybridize with human VEGFA mRNA. napRNAn is hfCas13X (containing two mutations Y672A+Y676A; see WO 2022 / 068912 (PCT / CN2021 / 121926), which is incorporated herein by reference in its entirety). The amino acid sequence of napRNAn is listed in SEQ ID NO: 27, and the polynucleotide encoding napRNAn is listed in SEQ ID NO: 20. Two different guide sequences are listed in SEQ ID NO: 1-2 (g1-g2), and the corresponding target sequences are listed in SEQ ID NO: 18-19 (T1-T2). The scaffold sequence is set forth in SEQ ID NO: 23. The gRNA is set forth in SEQ ID NO: 25 in the configuration of Scaffold-g1-Scaffold-g2-Scaffold (SGSGS)(DR-Spacer-DR-Spacer-DR).

[0563] Construction of transgenic plasmids for AAV packaging:

[0564] For the napRNAn system of the present disclosure described above, a therapeutic rAAV transgenic plasmid (designated "HG202") containing a therapeutic rAAV vector genome for packaging into wild-type AAV8 capsids to prepare integrated therapeutic rAAV8 particles was designed to include the elements in Table 1 below from 5' to 3'. An NLS was inserted between the N-terminal Met and the remaining amino acid sequence of napRNAn. In other words, the NLS coding sequence was inserted between the 5' in-frame start codon ATG of the napRNAn coding sequence and the remaining polynucleotide sequence.

[0565] Table 1. Elements of the therapeutic rAAV vector genome for therapeutic rAAV transgene plasmids.

[0566]

[0567] The full length of the 5' ITR to 3' ITR sequence of the therapeutic rAAV vector genome of the therapeutic rAAV transgene plasmid is set forth in SEQ ID NO:14.

[0568] For the control napRNAn system described above, a control rAAV transgenic plasmid (designated "HG203" or "HG-203") containing a control rAAV vector genome for packaging into wild-type AAV9 capsids to produce integrated control rAAV9 particles was designed to include the elements in Table 2 below from 5' to 3'. An NLS was inserted between the N-terminal Met and the remaining amino acid sequence of napRNAn. In other words, the NLS coding sequence was inserted between the 5' in-frame start codon ATG of the napRNAn coding sequence and the remaining polynucleotide sequence.

[0569] Table 2. Elements of the control rAAV vector genome of the control rAAV transgene plasmid.

[0570]

[0571]

[0572] The full length of the 5' ITR to 3' ITR sequence of the control rAAV vector genome of the control rAAV transgenic plasmid is set forth in SEQ ID NO:24.

[0573] rAAV particle preparation:

[0574] Using a conventional three-plasmid transfection system, with necessary modifications, therapeutic rAAV8 particles (HG202) with wild-type AAV8 capsids and control rAAV9 particles (HG203) with wild-type AAV9 capsids were produced by co-transfecting the therapeutic or control rAAV transgene plasmid, packaging plasmid, and helper plasmid into HEK293T cells at a weight ratio of 1:1:2. The therapeutic or control rAAV transgene plasmid was packaged by the AAV8 or AAV9 capsid to form a genome within the capsid, and the genome and AAV8 or AAV9 capsid together constituted the rAAV8 or rAAV9 particles.

[0575] Specifically, HEK293T cells were cultured in competent DMEM medium and plated 24 hours before plasmid transfection. Shortly before transfection, the medium was replaced with fresh DMEM containing 2% FBS.

[0576] PEI-MAX was used as a transfection reagent. Transfected HEK293T cells were harvested from the culture medium 72 hours after translation. rAAV particles were purified from the cells by iodixanol density gradient ultracentrifugation.

[0577] Experimental procedure:

[0578] Unless otherwise indicated herein, the laser-induced CNV mouse model, rAAV particle administration by subretinal injection ( Figure 13 ) and knockdown assays (e.g., inhibition of CNV growth).

[0579] result:

[0580] Comparison of VEGFA knockdown efficiency between HG202 and HG203

[0581] Therapeutic rAAV8 particles HG202 (having a wt AAV8 capsid and an ITR to ITR sequence of SEQ ID NO: 14) differ from control rAAV9 particles HG203 (having a wt AAV9 capsid and an ITR to ITR sequence of SEQ ID NO: 24) in multiple respects, including, among others: the selection of a wt AAV8 capsid rather than a wt AAV9 capsid, the selection of hfCas13Y (and a compatible scaffold sequence) rather than hfCas13X (and a compatible scaffold sequence), the selection of the Cbh promoter (SEQ ID NO: 6) for expression of hfCas13Y rather than the EFS promoter (SEQ ID NO: 18) for expression of hfCas13X, the selection of the NLS and polyA signal, and the selection of the positional relationship of the gRNA expression cassette to the Cas13 expression cassette in the 5'-3' or 3'-5' orientation.

[0582] refer to Figure 5 , the results of in vivo mVEGFA mRNA knockdown showed that:

[0583] (1) At the same dose of 1E9 vg / eye, the knockdown efficiency of mVEGFA mRNA by therapeutic rAAV8 particles (44%, 1E9 vg / eye) was significantly higher than that by control rAAV9 particles (29%, 1E9 vg / eye);

[0584] (2) Even at a dose of approximately 1 / 3.3, the mVEGFA mRNA knockdown efficiency of the therapeutic rAAV8 particles (44%, 3E8 vg / eye) was significantly higher than that of the control rAAV9 particles (29%, 1E9 vg / eye);

[0585] (3) even at approximately 1 / 10 the dose, the mVEGFA mRNA knockdown efficiency of the therapeutic rAAV8 particles (29%, 1E8 vg / eye) was comparable to that of the control rAAV9 particles (29%, 1E9 vg / eye); and

[0586] (4) Even at approximately 1 / 33 the dose, the mVEGFA mRNA knockdown efficiency of the therapeutic rAAV8 particles (30%, 3E7 vg / eye) was comparable to that of the control rAAV9 particles (29%, 1E9 vg / eye).

[0587] Overall, the knockdown results demonstrated that the therapeutic rAAV8 particles HG202 achieved a significant effect of knocking down VEGFA mRNA in vivo, and in particular, achieved significantly improved knockdown efficiency compared to the control rAAV9 particles HG203, as reflected by significantly higher knockdown efficiency at about 1 / 3.3 doses and comparable knockdown efficiency at about 1 / 10 doses to about 1 / 33 doses, which also highlights the superiority of the therapeutic rAAV8 particles HG202 over the control rAAV9 particles.

[0588] HG203 has a higher safety profile than HG203 because a lower dose can be used due to its effectiveness.

[0589] HG202-mediated RNA knockdown of Vegfa mRNA in vivo

[0590] HG202 was observed to highly express hfCas13Y mRNA and downregulate Vegfa mRNA in the eyes of treated mice.

[0591] Figure 9In vivo VEGFA knockdown is shown. Subretinal injection of different doses of HG202 resulted in significant dose-related expression of hfCas13Y mRNA in the eyes of treated mice, while a dose-dependent reduction in Vegfa mRNA expression was observed. Dose 1 = 3E+7 vg / eye; Dose 2 = 1E+8 vg / eye; Dose 3 = 3E+8 vg / eye; Dose 4 = 1E+9 vg / eye.

[0592] HG202 inhibits CNV (choroidal neovascularization) in mice

[0593] Figure 10 Figure 3 shows the reduction of CNV by HG202 in vivo. At week 8, the area of ​​laser-induced CNV was significantly reduced in the eyes of mice treated with HG202 ( Figure 10 A) Furthermore, HG202 reduced CNV significantly better at all doses (59% to 87%) than the anti-VEGF agent aflibercept (47%). Figure 10 B is a representative image of CNV staining at different doses of HG202. *P < 0.05; **P < 0.01; ****P < 0.0001.

[0594] HG202 is well tolerated in mice

[0595] It was observed that HG202 had no significant effects on retinal structure and function at effective doses.

[0596] Figure 11 The safety of HG202 was shown. OCT results showed that there was no significant effect on retinal structure 8 weeks after subretinal injection of HG202 at doses 1-3. Figure 11 A). OCT ( Figure 11 B) and ERG( Figure 11 C) Results show no significant changes in retinal structure and function 6 months after subretinal injection of HG202 at a dose of 3. NS = not significant; DA = dark adaptation.

[0597] This suggests that the hfCas13Y system, delivered with rAAV8 particles targeting VEGFA mRNA, may be a promising therapy for treating VEGFA-related diseases such as wet AMD and diabetic macular edema (DME). A single subretinal injection of HG202 enables long-term transgene expression and RNA editing, eliminating the need for 1- or 2-month repeat injections and providing lasting therapeutic benefit to patients (with or without anti-VEGF antibodies) for the treatment of nAMD and DME.

[0598] ***

[0599] Various modifications and variations of the products, methods and uses of the present disclosure will be clear to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in conjunction with specific embodiments, it will be understood that further modifications are possible and that the disclosure claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described manner for carrying out the present disclosure that are obvious to those skilled in the art are intended to fall within the scope of the present disclosure. This application is intended to cover any variation, use or adaptation of the present disclosure that generally follows the principles of the present disclosure, and includes such deviations from the present disclosure as are within the known customary practices in the art to which the present disclosure pertains and are applicable to the basic features heretofore shown.

[0600] Exemplary sequences

[0601] Guide sequence, SEQ ID NO: 1-2

[0602]

[0603]

[0604] AAV2 5' ITR sequence, SEQ ID NO: 3

[0605] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0606] U6 promoter sequence, SEQ ID NO: 4

[0607] GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG

[0608] hfCas13Y scaffold sequence, SEQ ID NO: 5

[0609] GCTGTGATAGACCTCGATTTGTGGGGTAGTAACAGC

[0610] Cbh promoter, SEQ ID NO:6

[0611] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGG

[0612] Kozak sequence, SEQ ID NO:7

[0613] GCCACC

[0614] bpSV40 NLS coding sequence 1, SEQ ID NO: 8

[0615] AAGCGCACAGCCGACGGCAGCGAGTTTGAAAGCCCTAAGAAGAAAAGAAA GGTG

[0616] napRNAn (hfCas13Y) coding sequence, SEQ ID NO: 9

[0617]

[0618] bpSV40 NLS coding sequence 2, SEQ ID NO:10

[0619] AAGAGAACCGCCGACGGCTCTGAGTTCGAAAGTCCTAAGAAGAAGCGGAA GGTG

[0620] WPRE3 sequence, SEQ ID NO:11

[0621] AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTT

[0622] Sequence encoding bGH polyA signal, SEQ ID NO:12

[0623] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0624] AAV2 3' ITR sequence, SEQ ID NO:13

[0625] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0626] ITR to ITR sequence, SEQ ID NO: 14

[0627]

[0628]

[0629]

[0630] bpSV40 NLS amino acid sequence, SEQ ID NO: 15

[0631] KRTADGSEFESPKKKRKV

[0632] napRNAn (hfCas13Y) amino acid sequence, SEQ ID NO: 16

[0633] MNGIELKKEEAAFYFNQAELNLKAIEDNIFDKERRKTLLNNPQILAKMENFIFNFRDVTKNAKGEIDCLLLKLRELRNFYSHYVHKRDVRELSKGEKPILEKYYQFAIESTGSENVKLEIIENDAWLADAGVLFFLCIFLKKSQANKLISGISGFKRNDATGQPRRNLFTYFSIREGYKVVPEMQKHFLLFSLVNHLSNQDDYIEKAHQPYDIGEGLFFHRIASTFLNISGILRNMKFYTYQSKRLVEQRGELKREKDIFAWEEPFQGNSYFEINGHKGVIGEDELKELCYAFLIGNQDANKVEGRITQFLEKFRNANSVQQVKDDEMLKPEYFPANYFAESGVGRIKDRVLNRLNKAIKSNKAKKGEIIAYDKMREVMAFINNSLPVDEKLKPKDYKRYLGMVRFWDREKDNIKREFETKEWSKYLPSNFWTAKNLERVYGLAREKNAELFNKLKADVEKMDERELEKYQKINDAKDLANLRRLASDFGVKWEEKDWDEYSGQIKKQITDSQKLTIMKQRITAGLKKKHGIENLNLRITIDINKSRKAVLNRIAIPRGFVKRHILGWQESEKVSKKIREAECEILLSKEYEELSKQFFQSKDYDKMTRINGLYEKNKLIALMAVYLMGQLRILFKEHTKLADITKTTVDFKISDKVTVKIPFSNAPSLVYTMSSKAVDNIGNYGFSNKDKDKPILGKIDVIEKQRMEFIKEVLGFEKYLFDDKIIDKSKFADTATHISFAEIVEELVEKGWDKDRLTKLKDARNKALHGEILTGTSFDETKSLINELKK

[0634] gRNA (S-g1-S-g2-S) of hfCas13Y, SEQ ID NO:17

[0635]

[0636] EFS promoter, SEQ ID NO:18

[0637] TAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGGTTGG

[0638] SV40 coding sequence, SEQ ID NO:19

[0639] CCCAAGAAGAAGCGGAAGGTG

[0640] napRNAn(hfCas13X) coding sequence, SEQ ID NO:20

[0641]

[0642] Sequence encoding the SV40 polyA signal, SEQ ID NO:21

[0643] AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATC

[0644] U6 promoter, SEQ ID NO:22

[0645] GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG

[0646] hfCas13X scaffold sequence, SEQ ID NO:23

[0647] GCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC

[0648] ITR to ITR (control), SEQ ID NO:24

[0649]

[0650] gRNA(S-g1-S-g2-S), SEQ ID NO:25

[0651]

[0652] SV40 NLS amino acid sequence, SEQ ID NO: 26

[0653] PKKKRKV

[0654] napRNAn (hfCas13X) amino acid sequence, SEQ ID NO: 27

[0655] MAQVSKQTSKKRELSIDEYQGARKWCFTIAFNKALVNRDKNDGLFVESLLRHEKYSKHDWYDEDTRALIKCSTQAANAKAEALRNYFSHYRHSPGCLTFTAEDELRTIMERAYERAIFECRRRETEVIIEFPSLFEGDRITTAGVVFFVSFFVERRVLDRLYGAVSGLKKNEGQYKLTRKALSMYCLKDSRFTKAWDKRVLLFRDILAQLGRIPAEAYEYYHGEQGDKKRANDNEGTNPRHKDKFIEFALHYLEAQHSEICFGRRHIVREEAGAGDEHKKHRTKKGVVDFSKKDEDQSYIISKNNVIVRIDKNAGPRSYRMGLNELKYLVLLSLQGKGDDAIAKLYRYRQHVENILDVVKVTDKDNHVFLPRFVLEQHGIGRKAF KQRIDGRVKHVRGVWEKKKAATNEMTLHEKARDILQYVNENCTRSFNPGEYNRLLVCLVGKDVENFQAGLKRLQLAERIDGRVYSIFAQTSTINEMHQVVCDQILNRLCRIGDQKLYDYVGLGKKDEIDYKQKVAWFKEHISIRRGFLRKKFWYDSKKGFAKLVEEHLESGGGQRDVGLDKKYYHIDAIGRFEGANPALYETLARDLCLMMAQYFLGSVRKELGNKIVWSNDSIELPVEGSVGNEKSIVFSVSDYGKLYVLDDAEFLGRICEYFMPHEKGKIRAHTVAEKGFRAYNDLQKKCVEAVLAFEEKVVKAKKMSEKEGAHYIDFREILAQTMCKEAEKTAVNKVRRAFFHHHLKFVIDEFGLFSDVMKKYGIEKEWKFPVK

[0656] 5'ITR,SEQ ID NO:28

[0657] CCTGCAGGCAGCTGCGGCCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0658] 3' ITR, SEQ ID NO: 29

[0659] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0660] The amino acid sequence of napRNAn (hfCas13Y) with N-terminal Met removed, SEQ ID NO: 32

[0661] NGIELKKEEAAFYFNQAELNLKAIEDNIFDKERRKTLLNNPQILAKMENFIFNFRDVTKNAKGEIDCLLLKLRELRNFYSHYVHKRDVRELSKGEKPILEKYYQFAIESTGSENVKLEIIENDAWLADAGVLFFLCIFLKKSQANKLISGISGFKRNDATGQPRRNLFTYFSIREGYKVVPEMQKHFLLFSLVNHLSNQDDYIEKAHQPYDIGEGLFFHRIASTFLNISGILRNMKFYTYQSKRLVEQRGELKREKDIFAWEEPFQGNSYFEINGHKGVIGEDELKELCYAFLIGNQDANKVEGRITQFLEKFRNANSVQQVKDDEMLKPEYFPANYFAESGVGRIKDRVLNRLNKAIKSNKAKKGEIIAYDKMREVMAFINNSLPVDEKLKPKDYKRYLGMVRFWDREKDNIKREFETKEWSKYLPSNFWTAKNLERVYGLAREKNAELFNKLKADVEKMDERELEKYQKINDAKDLANLRRLASDFGVKWEEKDWDEYSGQIKKQITDSQKLTIMKQRITAGLKKKHGIENLNLRITIDINKSRKAVLNRIAIPRGFVKRHILGWQESEKVSKKIREAECEILLSKEYEELSKQFFQSKDYDKMTRINGLYEKNKLIALMAVYLMGQLRILFKEHTKLADITKTTVDFKISDKVTVKIPFSNAPSLVYTMSSKAVDNIGNYGFSNKDKDKPILGKIDVIEKQRMEFIKEVLGFEKYLFDDKIIDKSKFADTATHISFAEIVEELVEKGWDKDRLTKLKDARNKALHGEILTGTSFDETKSLINELKK

[0662] The coding sequence of napRNAn (hfCas13Y) with the 5' start codon ATG removed, SEQ ID NO: 33

[0663]

[0664] Amino acid sequence of napRNAn (hfCas13X) with the N-terminal Met removed, SEQ ID NO:34

[0665] AQVSKQTSKKRELSIDEYQGARKWCFTIAFNKALVNRDKNDGLFVESLLRHEKYSKHDWYDEDTRALIKCSTQAANAKAEALRNYFSHYRHSPGCLTFTAEDELRTIMERAYERAIFECRRRETEVIIEFPSLFEGDRITTAGVVFFVSFFVERRVLDRLYGAVSGLKKNEGQYKLTRKALSMYCLKDSRFTKAWDKRVLLFRDILAQLGRIPAEAYEYYHGEQGDKKRANDNEGTNPKRHKDKFIEFALHYLEAQHSEICFGRRHIVREEAGAGDEHKKHRTKGKVVVDFSKKDEDQSYYISKNNVIVRIDKNAGPRSYRMGLNELKYLVLLSLQGKGDDAIAKLYRYRQHVENILDVVKVTDKDNHVFLPRFVLEQHGIGRKAFKQRIDGRVKHVRGVWEKKKAATNEMTLHEKARDILQYVNENCTRSFNPGEYNRLLVCLVGKDVENFQAGLKRLQLAERIDGRVYSIFAQTSTINEMHQVVCDQILNRLCRIGDQKLYDYVGLGKKDEIDYKQKVAWFKEHISIRRGFLRKKFWYDSKKGFAKLVEEHLESGGGQRDVGLDKKYYHIDAIGRFEGANPALYETLARDRLCLMMAQYFLGSVRKELGNKIVWSNDSIELPVEGSVGNEKSIVFSVSDYGKLYVLDDAEFLGRICEYFMPHEKGKIRAHTVAEKGFRAYNDLQKKCVEAVLAFEEKVVKAKKMSEKEGAHYIDFREILAQTMCKEAEKTAVNKVRRAFFHHHLKFVIDEFGLFSDVMKKYGIEKEWKFPVK

[0666] Coding sequence of napRNAn (hfCas13X) with the 5'-start codon ATG removed, SEQ ID NO:35

[0667]

Claims

1. A system comprising: (1) a guide nucleic acid comprising c) a scaffold sequence capable of forming a complex with the Cas13 endonuclease, and d) a guide sequence capable of hybridizing to a target sequence on a transcript of the VEGFA gene, thereby directing the complex to the transcript, or a polynucleotide encoding the guide nucleic acid; and (2) the Cas13 endonuclease or a polynucleotide encoding the Cas13 endonuclease; wherein the Cas13 endonuclease comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 16 or 32, comprises substitutions D160A, D642A, Y666A, and Y677A relative to a wild-type version of the Cas13 endonuclease of SEQ ID NO: 16, and has a guide sequence-specific RNA endonuclease activity substantially the same (e.g., at least about 80%, 90%, 95%, 99% or more) as the Cas13 endonuclease of SEQ ID NO: 16; wherein the guide sequence comprises (1) the sequence of SEQ ID NO: 1 or 2, or a 5' or 3' truncation thereof which is truncated by 1, 2, 3, 4, 5 or 6 nucleotides at the 5' or 3' end; or (2) a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO: 1 or 2, or a 5' or 3' truncation thereof which is truncated by 1, 2, 3, 4, 5 or 6 nucleotides at the 5' or 3' end; or (3) a sequence having at most 1, 2, 3, 4, 5 or 6 nucleotides that differ from SEQ ID NO: 1 or 2, whether contiguous or not.

2. The system of any preceding claim, wherein the scaffold sequence has a secondary structure that is substantially identical to the secondary structure of the sequence of SEQ ID NO: 5; or wherein the scaffold sequence comprises (1) the sequence of SEQ ID NO: 5, or a 5' or 3' truncation thereof that is truncated by 1, 2, 3, 4, 5, or 6 nucleotides at the 5' or 3' end; or (2) a sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 5, or a 5' or 3' truncation thereof that is truncated by 1, 2, 3, 4, 5, or 6 nucleotides at the 5' or 3' end; or (3) a sequence that differs from SEQ ID NO: 5 by at most 1, 2, 3, 4, 5, or 6 nucleotides, whether contiguous or not.

3. The system of any preceding claim, wherein the guide nucleic acid comprises from 5' to 3' one said scaffold sequence, one said guide sequence and one said scaffold sequence, wherein the scaffold sequences are the same or different.

4. The system of any preceding claim, wherein the guide nucleic acid comprises from 5' to 3' one said scaffold sequence, one said guide sequence, one said scaffold sequence, one said guide sequence and one said scaffold sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

5. The system of any preceding claim, wherein the guide nucleic acid comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of any one of SEQ ID NOs: 17 and 30-31; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of any one of SEQ ID NOs: 17 and 30-31.

6. The system of any preceding claim, wherein the transcript of the VEGFA gene is VEGFA pre-mRNA or VEGFA mRNA.

7. The system of any preceding claim, wherein the VEGFA gene is a human VEGFA gene.

8. A recombinant adeno-associated virus (rAAV) vector genome comprising: (1) a first polynucleotide sequence comprising a sequence containing or encoding a guide nucleic acid as defined in any preceding claim; and (2) a second polynucleotide sequence comprising a sequence encoding a Cas13 endonuclease as defined in any preceding claim, The rAAV vector genome is suitable for encapsulation into rAAV particles.

9. The rAAV vector genome of any preceding claim, wherein the first polynucleotide sequence comprises a U6 promoter (e.g., SEQ ID NO: 4 or 22) operably linked to a sequence comprising or encoding the guide nucleic acid.

10. The rAAV vector genome of any preceding claim, wherein the second polynucleotide sequence comprises a Cbh promoter operably linked to a sequence encoding the Cas13 endonuclease (e.g., SEQ ID NO: 6).

11. The rAAV vector genome of any preceding claim, wherein the second polynucleotide comprises a first sequence encoding a first NLS at the 5' end of the sequence encoding the Cas13 endonuclease and / or a second sequence encoding a second NLS at the 3' end of the sequence encoding the Cas13 endonuclease.

12. The rAAV vector genome of any preceding claim, wherein the first NLS and / or the second NLS is an SV40 NLS (e.g., SEQ ID NO: 26), a bpSV40 NLS (e.g., SEQ ID NO: 15), or a nucleoplasmin NLS (npNLS).

13. The rAAV vector genome of any preceding claim, wherein the second polynucleotide sequence comprises a WPRE sequence downstream of the sequence encoding the Cas13 endonuclease; optionally wherein the WPRE sequence is selected from the group consisting of a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), WPRE3 (e.g., SEQ ID NO: 11), and functional variants thereof (e.g., functional truncations).

14. The rAAV vector genome of any preceding claim, wherein the second polynucleotide sequence comprises a sequence encoding a bGH polyA signal downstream of a sequence encoding the Cas13 endonuclease (e.g., SEQ ID NO: 12).

15. The rAAV vector genome of any preceding claim, wherein the second polynucleotide sequence comprises, from 5′ to 3′, a promoter of SEQ ID NO: 6, a Kozak sequence of gccacc, an in-frame start codon ATG, a first sequence encoding a first NLS of SEQ ID NO: 15, a sequence encoding a Cas13 endonuclease of SEQ ID NO: 16 or 32, a second sequence encoding a second NLS of SEQ ID NO: 15, an in-frame stop codon, a WPRE sequence of SEQ ID NO: 11, and a sequence encoding the polyA signal of SEQ ID NO:

12.

16. The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises, from 5' to 3', the first polynucleotide sequence and the second polynucleotide sequence; or comprises, from 5' to 3', the second polynucleotide sequence and the first polynucleotide sequence.

17. The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises from 5' to 3': (1) 5' ITR of SEQ ID NO: 3; (2) the promoter of SEQ ID NO: 4; (3) a sequence encoding the scaffold sequence of SEQ ID NO: 5; (4) a sequence encoding the guide sequence of SEQ ID NO: 1; (5) a sequence encoding the scaffold sequence of SEQ ID NO: 5; (6) a sequence encoding the guide sequence of SEQ ID NO: 2; (7) a sequence encoding the scaffold sequence of SEQ ID NO: 5; (8) the promoter of SEQ ID NO: 6; (9) Kozak sequence of gccacc; (10) in-frame start codon ATG; (11) a first sequence of SEQ ID NO: 8 encoding the first NLS of SEQ ID NO: 15; (12) a sequence of SEQ ID NO: 33 encoding the Cas13 endonuclease of SEQ ID NO: 32; (13) a second sequence SEQ ID NO: 10 encoding a second NLS of SEQ ID NO: 15; (14) in-frame stop codon; (15) WPRE sequence of SEQ ID NO: 11; (16) a sequence encoding a polyA signal of SEQ ID NO: 12; and (17) 3' ITR of SEQ ID NO: 13; The rAAV vector genome comprises an optional linker between any two adjacent components among components (1) to (17).

18. The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises, consists essentially of, or consists of a polynucleotide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to the sequence of SEQ ID NO:

14.

19. The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises from 5' to 3': (1) 5' ITR of SEQ ID NO: 3; (2) the promoter of SEQ ID NO: 4; (3) a sequence encoding the scaffold sequence of SEQ ID NO: 5; (4) a sequence encoding the guide sequence of SEQ ID NO: 1; (5) a sequence encoding the scaffold sequence of SEQ ID NO: 5; (6) a sequence encoding the guide sequence of SEQ ID NO: 2; (7) a sequence encoding the scaffold sequence of SEQ ID NO: 5; (8) the promoter of SEQ ID NO: 6; (9) Kozak sequence of gccacc; (10) in-frame start codon ATG; (11) a first sequence of SEQ ID NO: 8 encoding the first NLS of SEQ ID NO: 15; (12) a sequence of SEQ ID NO: 33 encoding the Cas13 endonuclease of SEQ ID NO: 32; (13) a second sequence SEQ ID NO: 10 encoding a second NLS of SEQ ID NO: 15; (14) in-frame stop codon; (15) WPRE sequence of SEQ ID NO: 11; (16) a sequence encoding a polyA signal of SEQ ID NO: 12; and (17) 3' ITR of SEQ ID NO: 13; wherein the rAAV vector genome comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to the sequence of SEQ ID NO: 14; wherein the rAAV vector genome does not comprise any nucleotide difference in any of the components (1) to (17) compared to the sequence of SEQ ID NO: 14; wherein the rAAV vector genome comprises one or more nucleotide differences compared to the sequence of SEQ ID NO: 14 or does not comprise any nucleotide differences compared to the sequence of SEQ ID NO: 14 between any two adjacent components among the components (1) to (17).

20. The rAAV vector genome of any preceding claim, wherein the rAAV vector genome comprises, consists essentially of, or consists of the sequence of SEQ ID NO:

14.

21. A recombinant AAV (rAAV) particle comprising the rAAV vector genome of any preceding claim.

22. The rAAV particle of any preceding claim, wherein the rAAV particle comprises a capsid of serotype AAV8.

23. The rAAV particle of any preceding claim, wherein the AAV8 is wild-type AAV8 or a functional variant thereof.

24. A method for producing rAAV particles, comprising culturing a transgenic plasmid comprising the rAAV vector genome of any preceding claim in a host cell, thereby encapsidating the rAAV vector genome into a capsid of serotype AAV8.

25. A cell comprising a transgenic plasmid comprising the rAAV vector genome of any preceding claim.

26. Use of a cell comprising a transgenic plasmid comprising the rAAV vector genome of any preceding claim for producing rAAV particles.

27. A pharmaceutical composition comprising (1) the system of any preceding claim or the rAAV particle of any preceding claim and (2) a pharmaceutically acceptable excipient.

28. A method of downregulating expression of a VEGFA gene, comprising contacting a transcript of the VEGFA gene with the system of any preceding claim, the rAAV particle of any preceding claim, or the pharmaceutical composition of any preceding claim.

29. A cell or progeny thereof comprising the system of any preceding claim or the rAAV particle of any preceding claim.

30. A cell or progeny thereof comprising a VEGFA transcript modified by the system of any preceding claim, the rAAV particle of any preceding claim, or the method of any preceding claim.

31. A method for preventing, diagnosing and / or treating a VEGFA-related disease in a subject in need thereof, comprising administering to the subject the system of any preceding claim, the rAAV particle of any preceding claim, or the pharmaceutical composition of any preceding claim, wherein the Cas13 endonuclease modifies the transcript of the VEGFA gene, thereby resulting in downregulation of expression of the VEGFA gene, thereby treating the VEGFA-related disease.

32. The method of any preceding claim, wherein the VEGFA-related disease is neovascular age-related macular degeneration (nAMD), diabetic macular edema (DME), or cancer.

33. The method of any preceding claim, wherein the subject is unresponsive to anti-VEGF therapy, such as an anti-VEGF antibody.

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