WO2024213161A1

WO2024213161A1 - Guide nucleic acids targeting hbv and uses thereof

Info

Publication number: WO2024213161A1
Application number: PCT/CN2024/087814
Authority: WO
Inventors: Yingsi ZHOU
Original assignee: Huidagene Therapeutics Co Ltd; Huidagene Therapeutics Singapore Pte Ltd
Current assignee: Huidagene Therapeutics Co Ltd; Huidagene Therapeutics Singapore Pte Ltd
Priority date: 2023-04-14
Filing date: 2024-04-15
Publication date: 2024-10-17
Anticipated expiration: 2025-10-14

Abstract

Provided are guide nucleic acids targeting HBV cccDNA or transcript thereof, systems or LNP comprising or encoding the guide nucleic acids, and methods of using the same, e.g., method for treating HBV associated diseases.

Description

GUIDE NUCLEIC ACIDS TARGETING HBV AND USES THEREOF

REFERENCE TO RELATED APPLICATIONS

The instant application claims the priority to and the benefit of the filing date of PCT/CN2023/088478, filed on April 14, 2023, PCT/CN2023/091684, filed on April 28, 2023, PCT/CN2023/134857, filed on November 28, 2023, PCT/CN2023/142117, filed on December 26, 2023, and PCT/CN2024/079317, filed on February 29, 2024, the entire contents of which, including any drawings and sequence listing, are incorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The disclosure contains a Sequence Listing XML file which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 15, 2024, by software “WIPO Sequence” according to WIPO Standard ST. 26, is named HGP033PCT, and is 491, 227 bytes in size.

According to WIPO Standard ST. 26, symbol “t” is used to denote both T in DNA and U in RNA. Thus, in the instant sequence listing prepared according to ST. 26, wherever a sequence is an RNA, the T in the sequence shall be deemed as U.

BACKGROUND

Hepatitis B virus (HBV) is the causative agent of hepatitis B liver infection (which is also referred to as “hepatitis B” ) . After infection of Hepatitis B virus (HBV) into cells, the HBV DNA packaged in HBV is released and converted to covalently-closed-circular DNA (cccDNA) in the nucleus of the infected cells, which functions as templates for transcription of viral RNA (e.g., pregenomic mRNA, subgenomic mRNA) , translation of viral proteins (e.g., HBV surface antigen HBsAg, HBV core antigen HBcAg, HBV polymerase) , and reverse transcription of HBV DNA for the assembly of infectious HBV.

Current aim of treatment of HBV infection is undetectable HBV DNA and sustained loss of hepatitis surface antigen (HBsAg) after treatment ends. However, although current antiviral therapies with nucleoside analogues inhibit replication of HBV DNA, they fail to directly target cccDNA and reduce or eliminate cccDNA as well as cure HBV infection. An efficient cure of HBV infection will require elimination of the HBV cccDNA, which is hidden in nucleus, stable, and has a very long half-life. It would be desired to develop products and methods to treat HBV associated diseases on a more fundamental basis by directly functioning on cccDNA.

Citation or identification of any document in the disclosure is not an admission that such a document is available as prior art to the disclosure. Each of the references mentioned or cited in the disclosure is incorporated by reference in its entirety.

SUMMARY

Provided in the disclosure are products and methods for the treatment of HBV associated diseases, such as, Hepatis B, by targeting and modifying HBV cccDNA through the use of CRISPR-Cas12 systems suitable for delivery by lipid nanoparticle (LNP) . Guide sequences are designed to target cccDNA so as to guide a Cas12 polypeptide or a fusion comprising a Cas12 polypeptide and a functional domain to the cccDNA, which then functions on the cccDNA in a guide sequence-specific manner, leading to a decreased level of HBV infection biomarkers including HBV cccDNA, HBV DNA, and HBsAg and providing a promising treatment for treating and even curing HBV associated diseases. The HBV cccDNA-targeting CRISPR-Cas12 systems of the disclosure have high editing efficiency that in some embodiments are better than Cas9 and Cpf1 systems.

In an aspect, the disclosure provides a system comprising:

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

(a) a scaffold sequence capable of forming a complex with a Cas12i endonuclease, and

(b) a guide sequence capable of hybridizing to a target sequence on a target strand of an HBV cccDNA, thereby guiding the complex to the HBV cccDNA; and

(2) the Cas12i endonuclease or a polynucleotide encoding the Cas12i endonuclease;

wherein the guide sequence comprises (1) a sequence of SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 1-92 and 133-547; and

wherein the Cas12i endonuclease comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 94 or a N-terminal truncation thereof without the first N-terminal Methionine.

In some embodiments, the Cas12i endonuclease comprises substitutions N243R+E336R+D892R relative to the wild type of the Cas12 endonuclease of SEQ ID NO: 94, and 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: 94.

In some embodiments, the scaffold sequence has substantially the same secondary structure as the secondary structure of the sequence of SEQ ID NO: 93 or 129; or wherein the scaffold sequence or the additional scaffold sequence comprises (1) a sequence of SEQ ID NO: 93 or 129 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 93 or 129 or a 5’ or 3’end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO:93 or 129.

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

In some embodiments, the guide nucleic acid is a guide RNA, wherein the scaffold sequence of the guide RNA is modified to comprises a 2’-O-methyl 3’phosphorothioate modification on each of its first three 5’ end nucleotides.

In some embodiments, the guide nucleic acid is a guide RNA, wherein the guide RNA comprises a modified 3’ poly U tail comprising, consisting essentially of, or consisting of four uracils with 2’-O-methyl 5’ phosphorothioate modifications on its first three 5’ end uracils.

In some embodiments, the guide nucleic acid comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of any one of SEQ ID NOs: 103-105 and 118-120; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of any one of SEQ ID NOs: 103-105 and 118-120.

In some embodiments, the system comprises two, three, or more guide nucleic acids, each of the guide nucleic acids comprising:

(1) a scaffold sequence capable of forming a complex with a Cas12i endonuclease, and

(2) a guide sequence capable of hybridizing to a target sequence on a target strand of an HBV cccDNA, thereby guiding the complex to the HBV cccDNA,

wherein the guide sequence comprises (1) a sequence of SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% to SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 1-92 and 133-547;

wherein the guide sequences of the guide nucleic acids are different.

In some embodiments, wherein the system comprises three guide nucleic acids comprising guide sequences of SEQ ID NOs: 1, 2, and 3, respectively.

In some embodiments, the polynucleotide encoding the Cas12i endonuclease is a mRNA.

In some embodiments, the mRNA encoding the Cas12i endonuclease comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 102.

In some embodiments, the editing efficiency for the protospacer sequence, the target sequence, or the guide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

In another aspect, the disclosure provides a method of modifying an HBV cccDNA, comprising contacting the HBV cccDNA with a system of the disclosure.

In some embodiments, guiding the complex to the HBV cccDNA enables the Cas12i endonuclease to specifically cleave the HBV cccDNA in a guide sequence-specific manner.

In some embodiments, the specific cleavage of the HBV cccDNA leads to degradation of the HBV cccDNA.

In some embodiments, the specific cleavage of the HBV cccDNA generates an in-frame stop codon in the HBV cccDNA.

In yet another aspect, the disclosure provides a lipid nanoparticle (LNP) comprising the system of the disclosure.

In some embodiments, the lipid mixture for preparing the LNP comprises ALC-0315, Cholesterol, DMG-PEG, and DSPC.

In some embodiments, the lipid mixture for preparing the LNP comprises about 50 mM of ALC-0315, about 50 mM of Cholesterol, about 10 mM of DMG-PEG, and about 20 mM of DSPC.

In some embodiments, the nucleic acid (e.g., RNA) mixture for preparing the LNP comprises the polynucleotide encoding the Cas12i endonuclease and the guide nucleic acid.

In some embodiments, the nucleic acid (e.g., RNA) mixture for preparing the LNP comprises the polynucleotide encoding the Cas12i endonuclease and the guide nucleic acid in a ratio of about 1: 1, 1: 1.1, 1: 1.2, 1: 1.3, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.7, 1: 1.8, 1: 1.9, or about 1: 2.

In some embodiments, the lipid mixture and the nucleic acid (e.g., RNA) mixture is packaged in a ratio of about 1: 1, 1: 2 1: 3, 1: 4, or about 1: 5.

In yet another aspect, the disclosure provides a pharmaceutical composition comprising (1) the system of the disclosure or the LNP of the disclosure and (2) a pharmaceutically acceptable excipient.

In yet another aspect, the disclosure provides a cell or a progeny thereof comprising the guide nucleic acid of the disclosure, the system of the disclosure, or the LNP of the disclosure.

In yet another aspect, the disclosure provides a cell or a progeny thereof comprising HBV cccDNA or transcript thereof modified by the system of the disclosure or the method of the disclosure.

In yet another aspect, the disclosure provides a method for preventing, diagnosing, and/or treating an HBV associated disease in a subject in need thereof, comprising administering to the subject the system of the disclosure, the LNP of the disclosure, or the pharmaceutical composition of the disclosure, wherein the Cas12i endonuclease modifies HBV cccDNA, and wherein the modification of the HBV cccDNA treats the disease.

In some embodiments, the HBV associated disease is selected from the group consisting of Hepatitis B, acute hepatitis B, chronic hepatitis B (CHB) , cirrhosis, hepatocellular carcinoma (HCC) , liver cancer, and liver failure.

In yet another aspect, the disclosure provides a guide nucleic acid, or a polynucleotide (e.g., a DNA, an RNA, a DNA/RNA mixture) encoding the guide nucleic acid, comprising a guide sequence capable of hybridizing to a target sequence on a target strand of an HBV cccDNA or a target sequence on a transcript of an HBV cccDNA, thereby guiding the complex to the HBV cccDNA or the transcript;

wherein the guide sequence comprises (1) a sequence of SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 1-92 and 133-547.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the disclosure will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. It is understood that any aspect or embodiment of the disclosure can be combined with any other one or more aspects or embodiments of the disclosure, including aspects or embodiments only described in one sub-section, only in the examples, or only in the claims, to constitute another embodiment explicitly or implicitly disclosed herein unless otherwise indicated.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure may be utilized, and the accompanying drawings of which:

FIG. 1 is a schematic showing an exemplary target dsDNA, an exemplary guide RNA, and an exemplary napDNAbp.

FIG. 2 is another schematic showing an exemplary target dsDNA, an exemplary guide RNA, and an exemplary napDNAbp.

FIG. 3 is a schematic showing an exemplary target dsDNA, an exemplary transcript (target RNA) transcribed from the target dsDNA, an exemplary guide RNA, and an exemplary napRNAbp.

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

FIG. 5 illustrates a representative Cas12 endonuclease based therapeutic strategy.

FIG. 6 shows the results of screening gRNAs in PB-HBV-HEK293T cell line.

FIG. 7 shows the multiplexing of three sgRNAs with hfCas12Max nuclease reduced HBV viral parameters.

FIG. 8 shows that LNP-mediated delivery of hfCas12Max mRNA and triple gRNA combination (G47, G82, and G116) led to sustained reduction of HBV viral markers in primary human hepatocytes (PHHs) .

FIG. 9 shows that LNP-mediated delivery of hfCas12Max mRNA and gRNAs led to sustained reduction of HBV viral markers in AAV-HBV mouse model..

FIG. 10 shows an exemplary RNA ( “RNA” ) ; an exemplary modified RNA carrying a 2’-O-methyl ( “2’-OMe” or “M”) modification on the ribose of a modified nucleotide of the modified RNA; an exemplary modified RNA carrying a 2’-O-methyl modification on the ribose of a modified nucleotide of the modified RNA plus a 3’ phosphorothioate ( “PS” ) inter-nucleotide linkage 3’ to the ribose of the modified nucleotide (collectively, “2’-O-methyl 3’ phosphorothioate modification” or “MS” ) (wherein the modified ribonucleotide is denoted with Nms (N = A, U, G, or C) throughout the disclosure) ; an exemplary modified RNA carrying a 2’-O-methyl modification on the ribose of a modified nucleotide of the modified RNA plus a 3’ thioPACE inter-nucleotide linkage 3’ to the ribose of the modified nucleotide (collectively, “MCP” ) ; and an exemplary modified RNA carrying a 2’-fluoro (2’-F) modification on the ribose of a modified nucleotide (wherein the modified ribonucleotide is denoted with fN (N = A, U, G, or C) throughout the disclosure) of the modified RNA.

FIG. 11 illustrates the modifications of an embodiment of the modified guide RNA of the disclosure, carrying a 2’-O-methyl 3’ phosphorothioate modification on each of the three 5’ end nucleotides AGA, and a modified 3’poly U tail consisting of 4 uracil with a 2’-O-methyl 3’ phosphorothioate modification on each of the first three 5’ end uracil. “- (S) -” denotes a phosphorothioate inter-nucleotide linkage. “-O-Methyl” denotes a 2’-O-Methyl modification on ribose of the nucleotide.

The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

Definitions

The disclosure will be described with respect to particular embodiments, but the disclosure is not limited thereto in any respect. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms as set forth hereinafter are generally to be understood in their plain and ordinary meaning or common sense unless indicated otherwise.

Overview

Nucleic acid programmable binding protein (napBP) , for example, nucleic acid programmable DNA binding protein, (napDNAbp) , such as Cas9, Cas12, IscB, nucleic acid programmable RNA binding protein (napRNAbp) , such as, Cas13, is capable of binding to a target nucleic acid (e.g., dsDNA, mRNA) as guided by a guide nucleic acid (e.g., a guide RNA) comprising a guide sequence targeting the target nucleic acid. In some embodiments, the target nucleic acid is eukaryotic.

Without wishing to be bound by theory, in some embodiments, the guide nucleic acid comprises a scaffold sequence responsible for forming a complex with the napBP, and a guide sequence that is intentionally designed to be responsible for hybridizing to a target sequence of the target nucleic acid, thereby guiding the complex comprising the napBP and the guide nucleic acid to the target nucleic acid.

Referring to FIG. 1, an exemplary target dsDNA (e.g., HBV cccDNA) is depicted to comprise a 5’ to 3’ single DNA strand and a 3’ to 5’ single DNA strand.

An exemplary guide nucleic acid (e.g., a guide RNA) is depicted to comprise a guide sequence and a scaffold sequence. The guide sequence is designed to hybridize to a part of the 3’ to 5’ single DNA strand, and so the guide sequence “targets” that part. And thus, the 3’ to 5’ single DNA strand is referred to as a “target strand (TS) ” of the target dsDNA, while the opposite 5’ to 3’ single DNA strand is referred to as a “nontarget strand (NTS) ” of the target dsDNA. That part of the target strand based on which the guide sequence is designed and to which the guide sequence may hybridize is referred to as a “target sequence” , while the opposite part on the nontarget strand corresponding to that part is referred to as the “protospacer sequence” , which is 100% (fully) reversely complementary to the target sequence and is said to be “corresponding to” the target sequence in the disclosure.

Referring to FIG. 3, an exemplary target dsDNA (e.g., HBV cccDNA) is depicted to comprise a 5’ to 3’ single DNA strand and a 3’ to 5’ single DNA strand. According to conventional transcription process, an exemplary target RNA (transcript, e.g., a pre-mRNA) may be transcribed using the 3’ to 5’ single DNA strand as a synthesis template, and thus the 3’ to 5’ single DNA strand is referred to as a “template strand” or a “antisense strand” . The transcript so transcribed has the same primary sequence as the 5’ to 3’ single DNA strand except for the replacement of T with U, and thus the 5’ to 3’ single DNA strand is referred to as a “coding strand” or a “sense strand” .

An exemplary guide nucleic acid (e.g., a guide RNA) is depicted to comprise a guide sequence and a scaffold sequence. The guide sequence is designed to hybridize to a part of the transcript (target RNA) , and so the guide sequence “targets” that part. And thus, that part of the target RNA based on which the guide sequence is designed and to which the guide sequence may hybridize is referred to as a “target sequence” . In some embodiments, the guide sequence is 100% (fully) reversely complementary to the target sequence. In some other embodiments, the guide sequence is reversely complementary to the target sequence and contains a mismatch with the target sequence (as exemplified in FIG. 4) .

Generally, as is conventional in the art, a nucleic acid sequence (e.g., a DNA sequence, an RNA sequence) is written in 5’ to 3’ direction /orientation unless explicitly indicated otherwise.

For example, for a DNA sequence of ATGC, it is usually understood as 5’-ATGC-3’ unless otherwise indicated. Its reverse sequence is 5’-CGTA-3’. Its fully complementary sequence is 5’-TACG-3’. Its fully reverse complementary sequence is 5’-GCAT-3’. Note that the fully complementary sequence usually does not have the ability to base-pair /hybridize with the original sequence.

Generally, the double-strand sequence of a dsDNA may be represented with the sequence of its 5’ to 3’ single DNA strand conventionally written in 5’ to 3’ direction /orientation unless otherwise indicated.

For example, for a 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 may be simply represented as 5’-ATGC-3’.

5’-----ATGC -----3’

3’-----TACG -----5’

It should be noted that either the 5’ to 3’ single DNA strand or the 3’ to 5’ single DNA strand of a dsDNA can be a nontarget strand from which a protospacer sequence is selected.

Generally, for a gene as a 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. It should be noted that either the sense strand or the antisense strand of a gene can be a nontarget strand from which a protospacer sequence is selected.

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

To hybridize to a target dsDNA, in one embodiment, the guide sequence of a guide nucleic acid is designed to have a sequence of 5’-AUGC-3’ that is fully reversely complementary to the 3’ to 5’ strand of the target dsRNA, which would be set forth in ATGC in the electric sequence listing but marked as an RNA sequence; and in another embodiment, the guide sequence of a guide nucleic acid is designed to have a sequence of 5’-GCAU-3’ that is fully reversely complementary to the 5’ to 3’ strand of the target dsRNA, which would be set forth in GCAT in the electric sequence listing but marked as an RNA sequence.

In the case that the guide sequence of a guide nucleic acid is fully reversely complementary to the target sequence and the target sequence is fully reversely 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 correspondingly the T in the protospacer sequence due to its DNA nature. According to WIPO standard ST. 26, symbol “t” is used to denote both T in DNA and U in RNA (See “Table 1: List of nucleotides symbols” , the definition of symbol “t” is “thymine in DNA/uracil in RNA (t/u) ” ) . Thus, in the electronic sequence listing of the disclosure prepared according to ST. 26, such a guide sequence could be set forth in the same sequence as a corresponding protospacer sequence. For convenience, a single SEQ ID NO in the electronic sequence listing can be used to denote both such guide sequence and protospacer sequence, regardless whether such a single SEQ ID NO is marked as DNA or RNA in the electronic sequence listing. When a reference is made to such a SEQ ID NO that sets forth a protospacer /guide sequence, it refers to either a protospacer sequence that is a DNA sequence or a guide sequence that is an RNA sequence depending on the context, no matter whether it is marked as a DNA or an RNA in the electronic sequence listing.

To hybridize to the target RNA, in one embodiment, the guide sequence of a guide nucleic acid is designed to have a sequence of 5’-GCAU-3’ that is fully reversely complementary to the (target) sequence of the target RNA, which would be set forth in GCAT in the electric sequence listing but marked as an RNA sequence.

Term

As used herein, if a DNA sequence, for example, 5’-ATGC-3’ is transcribed to an RNA sequence, with each dT (deoxythymidine, or “T” for short) in the primary sequence replaced with a U (uridine) and other dA (deoxyadenosine, or “A” for short) , dG (deoxyguanosine, or “G” for short) , and dC (deoxycytidine, or “C” for short) replaced with A (adenosine) , G (guanosine) , and C (cytidine) , respectively, for example, 5’-AUGC-3’, it is said in the disclosure that the DNA sequence “encodes” the RNA sequence.

As used herein, the term “activity” refers to a biological activity. In some embodiments, the activity includes enzymatic activity, e.g., catalytic ability of an effector. For example, the activity can include nuclease activity, e.g., dsDNA endonuclease activity, RNA endonuclease activity.

As used herein, the term “nucleic acid programmable binding protein (napBP) ” may be used interchangeably with “nucleic acid programmable binding domain (napBD) ” to refer to a protein that can associate (e.g., bind) with a programmable nucleic acid (e.g., DNA or RNA) , such as a guide nucleic acid (e.g., gRNA) , that is able to be programmed to guide the protein to a specific sequence of a target nucleic acid via the interaction (e.g., hybridization) between the programmable nucleic acid (e.g., the guide sequence of the programmable nucleic acid) and the target nucleic acid (e.g., the target sequence of the target nucleic acid) . The napBP may be indirectly associated with (e.g., bound to) the target nucleic acid via the interaction (e.g., binding) between the napBP and the programmable nucleic acid (e.g., scaffold sequence of the programmable nucleic acid) and the interaction (e.g., hybridization) between the programmable nucleic acid (e.g., the guide sequence of the programmable nucleic acid) and the target nucleic acid (e.g., the target sequence of the 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) .

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 interacting with (e.g., binding to, coming into contact with, adhering to) one another. As used herein, the term “complex” can refer to a grouping of a guide nucleic acid and a polypeptide (e.g., a napBP) . As used herein, the term “complex” can refer to a grouping of a guide nucleic acid, a polypeptide (e.g., a napBP) , and a target nucleic acid.

As used herein, the term “protospacer adjacent motif’ or “PAM” refers to a short DNA sequence (or a DNA motif) adjacent to a protospacer sequence on the nontarget strand of a dsDNA. As used herein, the term “adjacent” includes instances wherein there is no nucleotide between the protospacer sequence and the PAM and also instances wherein there are a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides between the protospacer sequence and the PAM. As used herein, A “immediately adjacent (to) ” B, A “immediately 5’ to” B, and A “immediately 3’ to” B mean that there is no nucleotide between A and B. In some embodiments, the PAM is immediately 5’ to a protospacer sequence. In some embodiments, the PAM is immediately 3’ to a protospacer sequence.

As used herein, the term “guide nucleic acid” refers to any nucleic acid that facilitates the targeting of a napBP to a target nucleic acid. For this purpose, the guide nucleic acid may be designed to include a guide sequence capable of hybridizing to a specific sequence of a target nucleic acid, and the guide nucleic acid may also comprise a scaffold sequence facilitating the guiding of a 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 encoding a guide RNA.

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.

As used in the context of CRISPR-Cas techniques (e.g., CRISPR-Cas12 techniques) , 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” .

As described herein, the guide sequence is so designed to be capable of hybridizing to a target sequence. 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 bonding between the bases of the polynucleotide sequences. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. A polynucleotide sequence capable of hybridizing to a given polynucleotide sequence is referred to as the “complement” of the given polynucleotide sequence. As used herein, the hybridization of a guide sequence and a target sequence is so stabilized to permit an effector polypeptide (e.g., a napBP) that is complexed with a nucleic acid comprising the guide sequence or a function domain associated (e.g., fused) with the effector polypeptide to act (e.g., cleave, deaminize) on the target sequence or its complement or nearby sequence.

For the purpose of hybridization, in some embodiments, the guide sequence is reversely complementary to a target sequence. As used herein, the term “reverse complementary” refers to the ability of nucleobases of a first polynucleotide sequence, such as a guide sequence, to base pair with 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, a first polynucleotide sequence (e.g., a guide sequence) comprises 100% (fully) reverse complementarity to a second nucleic acid (e.g., a target sequence) . In some embodiments, a first polynucleotide sequence (e.g., a guide sequence) is reverse complementary to a second polynucleotide sequence (e.g., a target sequence) if the first polynucleotide sequence comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%complementarity to the second nucleic acid (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 the nucleotides of the second polynucleotide sequence) . As used herein, the term “substantially complementary” refers to a first polynucleotide sequence (e.g., a guide sequence) that has a certain level of complementarity to 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 base-pair with the nucleotides of the second polynucleotide sequence, or at most 1, 2, 3, 4, or 5 contiguous or non-contiguous nucleotides of the first polynucleotide sequence mismatch the nucleotides of the second polynucleotide sequence) . In some embodiments, the level of complementarity is such that the first polynucleotide sequence (e.g., a guide sequence) can hybridize to the second polynucleotide sequence (e.g., a target sequence) with sufficient affinity to permit an effector polypeptide (e.g., a napBP) that is complexed with a nucleic acid comprising the first polynucleotide sequence or a function domain associated (e.g., fused) with the effector polypeptide to act (e.g., cleave, deaminize) on the target sequence or its complement or nearby sequence. In some embodiments, a guide sequence that is substantially complementary to a target sequence has less than 100%complementarity to the target sequence. In some embodiments, a guide sequence that is substantially complementary to a target sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the target sequence, and/or has at most 1, 2, 3, 4, or 5 contiguous or non-contiguous nucleotide mismatches from the target sequence.

As used herein, the term “sequence identity” is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percentage sequence identity (%) between two or more sequences (polypeptide or polynucleotide sequences) . Sequence homologies may be generated by any of a number of computer programs known in the art, for example, BLAST, FASTA. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12: 387) . Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid-Chapter 18) , FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) , and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60) . A commonly used online tool to calculate percentage sequence identity between two or more sequences (polypeptide or polynucleotide sequences) is available on the website of EMBL's European Bioinformatics Institute (www dot ebi dot ac dot uk slash jdispatcher slash) , allowing fast online calculation of percentage sequence identity by global alignment or local alignment.

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

As used herein, a “variant” is interpreted to mean a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties, e.g., binding property of a napBP. A typical variant of a polynucleotide differs in nucleic acid sequence from another reference polynucleotide. A change in the nucleic acid sequence of the polynucleotide variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. A change in the nucleic acid sequence of the polynucleotide variant may result in an amino acid substitution, addition, and/or deletion in the polypeptide encoded by the reference polynucleotide. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the difference is limited so that the sequences of the reference polypeptide and the polypeptide variant are closely similar overall and, in many regions, identical. The polypeptide variant and 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 that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.

As used herein, the terms “upstream” and “downstream” refer to the relative positions of two or more elements within a nucleic acid in 5’ to 3’ direction. A first sequence is upstream of a second sequence when the 3’ end of the first sequence is present at the left side of the 5’ end of the second sequence. A first sequence is downstream of a second sequence when the 5’ end of the first sequence is present at the right side of the 3’ end of the second sequence. In some embodiments, the PAM is upstream of a napBP-induced indel, and a napBP-induced indel is downstream of the PAM. In some embodiments, the PAM is downstream of a napBP-induced indel, and a napBP-induced indel is upstream of the PAM.

As used herein, the term “wild type” has the meaning commonly understood by those skilled in the art to mean a typical form of an organism, a strain, a gene, or a feature that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from sources in nature and not intentionally modified.

As used herein, the terms “non-naturally occurring” and “engineered” are used interchangeably and refer to artificial participation. When these terms are used to describe a nucleic acid or a polypeptide, it is meant that the nucleic acid or polypeptide is at least substantially freed from at least one other component of its association in nature or as found in nature.

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 a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) . Regulatory elements may also direct expression in a time-dependent manner, e.g., in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific.

As used herein, the term “cell” is understood to refer not only to a particular individual cell, but to the progeny or potential progeny of the cell. Because certain modifications may occur in succeeding generations due to either 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.

As used herein, the term “in vivo” refers to inside the body of an organism, and the terms “ex vivo” or “in vitro” means outside the body of an organism.

As used herein, the term “treat” , “treatment” , or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of the disclosure, the beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, diminishing the extent of a disease, stabilizing a disease (e.g., delaying the worsening of a disease) , delaying the spread (e.g., metastasis) of a disease, delaying the recurrence of a disease, reducing recurrence rate of a disease, delay or slowing the progression of a disease, ameliorating a disease state, providing a remission (partial or total) of a disease, decreasing the dose of one or more other medications required to treat a disease, delaying the progression of a disease, increasing the quality of life, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of a disease (such as cancer) . The methods of the disclosure contemplate any one or more of these aspects of treatment.

As used herein, the term “disease” includes the terms “disorder” and “condition” and is not limited to those have been specifically medically defined.

As used herein, the term “transcript” includes any transcription product by transcription from a DNA (e.g., HBV cccDNA) , including subgenomic RNA, mRNA, non-coding RNA, and any variants, derivatives, or ancestors thereof, for example, pre-mRNA, and any transcripts or isoforms produced from the DNA or the pre-mRNA by, e.g., alternative promoter usage, alternative splicing, alternative initiation, and any naturally occurring variants thereof or processed products therefrom.

As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method may be used to treat cancer of types other than X.

As used herein, the singular forms “a” , “an” , and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, the term “and/or” in a phrase such as “A and/or B” is intended to include both A and B; A or B; A (alone) ; and B (alone) . Likewise, the term “and/or” in a phrase 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) .

As used herein, when the term “about” is ahead of a serious of numbers (for example, about 1, 2, 3) , it is understood that each of the serious of numbers is modified by the term “about” (that is, about 1, about 2, about 3) . The term “about X-Y” or “about X to Y” used herein has the same meaning as “about X to about Y. ”

It is understood that embodiments of the disclosure described herein include “consisting” and/or “consisting essentially of” embodiments.

It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely” , “only” , and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

DETAILED DESCRIPTION

Overview

Provided in the disclosure are products and methods for the treatment of HBV associated diseases, such as, Hepatis B, by targeting and modifying HBV cccDNA through the use of CRISPR-Cas12 systems suitable for delivery by lipid nanoparticle (LNP) . Guide sequences are designed to target cccDNA so as to guide a Cas12 polypeptide or a fusion comprising a Cas12 polypeptide and a functional domain to the cccDNA, which then functions on the cccDNA in a guide sequence-specific manner, leading to a decreased level of HBV infection biomarkers including HBV cccDNA, HBV DNA, and HBsAg and providing a promising treatment for treating and even curing HBV associated diseases.

Guide nucleic acid

In an aspect, the disclosure provides a guide nucleic acid comprising a guide sequence capable of hybridizing to a target sequence on a target strand of an HBV cccDNA or a target sequence on a transcript of an HBV cccDNA.

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 the hybridization of the guide sequence to the target sequence guides the complex to the HBV cccDNA or the transcript.

The components of the guide nucleic acid are described more specifically in the other sub-sections herein.

System

In another aspect, the disclosure provides a system comprising:

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

(b) a guide sequence capable of hybridizing to a target sequence on a target strand of an HBV cccDNA or a target sequence on a transcript of an HBV cccDNA, thereby guiding the complex to the HBV cccDNA or the transcript; and

(2) the napBP or a polynucleotide encoding the napBP.

In some embodiments, the system is a complex comprising the napBP complexed with the guide nucleic acid. In some embodiments, the complex further comprises the HBV cccDNA or transcript thereof hybridized with the guide sequence.

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

The components of the system are described more specifically in the other sub-sections herein.

LNP

In yet another aspect, the disclosure provides a lipid nanoparticle (LNP) comprising a system comprising:

(2) a polynucleotide encoding the napBP.

The components of the LNP are described more specifically in the other sub-sections herein.

rAAV vector genome

The disclosure provides various delivery of the system of the disclosure, for example, delivery via a rAAV vector. In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) vector genome encoding or comprising the system of the disclosure. In some embodiments, the rAAV vector genome is a DNA (e.g., a ssDNA, a dsDNA) or an RNA.

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

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

(2) a second polynucleotide sequence comprising a sequence encoding the napBP,

wherein the rAAV vector genome is adapted to be encapsulated into a rAAV particle (e.g., a DNA-encapsulated rAAV particle, an RNA-encapsulated rAAV particle) .

The components of the rAAV vector genome are described more specifically in the other sub-sections herein.

Modification method

In yet another aspect, the disclosure provides a method of modifying an HBV cccDNA or transcript thereof, comprising contacting the HBV cccDNA or transcript thereof with a system comprising:

(2) the napBP or a polynucleotide encoding the napBP.

In some embodiments, the modification of the HBV cccDNA or transcript thereof treats, detects, or diagnose an HBV associated disease. In some embodiments, the modification of the HBV cccDNA or transcript thereof detects or diagnose the presence, progress, or development of an HBV associated disease.

The components of the method are described more specifically in the other sub-sections herein.

Mechanism

The disclosure provides various mechanisms for modifying HBV cccDNA for the purpose of treating or even curing HBV associated diseases.

HBV cccDNA in cell nucleus is a source for continuous generation of new infectious HBVs. First, HBV cccDNA functions as a replication template, from which HBV viral RNAs (e.g., pregenomic RNA, subgenomic RNA) are transcribed. Then, HBV viral proteins (e.g., HBV surface antigen HBsAg, HBV core antigen HBcAg, HBV polymerase) are translated from the HBV viral RNAs, and HBV DNA (viral genome) is reversely transcribed from the HBV viral RNAs. Together the HBV viral proteins and HBV DNA assemble to produce new infectious HBVs (viral particles) .

Provided in the disclosure is a guide nucleic acid comprising a guide sequence designed to be capable of hybridizing to a target sequence of HBV cccDNA, thereby guiding a napDNAbp to the target sequence to function there.

In some embodiments, the HBV cccDNA is modified by dsDNA cleavage. FIG. 5 illustrates a representative Cas12 endonuclease based therapeutic strategy. FIG. 5A shows schematic of a therapeutic vector encoding a Cas12i endonuclease (hfCas12Max) , which is a representative high efficiency, high fidelity Cas12 endonuclease developed by HuidaGene Therapeutics Co., Ltd. (PCT/CN2023/090695, incorporated herein by reference in its entirety) . FIG. 5B shows schematic of one or more (e.g., two, three) endonuclease-mediated double-stranded break (s) (DSB (s) ) in HBV cccDNA. In some embodiments, the DSB(s) in the HBV cccDNA leads to the degradation of HBV cccDNA by, for example, exonuclease in the infected cells. For this purpose, two or more (e.g., three) guide nucleic acids containing different guide sequences, or a guide nucleic acid containing two or more (e.g., three) different guide sequences, capable of guiding a napDNAn to different target sites of HBV cccDNA to cleave at the multiple target sites can be applied. The introduction of multiple DSBs in HBV cccDNA can facilitate the degradation of HBV cccDNA. As a result, HBV DNA and/or HBsAg in a subject is decreased or eliminated.

In some embodiments, the DSB in the HBV cccDNA leads to the generation of Indel mutation in HBV cccDNA via DNA repair (e.g., error-prone non-homologous end-joining (NHEJ) mechanism) , which may lead to the generation of a mutated, replication-incompetent HBV cccDNA. For example, the Indel mutation contains a premature stop codon; or alternatively, the Indel mutation contains a frameshift mutation that results in a premature stop codon downstream of the frameshift mutation. The premature stop codon would interference the proper translation of HBV viral proteins from the HBV viral RNA transcribed from the HBV cccDNA containing the Indel mutation. As a result, HBV DNA and/or HBsAg in a subject is decreased or eliminated.

In some embodiments, the HBV cccDNA is modified by DNA epigenomic modification, e.g., methylation. In some embodiments, the DNA epigenomic modification of the HBV cccDNA decreases or eliminates the transcription of the HBV cccDNA. As a result, HBV DNA and/or HBsAg in a subject is decreased or eliminated.

HBV infected individual cannot be completely cured without a clearance of cccDNA from infected hepatocytes. In the disclosure, the clearance is achieved by, for example, the degradation of cccDNA, which is straightforward clearance of cccDNA, the generation of a mutated, replication-incompetent cccDNA or a transcriptionally silenced cccDNA, which can also be regarded as clearance of cccDNA since the modified cccDNA is “dead” in a sense of virus replication.

In the case that the generation of HBV viral RNA and/or viral protein from HBV cccDNA is properly downregulated, it is reasonably expected that the syndromes (e.g., syndromes of Hepatitis B) caused by the generation of HBV viral RNA and/or viral protein from HBV cccDNA can be alleviated.

DNA modification

Provided in the disclosure are multiple ways to modify HBV cccDNA on DNA level. In some embodiments, the napBP is a nucleic acid programmable DNA binding protein (napDNAbp) .

DNA cleavage

Provided in the disclosure are tools and methods using DNA cleavage to modify HBV cccDNA.

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

In some embodiments, guiding the complex to the HBV cccDNA enables the napDNAn to specifically cleave the HBV cccDNA in a guide sequence-specific manner.

In some embodiments, the specific cleavage of the HBV cccDNA leads to incorporation of an insertion and/or deletion (an indel) mutation into the HBV cccDNA.

In some embodiments, the specific cleavage of the HBV cccDNA or the insertion and/or deletion mutation generates an in-frame stop codon in the HBV cccDNA. As is well understood, the presence of an in-frame stop codon in a DNA and/or in the transcript transcribed from a DNA typically stops the translation of a protein from the transcript. Typically, stop codon is TAG, TAA, or TGA in DNA, and UAG, UAA, or UGA in RNA.

In some embodiments, the specific cleavage of the HBV cccDNA or the insertion and/or deletion mutation generates a 3n+1 frameshift mutation, a 3n+2 frameshift mutation, a 3n-1 frameshift mutation, or a 3n-2 frameshift mutation in the HBV cccDNA, wherein n is 0 or a positive integer (e.g., 1, 2, 3) .

In some embodiments, the frameshift mutation decreases or eliminates transcription of the HBV cccDNA and/or translation of a transcript of the HBV cccDNA. In some embodiments, the frameshift mutation generates an in-frame stop codon in the HBV cccDNA.

DNA epigenomic modification

In some aspects, the disclosure provides a way to epigenomic modification of HBV cccDNA, e.g., methylation.

In some embodiments, the napBP comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 548.

In some embodiments, the fusion protein comprises a transcription inhibiting domain (e.g., KRAB domain or SID domain) .

In some embodiments, the fusion protein comprises a KRAB domain.

In some embodiments, the KRAB domain comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 549.

In some embodiments, the fusion protein comprises a DNA methyltransferase, such as, DNMT3l, DNMT3a.

In some embodiments, the fusion protein comprises a DNMT3l domain and a DNMT3a domain.

In some embodiments, the DNMT3l domain comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 550.

In some embodiments, the DNMT3a domain comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 551.

In some embodiments, the fusion protein comprises the napBP, a DNMT3l domain, a DNMT3a domain, and a KRAB domain.

In some embodiments, the fusion protein comprises, from N-terminal to C-terminal, the napBP, the KRAB domain, the DNMT3l domain, and the DNMT3a domain.

In some embodiments, the fusion protein comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 552.

napBP

For the purpose of the disclosure, the napBP is capable of forming a complex with the guide nucleic acid of the disclosure by complexing with the scaffold sequence of the guide nucleic acid and is thereby guided to the HBV cccDNA or transcript thereof via the hybridization of the guide sequence of the guide nucleic acid to the target sequence of the HBV cccDNA or transcript. When the napBP is guided to the HBV cccDNA or transcript, the activity of the napBP (or the functional domain associated with (e.g., bound to) the napBP) functions to modify the HBV cccDNA or transcript thereof, leading to modification of the HBV cccDNA. Generally, any such napBP can be used with the guide nucleic acid of the disclosure, and when a napBP is selected, the scaffold sequence compatible to the napBP for complexing with the napBP can also be selected accordingly. The scaffold sequence is generally conserved.

In some embodiments, the napBP (e.g., napDNAbp) is capable of recognizing a protospacer adjacent motif (PAM) on the nontarget strand of the HBV cccDNA, wherein the PAM is immediately 5’ or 3’ to a protospacer sequence on the nontarget strand of the HBV cccDNA, and wherein the protospacer sequence is fully reversely complementary to the target sequence.

In some embodiments, the PAM comprises 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.

Non-limiting examples of the napBP include CRISPR-associated (Cas) protein, IscB, TAL nuclease, meganuclease, and zinc-finger nuclease. Non-limiting examples of CRISPR-associated (Cas) protein 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 protein 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, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, IscB, homologues thereof, or modified or engineered versions thereof. Other napDNAbp are also within the scope of this disclosure, e.g., IscB, IsrB, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here? ” CRISPR J. 2018 October; 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 are hereby incorporated by reference.

In some embodiments, the Cas protein is an endonuclease, a nickase, or a dead Cas.

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

In some embodiments, the PAM comprises 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) , e.g., SaCas9, SpCas9, or a mutant thereof.

In some embodiments, the PAM comprises 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 a IscB protein, e.g., OgeuIscB or a mutant thereof.

In some cases, the recognizing ability of the napBP to a target nucleic acid may not be limited to any specific PAM, which means that the napBP can recognize any PAM, such that the PAM is not a substantial restriction on the selection of a protospacer sequence or a target sequence. Such a napBP is called “PAMless” , for example, a PAMless SpCas9 mutant. In some embodiments, the napBP is PAMless.

In some embodiments, the napBP is a Class 2, Type VI CRISPR-associated protein (Cas13) . Cas13 can be targeted to RNA by a guide nucleic acid. Cas13 is particularly useful since there is no PAM restriction for eukaryotic transcripts when Cas13 is used as the napBP. In some embodiments, the Cas13 is Cas13a (C2c2) , Cas13b (such as, Cas13b1, Cas13b2) , Cas13c, Cas13d, Cas13e (Cas13X) , Cas13f (Cas13Y) , or a mutant thereof. In some embodiments. The napBP is hfCas13Y (hfCas13f /hfCas13f. 1) in PCT/CN2022/122833. As used herein, the term “Cas13Y” is used interchangeably with “Cas13f” , and the term “hfCas13Y” is used interchangeably with “hfCas13f” and “hfCas13f. 1” .

Typically, a Cas protein (e.g., Cas9, Cas12, Cas13) can associate with a CRISPR RNA (crRNA) comprising a spacer sequence that guides the Cas protein to a specific sequence that is reversely complementary and capable of hybridizing to the spacer sequence of the crRNA. The crRNA also comprises a scaffold sequence capable of complexing with the Cas protein.

In some embodiments, the napBP comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 94 or 548 or a N-terminal truncation thereof without the first N-terminal Methionine. 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: 94.

In some embodiments, the napBP comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 94 or a N-terminal truncation thereof without the first N-terminal Methionine, comprises substitutions N243R+E336R+D892R relative to the wild type of the Cas12 endonuclease of SEQ ID NO: 94, and retaining 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: 94.

In some embodiments, the napBP comprises a sequence of SEQ ID NO: 94 or 548.

In some embodiments, the sequence encoding the napBP comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 95 or a 5’ end truncation thereof without the first 5’ ATG codon. 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: 95.

In some embodiments, the sequence encoding the napBP comprises a sequence of SEQ ID NO: 95.

Design of protospacer sequence/target sequence; Target site

For the purpose of the disclosure, in some embodiments, the protospacer sequence or target sequence is located such that the HBV cccDNA or transcript thereof can be specifically modified by the napBP or a functional domain associated with the napBP.

To facilitate the evaluation of selected protospacer sequences or target sequence and designed guide sequences in mouse models, in some embodiments, the protospacer sequence or target sequence is located such that a mouse HBV cccDNA or transcript thereof can be specifically modified by the napBP or a functional domain associated with the napBP. In some embodiments, the protospacer sequence or target sequence is located such that both a human HBV cccDNA or transcript thereof and a mouse HBV cccDNA or transcript thereof can be specifically modified by the napBP or a functional domain associated with the napBP. That is, the protospacer sequence or target sequence is selected to be cross-reactive to both human and mouse species. Alternatively, the protospacer sequence or target sequence is selected to be conservative across various species.

In some embodiments, the protospacer sequence is a stretch of contiguous nucleotides identified from the nontarget strand of the HBV cccDNA by identifying the stretch of contiguous nucleotides immediately 5’ or 3’, and optionally, 3’, to the PAM on the nontarget strand.

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 contiguous nucleotides on the nontarget strand of the HBV cccDNA, or a stretch of contiguous nucleotides on the nontarget strand of the HBV cccDNA in a numerical range between any two of the preceding values, e.g., a stretch of from about 16 to about 50 contiguous nucleotides. In some embodiments, the protospacer sequence is a stretch of about 20, 30, or 50 contiguous nucleotides on the nontarget strand of the HBV cccDNA.

In some embodiments, the protospacer sequence on the nontarget strand of the HBV cccDNA corresponding to the target sequence is immediately 3’ to PAM of 5’-NTN-3’, wherein N is A, T, G, or C.

In some embodiments, the protospacer sequence on the nontarget strand of the HBV cccDNA corresponding to the target sequence is immediately 5’ to PAM of 5’-NGG-3’, wherein N is A, T, G, or C.

In some embodiments, the protospacer sequence on the nontarget strand of the HBV cccDNA corresponding to the target sequence is immediately 5’ to PAM of 5’-NNNGAN-3’, wherein N is A, T, G, or C.

In some embodiments, the protospacer sequence is a stretch of about 20, 30, or 50 contiguous nucleotides of the nontarget strand of the HBV cccDNA immediately 3’ to PAM of 5’-NTN-3’, wherein N is A, T, G, or C.

In some embodiments, the protospacer sequence is a stretch of about 20, 30, or 50 contiguous nucleotides of the nontarget strand of the HBV cccDNA immediately 5’ to PAM of 5’-NGG-3’, wherein N is A, T, G, or C.

In some embodiments, the protospacer sequence is a stretch of about 20, 30, or 50 contiguous nucleotides of the nontarget strand of the HBV cccDNA immediately 5’ to PAM of 5’-NNNGAN-3’, wherein N is A, T, G, or C.

In some embodiments, the target sequence is a stretch of contiguous nucleotides identified from the target strand of the HBV cccDNA or from the transcript thereof.

In some embodiments, the target 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 contiguous nucleotides on the target strand of the HBV cccDNA or on the transcript thereof, or a stretch of contiguous nucleotides on the target strand of the HBV cccDNA or on the transcript thereof in a numerical range between any two of the preceding values, e.g., a stretch of from about 16 to about 50 contiguous nucleotides. In some embodiments, the target sequence is a stretch of about 20, 30, or 50 contiguous nucleotides on the target strand of the HBV cccDNA or on the transcript thereof.

In some embodiments, the nontarget strand is the sense strand of the HBV cccDNA.

In some embodiments, the nontarget strand is the antisense strand of the HBV cccDNA.

In some embodiments, the target strand is the sense strand of the HBV cccDNA.

In some embodiments, the target strand is the antisense strand of the HBV cccDNA.

In some embodiments, the protospacer sequence on the nontarget strand of the HBV cccDNA corresponding to the target sequence or target sequence on the target strand of the HBV cccDNA is located at or within an exon of the HBV cccDNA or transcript thereof, or at or within a splice donor or a splice acceptor of the exon.

In some embodiments, the protospacer sequence on the nontarget strand of the HBV cccDNA corresponding to the target sequence or target sequence on the target strand of the HBV cccDNA is located at or within a regulatory element of the HBV cccDNA.

In some embodiments, the protospacer sequence on the nontarget strand of the HBV cccDNA corresponding to the target sequence or target sequence on the target strand of the HBV cccDNA is located at or within a coding sequence of the HBV cccDNA.

In some embodiments, the HBV cccDNA is human HBV cccDNA, non-human primate HBV cccDNA, or mouse HBV cccDNA.

In some embodiments, the HBV cccDNA or transcript thereof is in a eukaryotic cell, for example, a human cell, a non-human primate cell, or a mouse cell. In some embodiments, the HBV cccDNA or transcript thereof is in a hepatocyte.

Design of guide sequence according to protospacer/target sequence

In some embodiments, the guide sequence is in a length of about, at least about, or at most about 14 nucleotides, e.g., 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 nucleotides, or in a length of nucleotides in a numerical range between any two of the preceding values, e.g., in a length of from about 16 to about 50 nucleotides. In some embodiments, the guide sequence is in a length of about 20, 30, or 50 nucleotides.

In some embodiments, (1) the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (fully) , optionally about 100% (fully) , reversely complementary to the target sequence; (2) the guide sequence contains no more than 5, 4, 3, 2, or 1 mismatch or contains no mismatch with the target sequence; or (3) the guide sequence comprises no mismatch with the target sequence in the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 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, or 70 nucleotides at the 5’ end of the guide sequence when the PAM is immediately 5’ to the protospacer sequence or at the 3’ end of the guide sequence when the PAM is immediately 3’ to the protospacer sequence. In some embodiments, the guide sequence is about 100% (fully) , reversely complementary to the target sequence.

In some embodiments, the guide sequence contains 1 mismatch with the target sequence. In some embodiments, the guide sequence is about 98%reversely complementary to the target sequence. In some embodiments, the 1 mismatch in the guide sequence is at a position corresponding the nucleotide of the target sequence that is intended to be substituted.

Selection of protospacer/target/guide sequence; Effect of system

In some embodiments, the protospacer sequence, the target sequence, or the guide sequence is selected according to the editing efficiency of the napDNAn guided to the HBV cccDNA by the guide sequence. The editing efficiency can be represented by, for example, indel percentage for a particular protospacer sequence, target sequence, or guide sequence as measured by sequencing. The higher the editing efficiency is, the more suitable the protospacer sequence, the target sequence, or the guide sequence is.

In some embodiments, the protospacer sequence, the target sequence, or the guide sequence is selected according to the level of HBV cccDNA, HBV DNA, or HBsAg in vitro or in vivo. The lower the level is, the more suitable the protospacer sequence, the target sequence, or the guide sequence is.

In some embodiments, the level of HBV cccDNA, HBV DNA, or HBsAg is decreased in a cell model (e.g., HEK293T cell model) or an animal model (e.g., a mouse model, a non-human primate model) 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, upon administration of the system of the disclosure to the cell model or the animal model, compared to the level of HBV cccDNA, HBV DNA, or HBsAg in the same cell model or animal model that does not receive the administration.

Overall structure of guide nucleic acid

In some embodiments, the guide nucleic acid comprises a scaffold sequence 5’ to a guide sequence. In some embodiments, the guide nucleic acid comprises a scaffold sequence 3’ to a guide sequence.

In some embodiments, the guide nucleic acid comprises one scaffold sequence and one guide sequence.

In some embodiments, the guide nucleic acid comprises one scaffold sequence 5’ to one guide sequence. In some embodiments, the guide nucleic acid comprises one scaffold sequence 3’ to one guide sequence.

In some embodiments, the guide nucleic acid comprises one or more scaffold sequence and/or one or more guide sequence, provided that the guide nucleic acid does not comprise one scaffold sequence and one guide sequence.

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

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

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 scaffold sequences are the same or different, and wherein guide sequences are the same or different.

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 scaffold sequences are the same or different, and wherein guide sequences are the same or different.

In some embodiments, the 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 scaffold sequences are the same or different, and wherein guide sequences are the same or different.

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 scaffold sequences are the same or different, and wherein guide sequences are the same or different.

In some embodiments, the 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 scaffold sequences are the same or different, and wherein guide sequences are the same or different.

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 scaffold sequences are the same or different, and wherein guide sequences are the same or different.

In some embodiments, the guide nucleic acid comprises a linker or no linker between any adjacent scaffold sequence and guide sequence. In some embodiments, the guide nucleic acid comprises no linker between any adjacent scaffold sequence and guide sequence.

Multiple guide nucleic acid

The system or rAAV vector genome of the disclosure may comprise or encode one guide nucleic acid or 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.

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 additional guide nucleic acids comprising:

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

(2) an additional guide sequence capable of hybridizing to an additional target sequence on a target strand of the HBV cccDNA or an additional target sequence on the transcript thereof, thereby guiding the complex to the HBV cccDNA or the transcript.

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

In some embodiments, the additional protospacer sequence is on the different strand from the protospacer sequence.

In some embodiments, the additional protospacer sequence is the same or different from the protospacer sequence.

In some embodiments, the additional target sequence is the same or different from the target sequence.

In some embodiments, the additional guide sequence is the same or different from the guide sequence.

In some embodiments, the additional scaffold sequence is the same or different from the scaffold sequence. In some embodiments wherein the system comprises the same napBP and multiple guide nucleic acids, the scaffold sequences of the multiple guide nucleic acids may be the same or different (e.g., different by no more than 5, 4, 3, 2, or 1 nucleotide) to be compatible to the same napBP. In some embodiments wherein that the system comprises different napBP (e.g., a Cas12i and a Cas9) and multiple guide nucleic acids, the scaffold sequences of the multiple guide nucleic acids may be different to be compatible to the different napBP, respectively.

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) .

Nature and modification of guide nucleic acid

In some embodiments, the guide nucleic acid (e.g., the guide nucleic acid, the additional guide nucleic acid) is an RNA, i.e., a 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 a modified ribonucleotide. In some embodiments, the guide nucleic acid is a modified RNA containing a deoxyribonucleotide. In some embodiments, the guide nucleic acid is a modified RNA containing a modified deoxyribonucleotide. In some embodiments, the guide nucleic acid comprises a modified or unmodified deoxyribonucleotide and a modified or unmodified ribonucleotide.

The disclosure provides modified guide RNAs that lead to improved DNA editing efficiency of CRISPR-Cas12 system comprising such modified guide RNAs. CRISPR-Cas12 system may be used for various DNA editing application, e.g., dsDNA cleavage, epigenomic modification, such as, gene activation, gene inhibition /suppression. The DNA editing efficiency then may be DNA endonuclease activity (may also be termed as “dsDNA cleavage activity” ) , epigenomic modification efficiency, such as, gene activation efficiency, gene inhibition /suppression efficiency.

In an aspect, the disclosure provides a modified guide RNA. The modified guide RNA comprises (1) a direct repeat (DR) sequence capable of forming a complex with a Cas12 polypeptide; and (2) a guide sequence capable of hybridizing to a target sequence of a target DNA, thereby guiding the complex to the target DNA; wherein the modified guide RNA comprises a chemically modified nucleotide (e.g., carrying a modification at the 2’ position of the ribose of the chemically modified nucleotide and/or at the inter-nucleotide linkage 3’ to the ribose of the chemically modified nucleotide) .

An exemplary nucleotide of an exemplary RNA is shown in FIG. 2 (the upper nucleotide) . The nucleotide comprises a base (B) , a ribose, and a phosphate linkage (an inter-nucleotide linkage 3’ to the ribose of the nucleotide that links a next 3’ nucleotide) . The ribose carries a hydroxy (-OH) substitute at the 2’ position close to the base.

As a type of nucleotide modification, the hydroxy substitute at the 2’ position of the ribose may be replaced with another substitute, e.g., 2’-fluoro (a2’-fluoro (2’-F) modification) , or 2’-H (2’-deoxy modification) . In some embodiments, the modified guide RNA comprises a chemically modified nucleotide carrying a modification at the 2’ position of the ribose of the chemically modified nucleotide.

As another type of nucleotide modification, the phosphate 3’ to the ribose of the nucleotide may be replaced with another inter-nucleotide linkage, e.g., a phosphorothioate linkage (a3’ phosphorothioate modification) , a thioPACE linkage (a3’ thioPACE modification) . In some embodiments, the modified guide RNA comprises a chemically modified nucleotide carrying a modification at the inter-nucleotide linkage 3’ to the ribose of the chemically modified nucleotide.

In some embodiments, the chemically modified nucleotide comprises an alkylated 2’-OH modification (such as 2’-O-Me modification, 2’-O-2-methoxyethyl (2’-O-MOE) ) , a 2’-F modification, or a 2’-deoxy modification.

For the alkylated 2’-OH modification, the original 2’-OH at the 2’ position of the ribose is alkylated, e.g., methylated.For example, the original 2’-OH is alkylated to 2’-O-Methyl, and hence the chemically modified nucleotide comprises 2’-O-Methyl ( “2’-OMe” or “M” ) at the 2’ position in place of the original 2’-OH.

For the 2’-F modification, the original 2’-OH is replaced with 2’-F, and hence the chemically modified nucleotide comprises 2’-F at the 2’ position in place of the original 2’-OH.

For the 2’-deoxy modification, the original 2’-OH is deoxidized, and hence the chemically modified nucleotide comprises 2’-H at the 2’ position in place of the original 2’-OH.

In some embodiments, the chemically modified nucleotide comprises a 3’ phosphorothioate ( “PS” ) linkage or 3’ thioPACE linkage This means that the linkage replaces the original phosphate linkage.

In some embodiments, the modified guide RNA comprises a string of 2-6 consecutive chemically modified nucleotides.

In some embodiments, the chemically modified nucleotide is at or near the 5’ end of the modified guide RNA, the 3’ end of the modified guide RNA, internal to the modified guide RNA, the 5’ end of the guide sequence, the 3’ end of the guide sequence, internal to the guide sequence, the 5’ end of the scaffold sequence, the 3’ end of the scaffold sequence, internal to the scaffold sequence, or a combination thereof.

The chemically modified nucleotide being at or near the 5’ end of the modified guide RNA, the guide sequence, or the scaffold sequence means that the chemically modified nucleotide is the first, the second, the third, the fourth, or the fifth 5’ end nucleotide of the modified guide RNA, the guide sequence, or the scaffold sequence. The 5’ end nucleotide refers to a nucleotide at or near the 5’ end but not necessary the most 5’ end nucleotide. The first 5’ end nucleotide refers to the most 5’ end nucleotide. Taking modified gRNA S131 in Example 1 as an example, the first, second, third, fourth, and fifth 5’ end nucleotide of the modified gRNA or the scaffold sequence are A, G, A, A, and A, respectively, and the first, second, third, fourth, and fifth 5’ end nucleotide of the guide sequence are T, A, G, A, and A, respectively.

The chemically modified nucleotide being at or near the 3’ end of the modified guide RNA, the guide sequence, or the scaffold sequence means that the chemically modified nucleotide is the first, the second, the third, the fourth, or the fifth 3’ end nucleotide of the modified guide RNA, the guide sequence, or the scaffold sequence. The 3’ end nucleotide refers to a nucleotide at or near the 3’ end but not necessary the most 3’ end nucleotide. The first 3’ end nucleotide refers to the most 3’ end nucleotide. Taking modified gRNA S131 in Example 1 as an example, the first, second, third, fourth, and fifth 3’ end nucleotide of the modified gRNA or the guide sequence are A, T, G, A, and G, respectively, and the first, second, third, fourth, and fifth 3’ end nucleotide of the scaffold sequence are C, A, C, A, and G, respectively.

The chemically modified nucleotide being internal to the modified guide RNA, the guide sequence, or the scaffold sequence means that the chemically modified nucleotide is not at or near the 5’ or 3’ end of the modified guide RNA, the guide sequence, or the scaffold sequence.

In some embodiments, the modified guide RNA does not comprise a tracrRNA or a tracr sequence. In some embodiments, the modified guide RNA comprises a tracrRNA or a tracr sequence.

In some embodiments, the guide sequence is 5’ or 3’ to the scaffold sequence. In some embodiments, the guide sequence is 3’ to the scaffold sequence.

Modified 3’ poly U tail

In some embodiments, the guide RNA comprises, at the 3’ end of the guide RNA, a 3’ poly U tail comprising more than one uracil. In other words, a poly U tail comprising more than one uracil is added /attached to the 3’ end of the guide RNA. In the case that guide sequence of the guide RNA is 3’ to the scaffold sequence, then the guide RNA comprises, at the 3’ end of the guide sequence, a 3’ poly U tail comprising more than one uracil. In other words, a poly U tail comprising more than one uracil is added /attached to the 3’ end of the guide sequence. In some embodiments, the modified 3’ poly U tail is 3’ to the guide sequence. One example of the addition of polyU tail is shown in the gRNA of SEQ ID NO: 118, where the poly U tail is composed of UUUU and attached to the 3’ end of the guide sequence of SEQ ID NO: 1 comprised in the gRNA of SEQ ID NO: 118. The poly U tail is designated as 3’ poly U tail because it is located at the 3’ end of the guide RNA of the disclosure.

The 3’ poly U tail may be modified to a modified 3’ poly U tail. In some embodiments, the modified 3’ poly U tail comprises a 2’-O-methyl 3’ phosphorothioate modification on an uracil of the modified 3’ poly U tail. In some embodiments, the modified 3’ poly U tail comprises two, three, four, five, six, seven, or more uracils. In some embodiments, the modified 3’ poly U tail comprises a 2’-O-methyl 3’ phosphorothioate modification on one, two, three, four, five, six, seven, or more (consecutive or non-consecutive) uracils of the modified 3’ poly U tail. In some embodiments, the modified 3’ poly U tail comprises a 2’-O-methyl 3’phosphorothioate modification on its first two, three, four, five, six, seven, or more 5’ end uracils. In some embodiments, the modified 3’ poly U tail comprises, consists essentially of, or consists of four uracils with 2’-O-methyl 5’ phosphorothioate modifications on its first three 5’ end uracils. For example, the modified 3’ poly U tail is composed of 5’-TmsTmsTmsT-3’.

By “2’-O-methyl 3’ phosphorothioate modification” it means, referring to FIG. 2, a 2’-O-methyl modification on the ribose of a modified nucleotide plus a 3’ phosphorothioate ( “PS” ) inter-nucleotide linkage 3’ to the ribose of the modified nucleotide (collectively, “2’-O-methyl 3’ phosphorothioate modification” or “MS” ) , wherein the modified ribonucleotide is denoted with Nms (N = A, U, G, or C) throughout the disclosure.

Modified scaffold sequence

The scaffold sequence of the modified gRNA of the disclosure may be modified as desired. In some embodiments, the scaffold sequence comprises a 2’-O-methyl 3’ phosphorothioate modification. In some embodiments, the scaffold sequence is 5’ to the guide sequence. In some embodiments, the scaffold sequence comprises a 2’-O-methyl 3’phosphorothioate modification on one, two, or three nucleotides of its first three, four, five, or six 5’ end nucleotides. In some embodiments, the scaffold sequence comprises a 2’-O-methyl 3’phosphorothioate modification on each of its first three 5’ end nucleotides.

In some embodiments, the scaffold sequence comprises motif 5’-AGA-3’ as its first three 5’ end nucleotides. In some embodiments, the scaffold sequence comprises a 2’-O-methyl 3’phosphorothioate modification on each nucleotide of the motif 5’-AGA-3’ as its first three 5’ end nucleotides. That is, the scaffold sequence comprise modified motif 5’-AmsGmsAms-3’ as its first three 5’ end nucleotides.

In some embodiments, the scaffold sequence has substantially the same secondary structure as the secondary structure of SEQ ID NO: 93 or 129. In some embodiments, the scaffold sequence is a N-terminal truncation of SEQ ID NO: 129, and wherein the N-terminal truncation eliminates a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the scaffold sequence comprises a polynucleotide sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the polynucleotide sequence of SEQ ID NO: 93 or 129, or wherein the scaffold sequence comprises a polynucleotide sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide sequence changes compared to the polynucleotide sequence of SEQ ID NO: 93 or 129. In some embodiments, the scaffold sequence comprises, consists essentially of, or consists of AmsGmsAmsAATGTGTCCCCAGTTGACAC, wherein Nms represents a ribonucleotide N (N = A, U, G, or C) with a 2’-O-methyl 5’ phosphorothioate modification.

Modified Guide Sequence

The guide sequence of the modified gRNA of the disclosure may be modified as desired. In some embodiments, the guide sequence comprises a 2’-fluoro modification. In some embodiments, the guide sequence is 3’ to the scaffold sequence. In some embodiments, the guide sequence comprises a 2’-fluoro modification on one or two nucleotides of its first two 3’ end nucleotides. In some embodiments, the guide sequence comprises a 2’-fluoro modification on each nucleotide of its first two 3’ end nucleotides.

In some embodiments, the guide sequence is in a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, and optionally about 20 nucleotides.

Characterization of Modified gRNA

In some embodiments, the guide RNA comprises, from 5’ to 3’:

(i) a modified scaffold sequence as described in the disclosure;

(ii) a guide sequence; and

(iii) a modified 3’ poly U tail as described in the disclosure.

In some embodiments, the guide RNA comprises, from 5’ to 3’:

(i) a modified scaffold sequence comprising a 2’-O-methyl 3’phosphorothioate modification on each of its first three 5’ end nucleotides;

(ii) a guide sequence; and

(iii) a modified 3’ poly U tail comprises, consists essentially of, or consists of four uracils with 2’-O-methyl 5’ phosphorothioate modifications on its first three 5’ end uracils.

In some embodiments, the guide RNA comprises, from 5’ to 3’:

(i) a scaffold sequence comprises, consists essentially of, or consists of: AmsGmsAmsAATGTGTCCCCAGTTGACAC;

(ii) a guide sequence; and

(iii) a modified 3’ poly U tail of TmsTmsTmsT;

wherein Nms represents a ribonucleotide N (N = A, U, G, or C) with a 2’-O-methyl 3’ phosphorothioate modification.

In some embodiments, the modified guide RNA has increased stability compared with an otherwise identical control guide RNA without the modification of the modified guide RNA, e.g., an increase by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more.

In some embodiments, the modified guide RNA has decreased immunogenicity compared with an otherwise identical control guide RNA without the modification of the modified guide RNA, e.g., a decrease by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Applications of Modified gRNA

In another aspect, the disclosure provides a system or composition comprising: (i) a Cas12 polypeptide or a polynucleotide encoding the Cas12 polypeptide; and (ii) the modified guide RNA of the disclosure. In some embodiments, (1) the system has an increased on-target DNA editing efficiency (e.g., DNA cleavage activity, gene activating or inhibiting efficiency) as compared with an otherwise identical control system comprising a guide RNA without the modification of the modified guide RNA, e.g., an increase by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more; or (2) the system has a decreased off-target DNA editing efficiency (e.g., DNA cleavage activity, gene activating or inhibiting efficiency) as compared with an otherwise identical control system comprising a guide RNA without the modification of the modified guide RNA, e.g., a decrease by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

In yet another aspect, the disclosure provides a lipid nanoparticle (LNP) comprising (i) an mRNA encoding a Cas12 polypeptide; and (ii) the modified guide RNA of the disclosure.

In yet another aspect, the disclosure provides a ribonucleoprotein (RNP) comprising (i) a Cas12 polypeptide; and (ii) the modified guide RNA of the disclosure.

In some embodiments, the Cas12 polypeptide does not comprise a Zinc finger. In some embodiments, the Cas12 polypeptide comprises a Zinc finger. In some embodiments, the Cas12 polypeptide is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas12l, Cas12m, or Cas12n. In some embodiments, the Cas12 polypeptide is a Cas12i polypeptide. In some embodiments, the Cas12i polypeptide comprises an amino acid sequence having a sequence identity of at least about 60% (e.g., at least about 60%, 65%, 70%, 75%, 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%) to the amino acid sequence of SEQ ID NO: 94 or the amino acid sequence of SEQ ID NO: 94 but lacking N-terminal starting Methionine (M) (coded by start codon ATG) . For example, the N-terminal starting Methionine (M) (coded by start codon ATG) at position 1 of SEQ ID NO: 94 may be removed.

In yet another aspect, the disclosure provides a method of modifying a target DNA comprising contacting the target DNA with the system, the LNP, or the RNP of the disclosure. In some embodiments, the method is ex vivo, in vivo, or in vitro. In some embodiments, the method is non-therapeutical.

In yet another aspect, the disclosure provides use of the system, the LNP, or the RNP of the disclosure in the manufacture of an agent for the modification of a target DNA. In some embodiments, the modification is ex vivo, in vivo, or in vitro. In some embodiments, the modification is non-therapeutical.

In yet another aspect, the disclosure provides a cell comprising the modified guide RNA, the system, the LNP, or the RNP of the disclosure. In some embodiments, the cell is not a human germ cell (i.e., an embryonic cell, an egg cell, a sperm cell) . In some embodiments, the cell is not a human embryonic stem cell.

Specific target and guide sequences

In some embodiments, the guide sequence or the additional guide sequence comprises (1) a sequence of SEQ ID NO:1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 1-92 and 133-547.

In some embodiments, the guide sequence or the additional guide sequence comprises a sequence of SEQ ID NO: 1-92 and 133-547.

Specific scaffold sequence

For the purpose of the disclosure, the scaffold sequence is compatible with the napBP of the disclosure and is capable of complexing with the napBP. The scaffold sequence may be a naturally occurring scaffold sequence identified along with the napBP, or a variant thereof maintaining the ability to complex with the napBP. Generally, the ability to complex with the napBP is maintained as long as the secondary structure of the variant is substantially identical to the secondary structure of the naturally occurring scaffold sequence. A nucleotide deletion, insertion, or substitution in the primary sequence of the scaffold sequence may not necessarily change the secondary structure of the scaffold sequence (e.g., the relative locations and/or sizes of the stems, bulges, and loops of the scaffold sequence do not significantly deviate from that of the original stems, bulges, and loops) . For example, the nucleotide deletion, insertion, or substitution may be in a bulge or loop region of the scaffold sequence so that the overall symmetry of the bulge and hence the secondary structure remains largely the same. The nucleotide deletion, insertion, or substitution may also be in the stems of the scaffold sequence so that the lengths of the stems do not significantly deviate from that of the original stems (e.g., adding or deleting one base pair in each of two stems correspond to 4 total base changes) .

In some embodiments, the scaffold sequence or the additional scaffold sequence has substantially the same secondary structure as the secondary structure of the sequence of SEQ ID NO: 93 or 129.

In some embodiments, the scaffold sequence or the additional scaffold sequence comprises (1) a sequence of SEQ ID NO: 93 or 129 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 93 or 129 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 93 or 129.

In some embodiments, the scaffold sequence or the additional scaffold sequence comprises the sequence of SEQ ID NO:93 or 129.

Specific guide nucleic acid sequence

In some embodiments, the guide nucleic acid comprises a sequence having a sequence identity of at least about 80%(e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of any one of SEQ ID NOs: 103-105 and 118-120; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of any one of SEQ ID NOs: 103-105 and 118-120.

In some embodiments, the guide nucleic acid comprises a sequence of any one of SEQ ID NOs: 103-105 and 118-120.

Regulation of guide nucleic acid

Also provided in the disclosure is a polynucleotide comprising or encoding the guide nucleic acid.

In some embodiments, the polynucleotide comprising or encoding the guide nucleic acid is a DNA, a RNA, or a DNA/RNA mixture. By “DNA/RNA mixture” it refers to a nucleic acid comprising both one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether consecutive or not. However, by “DNA” or “RNA” it may also refer to a DNA containing one or more modified or unmodified ribonucleotides, whether consecutive or not, or an RNA containing one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.

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

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

In some embodiments, the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter.

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

Regulation of napBP

In some embodiments, the polynucleotide encoding the napBP is a DNA, a RNA, or a DNA/RNA mixture. By “DNA/RNA mixture” it refers to a nucleic acid comprising both one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether consecutive or not. However, by “DNA” or “RNA” it may also refer to a DNA containing one or more modified or unmodified ribonucleotides, whether consecutive or not, or an RNA containing one or more modified or unmodified deoxyribonucleotides, whether consecutive or not.

In some embodiments, the polynucleotide encoding the napBP is a mRNA.

In some embodiments, the polynucleotide encoding the napBP comprises a sequence encoding the napBP and a promoter operably linked to the sequence encoding the napBP.

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

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

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

In some embodiments, the promoter is a T7 promoter.

In some embodiments, the second polynucleotide sequence comprises a Kozak sequence (gccacc) 5’ to the sequence encoding the napBP.

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

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

In some embodiments, the second polynucleotide sequence comprises a sequence encoding a first NLS 5’ to the sequence encoding the napBP and a second sequence encoding a second NLS 3’ to the sequence encoding the napBP.

In some embodiments, the NLS, the first NLS, and/or the second NLS is a SV40 NLS, a bpSV40 NLS, or a Nucleoplasmin NLS (npNLS) (SEQ ID NO: 96) .

In some embodiments, the napBP comprises a SV40 NLS at the N-terminal of the napBP and a npNLS at the C-terminal of the napBP.

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

In some embodiments, the second polynucleotide comprises a sequence encoding a first NLS at the 5’ end of the sequence encoding the napBP.

In some embodiments, the second polynucleotide comprises a sequence encoding a first NLS inserted between the 5’start codon ATG and the remaining polynucleotide sequence of the sequence encoding the napBP.

In some embodiments, the second polynucleotide comprises a sequence encoding a second NLS at the 3’ end of the sequence encoding the napBP.

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

In some embodiments, the WPRE sequence is selected from the group consisting of Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) , WPRE3 (ashortened WPRE) , and a functional variant (e.g., a functional truncation) thereof.

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

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

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

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

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

In some embodiments, the Kozak sequence is gccacc.

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

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

In some embodiments, the sequence encoding the polyA signal comprises a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 101; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of SEQ ID NO: 101.

In some embodiments, the mRNA encoding the napBP comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 102.

LNP formulation

In some embodiments, the lipid mixture further comprises ethanol.

In some embodiments, the nucleic acid (e.g., RNA) mixture for preparing the LNP comprises the polynucleotide encoding the napBP and the guide nucleic acid.

In some embodiments, the nucleic acid (e.g., RNA) mixture for preparing the LNP comprises the polynucleotide encoding the napBP and the guide nucleic acid in a ratio of about 1: 1, 1: 1.1, 1: 1.2, 1: 1.3, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.7, 1: 1.8, 1: 1.9, or about 1: 2.

In some embodiments, the nucleic acid (e.g., RNA) mixture further comprises NaAc buffer.

Full length rAAV vector genome

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

In some embodiments, the 5’ ITR sequence and the 3’ ITR sequence are both wild-type ITR sequences from 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 Clade to which any of the AAV1-AAV13 belong, or a functional variant (e.g., a functional truncation) thereof.

In some embodiments, the rAAV vector genome comprises, from 5’ to 3’, the first polynucleotide sequence and the second polynucleotide sequence.

In some embodiments, the rAAV vector genome comprises, from 5’ to 3’, the second polynucleotide sequence and the first polynucleotide sequence.

In some embodiments, the rAAV vector genome comprises, from the 5’ to 3’,

(1) a 5’ ITR as described in the disclosure;

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

(3) a sequence encoding a guide nucleic acid as described in the disclosure;

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

(5) optionally, a Kozak sequence as described in the disclosure;

(6) an in-frame start codon ATG;

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

(8) a sequence encoding the napBP as described in the disclosure;

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

(10) an in-frame stop codon,

(11) optionally, a WPRE sequence as described in the disclosure;

(12) a sequence encoding a polyA signal as described in the disclosure; and

(13) a 3’ ITR as described in the disclosure.

In some embodiments, the rAAV vector genome of the disclosure is a DNA rAAV vector genome or an RNA rAAV vector genome. By “DNA rAAV vector genome” it means that the rAAV vector genome is a DNA that can be encapsulated into a rAAV particle. By “RNA rAAV vector genome” it means that the rAAV vector genome is an RNA that can be encapsulated into a rAAV particle.

rAAV particle

In yet another aspect, the disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the disclosure. A simple introduction of AAV for delivery may refer to “Adeno-associated Virus (AAV) Guide” (addgene. org/guides/aav/) .

Adeno-associated virus (AAV) , when engineered to delivery, e.g., a protein-encoding sequence of interest, may be termed as a (r) AAV vector, a (r) AAV vector particle, or a (r) AAV particle, where “r” stands for “recombinant” . And the genome packaged in AAV vectors for delivery may be termed as a (r) AAV vector genome, vector genome, or vg for short, while viral genome may refer to the original viral genome of natural AAVs.

The serotypes of the capsids of rAAV particles can be matched to the types of target cells. For example, Table 2 of WO2018002719A1 lists exemplary cell types that can be transduced by the indicated AAV serotypes (incorporated herein by reference) .

In some embodiments, the rAAV particle comprising a capsid with a serotype suitable for delivery into hepatocytes. In some embodiments, the rAAV particle comprising a capsid with a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV. PHP. eB, a member of the Clade to which any of the AAV1-AAV13 belong, or a functional variant (e.g., a functional truncation) thereof, encapsidating the rAAV vector genome. In some embodiments, the serotype of the capsid is wild type serotype or a functional variant thereof.

General principles of rAAV particle production are known in the art. In some embodiments, rAAV particles may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650) .

The vector titers are usually expressed as vector genomes per ml (vg/ml) . In some embodiments, the vector titer is above 1×10⁹, above 5×10¹⁰, above 1×10¹¹, above 5×10¹¹, above 1×10¹², above 5×10¹², or above 1×10¹³ vg/ml.

Instead of packaging a single strand (ss) DNA sequence as a vector genome of a rAAV particle, systems and methods of packaging an RNA sequence as a vector genome into a rAAV particle is recently developed and applicable herein. See PCT/CN2022/075366, which is incorporated herein by reference in its entirety.

When the vector genome is RNA as in, for example, PCT/CN2022/075366, for simplicity of description and claiming, sequence elements described herein for DNA vector genomes, when present in RNA vector genomes, should generally be considered to be applicable for the RNA vector genomes 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 element in the DNA sequence is replaced with the corresponding element with a corresponding function in the RNA sequence or omitted because its function is unnecessary in the RNA sequence and/or an additional element necessary for the RNA vector genome is introduced.

As used herein, a coding sequence, e.g., as a sequence element of rAAV vector genomes herein, is construed, understood, and considered as covering and covers both a DNA coding sequence and an RNA coding sequence. When it is a DNA coding sequence, an RNA sequence can be transcribed from the DNA coding sequence, and optionally further a protein can be translated from the transcribed RNA sequence as necessary. When it is an RNA coding sequence, the RNA coding sequence per se can be a functional RNA sequence for use, or an RNA sequence can be produced from the RNA coding sequence, e.g., by RNA processing, or a protein can be translated from the RNA coding sequence.

For example, a Cas13 coding sequence encoding a Cas13 polypeptide covers either a Cas13 DNA coding sequence from which a Cas13 polypeptide is expressed (indirectly via transcription and translation) or a Cas13 RNA coding sequence from which a Cas13 polypeptide is translated (directly) .

For example, a gRNA coding sequence encoding a gRNA covers either a gRNA DNA coding sequence from which a gRNA is transcribed or a gRNA RNA coding sequence (1) which per se is the functional gRNA for use, or (2) from which a gRNA is produced, e.g., by RNA processing.

In some embodiments for rAAV RNA vector genomes, 5’-ITR and/or 3’-ITR as DNA packaging signals may be unnecessary and can be omitted at least partly, while RNA packaging signals can be introduced.

In some embodiments for rAAV RNA vector genomes, a promoter to drive transcription of DNA sequences may be unnecessary and can be omitted at least partly.

In some embodiments for rAAV RNA vector genomes, a sequence encoding a polyA signal may be unnecessary and can be omitted at least partly, while a polyA tail can be introduced.

Similarly, other DNA elements of rAAV DNA vector genomes can be either omitted or replaced with corresponding RNA elements and/or additional RNA elements can be introduced, in order to adapt to the strategy of delivering an RNA vector genome by rAAV particles.

In yet another aspect, the disclosure provides a method for production of a rAAV particle, comprising culturing in a host cell a transgene plasmid comprising the rAAV vector genome of the disclosure, thereby encapsulating the rAAV vector genome into a capsid with a serotype of AAV8. In yet another aspect, the disclosure provides a cell comprising a transgene plasmid comprising the rAAV vector genome of the disclosure. In yet another aspect, the disclosure provides use of the cell comprising a transgene plasmid comprising the rAAV vector genome of the disclosure for the production of rAAV particles.

Pharmaceutical composition

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

In some embodiments, the pharmaceutical composition comprises the rAAV particle in a concentration selected from the group consisting of about 1×10¹⁰ vg/mL, 2×10¹⁰ vg/mL, 3×10¹⁰ vg/mL, 4×10¹⁰ vg/mL, 5×10¹⁰ vg/mL, 6×10¹⁰ vg/mL, 7×10¹⁰ vg/mL, 8×10¹⁰ vg/mL, 9×10¹⁰ vg/mL, 1×10¹¹ vg/mL, 2×10¹¹ vg/mL, 3×10¹¹ vg/mL, 4×10¹¹ vg/mL, 5×10¹¹ vg/mL, 6×10¹¹ vg/mL, 7×10¹¹ vg/mL, 8×10¹¹ vg/mL, 9×10¹¹ vg/mL, 1×10¹² vg/mL, 2×10¹² vg/mL, 3×10¹² vg/mL, 4×10¹² vg/mL, 5×10¹² vg/mL, 6×10¹² vg/mL, 7×10¹² vg/mL, 8×10¹² vg/mL, 9×10¹² vg/mL, 1×10¹³ vg/mL, or in a concentration of a numerical range between any of two preceding values, e.g., in a concentration of from about 9×10¹⁰ vg/mL to about 8×10¹¹ vg/mL.

In some embodiments, the pharmaceutical composition is an injection formulation.

In some embodiments, the volume of the injection is selected from the group consisting of 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 a volume of a numerical range between any of two preceding values, e.g., in a concentration of from about 10 microliters to about 750 microliters.

Cells

In yet another aspect, the disclosure provides a cell or a progeny thereof comprising the guide nucleic acid of the disclosure, the system of the disclosure, the LNP of the disclosure, or the rAAV particle of the disclosure. In some embodiments, the cell is a eukaryote. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human hepatocyte.

In yet another aspect, the disclosure provides a cell or a progeny thereof comprising HBV cccDNA or transcript thereof modified by the system of the disclosure, the LNP of the disclosure, the rAAV particle of the disclosure, or the method of the disclosure. In some embodiments, the cell is a eukaryote. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human hepatocyte.

In some embodiments, the cell is not within the body of an organism, such as, 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.

Treatment method

In yet another aspect, the disclosure provides a method for preventing, diagnosing, and/or treating an HBV associated disease in a subject in need thereof, comprising administering to the subject the system of the disclosure, the LNP of the disclosure, the rAAV particle of the disclosure, or the pharmaceutical composition of the disclosure, wherein the napBP modifies HBV cccDNA or transcript thereof, and wherein the modification of the HBV cccDNA or transcript thereof treats the disease. In some embodiments, the napBP modifies the HBV cccDNA, leading to downregulated presence or transcription of HBV cccDNA, thereby treating the HBV associated disease.

As used herein, the term “HBV associated diseases” includes diseases that are caused by the infection of HBV into cells.

In some embodiments, the HBV cccDNA or transcript thereof is in a eukaryotic cell, for example, a human cell, a non-human primate cell, or a mouse cell, such as, a hepatocyte.

In some embodiments, the administrating comprises local administration or systemic administration.

In some embodiments, the administrating comprises subretinal administration, intrathecal administration, intramuscular administration, intravenous administration, transdermal administration, intranasal administration, oral administration, mucosal administration, intraperitoneal administration, intracranial administration, intracerebroventricular administration, or stereotaxic administration.

In some embodiments, the administration is injection or infusion, e.g., intravenous injection.

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

In some embodiments, the level of the HBV cccDNA, HBV DNA, or HBsAg is decreased in the subject 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 HBV cccDNA, HBV DNA, or HBsAg in the subject prior to the administration.

In some embodiments, the median survival of the subject suffering from the disease but receiving 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 year, 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 that of a subject or a population of subjects suffering from the disease and not receiving the administration.

The dose of the rAAV particle for treatment of the HBV associated diseases may be either via a single dose, or multiple doses. One skilled in the art understands that the actual dose may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation/modification sought, the administration routes, the administration modes, the types of transformation/modification sought, etc.

In some embodiments, the rAAV particle is administrated in a therapeutically effective dose. For example, the therapeutically effective dose of the rAAV particle may be about 1.0E+7 (1.0 × 10⁷) , 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, 4.0E+12, 6.0E+12, 8.0E+12, 1.0E+13, 2.0E+13, 3.0E+13, 4.0E+13, 6.0E+13, 8.0E+13, 1.0E+14, 2.0E+14, 3.0E+14, 4.0E+14, 6.0E+14, 8.0E+14, 1.0E+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 a range of any two of the those point values. vg stands for vector genomes of rAAV particles for administration.

Kit

In yet another aspect, the disclosure provides a kit comprising the system of the disclosure, the rAAV particle of the disclosure, or the pharmaceutical composition of the disclosure, or any one, two, or all components of the same.

In some embodiments, the kit further comprises an instruction to use the component (s) contained therein, and/or instructions for combining with additional component (s) that may be available or necessary elsewhere.

In some embodiments, the kit further comprises one or more buffers that may be used to dissolve any of the component (s) contained therein, and/or to provide suitable reaction conditions for one or more of the component (s) . Such buffers may include one or more of PBS, HEPES, Tris, MOPS, Na₂CO₃, NaHCO₃, NaB, or combinations thereof. In some embodiments, the reaction condition includes a proper pH, such as a basic pH. In some embodiments, the pH is between 7-10.

In some embodiments, any one or more of the kit components may be stored in a suitable container or at a suitable temperature, e.g., 4 degree Celsius.

Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the disclosure.

EXAMPLES

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

Example 1. Screening of HBV cccDNA-targeting guide sequences and evaluation

Effective guide RNAs (gRNAs) were initially selected and screened within the PiggyBac (PB) -HBV-HEK293 system. Furthermore, a functional gRNAs screen was performed in HBV-integrated cell lines and primary human hepatocytes (PHHs) infected with HBV. HBV cccDNA was generated and presented in the cell line. To evaluate the in vivo efficacy of the hfCas12Max endonuclease (SEQ ID NO: 94) guided by the HBV-targeting gRNAs, an episomal adeno-associated virus (AAV) mouse model harboring a 1.3-fold HBV genome was developed, which served as a surrogate for HBV cccDNA. Clinically relevant delivery was achieved through the systemic administration of LNPs encapsulating hfCas12Max encoding mRNA and HBV-

Upon delivery via the LNPs, the hfCas12Max system resulted in a substantial decrease in cccDNA, HBV-DNA, and HBsAg levels in HBV-infected PHHs. In the AAV-HBV mouse model, sustained reductions in HBsAg expression (up to 2.5 log) and serum HBV DNA (～3log) following systemic delivery of varying doses of the CRISPR-hfCas12Max system mediated by LNP was observed.

I. Screening gRNAs in PB-HBV-HEK293T cell line

FIG. 6 shows the results of screening gRNAs in PB-HBV-HEK293T cell line by co-transfection with hfCas12Max endonuclease (SEQ ID NO: 94) vector. FIG. 6A shows the schematic of the PB-HBV vector for transfection into HEK293T cells to establish PB-HBV-HEK293T cell line for gRNA screening. FIG. 6B shows Indel percentages (indicating editing efficiency) for a series of designed gRNAs in the PB-HBV-HEK293T cell line (n=2 or 3) . The inverted red triangle marks several gRNAs with relatively high editing efficiency (as denoted by “indel%” measured by sequencing) , and the combinations of the gRNAs were evaluated subsequently.

The gRNAs were each composed of one same scaffold sequence (SEQ ID NO: 93) 5’ to a different guide sequence (one of SEQ ID NOs: 1-92) . The sequences of representative gRNA are set forth in SEQ ID NO: 103-105, comprising guide sequences G47 (SEQ ID NO: 1) , G82 (SEQ ID NO: 2) , and G116 (SEQ ID NO: 3) , respectively.

II. Multiplexing three gRNAs with hfCas12Max endonuclease reduced HBV viral parameters

FIG. 7 shows the multiplexing of three gRNAs with hfCas12Max endonuclease reduced HBV viral parameters. HBsAg (FIG. 7A) and HBV DNA (FIG. 7B) were reduced in Hep2.2.15 and/or HepAD38 cells as induced by hfCas12Max coding mRNA (SEQ ID NO: 102) and various gRNA combinations delivered by LNP (n=3) . The combination of gRNAs-47, -82 and -116 (Red triangle) resulted in the highest potency of both HBsAg and HBV DNA reduction, and thus this gRNA combination was then tested in vivo.

The LNPs were prepared by packaging the lipid mixture (in the table below) and RNA mixture (in the table below) in a ratio of 1: 3.

The LNPs were delivered into PB-HBV-HEK293T cells to evaluate the cleavage activity against HBV cccDNA. Significant cleavage activity was observed for most of the treatment gRNA (FIG. 6B) . Guide sequences G47, G82, and G116 (SEQ ID NOs: 1-3, respectively) were selected for subsequent experiments.

The gRNAs for LNP delivery were modified gRNAs (FIG. 11) , comprising a modified scaffold sequence comprising a 2’-O-methyl 3’phosphorothioate modification on each of its first three 5’ end nucleotides (AGA) and comprising a modified 3’ poly U tail consisting of four uracils (UUUU) with 2’-O-methyl 5’ phosphorothioate modifications on its first three 5’ end uracils. The sequences of representative modified gRNA are set forth in SEQ ID NO: 118-120, comprising guide sequences G47 (SEQ ID NO: 1) , G82 (SEQ ID NO: 2) , and G116 (SEQ ID NO: 3) , respectively.

III. LNP-mediated delivery of hfCas12Max mRNA and triple gRNA combination (G47, G82, and G116) led to sustained reduction of HBV viral markers in primary human hepatocytes (PHHs)

FIG. 8 shows that LNP-mediated delivery of hfCas12Max mRNA and triple gRNA combination (G47, G82, and G116) led to sustained reduction of HBV viral markers in primary human hepatocytes (PHHs) . HBV replication assessed by HBsAg ELISA and HBV DNA qPCR in PHH supernatant were shown in FIG. 8A and FIG. 8B, respectively. FIG. 8C shows the viability of PHH cells in each group was unaffected on day 15, demonstrating safety of the treatment. FIG. 8D shows that the hfCas12Max nuclease treatment resulted in significant reduction in HBV cccDNA at day 15 post transduction in PHH and shows inhibition with dose dependency (1 dose vs. 2 doses for low concentration) . “low-c” refers to low concentration; “EVT” refers to Entecavir.

IV. LNP-mediated delivery of hfCas12Max mRNA and gRNAs led to sustained reduction of HBV viral markers in AAV-HBV mouse model

FIG. 9 shows that LNP-mediated delivery of hfCas12Max mRNA and gRNAs led to sustained reduction of HBV viral markers in AAV-HBV mouse model. The AAV-HBV mouse model was used in the pilot in vivo study. 4 weeks after hydrodynamic injection with rAAV8-1.3-fold HBV genome, mice received two doses (2x) of the Cas12-gRNA reagent (hfCas12Max mRNA & gRNA formulated into a lipid nanoparticle (LNP) , at 2 mg/kg) . The Cas12 endonuclease treatment resulted in sustained reduction in HBsAg expression (～ 2 log) (FIG. 9A) and HBV DNA (Maximum ～ 4 log) (FIG. 9B) in serum (n = 3 or 4) till Day 70 after LNP injection. In addition, the AAV-HBV mouse model received single (FIG. 9C) or two (FIG. 9D) doses of the Cas12-gRNA reagent (hfCas12Max mRNA & various combinations of gRNA formulated into LNP) . The Cas12 endonuclease treatment resulted in sustained reduction in HBV DNA (～ 4 log) in serum (n =3 or 4) till Day 42 or Day 49 after LNP injection.

V. Conclusion

Through LNP delivery, the Cas12 endonuclease treatment resulted in a significant reduction of HBV-DNA and HBsAg in HBV-infected PHHs. In the AAV-HBV mouse model, sustained reductions in HBsAg expression (～ 2 log) and serum HBV DNA (～ 4 log) after LNP-mediated systemic delivery of the Cas12 endonuclease system were observed. The data suggests that the Cas12 endonuclease system could disrupt HBV expressing templates both in vitro and in vivo, indicating its potential in eradicating HBV cccDNA and paving the way toward a potential cure for HBV associated diseases.

* * *

Various modifications and variations of the described products, methods, and uses of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure. This application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure come within known customary practice within the art to which the disclosure pertains and may be applied to the essential features herein before set forth.

EXEMPLARY SEQUENCES

Protospacer sequence /guide sequence, SEQ ID NOs: 1-92, 20 nt

Scaffold sequence compatible to hfCas12Max, SEQ ID NO: 93, 23 nt

Amino acid sequence of hfCas12Max (napDNAn) , SEQ ID NO: 94, 1080 aa; with N-terminal Met (corresponding to Start Codon ATG of hfCas12Max coding sequence of SEQ ID NO: 95)

hfCas12Max coding sequence, SEQ ID NO: 95, 3240 nt; with Start Codon ATG corresponding to N-terminal Met of the amino acid sequence of hfCas12Max of SEQ ID NO: 94

Nucleoplasmin NLS (npNLS, NP NLS) amino acid sequence, SEQ ID NO: 96

Nucleoplasmin NLS (npNLS, NP NLS) coding sequence, SEQ ID NO: 97

T7 promoter, SEQ ID NO: 98

hHBB-5’ UTR, SEQ ID NO: 99

hHBB-3'UTR, SEQ ID NO: 100

polyA signal, SEQ ID NO: 101

mRNA encoding napDNAn with T7 promoter, SEQ ID NO: 102

Representative gRNA (S-G) 1 (G47) , SEQ ID NO: 103

Representative gRNA (S-G) 1 (G82) , SEQ ID NO: 104

Representative gRNA (S-G) 1 (G116) , SEQ ID NO: 105

Kozak sequence

gRNA, SEQ ID NO: 107-128

Note: the boxedanddenote 2’-O-methyl 3’phosphorothioate modifications of the indicated nucleotides.

Scaffold sequence compatible to hfCas12Max, SEQ ID NO: 129, 30 nt

Protospacer sequence /guide sequence, SEQ ID NOs: 133-547

Protospacer sequence /guide sequence, SEQ ID NOs: 511-547

SEQ ID NO: 548, dCas12Max, xCas12i-N243R+E336R+D1049A, 1080 aa

SEQ ID NO: 549, KRAB, 62 aa

SEQ ID NO: 550, Mus DNMT3l, 214 aa

SEQ ID NO: 551, Mus DNMT3a, 302 aa

SEQ ID NO: 552, Epigenomic editor /Fusion (dCas12Max-KRAB-DNMT3l-DNMT3a)

Claims

A system comprising:

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

(a) a scaffold sequence capable of forming a complex with a Cas12i endonuclease, and

(b) a guide sequence capable of hybridizing to a target sequence on a target strand of an HBV cccDNA, thereby guiding the complex to the HBV cccDNA; and

(2) the Cas12i endonuclease or a polynucleotide encoding the Cas12i endonuclease;

wherein the guide sequence comprises (1) a sequence of SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 1-92 and 133-547; and

wherein the Cas12i endonuclease comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 94 or a N-terminal truncation thereof without the first N-terminal Methionine.
The system of any preceding claim, wherein the Cas12i endonuclease comprises substitutions N243R+E336R+D892R relative to the wild type of the Cas12 endonuclease of SEQ ID NO: 94, and 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: 94.
The system of any preceding claim, wherein the scaffold sequence has substantially the same secondary structure as the secondary structure of the sequence of SEQ ID NO: 93 or 129; or wherein the scaffold sequence or the additional scaffold sequence comprises (1) a sequence of SEQ ID NO: 93 or 129 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 93 or 129 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 93 or 129.
The system of any preceding claim, wherein the guide nucleic acid comprises one said scaffold sequence 5’ to one said guide sequence; wherein the guide nucleic acid comprises, from 5’ to 3’, one said scaffold sequence, one said guide sequence, one said scaffold sequence, and one said guide sequence, wherein scaffold sequences are the same or different, and wherein guide sequences are the same or different; or 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, one said scaffold sequence, and one said guide sequence, wherein scaffold sequences are the same or different, and wherein guide sequences are the same or different.
The system of any preceding claim, wherein the guide nucleic acid is a guide RNA, wherein the scaffold sequence of the guide RNA is modified to comprises a 2’-O-methyl 3’ phosphorothioate modification on each of its first three 5’ end nucleotides.
The system of any preceding claim, wherein the guide nucleic acid is a guide RNA, wherein the guide RNA comprises a modified 3’ poly U tail comprising, consisting essentially of, or consisting of four uracils with 2’-O-methyl 5’ phosphorothioate modifications on its first three 5’ end uracils.
The system of any preceding claim, wherein the guide nucleic acid comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of any one of SEQ ID NOs: 103-105 and 118-120; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether consecutive or not, compared to the sequence of any one of SEQ ID NOs: 103-105 and 118-120.
The system of any preceding claim, wherein the system comprises two, three, or more guide nucleic acids, each of the guide nucleic acids comprising:

(1) a scaffold sequence capable of forming a complex with a Cas12i endonuclease, and

(2) a guide sequence capable of hybridizing to a target sequence on a target strand of an HBV cccDNA, thereby guiding the complex to the HBV cccDNA,

wherein the guide sequence comprises (1) a sequence of SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO: 1-92 and 133- 547;

wherein the guide sequences of the guide nucleic acids are different.
The system of any preceding claim, wherein the system comprises three guide nucleic acids comprising guide sequences of SEQ ID NOs: 1, 2, and 3, respectively.
The system of any preceding claim, wherein the polynucleotide encoding the Cas12i endonuclease is a mRNA.
The system of claim 8, wherein the mRNA encoding the Cas12i endonuclease comprises a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 102.
The system of any preceding claim, wherein the editing efficiency for the guide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
A method of modifying an HBV cccDNA, comprising contacting the HBV cccDNA with a system of any preceding claim.
The method of claim 10, wherein said guiding the complex to the HBV cccDNA enables the Cas12i endonuclease to specifically cleave the HBV cccDNA in a guide sequence-specific manner.
The method of any preceding claim, wherein the specific cleavage of the HBV cccDNA leads to degradation of the HBV cccDNA.
The method of any preceding claim, wherein the specific cleavage of the HBV cccDNA generates an in-frame stop codon in the HBV cccDNA.
A lipid nanoparticle (LNP) comprising the system of any preceding claim.
The LNP of any preceding claim, wherein the lipid mixture for preparing the LNP comprises ALC-0315, Cholesterol, DMG-PEG, and DSPC.
The LNP of claim 15, wherein the lipid mixture for preparing the LNP comprises about 50 mM of ALC-0315, about 50 mM of Cholesterol, about 10 mM of DMG-PEG, and about 20 mM of DSPC.
The LNP of any preceding claim, wherein the nucleic acid (e.g., RNA) mixture for preparing the LNP comprises the polynucleotide encoding the Cas12i endonuclease and the guide nucleic acid.
The LNP of any preceding claim, wherein the nucleic acid (e.g., RNA) mixture for preparing the LNP comprises the polynucleotide encoding the Cas12i endonuclease and the guide nucleic acid in a ratio of about 1: 1, 1: 1.1, 1: 1.2, 1: 1.3, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.7, 1: 1.8, 1: 1.9, or about 1: 2.
The LNP of any preceding claim, wherein the lipid mixture and the nucleic acid (e.g., RNA) mixture is packaged in a ratio of about 1: 1, 1: 2 1: 3, 1: 4, or about 1: 5.
A pharmaceutical composition comprising (1) the system of any preceding claim or the LNP of any preceding claim and (2) a pharmaceutically acceptable excipient.
A cell or a progeny thereof comprising the guide nucleic acid of any preceding claim, the system of any preceding claim, or the LNP of any preceding claim.
A cell or a progeny thereof comprising HBV cccDNA or transcript thereof modified by the system of any preceding claim or the method of any preceding claim.
A method for preventing, diagnosing, and/or treating an HBV associated disease in a subject in need thereof, comprising administering to the subject the system of any preceding claim, the LNP of any preceding claim, or the pharmaceutical composition of any preceding claim, wherein the Cas12i endonuclease modifies HBV cccDNA, and wherein the modification of the HBV cccDNA treats the disease.
The method of claim 23, wherein the HBV associated disease is selected from the group consisting of Hepatitis B, acute hepatitis B, chronic hepatitis B (CHB) , cirrhosis, hepatocellular carcinoma (HCC) , liver cancer, and liver failure.
A guide nucleic acid, or a polynucleotide (e.g., a DNA, an RNA, a DNA/RNA mixture) encoding the guide nucleic acid, comprising a guide sequence capable of hybridizing to a target sequence on a target strand of an HBV cccDNA or a target sequence on a transcript of an HBV cccDNA;

wherein the guide sequence comprises (1) a sequence of SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to SEQ ID NO: 1-92 and 133-547 or a 5’ or 3’ end truncation thereof with 1, 2, 3, 4, 5, or 6 nucleotides truncated at the 5’ or 3’ end; or (3) a sequence having at most 1, 2, 3, 4, 5, or 6 nucleotide differences, whether consecutive or not, compared to SEQ ID NO:1-92 and 133-547.