Targeted cardiac myocyte aav capsid protein variants and uses thereof

Abstract

The present invention relates to an adeno-associated virus (AAV) capsid protein variant having an amino acid mutation between position 586 to 589 relative to a parent AAV capsid protein, wherein the mutation is a substitution of residues 587 and 588 of the parent AAV capsid protein to the amino acid sequence set forth in SEQ ID NO: 5 or 8, wherein the amino acid positions are numbered according to SEQ ID NO: 9. The present invention further relates to an isolated nucleic acid encoding said AAV capsid protein variant, a nucleic acid vector comprising the same, a host cell. The present invention further relates to a recombinant adeno-associated virus (rAAV) vector comprising said capsid protein variant, a pharmaceutical composition comprising said rAAV vector and the use thereof in the treatment or prevention of cardiovascular diseases.

Description

Technical Field

[0001] This invention relates to the field of gene therapy using recombinant adeno-associated virus vectors. Specifically, this invention relates to adeno-associated virus (AAV) capsid protein variants, isolated nucleic acid molecules encoding them, recombinant adeno-associated virus vectors having said AAV capsid protein variants, and their use in treating cardiovascular diseases (e.g., heart failure or genetic cardiomyopathy). Background Technology

[0002] Cardiovascular disease is one of the leading causes of morbidity, disability, and death worldwide. Furthermore, heart failure is recognized as one of the most devastating clinical outcomes. Gene therapy programs, based on the genetic modification of normal or damaged cardiomyocytes, hold promise for reversing cardiomyocyte damage, treating existing damaged heart tissue, or even preventing the occurrence and progression of new pathologies.

[0003] In the field of gene therapy, recombinant adeno-associated virus (rAAV) vectors have been approved by the FDA for the treatment of patients with specific gene mutations that cause retinal dystrophy or spinal muscular atrophy. However, there are currently no approved cardiovascular gene therapy regimens using AAV in humans.

[0004] Among currently available AAV vectors, adeno-associated virus (AAV) vectors with serotype 9 capsid protein (also known as AAV9 viral vectors) are considered one of the major serotypes for cardiac gene therapy. Studies in small animals have shown that cardiac gene delivery using AAV9 is highly efficient. However, studies in large animals (e.g., dogs or pigs) have shown that while direct cardiac administration can transduce adjacent cardiomyocytes relatively effectively, intravenous administration of AAV9 results in low cardiac gene delivery efficiency. Moreover, in cases where the therapeutic effect is mediated by transgenes encoding cell membrane-binding or intracellular proteins, the transduction efficiency of cardiomyocytes (i.e., the number of transduced cells) may have a greater impact on the therapeutic effect than the transgene expression level. On the other hand, studies on the biological distribution of AAV9 in vivo have also shown that the viral vector can be easily detected in all organs throughout the body after AAV9 administration; the biological distribution of AAV9 is not limited to cardiac tissue. Furthermore, the potential off-target effects resulting from this distribution characteristic of AAV9 can only be partially mitigated even with the use of heart-specific promoters and enhancers. See Physiol Genomics 54:316–318, 2022, https: / / doi.org / 10.1152 / physiolgenomics.00102.2022.

[0005] In order to expand the application potential of rAAV in the treatment of cardiovascular diseases, there is an urgent need in the field to develop adeno-associated virus (AAV) capsid protein variants with high transduction efficiency and high targeting to cardiomyocytes, as well as recombinant adeno-associated virus vectors containing said capsid protein variants. Summary of the Invention

[0006] In the course of developing novel capsid protein variants, the inventors obtained the capsid protein variants of this invention through capsid protein variant screening. Compared to the wild-type AAV9 capsid protein, the AAV capsid variants of this invention have an amino acid sequence of SEQ ID NO. 5 or 8 between amino acid positions 586 and 589 (amino acid positions according to SEQ ID NO: 9) replacing the original residues at positions 587-588. The capsid protein variants of this invention resulting from said substitution are, herein, referred to as CapX-020 (one example of which is shown in SEQ ID NO: 4) and CapX-123 (one example of which is shown in SEQ ID NO: 7), respectively. Animal experiments demonstrated that the capsid protein variants CapX-020 and CapX-123 enhance the transduction of adeno-associated virus vectors into cardiomyocytes while reducing transduction into kidney and skeletal muscle cells through comparison of reporter gene expression and fluorescence intensity. Therefore, by using the capsid protein variant according to the present invention, an AAV viral vector with cardiomyocyte targeting can be constructed to improve the treatment of cardiovascular diseases, while reducing the infection of skeletal muscle and the toxicity of the AAV-based drugs to the kidneys.

[0007] Therefore, in a first aspect, the present invention provides an adeno-associated virus (AAV) capsid protein variant relative to the parental AAV capsid protein, said variant having an amino acid sequence of SEQ ID NO:5 or 8 between amino acid positions 586 and 589, replacing amino acid residues at positions 587-588 of the parental AAV capsid protein, wherein the amino acid positions are numbered according to SEQ ID NO:9.

[0008] In some embodiments, the capsid protein variants of the present invention confer increased transduction of AAV viral particles containing them to cardiomyocytes compared to the parental AAV capsid protein. In other embodiments, AAV viral particles containing the capsid protein variants of the present invention exhibit increased transduction efficiency to cardiomyocytes and decreased transduction efficiency to skeletal muscle cells and / or kidney cells compared to the parental AAV capsid protein. In some embodiments, the increase in cardiomyocyte transduction efficiency is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 250%, or 300%. In other embodiments, the transduction efficiency of the skeletal muscle cells and / or kidney cells is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 250%, or 300%. In some embodiments, AAV viral particles comprising a variant of the capsid protein of the present invention have a higher cardiomyocyte / kidney cell transduction efficiency ratio compared to the parental AAV capsid protein. In some embodiments, AAV viral particles comprising a variant of the capsid protein of the present invention have a higher cardiomyocyte / skeletal muscle cell transduction efficiency ratio compared to the parental AAV capsid protein. In some embodiments, the transduction efficiency for a particular cell population can be measured by the proportion of cells in that population that are positive for AAV transduction. For example, if 1 × 10 6 One cell was exposed to AAV virus, and 0.5 × 10⁻⁶ cells were identified. 6 If each cell contains at least one copy of the AAV genome, then the transduction efficiency, measured by the proportion of positive cells, is 50%. A method used to determine this transduction efficiency is, for example, flow cytometry. For instance, when the AAV genome contains a polynucleotide encoding green fluorescent protein (GFP), GFP can be... + The percentage of cells transduced is used as a measure of transduction efficiency. In other embodiments, the transduction efficiency for a particular cell population can also be measured by measuring the average transgene expression level exhibited in that population transduced via AAV, wherein the transgene is contained in the genome of the AAV vector and is preferably expressed under constitutive promoter control. For example, when the AAV genome contains a polynucleotide encoding green fluorescent protein (GFP), the transduction efficiency can be measured by measuring the average GFP expression level of a cell population transduced with that AAV.

[0009] In some embodiments, the parental AAV capsid protein is selected from the capsid proteins of the AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In some embodiments, the parental AAV capsid protein has a surface-exposed hypervariable ring VIII that is structurally corresponding to that of the AAV9 capsid protein. In some preferred embodiments, the parental AAV capsid protein is the AAV9 capsid protein.

[0010] In some preferred embodiments, the parental AAV capsid protein is the AAV9 capsid protein, which comprises or consists of the following amino acid sequence:

[0011] (a) The amino acid sequence of amino acids 203 to 736 of SEQ ID NO:9, having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity with it;

[0012] (b) The amino acid sequence of amino acids 137 to 736 of SEQ ID NO:9, having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with it; or

[0013] (c) The amino acid sequence of SEQ ID NO:9, having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity with it.

[0014] In some embodiments, the capsid protein variants of the present invention are VP1, VP2, or VP3 capsid protein variants, for example, the capsid protein variants:

[0015] (a) An amino acid sequence comprising SEQ ID NO:4 or 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the amino acid sequence at positions 567 to 568 being the amino acid sequence shown in SEQ ID NO:5 or 8;

[0016] (b) Encoded by a nucleic acid containing a nucleotide sequence that is different from the nucleotide sequence of SEQ ID NO:3 or 6 due to the degeneracy of the genetic code;

[0017] (c) A transcript derived from the amino acid sequence encoding (a) or containing the nucleotide sequence of (b).

[0018] In some preferred embodiments, the capsid protein variants according to the invention are VP1, VP2, or VP3 capsid protein variants comprising the amino acid sequence of SEQ ID NO:4 or 7 or its N-terminal truncated sequence. In other embodiments, the capsid protein variants according to the invention are VP1, VP2, or VP3 capsid protein variants encoded by nucleic acids comprising the nucleotide sequence of SEQ ID NO:3 or 6.

[0019] In a second aspect, the present invention provides an isolated nucleic acid, wherein the nucleic acid comprises a multinucleotide sequence encoding a variant of the AAV capsid protein described in the present invention.

[0020] In some embodiments, the nucleic acid according to the present invention comprises:

[0021] (a) A polynucleotide encoding SEQ ID NO:4 or 7, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with that amino acid sequence; or

[0022] (b) The nucleotide sequence of SEQ ID NO:3 or 6, or the nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity with it.

[0023] In some embodiments, the nucleic acid according to the invention is capable of being transcribed and expressed from at least one, two, or all three AAV capsid proteins selected from the following: VP1, VP2, and VP3. In this document, the expression "capable of being transcribed and expressed" in relation to a nucleic acid means that, when the nucleic acid is placed under the control of a suitable promoter, it can be transcribed and expressed in a suitable cell in which the nucleic acid is introduced to produce the aforementioned gene product.

[0024] In a third aspect, the present invention provides a nucleic acid vector, such as a nucleic acid expression vector, comprising the nucleic acid described in the present invention. In some embodiments, the vector further comprises a nucleic acid encoding the AAV REP protein. In some embodiments, the vector is a plasmid.

[0025] In a fourth aspect, the present invention provides an isolated host cell containing the nucleic acid described herein, optionally wherein the nucleic acid stably transfects the host cell, and preferably, the host cell further contains nucleic acid encoding an AAV REP protein and / or nucleic acid encoding a co-herb virus function.

[0026] In some embodiments, the host cell is a recombinant adeno-associated virus vector (rAAV) production cell. In some embodiments, the nucleic acid is stably transfected into the production cell. In other embodiments, the production cell also contains nucleic acid encoding AAV REP proteins (especially the REP proteins of AAV9 or AAV2). In other embodiments, the production cell also contains nucleic acid capable of trans-providing the helper viral functions required for AAV replication and assembly (e.g., E1a and E1b helper genes from adenovirus, and / or E4, E2a, and VA helper genes). In other embodiments, the production cell also contains an rAAV genome carrying heterologous nucleic acid.

[0027] In a fifth aspect, the present invention provides uses and methods for preparing recombinant adeno-associated virus (rAAV) vectors by using a nucleic acid according to the invention encoding a variant of the AAV capsid protein according to the invention, a vector according to the invention, or a host cell or production cell according to the invention. In some embodiments, the uses or methods include:

[0028] (i) Providing production cells according to the invention;

[0029] (ii) The cells are cultured under conditions that allow the packaging to produce rAAV viral particles containing a recombinant AAV genome; and

[0030] (iii) Harvest the cultured host cells or culture medium to collect the resulting recombinant AAV viral vector.

[0031] In a sixth aspect, the present invention provides a recombinant adeno-associated virus (rAAV) vector comprising a capsid protein variant according to the invention or produced using production cells of the present invention comprising nucleic acids according to the invention.

[0032] In some embodiments, rAAV vectors containing a variant of the capsid protein according to the invention exhibit increased transduction to cardiomyocytes and optionally decreased transduction to skeletal muscle cells and / or kidney cells, relative to rAAV vectors containing wild-type AAV capsid proteins or to reference rAAV vectors containing parental capsid proteins.

[0033] In some embodiments, the rAAV vector according to the present invention contains in its genome:

[0034] a.5' and 3' AAV inverted terminal repeat (ITR) sequences, and

[0035] b. An expression construct containing a heterologous nucleic acid between the 5' and 3' ITRs, wherein the heterologous nucleic acid encodes the target gene product;

[0036] Preferably, the expression construct comprises the following elements functionally linked to each other in the transcriptional direction:

[0037] - Promoter,

[0038] - Heterologous nucleic acids,

[0039] - Transcription terminator.

[0040] In some implementations, the heterologous nucleic acid encodes a target gene product for gene substitution, gene repression, or gene editing; preferably, the target gene product is a protein or RNA.

[0041] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a recombinant AAV viral vector and a pharmaceutically acceptable vector according to the present invention.

[0042] In an eighth aspect, the present invention provides a method for delivering heterologous nucleic acids to a subject in need, wherein the method comprises administering to the subject a recombinant AAV viral vector according to the invention or a pharmaceutical composition according to the invention, for example, by intravenous injection, for example, for the treatment or prevention of cardiovascular disease.

[0043] In a ninth aspect, the present invention provides the use of the recombinant AAV viral vector according to the invention for preparing a pharmaceutical composition for treating or preventing cardiovascular diseases, preferably, the pharmaceutical composition is administered by intravenous injection. Attached Figure Description

[0044] Combine with the following appendix Figure 1 Reading this description will provide a better understanding of the preferred embodiments of the invention as detailed below. For illustrative purposes, the figures show presently preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the figures.

[0045] Figure 1 The pRDAV-CMV-EGFP-Cap9 backbone plasmid (construct 1) is shown schematically. ITR: AAV inverted terminal repeat (ITR); CMV promoter: cytomegalovirus promoter; EGFP: gene encoding enhanced green fluorescent protein; shortpA: short polyadenylation signal; p40: p40 promoter from AAV9; CAP9: gene encoding wild-type AAV9 serotype capsid protein.

[0046] Figure 2 The pRDAV-CMV-EGFP-Cap9Δ-588 backbone plasmid (construction 2) generated based on construct 1 is shown schematically.

[0047] Figure 3This diagram shows a representative illustration of EGFP fluorescence microscopy results in the heart, kidneys, and skeletal muscle of mice infected via tail vein injection with CapX-020 or CapX-123 capsid variant virus.

[0048] Figure 4 The results showed the proportion of EGFP-positive cells detected in mouse cardiomyocytes after intravenous infection with CapX-020 or CapX-123 capsid variant virus, compared to the control rAAV9 virus.

[0049] Figure 5 The results showed the change in the mean EGFP fluorescence intensity detected in mouse kidney cells compared to the control rAAV9 virus after intravenous infection of mice with CapX-020 or CapX-123 capsid variant virus.

[0050] Figure 6 The results showed the change in the mean EGFP fluorescence intensity detected in mouse skeletal muscle cells compared to the control rAAV9 virus after intravenous infection of mice with CapX-020 or CapX-123 capsid variant virus.

[0051] Figure 7 The results showed the change in the mean EGFP fluorescence intensity detected in mouse cardiomyocytes compared to the control rAAV9 virus after intravenous infection of mice with CapX-020 or CapX-123 capsid variant virus.

[0052] Figure 8 The results showed changes in the average ratio of EGFP fluorescence intensity detected in mouse cardiomyocytes / kidney cells and in mouse cardiomyocytes / skeletal muscle cells, relative to the control rAAV9 virus, after intravenous infection of mice with CapX-020 or CapX-123 capsid variant virus.

[0053] Figure 9 The image shows a sequence alignment of the capsid protein variants of SEQ ID NO:4 and SEQ ID NO:7 with the wild-type AAV9 capsid protein of SEQ ID NO:9. This sequence alignment reveals that the amino acid residues of SEQ ID NOs:4 and 7 correspond to the position numbers of SEQ ID NO:9. As can be seen from the figure, SEQ ID NOs:4 and 7 have mutated amino acid sequences between positions 586-589 of the amino acid residues corresponding to SEQ ID NO:9. Detailed Implementation

[0054] Unless otherwise defined below, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0055] I. Definition

[0056] In this document, the term "about" when used in conjunction with a numeric value means to cover a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value. The term is also intended to cover values ​​within ±1%, ±0.5%, or ±0.1% of the specified numeric value.

[0057] In this document, the expression “and / or” is used to refer to any one of the listed related items, or any and all possible combinations of multiple listed related items.

[0058] In this document, the terms "comprising" or "including" mean including the stated elements, integers, or steps, or groups of elements, integers, or steps, but do not exclude any other elements, integers, or steps, or other groups of elements, integers, or steps. When the terms "comprising" or "including" are used herein, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps. For example, when referring to a polypeptide / protein that "comprising" a specific sequence, it is also intended to cover polypeptides / proteins consisting of that specific sequence.

[0059] In this document, the terms "AAV capsid protein variant," "AAV variant capsid protein," and "AAV capsid variant" refer to an AAV capsid protein that has at least one modification (including deletion, insertion, and / or substitution) in its amino acid sequence relative to the parental AAV capsid protein. The variant AAV capsid protein may have at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the parental capsid protein.

[0060] In this document, "parental AAV capsid protein" or "parental capsid protein" refers to the AAV capsid protein that serves as a template for introducing the capsid protein mutation at positions 568-589 according to the present invention. The parent AAV capsid protein, as a variant, can be a naturally occurring or "wild-type" AAV capsid protein; it can also be a non-wild-type capsid protein, for example, a capsid protein modified with one or more (e.g., no more than 10, 5, 4, 3, 2, or 1) amino acids introduced into a natural AAV capsid protein; or a chimeric capsid protein formed by splicing together capsid proteins from different AAV serotypes, such as an AAV5 / AAV9 chimeric capsid protein or an AAV2 / AAV9 chimeric capsid protein. It should be understood that when the parental capsid protein is a non-natural capsid protein, the parental capsid protein does not contain the capsid protein mutation at positions 586-589 according to the present invention. In some embodiments, the parental capsid protein is a natural AAV9 serum capsid protein, such as the VP1 protein having the amino acid sequence SEQ ID NO:9, or the corresponding VP2 or VP3 protein having an N-terminal truncation.

[0061] In this document, when referring to the amino acid position of the AAV capsid protein or its segments, it refers to the amino acid position numbered according to SEQ ID NO:9. The amino acid position in the sequence of the described AAV capsid protein corresponding to the amino acid position of SEQ ID NO:9 can be determined by comparing the amino acid sequence of the described AAV capsid protein with that of SEQ ID NO:9. For example, when referring to position 586 of the capsid protein, it refers to the 586th amino acid residue of SEQ ID NO:9, or an amino acid residue that appears at the corresponding position in other capsid proteins after comparison. It should be understood that, in this disclosure, when the AAV capsid protein variant involves amino acid substitutions at certain specific amino acid positions (e.g., replacing the original sequence with an amino acid sequence of different numbers of residues), the alignment of the AAV capsid protein variant with SEQ ID NO:9 at that position can be visually detected. If necessary, vacancies can be introduced into one or both of the aligned sequences to ensure that, after the vacancies are introduced, that position of the AAV capsid protein variant aligns with the corresponding position of the corresponding segment of SEQ ID NO:9 and has the corresponding amino acid residue number. As an example of such an alignment, see [link to relevant documentation]. Figure 9 Sequence alignment performed to determine amino acid positions can be performed using the Basic LocalAlignment Search Tool available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi with default parameters.

[0062] In this document, the terms "AAV viral particle" and "AAV viral unit" are used interchangeably, referring to a complete viral unit containing an AAV capsid and an AAV nucleic acid genome (including wild-type AAV genome and recombinant AAV genome) packaged within the capsid. In this regard, the AAV nucleic acid molecular chain packaged into any AAV viral particle can be a sense (e.g., "sense") strand or an antisense strand, and both strands are equally infectious.

[0063] In this document, the terms "recombinant AAV vector," "recombinant AAV viral particle," and "recombinant AAV viral particle" are used interchangeably to refer to non-wild-type recombinant AAV viral particles capable of serving as delivery vehicles for target nucleic acids. Typically, the viral vector comprises a capsid and a viral genome packaged therein, and preferably, the viral genome contains the target nucleic acid to be delivered to target cells or tissues. In this document, "recombinant" may be abbreviated as "r," for example, recombinant AAV may be referred to as rAAV. Therefore, in this document, the terms "recombinant AAV viral particle" and "recombinant AAV vector" may also be used interchangeably with "rAAV viral particle," "rAAV viral particle," or "rAAV" or "rAAV vector." Typically, recombinant AAV vectors are infectious but replication-defective.

[0064] In this document, for the purposes of this disclosure, the rAAV capsid may be derived from or derived from various adeno-associated virus serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. For example, the rAAV may have a viral protein capsid composed of AAV capsid proteins from said serotypes, chimeric capsid proteins formed by splicing together portions of AAV capsid proteins from at least two said serotypes (e.g., AAV5 / AAV9 chimeric capsid protein, AAV2 / AAV9 chimeric capsid protein), and / or variants thereof.

[0065] In this document, for the purposes of this disclosure, the viral genome packaged in the rAAV vector is recombinant, i.e., AAV genomic DNA with genetic modifications relative to wild-type AAV genomic DNA. To generate recombinant AAV viral particles capable of delivering the target nucleic acid to tissues or cells, typically only the inverted terminal repeat (ITR) cis-elements need to be retained in the genome, while the remaining sequences required for viral packaging can be provided trans-. Thus, typically, rAAV may have one or more AAV wild-type genes wholly or partially deleted, for example, with the rep and / or cap genes wholly or partially deleted, and thereby be replication-defective; but retain the functional flanking ITR sequences necessary for the rescue, replication, and packaging of the AAV viral particles. More typically, the recombinant AAV viral genome packaged in the rAAV viral particles may retain only the functional ITR sequences and preferably contains or consists of one or more exogenous nucleotide sequences located between two AAV ITR sequences. It should be understood that, for the purposes of rAAV, the functional ITR sequence may be, but is not required to be, a wild-type nucleotide sequence, which can be altered, for example, by the insertion, deletion, or substitution of nucleotides, as long as it still provides the functions required for rescue, replication, and packaging. Therefore, in this paper, an rAAV vector is a viral vector that contains at least a functional ITR that provides the functions required for viral replication and packaging in cis. Furthermore, it should be understood that a recombinant AAV viral genome may contain two or more (e.g., three) ITR sequences, and these ITR sequences may be identical or different.

[0066] In this paper, the terms AAV “inverted terminal repeat” or “ITR” are used interchangeably to refer to the functional inverted terminal repeat cis-acting element from the AAV viral genome, and encompass both wild-type ITR sequences and variant ITR sequences.

[0067] Wild-type ITRs of natural AAV viruses contain a Rep binding site (RBS) and a terminal resolution site (trs) in their sequence. They can be recognized by Rep protein binding, resulting in a cleavage at the trs and forming a unique "T"-shaped secondary structure, playing a crucial role in the AAV virus life cycle. The earliest isolated AAV virus, AAV2, has a 145 bp palindromic hairpin-structured inverted terminal repeat (ITR) located at both ends of the genome. Subsequently, different ITR sequences have been found in various serotypes of AAV viruses, but all can form hairpin structures and possess Rep binding sites. Recombinant AAV viral vectors based on these wild-type ITR sequences are generally single-stranded AAV vectors (ssAAV).

[0068] Compared to the wild-type ITR sequence, a variant ITR can be a non-natural ITR sequence, for example, containing one or more nucleotide deletions, substitutions, and / or additions, and / or truncations, but still functional, i.e., capable of generating rAAV viral vectors. It has been found that, unlike ssAAV described above, by modifying the ITR by deleting the trs sequence and optionally the D sequence from one side of the AAV virus's ITR sequence, it is possible to package a recombinant AAV viral vector carrying self-complementary genomic DNA, thereby generating a virus called scAAV (self-complementary AAV). The packaging capacity of scAAV viral vectors is half that of ssAAV viral vectors, approximately 2.2kb-2.5kb, but its transduction efficiency after infecting cells is higher. See, for example, Self-complementary AAV Vectors; Advances and Applications, https: / / doi.org / 10.1038 / mt.2008.171. Such variant ITR sequences that can be used to generate scAAV viruses are also referred to as ΔITRs in this paper. This disclosure considers not only ssAAV vectors generated by combining two wild-type ITRs, but also scAAV vectors generated by combining a ΔITR sequence with a wild-type ITR.

[0069] As those skilled in the art will understand, the viral capsid protein and viral genome ITR sequence of recombinant adeno-associated virus can originate from the same or different AAV virus serotypes.

[0070] In this document, the term "host cell" refers to a cell to which exogenous polynucleotides have been introduced, including progeny cells of such cells. Examples of host cells include, but are not limited to, microbial, yeast, insect, and mammalian cells. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a cardiomyocyte. Depending on the context in which the term is used, the host cell may be an in vitro, ex vivo, or in vivo cell. In other embodiments, the host cell is a production cell used to produce the rAAV vector according to the present invention, such as "HEK293 cell" or "293 cell," or a cell line derived from said cell.

[0071] In this document, in some embodiments, the term "production cell" or "production cell line" refers to a population of cells capable of continuous or long-term growth and division in vitro. It is known in the art that during the storage or passage of such a clonal cell population, the karyotype can undergo spontaneous or induced changes, and thus may not be entirely identical to the ancestral cells or culture, but still retain the desired characteristics of the original production cell. Such production cells or cell lines are covered within the scope of this invention.

[0072] In this document, the terms "exogenous" or "heterogeneous" used to describe nucleic acids or proteins are used interchangeably. They refer to a nucleic acid or protein being exogenous or heterologous relative to a virus, host cell, subject, or other organism containing that nucleic acid or protein, or relative to a flanking nucleic acid or polypeptide linked thereto. That is, the nucleic acid or protein exists in a non-natural state in the virus, host cell, subject, or organism, or is linked to that flanking nucleic acid or polypeptide in a non-natural manner. For example, a nucleic acid introduced into a specific viral genome or host cell or subject via recombinant technology, thereby being linked to a sequence that is not naturally associated with it or is located in a non-natural chromosomal or cellular position or state, is heterologous relative to the viral genome, host cell, or subject. Therefore, a nucleic acid inserted into a host cell of the same origin or into an organism of the same origin, but existing in a non-natural state—for example, existing at a different copy number or under the control of different regulatory elements—is exogenous or heterologous.

[0073] In this document, "isolated" nucleic acid means a nucleic acid molecule that is artificially synthesized or isolated from at least some components of its natural environment containing it. For example, an isolated nucleic acid may be part of a larger nucleic acid, or part of a carrier or composition of substances, or may be contained within a cell and remain "isolated" provided that the larger nucleic acid, carrier, composition of substances, or specific cell is not the natural environment of the nucleic acid. In some embodiments, the "isolated" nucleic acid is enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more relative to the starting material.

[0074] In this paper, the term "functional connectivity," also known as "effective connectivity," refers to a relationship in which the specified components are positioned to allow them to function in the intended manner. For example, if a promoter or enhancer sequence stimulates or regulates transcription of a coding sequence in a suitable host cell or other expression system, then that promoter or enhancer sequence is effectively connected to the coding sequence. Generally, promoters effectively connected to a transcribed sequence are contiguous to that transcribed sequence; that is, they are cis-acting. However, some transcriptional regulatory sequences (such as enhancers) do not need to be physically adjacent to or closely proximate to the coding sequence that enhances transcription.

[0075] In this document, the term "sequence identity" is used to describe the sequence structural similarity between two amino acid sequences or polynucleotide sequences. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., vacancies can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0076] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. The particularly preferred set of parameters (and unless otherwise specified, a set of parameters to be used) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0077] Alternatively, the PAM120 weighted remainder table, gap length penalty of 12, and gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences or nucleotide sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0078] As used herein, the term "conservative" amino acid or nucleotide change refers to a neutral or near-neutral amino acid or nucleotide change that results in the protein or nucleic acid molecule containing the said amino acid or nucleotide change substantially retaining its original function. For example, a conserved amino acid substitution is the replacement of an amino acid with a different amino acid whose side chain has similar biochemical properties (e.g., charge, hydrophobicity, and size). Variations of such conserved modifications are additional to, and not exclusive to, polymorphic variants, interspecies homologs, and alleles. The following eight groups contain conserved amino acids: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)). Those skilled in the art can readily detect the conservation of amino acid or nucleotide changes in a specific polypeptide or nucleotide sequence using conventional techniques, such as functional assays.

[0079] In this article, "individual" or "subject" refers to a mammal. Examples of mammals include, but are not limited to, humans, non-human primates (e.g., cynomolgus monkeys, rhesus monkeys), rodents, and other mammals such as cattle, pigs, horses, and dogs. In this article, mammals include individuals at all stages of development (including embryonic and fetal stages).

[0080] In this article, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. The term "treatment" also encompasses modifying or improving at least one bodily parameter, including those that the patient may not be aware of.

[0081] In this article, the term "prevention" refers to preventing or delaying the onset, development, or progression of a disease or condition. In this article, "prevention" generally refers to hospital intervention implemented before at least one symptom of a disease occurs.

[0082] Several aspects of the present invention will be described below.

[0083] II. Capsid protein variants

[0084] Adeno-associated virus (AAV) is a non-pathogenic parvovirus consisting of a non-enveloped capsid and an internal 4.7 kb single-stranded DNA genome. The genome contains three open reading frames (ORFs), flanked by inverted terminal repeats (ITRs) that serve as the origin of viral replication and packaging signals. The rep ORF encodes four non-structural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and viral particle assembly. The cap ORF encodes three structural proteins (VP1-3) that assemble to form the viral capsid.

[0085] The AAV viral capsid consists of approximately 60 VP monomers from three VP capsid proteins, arranged in an icosahedral structure. The optimal molar ratio of the three capsid proteins VP1:VP2:VP3 is approximately 1:1:10. All three capsid proteins, VP1, VP2, and VP3, are encoded by the AAV cap gene, produced from the same transcript, and regulated by the p40 promoter. The three VP proteins share a common C-terminal sequence but different N-terminal start sites; VP2 and VP3, generated through alternative splicing, are two truncated forms of VP1. As an example, the capsid protein sequence of AAV9 is shown below, with the VP1-specific amino acid sequence shown in bold black (VP1 unique region). The amino acid sequence shared by VP1 and VP2 (“VP1 / VP2 shared region”) is underlined and italicized; the entire VP3, contained within VP1 and VP2, is the amino acid shared by all three capsid proteins (“shared VP3 region”), and is shown below in bold and italics.

[0086]

[0087]

[0088] VP1 and VP2 are mainly located in the cell nucleus, while the monomeric VP3 is distributed in both the nucleus and cytoplasm. The outer surface of the assembled AAV capsid is composed of the VP3 sequence (including VP3 and the C-terminus of VP1 and VP2), while the N-terminus of VP1 and VP2 are located inside the capsid.

[0089] The VP contains nine protruding loops called variable regions (VRs). VRs vary depending on the AAV serotype and are responsible for serotype-specific receptor binding differences. Due to their location of exposure and their function in receptor binding, the VRs that form protruding loops are ideal locations for capsid modification to redirect or extend AAV tropism (i.e., cell surface targeting).

[0090] To develop novel cell / tissue-targeting AAV capsid protein variants, the inventors screened for AAV capsid protein variants. Random sequences were inserted into the cap ORF at amino acid positions corresponding to the top of the VR-VIII loop (positions 586-588), and an AAV transfer plasmid containing a reporter gene and the cap ORF was constructed to couple the genotype and phenotype of the AAV capsid variant with reporter gene expression, generating a plasmid library. Subsequently, through capsid variant screening and in vivo tissue distribution assays, AAV capsid protein variants that could more efficiently and / or specifically transduce cardiomyocytes under intravenous administration and exhibited reduced transduction in skeletal muscle and kidney cells were identified. Thus, the inventors established the novel capsid protein variants of this invention.

[0091] Therefore, in one aspect, the present invention provides AAV capsid protein variants. In one embodiment, the AAV capsid protein variant according to the invention is derived from the parental AAV capsid protein by inserting a peptide of SEQ ID NO: 5 or 8 between amino acid positions 586 and 589 of the parental capsid protein to replace the original residues at positions 587-588 (AAV9VP1 according to SEQ ID NO: 9). For convenience, such substitution mutations of the invention are also referred to herein as “mutations at positions 586-589 according to the invention”. The capsid protein modification according to the invention does not interfere with capsid assembly and genome packaging, and allows rAAV to acquire modified cardiomyocyte targeting and optionally modified skeletal muscle cell and / or kidney cell targeting.

[0092] In some embodiments, the parental AAV capsid protein is a capsid protein selected from AAV serotypes of AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13, for example, the AAV9 serotype capsid protein. In other embodiments, the parental AAV capsid protein can be a chimeric capsid protein formed by splicing different AAV serotype capsid proteins, for example, an AAV5 / AAV9 chimeric capsid protein or an AAV2 / AAV9 chimeric capsid protein. In some embodiments, the parental AAV capsid protein is an AAV9 capsid protein containing the following amino acid sequence or an AAV9 capsid protein composed of the following amino acid sequence:

[0093] (a) The amino acid sequence of amino acids 203 to 736 of SEQ ID NO:9;

[0094] (b) The amino acid sequence of amino acids 137 to 736 of SEQ ID NO:9;

[0095] (c) The amino acid sequence of SEQ ID NO:9;

[0096] (d) The amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with any of (a)-(c); or

[0097] (e) Compared with the amino acid sequence of any one of (a)-(c), it has one or more (e.g., 1, 2, 3, 4, 5, 7, 8, 9 or 10) amino acid residue substitutions, deletions and / or additions (preferably conserved amino acid substitutions).

[0098] In other embodiments, the AAV capsid protein variant according to the invention is derived from a transcript encoding an amino acid sequence wherein the amino acid sequence is an AAV9 capsid protein sequence having the amino acid sequence shown in SEQ ID NO: 5 or 8 between amino acid positions 586 and 589 (e.g., the sequence of SEQ ID NO: 4 or 7). In this document, the expression “derived from” a transcript associated with a capsid protein variant means that the transcript, when under conditions suitable for its expression to produce the protein, is capable of producing the capsid protein variant, including different forms of capsid protein variants, such as VP1, VP2, or VP3 capsid protein variants, derived from the transcript by using alternative translation initiation sites and / or alternative splicing. In some embodiments, therefore, the AAV capsid protein of the invention derived from the transcript may be a capsid protein variant selected from VP1, VP2, and VP3. In some preferred embodiments, the AAV capsid protein variant according to the invention is derived from a transcript encoding the amino acid sequence shown in SEQ ID NO: 4 or 7. In some preferred embodiments, the AAV capsid protein variant according to the invention is derived from a transcript containing the nucleotide sequence shown in SEQ ID NO: 3 or 6.

[0099] In some embodiments, in addition to the mutation at positions 586-589 according to the invention, the capsid protein variants according to the invention may include or exclude amino acid substitutions (preferably, conservative substitutions) at one or more other amino acid positions, for example, 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0100] In some embodiments, the AAV capsid protein variants according to the invention may be further modified to, for example, enhance or extend the delivery of the recombinant vector. Methods for constructing and screening recombinant AAV capsid protein libraries are well known in the art. See, for example, US2005 / 0053922 and US2009 / 0202490, the disclosures of which are incorporated herein by reference in their entirety.

[0101] In some embodiments, the variant capsid proteins disclosed herein confer increased cardiomyocyte transduction with rAAV viral particles containing the corresponding parental AAV capsid protein or wild-type AAV capsid protein compared to transducing cardiomyocytes with rAAV viral particles containing the same protein. For example, the variant capsid proteins of the present invention result in an average increase in AAV viral particle uptake per cardiomyocyte compared to AAV viral particles containing the parental AAV capsid protein or wild-type AAV; and / or the variant capsid proteins of the present invention result in an increase in AAV viral particle uptake per cardiomyocyte. In some implementations, AAV viral particles containing the variant capsid protein of the present invention preferentially transduce cardiomyocytes relative to other cells, for example, having an increased cardiomyocyte / kidney cell transduction efficiency ratio and / or an increased cardiomyocyte / skeletal muscle cell transduction efficiency ratio compared to using rAAV viral particles containing the corresponding parental AAV capsid protein or wild-type AAV capsid protein, wherein preferably, the transduction efficiency ratio is increased by about 1.0-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5.0-fold, about 6.0-fold, about 7.0-fold, about 8.0-fold, about 9.0-fold, or about 10.0-fold or more.

[0102] Various methods in the art for measuring gene expression can be used to assess, in vitro or in vivo, the transduction efficiency of the AAV viral particles to cardiomyocytes and other cells (e.g., skeletal muscle cells and / or kidney cells), such as increased transduction efficiency, cell type selection, transduction priority, etc. For example, an rAAV genome containing a reporter gene (e.g., a fluorescent protein under constitutive promoter control) can be packaged using AAV capsid proteins, and transduction efficiency and / or cell type selectivity / priority can be assessed by detecting reporter gene expression (e.g., fluorescence microscopy) in in vitro cell-based assays (e.g., after transfection of target cells or a group of target cells) or in animal model-based assays. In some embodiments, such as the fluorescence reporter molecule assays described in the examples, after transduction with the AAV particles, the transduction efficiency of the AAV particles for different cells and the ratio of transduction efficiency between different cells are measured by measuring the average expression level of the transgene carried by the AAV particles in different cells (e.g., cardiomyocytes, kidney cells, and / or skeletal muscle cells).

[0103] III.rAAV Production

[0104] In some aspects, the present invention provides the use of nucleic acids encoding variants of the AAV capsid protein of the present invention, vectors containing such variants, and host cells in the preparation of rAAV vectors.

[0105] The general principles of rAAV production are summarized in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, CUM Topics in Microbial. and Immunol., 158:97-129. Various methods are described in the following documents: WO 95 / 13365; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441; WO 97 / 08298; WO 97 / 21825; WO 97 / 06243; WO 99 / 11764; US 5,786,211; US ​​5,871,982; and US 6,258,595. These documents, especially those relating to rAAV production, are incorporated herein by reference in their entirety.

[0106] It has been confirmed that the two terminal inverted repeat (ITR) sequences in the AAV genome are essential cis-acting elements for AAV integration, replication, rescue, and packaging. The two open reading frames located between the ITR sequences in the wild-type AAV genome, called Rep and Cap, encode four Rep proteins (Rep78, Rep68, Rep52, and Rep40), three Cap proteins (VP1, VP2, and VP3), and one assembly activator protein (AAP) involved in AAV replication and packaging, respectively. Transcription of Rep78 and Rep68 is controlled by the p5 promoter, transcription of Rep52 and Rep40 is initiated by the p19 promoter, and the p40 promoter regulates the transcription of Cap proteins VP1, VP2, and VP3.

[0107] Typically, to produce rAAV viral particles, the cis-acting element ITR needs to be retained in the rAAV genome, but the REP and CAP proteins required for replication and packaging can be provided trans-. Therefore, typically, an infectious rAAV viral particle can be produced by transfecting a suitable host cell (e.g., a suitable packaging cell) capable of trans-providing the missing AAV functions (e.g., the rep and / or cap proteins) with a vector (e.g., a plasmid) carrying an rAAV genome that has deleted all or part of the rep and / or cap genes.

[0108] In this document, the nucleic acid vector carrying the rAAV genome to be transferred to target cells, also referred to as an "AAV transfer vector," can be any suitable form of nucleic acid vector for gene transfer, such as a plasmid. In this document, the AAV function missing from the rAAV genome that needs to be trans-provided to assist in the packaging of the rAAV genome into productive AAV viral particles, also referred to as an "AAV helper function," is such as the rep and / or cap genes missing from the rAAV genome, or nucleic acids encoding AAV rep and / or cap expression products or functional variants thereof. In some embodiments, the AAV helper function can be provided by a vector encoding the helper function, such as a plasmid, bacteriophage, transposon, granule, virus, or viral particle. Typically, such vectors provide transient expression of the AAV rep and / or cap nucleic acids but lack the AAV ITR and therefore cannot replicate or package themselves. In other embodiments, the AAV helper function can be provided by cells or cell lines that stably express the helper function.

[0109] Adeno-associated virus (AAV) is a non-enveloped virus of the Parvoviridae family, belonging to the Dependoparvovirus genus. It requires the presence of helper viruses to replicate and assemble, producing infectious viral particles. Viruses that allow AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells are also referred to herein as “AAV helper viruses.” A wide variety of helper viruses used for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses. The functions encoded by the helper virus genome for AAV replication and packaging, also referred to herein as “helper virus functions,” include, for example, the E1a and E1b helper genes from adenovirus, and the E4, E2a, and VA helper genes. Helper virus functions can be provided in plasmid or other vector form, and / or in stable cell lines expressing said helper genes. Furthermore, as those skilled in the art will understand, AAV helper functions (rep / cap genes) and helper virus functions (e.g., E4, E2a, and VA helper genes) can be provided on single or separate vectors. In some aspects, helper virus functions can also be provided by wild-type adenovirus. For example, wild-type adenovirus and AAV transfer vectors can be used to transduce cell lines containing and stably expressing AAV Rep and Cap proteins, thereby generating infectious rAAV viral particles.

[0110] In some aspects, techniques for producing rAAV particles involve providing cells with the rAAV genome to be packaged, the rep and cap genes, and helper viral functions. Such techniques are well known in the art. For example, rAAV production cells (or packaging cells) comprising the following components: the rAAV genome, the AAV rep and cap genes separate from the rAAV genome (i.e., not within the rAAV genome), and helper viral functions. These production cells can then be used to produce the desired infectious rAAV particles.

[0111] In some aspects, therefore, the present invention provides a production cell (or packaging cell) for preparing a recombinant adeno-associated virus (rAAV) vector, comprising a cap gene encoding a variant of the capsid protein of the present invention, and optionally also comprising nucleic acids encoding an AAV rep gene and / or nucleic acids encoding helper viral functions. In some embodiments, the production cell further comprises an rAAV genome. In some embodiments, the rAAV genome comprises either the cap gene or the rep gene. In other embodiments, the rAAV genome does not comprise the cap gene and the rep gene.

[0112] In other aspects, the present invention provides a method for preparing a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the method includes:

[0113] (i) Providing production cells according to the invention;

[0114] (ii) The cells are cultured under conditions that allow the packaging to produce rAAV viral particles containing the recombinant AAV genome; and

[0115] (iii) Harvest the cultured host cells or culture medium to collect the resulting recombinant AAV viral vector.

[0116] In some embodiments, the production cells contain a nucleic acid according to the invention encoding a variant of the AAV capsid protein according to the invention. In some embodiments, the nucleic acid according to the invention comprises:

[0117] (a) A polynucleotide encoding SEQ ID NO:4 or 7, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with it and having an amino acid sequence as shown in SEQ ID NO:5 or 8 between amino acid positions 586 and 589; or

[0118] (b) A nucleotide sequence of SEQ ID NO:3 or 6, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the nucleotide sequence of SEQ ID NO:5 or 8 and encoding an amino acid sequence between amino acid positions 586 and 589.

[0119] In some embodiments, the nucleic acid according to the invention is capable of being transcribed and expressing at least one, any two, or all three AAV capsid proteins selected from the following: VP1, VP2, and VP3.

[0120] In some embodiments, the nucleic acid according to the invention is introduced into the production cells in the form of a nucleic acid vector. In some embodiments, the nucleic acid vector further comprises a nucleic acid encoding the AAV REP protein. In some embodiments, the nucleic acid vector is a plasmid.

[0121] In some embodiments, the nucleic acid according to the invention is stably transfected and integrated into the genome of the production cell.

[0122] In other embodiments, the production cells also contain (e.g., stably integrated) nucleic acids encoding AAV REP proteins (especially the REP proteins of AAV9 or AAV2). In other embodiments, the production cells also contain (e.g., stably integrated or introduced via a vector) nucleic acids capable of trans-providing helper viral functions (e.g., E1a and E1b helper genes from adenovirus, and / or E4, E2a, and VA helper genes).

[0123] In some embodiments, step (i) of the method according to the invention includes introducing a recombinant AAV genome containing heterologous nucleic acids into a production cell according to any of the above embodiments. In some embodiments, the rAAV genome to be introduced into the host cell lacks AAV rep and cap DNA. In some embodiments, the rAAV genome to be introduced into the host cell contains cap DNA encoding a variant of the capsid protein of the present invention, but lacks AAV rep DNA. In some embodiments, the rAAV genome is introduced into the host cell via an AAV transfer vector.

[0124] In some embodiments, the rAAV production method according to the invention further includes the step of generating production cells (or cell lines). In some embodiments, the production cells (or cell lines) express one or more of the following components necessary for rAAV genome packaging: an rAAV genome lacking the AAV rep and cap genes, an AAV rep gene separate from the rAAV genome, and a cap gene encoding a variant of the capsid protein according to the invention. Subsequently, the production cells can be infected with a helper virus (such as adenovirus) to produce infectious rAAV viral particles.

[0125] In some preferred embodiments, the rAAV production method according to the present invention includes: co-transfecting production cells (or cell lines) with three plasmids, wherein the three plasmids are: a plasmid (transfer plasmid) providing an rAAV genome containing heterologous nucleic acids and flanking ITRs; a plasmid providing AAV helper functions (cap and / or rep proteins); and a plasmid providing helper viral (e.g., adenovirus or herpes simplex virus) functions. In other embodiments, the present invention also considers using adenovirus or baculovirus instead of the above-described plasmids to introduce AAV helper functions and helper viral functions.

[0126] IV.rAAV carrier

[0127] In some aspects, the present invention provides a viral vector, which is an artificial recombinant viral particle having an rAAV genome packaged in a viral capsid composed of a variant of the capsid protein of the present invention. In some embodiments, the rAAV genome according to the present invention is an ssAAV genome or a scAAV genome.

[0128] In some embodiments, the recombinant AAV vector according to the present invention contains in its genome:

[0129] a. AAV inverted terminal repeat (ITR) sequence, and

[0130] b. An expression construct containing a heterologous nucleic acid, wherein the heterologous nucleic acid encodes the target gene product.

[0131] Preferably, the expression construct comprises the following elements functionally linked to each other in the transcriptional direction:

[0132] - Promoter,

[0133] - Heterologous nucleic acids encoding the product of the target gene,

[0134] - Transcription terminator.

[0135] In some implementations, the genome lacks AAV rep and cap DNA, meaning that AAVrep and cap DNA are not present in the genome.

[0136] In some embodiments, the rAAV genome disclosed herein contains two AAV ITRs flanking the expression construct. In some embodiments, the ITR sequences flanking the expression construct in the rAAV genome are natural, variant, or modified AAV ITR sequences. In some embodiments, at least one ITR sequence is a natural, variant, or modified AAV ITR sequence. In some embodiments, both ITR sequences are natural, variant, or modified AAV ITR sequences. In some embodiments, one of the two flanking ITRs is a modified AAV ITR sequence that allows for the generation of a complementary genome, while the other ITR is a natural AAV ITR sequence.

[0137] In the rAAV genome according to the present invention, the AAV ITR can be derived from or derived from any AAV serotype, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. Genomic sequences of various AAV serotypes, as well as natural inverted terminal repeats (ITRs), can be found in literature or public databases such as GenBank. See, for example, GenBank login numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), and AY530579 (AAV9). Publications describing AAV include Srivastava et al. (1983) J. Virol. 45:555; Chiorini et al. (1998) J. Virol. 71:6823; Chiorini et al. (1999) J. Virol. 73:1309; Bantel-Schaal et al. (1999) J. Virol. 73:939; Xiao et al. (1999) J. Virol. 73:3994; Muramatsu et al. (1996) Virol. 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; International Patent Publications WO2018 / 222503A1, WO2012 / 145601A2, WO2000 / 028061A2, WO1999 / 61601A2 and WO1998 / 11244A2; U.S. Patent Applications 15 / 782,980 and 15 / 433,322; and U.S. Patent No. 10,036,016 9,790,472, 9,737,618, 9,434,928, 9,233,131, 8,906,675, 7,790,449, 7,906,111, 7,718,424, 7,259,151, 7,198,951, 7,105,345, 6,962,815, 6,984,517 and 6,156,303.

[0138] In some implementations, the heterologous nucleic acid contained in the rAAV genome is efficiently linked to transcriptional control DNA, particularly to functional promoter DNA, enhancer DNA, and polyadenylation signaling sequence DNA in the target cell, to form an expression construct. The expression construct may also include intron sequences to facilitate the processing of the RNA transcript during expression in mammalian cells.

[0139] There are no particular limitations on the heteronucleotides contained in the rAAV according to the present invention. In some embodiments, the heteronucleotide encodes a gene product to be expressed in a target tissue / cell (e.g., cardiomyocytes). In some embodiments, the target tissue / cell is cardiomyocyte. In some embodiments, the cardiomyocyte includes atrial cardiomyocytes and / or ventricular cardiomyocytes. In some embodiments, the cardiomyocyte is a cardiomyocyte carrying a gene mutation associated with cardiovascular disease or condition. In some embodiments, the cardiomyocyte is an in vitro or ex vivo cell. In other embodiments, the cardiomyocyte is an in vivo cardiomyocyte. In some embodiments, expression of the gene product in the target cells / tissue will be beneficial for the treatment or prevention of a disease or condition (e.g., cardiovascular disease) associated with a genetic defect in that tissue. In other embodiments, the heteronucleotide contains a polynucleotide encoding a marker or reporter protein.

[0140] In some implementations, the heterologous nucleic acid encodes a target gene product for gene substitution, gene repression, or gene editing; preferably, the target gene product is a protein or RNA.

[0141] Gene replacement is one of the most direct forms of gene therapy, typically involving the introduction of a nucleic acid encoding a functional protein into a host cell and its expression within the host cell to compensate for the absence of that functional protein caused by a defective gene in the host cell. As those skilled in the art will understand, depending on the size and nature of the functional protein, a full-length functional protein can be expressed by introducing a single nucleic acid molecule encoding the protein into the host cell, or by introducing multiple nucleic acids, each encoding a portion of the protein, into the same host cell. In some embodiments, the rAAV according to the invention comprises an expression construct containing at least one heterologous nucleic acid operatively linked to a promoter for gene replacement. In some embodiments, the at least one heterologous nucleic acid encodes a therapeutic protein.

[0142] Gene repression can be achieved by using RNA interference (RNAi) techniques, such as antisense nucleic acids, siRNA, or microRNA, to suppress the expression or translation of defective genes in host cells, thereby treating or preventing functional disorders caused by the defective gene. In some embodiments, the rAAV according to the invention comprises an expression construct containing at least one heterologous nucleic acid operatively linked to a promoter for gene repression. In some embodiments, the at least one heterologous nucleic acid, upon introduction into host cell expression, results in the production of siRNA or microRNA to suppress the expression or translation of the defective gene in the host cell.

[0143] Gene editing is another gene therapy approach that targets and repairs gene defects, including but not limited to ZFN, TALEN, and CRISP / Cas editing. In some embodiments, the rAAV according to the invention comprises an expression construct containing at least one heterologous nucleic acid operatively linked to a promoter for gene editing (especially CRISP / Cas9 editing). In some embodiments, expression of this heterologous nucleic acid in target cells / tissues will target and repair gene defects in those cells / tissues.

[0144] Without being bound by any theory, cardiovascular diseases can be treated after heterologous nucleic acid expression in rAAV according to the present invention. Non-limiting examples of cardiovascular diseases and exemplary proteins or genes that can be targeted for each cardiovascular disease are listed below:

[0145] - Heart failure. Examples of potential targets for AAV gene therapy for this type of disease include: SERCA2a, SUMO1, S100A1, I1c (constitutively active inhibitor-1), shRNA-PLB, βARKct, VEGF / Ang1, VEGF-A / PDGF-B, VEGF-B, and Heme Oxygenase-1;

[0146] - Hereditary cardiomyopathy (CM), including but not limited to hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), restrictive cardiomyopathy (RCM), and left ventricular noncompacting cardiomyopathy (LVNC). Examples of potential targets for AAV gene therapy in this type of disease include: TTN (Titin), LMNA (Lamin-A / C), MYH7 (Myosin Heavy Chain 7), MYH6 (Myosin Heavy Chain 6), SCN5a (Sodium Channel Protein Type 5, α Subunit), MYBPC3 (Cardiac-Type Myosin-Binding Protein C), TNNT2 (Cardiac Muscle Troponin T), RBM20 (RNA-Binding Protein 20), TNNI3 (Cardiac Troponin I), MYL2 (Regulatory Light Chain of Cardiac Myosin β), MYL3 (Myosin Light Chain, Ventricular Isoform), PKP2 (Plakophilin 2), DSP (Desmoplakin), DSG2 (Desmoglein 2), and DSC2.

[0147] (Desmocolin 2), JUP (Junction Plakoglobin).

[0148] Other examples of cardiovascular diseases and therapeutic nucleic acids that can be used include, but are not limited to, Barth syndrome (Tafazzin); myocardial hypertrophy and heart failure (dODN, decoyoligodeoxynucleotides); and cardiomyocyte loss or dysfunction (miRNAi-LRP6).

[0149] See, e.g., Kyle Chamberlain et al., Cardiac Gene Therapy with Adeno-Associated Virus-Based Vectors, 2017, Curr Opin Cardiol. 2017 May; 32(3):275–282; Huili Zhang et al., AAV-mediated gene therapy: Advancing cardiovascular disease treatment, Front. Cardiovasc. Med. 9:952755.

[0150] In some embodiments, the heterologous nucleic acid encodes angiogenic growth factor for the treatment of cardiovascular disease, for example, in individuals requiring the induction of therapeutic angiogenesis (e.g., individuals with coronary artery disease or peripheral vascular disease). In some embodiments, the angiogenic growth factor is, for example, fibroblast growth factor, platelet-derived growth factor, hepatocyte growth factor, or hypoxia-inducible factor.

[0151] In other embodiments, the heterologous nucleic acid encodes SERCA2a, S100A1, I1c, or βARKct for, for example, treating individuals with heart failure.

[0152] There are no particular limitations on the promoters used to link heterologous nucleic acids in the expression construct. In some embodiments of the rAAV vectors disclosed herein, the heterologous nucleic acid encoding the gene product is efficiently linked to a constitutive promoter. Suitable constitutive promoters include, for example, cytomegalovirus (CMV) promoters, CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG)7, human elongation factor 1α promoter (EF1α), human phosphoglycerate kinase (PGK) promoter, mitochondrial heavy chain promoters, and ubiquitin promoters. In other embodiments, the heterologous nucleic acid encoding the gene product is efficiently linked to an inducible promoter. In some cases, the heterologous nucleic acid encoding the gene product is efficiently linked to muscle tissue-specific or muscle cell type-specific regulatory elements.

[0153] In addition to the promoter and the heteronucleotide, the expression construct may also include other regulatory sequences as needed. The terms "regulatory sequence" and "expression control sequence" are used interchangeably herein, referring to a nucleic acid sequence that induces, inhibits, or otherwise controls the transcription of a protein encoding a validly linked nucleic acid sequence. Regulatory sequences can be, for example, initiation sequences, enhancer sequences, intron sequences, and transcription termination sequences. In some embodiments, the expression construct also includes a Kozak sequence located upstream (i.e., 5') of the heteronucleotide.

[0154] V. Pharmaceutical Composition

[0155] In one aspect, the present invention provides pharmaceutical compositions comprising the rAAV vector of the present invention. The rAAV viral particles according to this disclosure, after purification, can be formulated according to known methods to prepare pharmaceutically useful compositions. The compositions of this disclosure can be formulated using techniques known in the art for administration to mammalian subjects, such as humans.

[0156] When the delivery system is formulated into a solution or suspension, it is contained in an acceptable carrier, such as an aqueous carrier. Various aqueous carriers can be used, such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized using conventional, well-known sterilization techniques, or they can be aseptically filtered. The resulting aqueous solution can be packaged for use as is, or it can be lyophilized and combined with a sterile solution prior to administration.

[0157] The pharmaceutical compositions according to the present invention may contain pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, tension modifiers, humectants, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitol monolaurate, triethanolamine oleate, etc. The pharmaceutical compositions according to the present invention may or may not contain preservatives.

[0158] The genomic titer of a viral vector, such as that in the compositions and preparations disclosed herein, can be determined using a variety of standard methods, including, for example, PCR. qPRC and ddPCR can also be used to determine the genomic titer of a viral vector.

[0159] In some embodiments, the pharmaceutical compositions of the present invention are formulated into a pharmaceutical preparation for delivery of the AAV carrier to the myocardium. The delivery may be by direct intramyocardial injection or by intravascular administration.

[0160] In some embodiments, the pharmaceutical compositions of the present invention may be formulated in a form suitable for administration via intramyocardial injection, vascular delivery via the coronary artery, systemic delivery via the superior vena cava, or systemic delivery via a peripheral vein.

[0161] In a preferred embodiment, the pharmaceutical composition of the present invention is formulated into a pharmaceutical preparation for intravenous delivery.

[0162] VI. Uses of Viral Vectors

[0163] As a gene therapy, the viral vector of the present invention can be used to physically introduce an AAV viral vector having a variant of the capsid protein of the present invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including but not limited to intramyocardial injection, vascular delivery via the coronary artery, systemic delivery via the superior vena cava or systemic delivery via peripheral veins, intramuscular, subcutaneous, intraperitoneal, spinal or other non-parenteral administration, such as by injection or infusion.

[0164] AAV viral vectors containing the capsid protein variants of the present invention exhibit enhanced targeting and transduction efficiency against cardiomyocytes; thus providing an effective means of gene-based delivery for the treatment of cardiovascular diseases. In some aspects, the AAV viral vectors of the present invention preferably also exhibit reduced transduction efficiency against skeletal muscle and / or kidney cells, thereby being suitable for formulation as AAV therapeutics targeting the heart while requiring reduction in skeletal muscle infection and nephrotoxicity.

[0165] In some embodiments, cardiovascular diseases or conditions treatable with the viral vector of the present invention include coronary artery disease, cardiomyopathy, endocarditis, congenital cardiovascular defects, and congestive heart failure. In other embodiments, cardiovascular diseases or conditions treatable with the viral vector of the present invention include hypertrophic cardiomyopathy; valvular heart disease; myocardial infarction; congestive heart failure; long QT syndrome; atrial arrhythmias; ventricular arrhythmias; diastolic heart failure; systolic heart failure; valvular heart disease; valvular calcification; left ventricular non-compactment; ventricular septal defect; and ischemia. In some preferred embodiments, the cardiovascular disease is, for example, heart failure or genetic cardiomyopathy.

[0166] Example

[0167] The present invention will be further described and illustrated below with reference to embodiments thereof. Obviously, the described embodiments are merely some, not all, implementations of the present invention. Based on the embodiments of the present invention, modifications or variations made by those skilled in the art to the implementation schemes of the present invention without departing from the spirit of the present invention are all within the scope of protection of the present invention. Unless otherwise specified, all reaction reagents involved in the embodiments can be purchased commercially.

[0168] Example 1. Construction of a backbone plasmid for an AAV9 capsid protein variant library

[0169] 1. Constructing the pRDAV-CMV-EGFP-Cap9 backbone plasmid (Constructor 1)

[0170] The original plasmid pRDAV-CMV-EGFP (preserved from the pRDAV-CMV gene) contains EcoRI and MluI restriction sites after EGFP. Construct 1 expressing the AAV9 capsid protein (Cap9) is generated as follows. Figure 1 (Illustrative illustration).

[0171] Cap9 complete ORF fragment amplification

[0172] Using the complete AAV9 genome as a template, the complete Cap9 ORF fragment was amplified by PCR and restriction enzyme sites were introduced. The complete Cap9 ORF fragment refers to an ORF fragment with a length of 2637 bp, starting from the P40 promoter and ending at the Cap9 stop codon TAA. The restriction endonuclease MluI was introduced at the P40 end and the restriction endonuclease EcoRI was introduced at the Cap9 stop codon end.

[0173] The primer sequences used for the PCR amplification are shown below:

[0174] Upstream primer (SEQ ID NO:10): ACGCGTacgtcaaaaagggtggagc

[0175] Downstream primer (SEQ ID NO:11): GAATTCttacagattacgagtcaggtatctg

[0176] The PCR amplification program was as follows: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 30 s, repeated denaturation to extension for 35 cycles, 72℃ finishing extension for 2 min, and termination at 4℃.

[0177] Generate builder 1

[0178] The PCR product was digested with EcoRI and MluI, and the fragment was recovered by gel electrophoresis. The original plasmid pRDAV-CMV-EGFP was then digested with both EcoRI and MluI. The resulting target fragment was ligated to the digested plasmid backbone to obtain the plasmid backbone pRDAV-CMV-EGFP-Cap9, i.e., construct 1. Figure 1 As shown in the figure, the size is 10191bp, and the sequence is shown in SEQ ID NO.1.

[0179] 2. Construct the pRDAV-CMV-EGFP-Cap9Δ-588 backbone plasmid (constructor 2)

[0180] A fragment from base position 881 (c.881) to base position 1985 (c.1985) of the wild-type Cap9 gene was selected. The alanine codon corresponding to amino acid position 587 was mutated to a tyrosine codon (A587Y). Simultaneously, the three bases encoding the original amino acid residue at position 588 were replaced with the GGTAACCGTTAACTT sequence (which contains BestEII and HpaI restriction sites), thus obtaining the Cap9Δ-588 sequence encoding the mutated 587 site and the deleted 588 site. A DraIII restriction site was introduced at position c.881 and a BamHI restriction site was introduced at position c.1985 of the Cap9Δ-588 sequence. Gene synthesis was commissioned to Qingke Biotechnology Co., Ltd. The synthesized product was incorporated into construct 1 via restriction enzyme ligation to replace the corresponding fragment between the DraIII and BamHI restriction sites in the original Cap9, resulting in construct 2, namely, pRDAV-CMV-EGFP-Cap9Δ-588. The sequence of construct 2 is shown in SEQ ID NO.2.

[0181] Example 2: Construction of AAV capsid variant virus library

[0182] Linearized skeleton carrier

[0183] Using construct 2 from Example 1 as the backbone vector, the backbone vector was linearized by double digestion with BestEII and HpaI. The digestion system consisted of: construct 2 (3 μg), CutSmart Buffer (5 μL), restriction endonuclease BestEII (0.5 μL), restriction endonuclease HpaI (0.5 μL), and water added to a final volume of 50 μL. The mixture was incubated at 37°C for 3–4 hours. After digestion, the linearized vector was recovered by gel excision.

[0184] Synthetic Oligo Library

[0185] The Oligo sequence used is as follows:

[0186] Oligo-F: GTAACNNNNNNNNN

[0187] Oligo-R: NNNNNNNNN

[0188] Oligo was denatured at 95 degrees Celsius and then slowly annealed at room temperature to obtain the annealed product.

[0189] Library construction and filtering

[0190] The Oligo annealing product and the linearized backbone (constructor 2) were ligated using T4 DNA ligase to obtain a recombinant plasmid. The ligation system was as follows: annealing product (2 μL), linearized backbone vector (25 ng), T4 DNA ligase (0.5 μL), ligation buffer (5 μL), water added to a final volume of 10 μL, incubated overnight at 16°C to obtain a recombinant plasmid containing Oligo.

[0191] The recombinant plasmid was transformed into E. coli HB101 competent cells as described below.

[0192] 1) Add 10 μl of recombinant plasmid to each Ep tube containing 100 μl of HB101 competent cells, gently tap the tube wall several times to mix thoroughly, and incubate on ice for 30 min;

[0193] 2) Place the Ep tube in a 42℃ water bath for 90 seconds;

[0194] 3) Slowly add 0.5 mL of LB medium to the Ep tube, and shake at 37°C and 80 rpm for 45 min;

[0195] 4) Spread the bacterial culture on LB agar plates containing ampicillin (0.1 g / L) and incubate overnight at 37°C.

[0196] Randomly select a single colony and inoculate it into a culture tube (LB medium containing 0.1 g / L ampicillin), and incubate overnight at 37°C with shaking at 200 rpm.

[0197] Perform plasmid extraction as described below.

[0198] 1) Centrifuge the bacterial culture obtained above at 12000 rpm for 1 minute and discard the supernatant culture medium;

[0199] 2) Add 250 μL of buffer P1 / RNaseA mixture and resuspend the bacteria by high-speed vortexing;

[0200] 3) Add 250μL of buffer P2 and invert the container 8-10 times;

[0201] 4) Add 350 μL of buffer P3 and immediately invert and mix 8-10 times to completely neutralize the solution;

[0202] 5) Centrifuge at 13000 rpm for 10 minutes, and pass the supernatant through the column;

[0203] 6) Centrifuge at 12000 for 1 minute, discard the waste liquid, add 500 μL of PW1, centrifuge at 12000 for 1 minute, and discard the waste liquid;

[0204] 7) Add 600 μL of PW2, centrifuge at 12000 for 1 minute, and discard the supernatant;

[0205] 8) Repeat the washing process once: Repeat step 7;

[0206] 9) Idle at 12000 rpm for 2 minutes;

[0207] 10) Add 30-50 μL of preheated elution buffer at 55℃, let stand for 2 minutes, and centrifuge at 12000 rpm for 1 minute.

[0208] Positive plasmids identified by concentration detection and enzyme digestion were numbered and 10 μL was sequenced. The positive plasmids were stored at -20°C to obtain the AAV capsid library.

[0209] Example 3: Packaging produces mutant AAV virus and control AAV virus

[0210] Reference (Xiao X, et al. Production of High-Tier Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus. J Virol. 1998; 72(3):2224-2232), a three-plasmid packaging system was used to package and purify recombinant AAV virus. In short, the packaging plasmid expressing the Rep gene of AAV, the helper plasmid providing the AdV helper function, and the ITR carrying AAV, as well as the transfer plasmid expressing the CAP9 variant and the green fluorescent protein eGFP (inserted into the CAP9 variant) were used. Figure 2 The plasmid shown) or the transfer plasmid expressing wild-type CAP9 and green fluorescent protein eGFP ( Figure 1 The plasmid (shown) was mixed at a 1:1:1 molar ratio and co-transfected into HEK293 cells using the calcium phosphate method. After 72 hours of transfection, cells and culture supernatant were collected, and the recombinant AAV virus was purified using PEG8000-NaCl & CH3Cl extraction. The AAV virus product was dissolved in 0.01% Pluronic F68-PBS, and the rAAV titer was determined using dot blot hybridization. Preliminary experiments selected recombinant AAV viruses that effectively transduced cardiomyocytes, obtaining AAV capsid variants numbered CapX-020 and CapX-123.

[0211] The coding sequences of the CAP9 variants in recombinant AAV viruses with sequencing numbers CapX-020 and CapX-123 are shown in SEQ ID NO. 3 or 6, and their corresponding nucleotide sequences are shown in SEQ ID NO. 4 or 7. The sequenced information was compared with the original capsid protein (CAP9) sequence, and the variants showed mutations at positions 587 and 588 corresponding to the original sequence (SEQ ID NO: 9). Figure 9 As shown, residues 587 and 588 (AQ) of the original sequence were replaced in the CAP9 variants of CapX-020 and CapX-123 with amino acid sequences as shown in SEQ ID NO.5 (YGNAKTQL) or as shown in SEQ ID NO.8 (YGNSPSKL), respectively.

[0212] Example 4: Biodistribution patterns of AAV capsid variant virus in mice

[0213] AAV virus injection

[0214] Male SPF-grade C57 mice, aged 6-8 weeks and weighing 18-20g, were selected. rAAV9 virus encoding the fluorescent protein EFGP and possessing the wild-type CAP9 capsid protein was used as a control. Recombinant AAV capsid variants, CapX-020 and CapX-123 obtained in Example 3, were used as experimental groups, with at least three mice in each group. rAAV9 virus and the capsid variants CapX-020 and CapX-123 were injected via the tail vein at a dose of 2E13 gc / kg. Twenty-eight days after injection, the mice were necropsy to collect tissue samples, and frozen sections were prepared for EGFP fluorescence observation.

[0215] Mouse tissue sections and immunofluorescence comparison

[0216] A. Preparation of frozen sections

[0217] 1) Tissue fixation: Fix fresh tissue with fixative for more than 24 hours, remove the tissue from the fixative, and use a scalpel to trim the tissue at the target site.

[0218] 2) Dehydration: Place the trimmed tissue in a 15% sucrose solution and dehydrate it at 4°C until it settles to the bottom. Then transfer it to a 30% sucrose solution and dehydrate it at 4°C until it settles to the bottom.

[0219] 3) OCT embedding: Remove the dehydrated tissue, gently blot the surface water with filter paper, place it cut-side up on the embedding stage, drop OCT embedding medium around the tissue, and place the embedding stage on the quick-freezing stage of a cryostat for quick freezing and embedding. Once the OCT turns white and hardens, it is ready for sectioning. For fresh tissue directly frozen for sectioning, fixation and dehydration are not required. Simply trim the target area of ​​tissue with a scalpel, embed it with OCT embedding medium, and then section it.

[0220] 4) Sectioning: Fix the embedding stage on the microtome, first make a rough cut to smooth the tissue surface, and then start slicing. The section thickness is 8-10μm. Place a clean glass slide on top of the cut tissue section, attach the tissue to the glass slide, and write a label.

[0221] B. Observe fluorescence

[0222] The frozen sections were protected with anti-fading mounting medium (ProLong, Thermo Fischer), and EGFP protein was observed and images were acquired under a fluorescence microscope using a blue excitation band module (488nm). Statistical analysis of the images was performed using ImageJ software.

[0223] result

[0224] Experimental results showed that, compared with the control group, AAV capsid variants CapX-020 and CapX-123, after tail vein injection, significantly enhanced the fluorescence intensity of mouse cardiomyocytes and increased the number of infected cells; while significantly reduced the fluorescence intensity of kidney cells and skeletal muscle cells and decreased the number of infected cells. Figure 3 CapX-020 and CapX-123 viruses achieved transduction rates (i.e., the proportion of EGFP-positive cells) of up to 95% and 100% in cardiomyocytes, respectively, significantly higher than the control rAAV9 virus. Figure 4 Fluorescence analysis results from ImageJ software on kidney cells, skeletal muscle cells, and cardiomyocytes showed that the average fluorescence intensity induced by CapX-020 and CapX-123 viruses in kidney cells was significantly lower than that of the control rAAV9, while the average fluorescence intensity induced by CapX-020 and CapX-123 viruses in cardiomyocytes was significantly higher than that of the control rAAV9, reaching 2 to 4 times or more. Figure 5-7 ),Depend on Figure 8 The fluorescence intensity ratios of cardiomyocytes, kidney cells, and skeletal muscle cells in mice were compared. It can be seen that the fluorescence intensity ratios of the control rAAV9 virus in cardiomyocytes / kidney cells and cardiomyocytes / skeletal muscle cells were low, while the corresponding ratios of CapX-020 and CapX-123 viruses were high, increasing by 5-10 times compared with the control rAAV9. This indicates that CapX-020 and CapX-123 significantly improved the specificity of cardiomyocyte transduction.

[0225] In summary, the experimental results indicate that the CapX-020 and CapX-123 capsid protein variants have stronger tissue specificity for cardiomyocytes than the AAV9 capsid protein. While enhancing cardiomyocyte transduction efficiency, they reduce transduction in the kidneys and skeletal muscle, making them a better choice for AAV therapeutics that target the heart while also reducing skeletal muscle infection and nephrotoxicity.

[0226] sequence list

[0227] SEQ ID NO:1: Construct 1:

[0228]

[0229] SEQ ID NO:2: Construct 2

[0230]

[0231] SEQ ID No. 3: DNA sequence encoding the CAP9 VP1 variant in plasmid CapX-020

[0232]

[0233] SEQ ID No.4: Amino acid sequence of CAP9 VP1 variant in CapX-020 plasmid

[0234] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSYGNAKTQLAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*

[0235] SEQ ID No.5: Amino acid sequence of the 587th to 588th sites of CAP9 VP1 variant in CapX-020 plasmid

[0236] YGNAKTQL

[0237] SEQ ID No. 6: DNA sequence encoding the CAP9 VP1 variant in the CapX-123 plasmid

[0238]

[0239] SEQ ID No. 7: Amino acid sequence of the CAP9 VP1 variant in the CapX-123 plasmid

[0240] *

[0241] SEQ ID No. 8: Amino acid sequence at positions 587 and 588 of the CAP9 VP1 variant in the CapX-123 plasmid.

[0242] YGNSPSKL

[0243] SEQ ID No. 9: Amino acid sequence of wild-type CAP9 VP1

[0244] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*

[0245] SEQ ID NO:10:ACGCGTacgtcaaaaagggtggagc

[0246] SEQ ID NO:11:GAATTCttacagattacgagtcaggtatctg

Claims

1. An adeno-associated virus (AAV) capsid protein variant having an amino acid sequence mutation between amino acid positions 586 and 589 relative to the parental AAV capsid protein, wherein said mutation is the substitution of amino acid residues 587-588 of the parental AAV capsid protein with the amino acid sequence shown in SEQ ID NO: 5 or 8, wherein the amino acid positions are numbered according to SEQ ID NO:

9. The parental AAV capsid protein therein is an AAV9 capsid protein composed of the amino acid sequence of SEQ ID NO:

9.

2. The AAV capsid protein variant according to claim 1, wherein, Compared to the parental AAV capsid protein, the capsid protein variant confers increased transduction of AAV viral particles containing it to cardiomyocytes.

3. The capsid protein variant according to claim 1, wherein the amino acid sequence of the capsid protein variant is SEQ ID NO: 4 or 7.

4. An isolated nucleic acid, wherein the nucleic acid encodes a variant of the AAV capsid protein according to any one of claims 1-3.

5. The nucleic acid according to claim 4, wherein it is a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4 or 7.

6. The nucleic acid according to claim 4, wherein it is a polynucleotide with the nucleotide sequence of SEQ ID NO: 3 or 6.

7. The nucleic acid according to any one of claims 4-6, wherein the nucleic acid is capable of transcribing and expressing three AAV capsid proteins: VP1, VP2 and VP3.

8. A nucleic acid vector comprising the nucleic acid of any one of claims 4-7.

9. The nucleic acid vector according to claim 8, wherein it is a nucleic acid expression vector.

10. The nucleic acid vector according to claim 8 or 9, further comprising a polynucleotide encoding the AAV REP protein.

11. An isolated host cell comprising the nucleic acid of any one of claims 4-7.

12. The host cell of claim 11, wherein the nucleic acid stably transfects the host cell.

13. The host cell according to claim 11 or 12, wherein the host cell further comprises nucleic acid encoding the AAV REP protein and / or nucleic acid encoding helper viral functions.

14. Use of the nucleic acid according to any one of claims 4-7, or the nucleic acid vector according to any one of claims 8-10, or the host cell according to any one of claims 11-13, for the preparation of a recombinant adeno-associated virus (rAAV) vector.

15. A recombinant adeno-associated virus (rAAV) vector comprising a capsid protein variant according to any one of claims 1-3.

16. The rAAV vector of claim 15, wherein the rAAV exhibits increased transduction of cardiomyocytes relative to wild-type AAV.

17. The rAAV vector of claim 15, wherein the rAAV exhibits reduced transduction of skeletal muscle cells and / or kidney cells relative to wild-type AAV.

18. The rAAV vector of claim 15, wherein the rAAV vector comprises in its genome: a. 5' and 3' AAV inverted terminal repeat (ITR) sequences, and b. An expression construct containing a heterologous nucleic acid between the 5' and 3' ITRs, wherein the heterologous nucleic acid encodes the target gene product.

19. The rAAV vector of claim 18, wherein the expression construct comprises elements functionally linked to each other in the transcriptional direction: - Promoter, - Heterologous nucleic acids, - Transcription terminator.

20. The rAAV vector according to claim 18 or 19, wherein the heterologous nucleic acid encodes a target gene product for gene substitution, gene repression, or gene editing.

21. The rAAV vector of claim 20, wherein the target gene product is a protein or RNA.

22. A pharmaceutical composition comprising the recombinant AAV viral vector and the pharmaceutically acceptable vector as described in any one of claims 15-21.

23. The pharmaceutical composition according to claim 22, for direct intramyocardial injection or for intravenous injection.

24. A method for delivering heterologous nucleic acids to cells cultured in vitro or in vitro, comprising contacting the cells with a recombinant AAV viral vector according to any one of claims 15-21, wherein the cells are cardiomyocytes.

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