Recombinant adeno-associated virus capsid protein

Abstract

The present disclosure generally relates to the field of biotechnology, in particular to a recombinant adeno-associated virus capsid protein. The invention also relates to a recombinant adeno-associated virus, which comprises: (a) a capsid comprising the recombinant adeno-associated virus capsid protein as shown in SEQ ID NO: 1; and (b) a recombinant adeno-associated viral genome. The recombinant adeno-associated virus containing the capsid protein of the recombinant adeno-associated virus or the recombinant adeno-associated virus obtained by packaging the capsid protein of the recombinant adeno-associated virus shows stronger expression in animal livers compared with wild type AAV8.

Description

Technical Field

[0001] The present disclosure generally relates to the field of biotechnology, and more particularly, to a recombinant adeno-associated virus capsid protein. Background Art

[0002] As a gene therapy vector, recombinant adeno-associated virus (rAAV) has become a popular tool in the field of gene therapy due to its low immunogenicity, broad host range, and high gene transfer ability. AAV vectors have shown great potential in the treatment of various diseases, especially in the treatment of liver-related diseases. Its ability to efficiently transduce hepatocytes makes it an ideal gene delivery system.

[0003] As an important organ for human metabolism and synthesis, the liver has a strong regenerative ability and can tolerate the long-term presence of foreign substances, so it has become an ideal target organ for gene therapy. AAV vectors have significant advantages in the treatment of liver diseases, especially in liver metabolic disorders and genetic liver diseases. For example, AAV8 and AAV9 serotypes have been widely used in liver gene therapy, but their high expression in the liver may lead to immune responses and other side effects. Recent studies have simulated the human physiological state through normothermic machine perfusion (NMP) technology to test the transduction efficiency of different AAV vectors in normal and fatty liver livers, and found that fatty liver has a significant impact on the transduction efficiency and specificity of AAV vectors. Different liver pathological states (such as fatty liver) have a significant impact on the transduction efficiency of AAV vectors, which provides a theoretical basis for personalized gene therapy.

[0004] In recent years, with the rapid development of gene therapy technology, higher requirements have been put forward for the tissue specificity and delivery efficiency of AAV vectors. Although traditional AAV serotypes (such as AAV8, AAV9) show high transduction ability in the liver, there are problems such as delivery efficiency, immune response, and non-specific expression. In the future, it is expected to achieve personalized treatment strategies for different liver diseases by precisely selecting AAV vectors. Therefore, developing new AAV serotypes or modifying existing serotypes to achieve more efficient and safer liver-targeted gene therapy has become a research hotspot.

[0005] The transduction efficiency of existing adeno-associated viruses in liver tissue needs to be improved. Therefore, in order to improve the virus transduction efficiency, the existing capsid proteins are usually modified, but the existing serotypes (such as AAV8) or the selected capsids still cannot meet the requirements of gene therapy clinical trials. Summary of the Invention

[0006] The present disclosure encompasses the following technical solutions:

[0007] One aspect of the present disclosure relates to a recombinant adeno-associated virus capsid protein having the sequence shown in SEQ ID NO: 1.

[0008] According to another aspect of the present disclosure, it relates to an isolated polynucleotide encoding the recombinant adeno-associated virus capsid protein as described above.

[0009] According to yet another aspect of the present disclosure, it relates to a vector comprising the polynucleotide as described above.

[0010] According to still another aspect of the present disclosure, it relates to a host cell comprising the polynucleotide as described above or the vector as described above.

[0011] According to still another aspect of the present disclosure, it relates to a method for producing a recombinant adeno-associated virus capsid protein, the method comprising culturing the host cell as described above under conditions for expressing the polynucleotide as described above and producing the capsid protein.

[0012] According to still another aspect of the present disclosure, it relates to a recombinant adeno-associated virus packaging vector combination product, which comprises:

[0013] A first nucleotide encoding one or more AAV Rep proteins;

[0014] A second nucleotide encoding a capsid protein and comprising the polynucleotide as claimed in claim 2;

[0015] A third nucleotide comprising a recombinant adeno-associated virus genome;

[0016] Wherein the packaging vector combination product is operable in a cell to encapsulate the recombinant adeno-associated virus genome in a capsid to form a recombinant adeno-associated virus.

[0017] According to still another aspect of the present disclosure, it relates to a kit comprising the vector as described above or the packaging vector combination product as described above.

[0018] According to still another aspect of the present disclosure, it relates to a recombinant adeno-associated virus comprising: (a) a capsid comprising the recombinant adeno-associated virus capsid protein as described above; and (b) a recombinant adeno-associated virus genome.

[0019] According to still another aspect of the present disclosure, it relates to a method for delivering a transgene to a cell, the method comprising contacting the cell with the recombinant adeno-associated virus as described above under conditions for transducing the cell and expressing the transgene.

[0020] According to still another aspect of the present disclosure, it relates to a pharmaceutical composition comprising the recombinant adeno-associated virus as described above.

[0021] The recombinant adeno-associated virus capsid protein of the present disclosure or the recombinant adeno-associated virus packaged by this recombinant adeno-associated virus capsid protein exhibits stronger expression in the animal liver compared to wild-type AAV8. Description of the Drawings

[0022] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 : Flow chart for screening and verification of new serotypes;

[0024] Figure 2 : Flow chart of the third-generation sequencing experiment;

[0025] Figure 3 : Map of the pAAV-CBh-Luc2-WPRE vector;

[0026] Figure 4 : Map of the pscAAV-TBG-EGFP-tWPA vector;

[0027] Figure 5 : Map of the pAAV-TBG-EGFP-WPRE vector;

[0028] Figure 6 : Map of the pAAV-CBh-EGFP-WPRE vector;

[0029] Figure 7 : Map of the AAV-AL04 serotype vector;

[0030] Figure 8 : Results of in vivo imaging of 3-week-old mice;

[0031] Figure 9 : Fluorescence results of liver sections of mice injected with new serotype viruses packaged by Shuttle 1;

[0032] Figure 10 : Fluorescence results of liver sections of mice injected with new serotype viruses packaged by Shuttle 2;

[0033] Figure 11 : Fluorescence results of liver sections of mice injected with new serotype viruses packaged by Shuttle 3. Specific Embodiments

[0034] Reference will now be made in detail to embodiments of the disclosure, one or more examples of which are described below. Each example is provided by way of explanation, and not limitation, of the disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the disclosure without departing from the scope or spirit of the disclosure. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0035] Unless otherwise noted, all terms used to disclose the disclosure (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Through further guidance, the following definitions are used to better understand the teachings of the disclosure. The terms used in the specification of the disclosure herein are for the purpose of describing particular embodiments only and are not intended to limit the disclosure.

[0036] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operation procedures used herein are all widely used terms and conventional procedures in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

[0037] As used herein, the terms “and / or,” “or / and,” and “and / or” include any one of the two or more related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include any two of the related listed items, any more of the related listed items, or the combination of all the related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from “and / or,” “or / and,” and “and / or,” it should be understood that in the present disclosure, this technical solution undoubtedly includes the technical solution connected by “logical AND,” and also undoubtedly includes the technical solution connected by “logical OR.” For example, “A and / or B” includes three parallel solutions: A, B, and A + B. Another example, the technical solution of “A, and / or, B, and / or, C, and / or, D” includes any one of A, B, C, and D (i.e., the technical solution connected by “logical OR”), and also includes any and all combinations of A, B, C, and D, that is, it includes the combination of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (i.e., the technical solution connected by “logical AND”).

[0038] The terms “comprising,” “including,” and “containing” used in the present disclosure are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0039] In the present disclosure, a numerical range represented by endpoints includes all the numerical values and fractions contained within that range, as well as the recited endpoints.

[0040] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly specifying the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0041] In the present disclosure, for concentration values, their meanings include fluctuations within a certain range. For example, they can fluctuate within the corresponding precision range. For example, for 2%, a fluctuation within the range of ±0.1% is allowed. For larger numerical values or those that do not require overly precise control, their meanings are also allowed to include larger fluctuations. For example, for 100 mM, fluctuations within the ranges of ±1%, ±2%, ±5%, etc. are allowed. Regarding molecular weight, its meaning is allowed to include a fluctuation of ±10%.

[0042] As used herein, unless otherwise indicated, the singular forms of the articles "a", "an", and "the" include plural referents.

[0043] In the present disclosure, descriptions such as "a plurality of" and "a variety of", unless otherwise specified, refer to a quantity greater than or equal to 2.

[0044] In the present disclosure, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the recited features, as well as an open technical solution containing the recited features.

[0045] In the present disclosure, "preferably", "more preferably", "even more preferably", and "it is advisable" are only for describing embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present disclosure. In the present disclosure, "optionally", "optional", and "optional" mean that it can be either present or absent, that is, it refers to any one of the two alternative options of "present" or "absent". If "optional" appears multiple times in a technical solution, unless otherwise specified and there are no contradictions or mutual restrictions, each "optional" is independent of each other.

[0046] All documents mentioned in this disclosure are incorporated herein by reference as if each document was individually incorporated by reference. Unless it conflicts with the inventive purpose and / or technical solution of this disclosure, the cited documents involved in this disclosure are incorporated by reference in their entirety and for all purposes. When this disclosure refers to a cited document, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated by reference. When this disclosure refers to a cited document, examples and preferred modes of the relevant technical features cited can also be incorporated by reference into this disclosure as a reference, but only to the extent that the disclosure can be implemented. It should be understood that when the cited content conflicts with the description in this disclosure, this disclosure shall prevail or be modified adaptively according to the description in this disclosure.

[0047] As used herein, the term "recombinant adeno-associated virus" or "rAAV" refers to an AAV that contains a genome lacking functional rep and cap genes.

[0048] As used herein, the term "cap gene" refers to a nucleic acid sequence encoding a capsid protein. For AAV, the capsid proteins can be VP1, VP2, or VP3. The VP1, VP2, and / or VP3 capsid proteins assemble into a capsid surrounding the rAAV genome.

[0049] As used herein, the term "rep gene" refers to a nucleic acid sequence encoding non-structural proteins (such as rep78, rep68, rep52, and rep40) required for the replication and production of AAV.

[0050] As used herein, the term "rAAV genome" refers to a nucleic acid molecule (e.g., DNA and / or RNA) containing the genomic sequence of rAAV. Those skilled in the art will understand that in the case where the rAAV genome contains a transgene (e.g., a heavy or light chain coding sequence of an antibody operably linked to a transcriptional regulatory element), the rAAV genome can be in a sense or antisense orientation relative to the transcriptional direction of the transgene.

[0051] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been isolated from one or more nucleic acid molecules present in its natural source.

[0052] As used herein, a "vector" refers to a nucleic acid molecule that serves as a vehicle for introducing a nucleic acid molecule (e.g., a polynucleotide disclosed herein) into a cell.

[0053] As used herein, an "expression vector" refers to a vector (e.g., a polynucleotide disclosed herein) that contains a transcriptional regulatory element operably linked to a gene of interest, which promotes the expression of the gene of interest in a cell and / or a cell-free expression system.

[0054] As used herein, the term "transgene" refers to a non-AAV nucleic acid sequence encoding a polypeptide (e.g., a protein, an antibody, or an scFv) or a non-coding RNA (e.g., an miRNA, an shRNA, an siRNA, an antisense RNA, a gRNA, an antagomir, an miRNA sponge, an RNA aptazyme, an RNA ribozyme, or an RNA aptamer).

[0055] As used herein, "nucleic acid" has the meaning well-known in the art and is also referred to as "polynucleotide", which is a molecule formed by multiple nucleotide monomers.

[0056] As used herein, unless otherwise specified, "protein" has the same meaning as "protein", both corresponding to "Protein" and can be used interchangeably. "Protein", "multi-amino acid", "peptide" and "polypeptide" have the same meaning and can be used interchangeably, and all include at least two amino acid units.

[0057] The first aspect of the present disclosure relates to a recombinant adeno-associated virus capsid protein having the sequence shown in SEQ ID NO: 1.

[0058] According to the conventional understanding of those skilled in the art, it should be considered that functional variants of the above amino acid sequences are also within the scope of the present disclosure. Compared with the sequence shown in SEQ ID NO:1, the functional variant has substantially the same function of showing stronger expression in animal liver. The functional variant has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO:1. Compared with the sequence shown in SEQ ID NO:1, the mutations of the functional variant are preferably substitutions, deletions or additions of amino acids or any combination thereof; preferably, the mutations are conservative substitutions. "Conservative substitution" means the substitution of an amino acid in a protein by another amino acid having similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), so that changes can be frequently made without changing the biological activity of the protein. Substitutions that are generally regarded as conservative substitutions are the substitutions among aliphatic amino acids Ala, Val, Leu and Ile, the interchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the substitution between amide residues Asn and Gln, the exchange of basic residues Lys and Arg, and the substitution between aromatic residues Phe and Tyr, etc. Those skilled in the art know that generally, a single amino acid substitution in a non-essential region of a polypeptide basically does not change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224, (4th edition)). In addition, the substitution of amino acids with similar structures or functions is unlikely to destroy the biological activity.

[0059] The second aspect of the present disclosure relates to an isolated polynucleotide encoding the recombinant adeno-associated virus capsid protein as described above.

[0060] The isolated nucleic acid includes RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and moreover, unless otherwise specified, the polypeptide also includes natural polynucleotides, sugars, or analogs with base alterations.

[0061] The isolated polynucleotide includes a nucleotide sequence encoding the amino acid sequence of the recombinant adeno-associated virus capsid protein, and also includes a nucleotide sequence complementary thereto. The complementary sequence includes a completely complementary sequence and a substantially complementary sequence, which refers to a sequence that can hybridize with the nucleotide sequence encoding the amino acid sequence of the protein complex under stringent conditions known in the art.

[0062] In addition, the codons of the nucleic acid corresponding to the capsid protein can be optimized according to the species of the host cell.

[0063] The third aspect of the present disclosure relates to a vector, which comprises the polynucleotide as described above;

[0064] In some embodiments, the vector is a plasmid or a viral vector.

[0065] In some embodiments, the vector is an expression vector.

[0066] The vector (e.g., an expression vector) can be introduced into cells (using any technique known in the art) for the proliferation of the vector and / or for the expression of the AAV capsid protein encoded by the vector. Thus, in the fourth aspect of the present disclosure, a host cell is provided, which comprises the polynucleotide as described above or the vector as described above (e.g., an expression vector). Further, in the fifth aspect of the present disclosure, a method for producing a recombinant adeno-associated virus capsid protein is provided, the method comprising culturing the host cell as described above under conditions for expressing the polynucleotide as described above and producing the capsid protein.

[0067] A variety of host cells and expression vector systems can be used to express the capsid proteins described herein. In these expression systems, the coding sequences of interest can be produced and subsequently purified. These expression systems also represent cells that can express the capsid proteins described herein in situ when transformed or transfected with the appropriate nucleotide coding sequences. These include but are not limited to microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing the capsid protein coding sequence; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing, for example, the capsid protein coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing, for example, the capsid protein coding sequence; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing, for example, the capsid protein coding sequence; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, NIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, and BMT10 cells) containing a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., the metallothionein promoter) or a promoter derived from a mammalian virus (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In an embodiment, the cells used to express the capsid proteins described herein are human cells, such as a human cell line. In an embodiment, the mammalian expression vector is pOptiVEC TM or pcDNA3.3. In an embodiment, bacterial cells (such as Escherichia coli) or eukaryotic cells (e.g., mammalian cells) are used for the expression of the capsid protein. For example, mammalian cells (such as CHO or HEK293 cells) in combination with a vector (such as the major immediate early gene promoter element from human cytomegalovirus) are an effective expression system for the capsid proteins disclosed herein. In an embodiment, insect cells (e.g., Sf9 cells) are used for the expression of the capsid protein.

[0068] For example, in an insect system, Autographa californica nucleopolyhedrovirus (AcNPV) can be used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The capsid protein coding sequence can be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0069] In mammalian host cells, many virus-based expression systems can be utilized. In the case of using adenovirus as an expression vector, the capsid protein coding sequence of interest can be ligated to an adenovirus transcriptional / translational control complex, e.g., a late promoter and a tripartite leader sequence. Then this chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the virus genome (e.g., the E1 region or the E3 region) will generate a recombinant virus that is viable and capable of expressing the capsid protein molecule in the infected host (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655 - 9, which is incorporated herein by reference in its entirety). Efficient translation of the inserted capsid protein coding sequence may also require specific initiation signals. These signals include the ATG start codon and adjacent sequences. Additionally, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These foreign translational control signals and start codons can have multiple sources, including natural and synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516 - 544, which is incorporated herein by reference in its entirety). Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O, T - 47D, NS0, CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK - 293T, HepG2, SP210, R1.1, B - W, L - M, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells.

[0070] For long - term, high - yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines that stably express the capsid proteins described herein can be engineered.

[0071] In an embodiment, instead of using an expression vector containing a viral replication origin, a host cell can be transformed with a polynucleotide (e.g., DNA or RNA) controlled by appropriate transcriptional regulatory elements (e.g., promoters, enhancers, sequences, transcriptional terminators, polyadenylation sites, etc.) and a selectable marker.

[0072] In a preferred embodiment, the promoter is a liver-specific promoter, such as TBG (Thyroxine binding globulin), Alb (albumin), ApoE (apolipoprotein E).

[0073] The sixth aspect of the present disclosure relates to a recombinant adeno-associated virus packaging vector combination product, which comprises:

[0074] A first nucleotide encoding one or more AAV Rep proteins;

[0075] A second nucleotide encoding a capsid protein and comprising the polynucleotide as described above;

[0076] A third nucleotide comprising a recombinant adeno-associated virus genome;

[0077] Wherein the packaging vector combination product is operable in a cell to encapsulate the recombinant adeno-associated virus genome in a capsid to form a recombinant adeno-associated virus.

[0078] In some embodiments, the combination product comprises a first vector and a second vector;

[0079] The first vector contains the first nucleotide and the second nucleotide, and the second vector contains the third nucleotide.

[0080] In some embodiments, the combination product comprises a fourth nucleotide, which contains one or more helper virus genes. In an embodiment of the combination product, the helper virus is selected from the group consisting of adenovirus, herpes virus (including herpes simplex virus (HSV), but excluding herpes simplex virus type 1), poxvirus (such as vaccinia virus, cowpox virus), cytomegalovirus (CMV), and baculovirus. In an embodiment where the helper virus is adenovirus, the adenovirus genome contains one or more adenovirus RNA genes selected from the group consisting of E1, E2, E4, and VA. In an embodiment, the adenovirus genome contains one or more adenovirus RNA genes selected from the group consisting of E2, E4, and VA. In an embodiment where the helper virus is HSV, the HSV genome contains one or more HSV genes selected from the group consisting of UL5 / 8 / 52, ICP0, ICP4, ICP22, and UL30 / UL42.

[0081] In some embodiments, the combination product comprises a third vector, and the third vector comprises the fourth nucleotide. The third vector can be an independent third vector, integrated with the first vector, or integrated with the second vector.

[0082] In some embodiments, the third nucleotide comprises a transgene.

[0083] In some embodiments, the transgene encodes a polypeptide or non-coding RNA.

[0084] The coding product of the transgene can be arranged as needed. For example, in some embodiments, the transgene encodes a therapeutic protein. In some embodiments, the transgene encodes an antibody or a fragment thereof (e.g., Fab or full-length antibody).

[0085] In some embodiments, the transgene encodes a scFv, nanobody or VHH.

[0086] In some embodiments, the transgene encodes coagulation factor VIII. AAV vectors have been used to introduce the gene for coagulation factor VIII into the liver of patients, by which patients can continuously produce coagulation factor VIII, significantly reducing the clinical symptoms of hemophilia.

[0087] In some embodiments, the transgene encodes FOXA2. Upregulation of FOXA2 in hepatocytes can significantly alleviate liver fibrosis.

[0088] In some embodiments, the transgene encodes a Cas nuclease and / or a CRISPR sgRNA.

[0089] In an embodiment of the combination product, the first vector, the second vector, and / or the third vector are contained within one or more plasmids. In an embodiment, the first vector and the third vector are contained within the first plasmid. In an embodiment, the second vector and the third vector are contained within the second plasmid.

[0090] In an embodiment of the packaged vector combination product, the first vector, the second vector, and / or the third vector are contained within one or more recombinant helper viruses. In an embodiment, the first vector and the third vector are contained within the recombinant helper virus. In an embodiment, the second vector and the third vector are contained within the recombinant helper virus.

[0091] The seventh aspect of the present disclosure relates to a kit comprising the vector as described above, or the packaged vector combination product as described above.

[0092] As used herein, the term "kit" may refer to any article (e.g., a package or container) that includes at least one device and contains a detection agent as described herein. The kit may further include instructions for use, additional reagents and / or components or assemblies used in the methods or steps described herein.

[0093] The components within the reagent or kit may be packaged in the form of a solution or a solid. In some preferred embodiments, at least one component of the reagent or kit is a solid, and the solid includes at least one of lyophilized microspheres, lyophilized cakes, lyophilized powders, and spots that rely on the presence of a solid medium.

[0094] The eighth aspect of the present disclosure relates to a recombinant adeno-associated virus, which comprises: (a) a capsid that contains a recombinant adeno-associated virus capsid protein as described above; and (b) a recombinant adeno-associated virus genome.

[0095] The rAAV genome can be of any type capable of being packaged within the AAV capsids disclosed herein. For example, in an embodiment, the rAAV genome is a single-stranded DNA genome. In an embodiment, the rAAV genome is a self-complementary genome as described, for example, in US7790154, which is incorporated herein by reference in its entirety.

[0096] In some embodiments, a transgene is included in the recombinant adeno-associated virus genome.

[0097] The transgene can be defined as the transgene in the sixth aspect of the present disclosure.

[0098] The ninth aspect of the present disclosure relates to a method for delivering a transgene to a cell, the method comprising contacting the cell with a recombinant adeno-associated virus as described above under conditions that transduce the cell and express the transgene.

[0099] In some embodiments, the cell is a hepatocyte.

[0100] The hepatocyte can be a healthy cell or a diseased cell, such as a cirrhotic cell or a hepatocyte-derived cancer cell.

[0101] The tenth aspect of the present disclosure relates to a pharmaceutical composition that contains a recombinant adeno-associated virus as described above. The pharmaceutical composition contains the rAAV disclosed herein and a pharmaceutically acceptable excipient, adjuvant, diluent, carrier, or vehicle, or a combination thereof. "Pharmaceutically acceptable carrier" includes any material that, when combined with the active ingredient of the composition, allows the ingredient to retain its biological activity and does not cause a destructive physiological reaction (such as an unexpected immune response). Pharmaceutically acceptable carriers include water, phosphate-buffered saline, emulsions (such as oil / water emulsions), and wetting agents.

[0102] The pharmaceutical composition may be contained in a container, package, or dispenser, such as a syringe, together with instructions for administration.

[0103] In other aspects of the present disclosure, there is provided a method of preventing or treating a disease or condition in a subject in need thereof, the method comprising delivering a therapeutically effective amount of the rAAV virus or pharmaceutical composition described herein to the subject.

[0104] The rAAV described in the present disclosure can be delivered to a subject by any suitable method known in the art. For example, it is preferred to administer rAAV suspended in a physiologically compatible carrier (e.g., in a composition) to a subject, i.e., a host animal such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque). In some embodiments, the host animal does not include humans. In some embodiments, the subject is human.

[0105] "Therapeutically effective amount" means an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the rAAV virus or pharmaceutical composition can vary according to factors such as the disease state, age, sex, and weight of the subject to be treated, as well as the ability of the rAAV virus or pharmaceutical composition to elicit the desired response in the subject. The dosing regimen can be adjusted to provide the optimal therapeutic response. A therapeutically effective amount is generally also an amount in which any toxic or detrimental effects of the rAAV virus or pharmaceutical composition are outweighed by the therapeutic beneficial effects. "Prophylactically effective amount" means an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result, such as preventing or inhibiting various conditions. Prophylactic doses can be used in subjects prior to or at an early stage of disease onset. In some cases, the prophylactically effective amount can be greater than or less than the therapeutically effective amount. The dosage administered depends to a large extent on the condition and size of the subject being treated, as well as the treatment formulation, treatment frequency, and route of administration. The regimen for continuous treatment, including dosage, formulation, and frequency, can be guided by the initial response and clinical judgment.

[0106] In some embodiments, the rAAV virus or pharmaceutical composition is administered to the subject once a day, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months, once a year, once every two years, once every five years, or once in a lifetime.

[0107] Exemplary routes of administration and delivery include intravenous (I.V.), intra-articular, intraperitoneal (I.P.), intra-arterial, intramuscular, parenteral, subcutaneous, intrapleural, dermal, transdermal, parenteral, e.g., transmucosal, intracranial, intraspinal, oral (enteral), mucosal, respiratory, intranasal, intubation, intralung, intralung instillation, buccal, sublingual, intravascular, intrathecal, intracavitary, iontophoresis, intraocular, intra-glandular, intra-organ, intra-fallopian.

[0108] In some embodiments, delivery of rAAV to a mammalian subject can be achieved, for example, by intramuscular injection into the mammalian subject.

[0109] In some embodiments, delivery of rAAV to a mammalian subject can be achieved, for example, by intravenous injection into the mammalian subject.

[0110] In some embodiments, delivery of rAAV to a mammalian subject can be achieved, for example, by intra-articular injection into the mammalian subject. "Intra-articular injection" is defined herein as an injection or infusion into a joint. Intra-articular injection is commonly used to administer drugs to joints affected by inflammation.

[0111] The effects of administering the rAAV or pharmaceutical composition described in the present application may include preventing the development of a disease condition, halting the progression of a disease condition, reversing the progression of a disease condition, etc. The disease or disorder is related to the target gene loaded in the rAAV and the gene product expressed.

[0112] Exemplary diseases or disorders include: hepatitis (such as viral hepatitis), cirrhosis, liver cancer, hemophilia.

[0113] The embodiments of the present disclosure will be described in detail below in conjunction with examples. It should be understood that these examples are only for illustrating the present disclosure and not for limiting the scope of the present disclosure. For the experimental methods without specific conditions noted in the following examples, preference is given to the guidance provided in the present disclosure, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0114] In the following specific examples, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0115] Example 1 Screening of New AAV Serotypes

[0116] The process for screening and validating new serotypes is as Figure 1 shown. Specifically:

[0117] 1. Construction of AAV New Capsid Mutation Library

[0118] The library consists of the following vectors:

[0119] 1) Using serotypes from more than 40 different species, a certain amount of viable capsid sequences were generated by family shuffling. A brand-new capsid sequence library was constructed using this method, and the generation process is as Figure 2 shown.

[0120] 2) A certain amount (millions of species) of brand-new capsid sequences generated by enzymatic digestion and ligation are then constructed onto the library-based shuttle vector using the method of enzymatic digestion and ligation, and a brand-new capsid library is constructed using the library shuttle vector (pAAV-UBC-EGFP-Cap(replacement)-SV40 polyA).

[0121] 3) Add 10 μL of the ligation product to 50 μL of electrocompetent cells specifically for the library (purchased from Lucigen Corporation), mix well and let it stand, then transfer it into a pre-cooled electrode cup, and perform electroporation using the electroporator from Bio-Rad. After electroporation, add 1 mL of SOC liquid medium pre-warmed at 37°C, and then resuscitate at 37°C for 1 hour, followed by centrifugation and plating to obtain a brand-new capsid library.

[0122] 4) Use the QIAGEN large-scale extraction kit (QIAGEN Plasmid Plus Midi Kit(25), catalog number 12943) to extract the plasmid of the brand-new capsid library for packaging.

[0123] 2. Packaging and screening of the AAV new capsid library

[0124] (1) Viral packaging of the AAV new capsid library

[0125] Inoculate 293AAV packaging cells at a density of 1.0×10 7 / 10 cm dish. After 18 - 24 hours of inoculation, when the cells adhere to the dish, AAV packaging plasmid transfection can be started. Use the polyetherimide (PEI) transfection reagent to transfect the AAV library plasmid, packaging Rep plasmid, and pHelper helper plasmid into the inoculated 293AAV packaging cells. After 72 hours of transfection, observe the packaging efficiency through a fluorescence microscope, and use a pipette to blow down the packaging cells to completely detach all cells from the culture dish, and collect the cell pellet samples.

[0126] (2) Purification of the AAV new capsid library virus

[0127] Perform repeated freezing and thawing of the collected cell pellet samples at -80°C and 37°C, centrifuge, collect the cell supernatant, and remove cell debris through a 0.45 μm PVDF filter. Subsequently, further purify using an AAV purification kit to harvest the recombinant AAV virus library.

[0128] (3) Determination of the titer of the AAV new capsid library virus

[0129] Take 20 μL of the concentrated virus solution, add 1 μL of RNase-free DNase, vortex, incubate at 37 °C for 30 min. After incubation, centrifuge at 10,000 rpm for 10 min. Add the centrifuged concentrated virus solution to 80 μL of Buffer for dilution, perform a 10-min metal bath at 100 °C. After cooling to room temperature, add 3 μL of proteinase K, incubate at 37 °C for 60 min, then perform a 10-min metal bath reaction at 100 °C. Subsequently, cool the sample to room temperature, and the diluted sample is used for titer detection by real-time quantitative PCR (qPCR). The detection uses a Taqman detection kit (TaqMan TM Fast Advanced Premix, catalog number: 4444963; brand: Applied Biosystems TM ). The qPCR reaction conditions are as follows: 95 °C for 10 min; 95 °C for 30 s; 60 °C for 30 s, for 35 cycles.

[0130] (4) Screening of AAV new capsid library in mice

[0131] Intravenously inject 200 μL of the AAV new capsid library virus stock solution into C57BL / 6 mice (source: Vital River). Three weeks after injection, collect the main organs such as the heart, liver, and brain of the mice. Use a genomic DNA extraction kit (catalog number DP304, brand Tiangen) to extract genomic DNA from each tissue. Perform third-generation sequencing on the extracted genomic DNA and the virus stock solution before injection, and analyze the sequencing results. Specific method:

[0132] Perform PCR amplification on the extracted genomic DNA and the purified AAV capsid library virus to obtain PCR products, which are used for third-generation sequencing. The library is sequenced on the PacBio Sequel platform of a third-generation sequencer. The PacBio sequencing experimental process includes genomic DNA extraction, genomic fragmentation, end repair, adapter ligation, DNA purification, and sequencing on the machine. The flow chart is as Figure 2 shown.

[0133] After sequencing, the raw data is quality controlled and filtered using fastq software to remove low-quality reads, adapter contamination sequences, and merge paired-end sequencing data. Subsequently, use third-party open-source packages in Python to build self-written analysis scripts, such as Biopython, pysam, pandas, etc., to classify the sequencing data tags to count the number of reads of different capsid mutants in different tissues and viruses. The naming method for the selected mutants is: AAV-AL xx; for example, the 01st Cap candidate sequence is: AAV-AL 01.

[0134] 3. Packaging Verification of Candidate New AAV Capsids

[0135] (1) Construction of Candidate New AAV Capsids

[0136] Replace the Cap sequence region of wild-type AAV with the candidate AAV-AL xx fragment to obtain the screened AAV capsid plasmid.

[0137] (2) AAV Viral Packaging of Candidate AAV Capsids

[0138] Use 4 different AAV shuttle vectors, pAAV-CBh-Luc2-WPRE, pscAAV-TBG-EGFP-tWPA, pAAV-CBh-EGFP-WPRE, pAAV-TBG-EGFP-WPRE (plasmid maps are shown in Figures 3 - 6 ), and use AAV-AL xx, etc. as serotype vectors to package AAV viruses of various candidate serotypes. The packaging and purification methods are as described in Step 2 above. Use the WPRE primer to measure the virus titer of the above finished viruses, and determine the volume of the virus stock solution for tail vein injection of mice according to the titer detection results.

[0139] Among them, taking AAV-AL04 as an example, the detailed packaging and AAV virus titer measurement process are described as follows:

[0140] AAV-AL04: It is a serotype plasmid. The construction method is to replace the Cap8 sequence in the original serotype plasmid AAV8 with the sequence of AL04 (which is the Cap sequence), and then obtain the AAV-AL04 serotype plasmid. The map is as Figure 7 shown.

[0141] Inoculate 293AAV packaging cells at 1.0×10 7 / 10 cm dish. After 18 - 24 h of inoculation, when the cells adhere to the wall, AAV packaging plasmid transfection can be started. Use the polyetherimide (PEI) transfection reagent to transfect the AAV shuttle plasmid, packaging serotype plasmid (AAV-AL04), and pHelper helper plasmid into the inoculated 293AAV packaging cells at a ratio of 1:1:1. After 72 h of transfection, observe the packaging efficiency through a fluorescence microscope, and use a pipette to blow down the packaging cells to completely detach all cells from the culture dish, and collect the cell pellet samples.

[0142] Perform repeated freezing and thawing of the collected cell pellet samples at -80 °C and 37 °C, centrifuge, collect the cell supernatant, and remove cell debris through a 0.45 μm PVDF filter. Subsequently, use an AAV purification kit for purification to harvest AAV viruses.

[0143] In vivo imaging verification of candidate new capsids in Example 2

[0144] The AAV8-WT, AAV-AL01 to AAV-AL10 viruses packaged with pAAV-CBh-Luc2-WPRE in Example 1 were injected into Balb / c mice (from Vital River) at a virus dose of 1E+11 vg / mouse and a volume of 200 μL. Three weeks after injection, in vivo imaging was performed on the experimental mice using a PE in vivo imaging instrument.

[0145] By intraperitoneal injection of D-luciferin potassium salt working solution (150 mg / kg body weight), the anesthetized mice were placed on the animal platform of the device, with the detection site facing upward (the direction of the camera lens). Generally, the supine position was adopted for shooting. After the exposure ended, images were obtained. The system would perform fusion of bright field images and luminescence images or fluorescence images, adjust the gray scale to optimize the display effect, and perform ROIs quantitative analysis.

[0146] The in vivo imaging results of mice injected with AAV8-WT, AAV-AL01 to AAV-AL10 viruses three weeks later are as Figure 8 shown. Compared with the control AAV8 (AAV8-WT), the serum types AAV-AL02, AAV-AL04, AAV-AL05, AAV-AL09, and AAV-AL10 had stronger fluorescence signals in the abdomen of mice.

[0147] According to the in vivo imaging results, the serum types AAV8-WT, AAV-AL02, AAV-AL04, and AAV-AL10 were selected. It was reported that self-complementary AAV (scAAV) drives stronger gene expression compared to single-stranded AAV (ssAAV). Using the scAAV shuttle plasmid (pscAAV-TBG-EGFP-tWPA) with the liver-specific promoter TBG, virus packaging was carried out referring to the packaging and purification steps in Example 1. Mice were injected via the tail vein at a dose of 5E+10 vg / mouse and a volume of 200 μL. Three days later, the livers in the mice were harvested for section verification. The fresh liver tissue was placed in 4% paraformaldehyde fixative (Sangon Biotech) and fixed overnight at 4°C. The next day, the paraformaldehyde was replaced with sucrose solution for dehydration until the tissue sank. Then, the liver tissue was embedded with OCT (Opti-mum cutting temperature compound, product number: 4583, brand: Sakura), sliced using a Leica microtome, and the cell nuclei were stained with DAPI (product number: 62247, brand: ThermoFisher) after slicing. Finally, an inverted fluorescence microscope (OLYMPUS) was used for observation and photography.

[0148] Using shuttle vector 1 ( Figure 4Package AAV8-WT, AAV-AL02, AAV-AL04, and AAV-AL10, and take liver sections of the virus in mice. The results are as Figure 9 shown. Compared with the slice fluorescence of the control group AAV8 (AAV8-WT), the new serotype AAV-AL04 has stronger expression in mouse liver tissue, and the fluorescence brightness statistics are about 1.5 times higher.

[0149] Considering that the loading capacity of single-stranded AAV (about 4.7 kb) is larger than that of self-complementary AAV vectors and has a wider application in the field of gene therapy. We further used shuttle 2 ( Figure 5 shown), the single-stranded AAV shuttle vector (pAAV-TBG-EGFP-WPRE) with the liver-specific promoter TBG, and shuttle 3 ( Figure 6 shown), the broad-spectrum promoter Cbh (pAAV-CBh-EGFP-WPRE) to package AAV8-WT and the new serotype AAV-AL04 for expression verification, and take liver sections of the virus in mice. The results are as Figures 10 - 11 shown. Compared with the slice fluorescence of the control AAV8 (AAV8-WT), when changing another two different shuttles, the new serotype AAV-AL04 still has stronger expression in mouse liver tissue.

[0150] The above-described embodiments only represent several implementation manners of the present disclosure. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present disclosure, several modifications and improvements can still be made, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure patent shall be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A recombinant adeno-associated virus capsid protein having the sequence shown in SEQ ID NO:

1.

2. An isolated polynucleotide encoding the recombinant adeno-associated virus capsid protein according to claim 1.

3. A vector comprising the polynucleotide according to claim 2; Optionally, the vector is a plasmid or a viral vector; Optionally, the vector is an expression vector. A host cell comprising the polynucleotide of claim 2 or the vector of claim 3.

5. A method for producing a recombinant adeno-associated virus capsid protein, comprising: The host cell of claim 4 is cultured under conditions where the polynucleotide of claim 2 is expressed and capsid protein is produced.

6. A recombinant adeno-associated virus packaging vector combination product, comprising: a first nucleotide encoding one or more AAV Rep proteins; A second nucleotide, which encodes a capsid protein and comprises the polynucleotide of claim 2; a third nucleotide comprising a recombinant adeno-associated virus genome; The packaging vector combination product is operably used in cells to encapsulate the recombinant adeno-associated virus genome in a capsid to form a recombinant adeno-associated virus.

7. The combination product according to claim 6, comprising a first carrier and a second carrier; The first vector comprises the first nucleotide and the second nucleotide, and the second vector comprises the third nucleotide; Optionally, the combination product comprises a fourth nucleotide, and the fourth nucleotide comprises one or more helper virus genes; further optionally, the combination product comprises a third vector, and the third vector comprises the fourth nucleotide; Optionally, the third nucleotide comprises a transgene, wherein preferably the transgene encodes a polypeptide or a non-coding RNA. Optionally, the third nucleic acid comprises a recombinant adeno-associated virus genome of a self-complementary AAV.

8. A kit comprising the vector according to claim 3, or the packaging vector combination product according to claim 6 or 7.

9. A recombinant adeno-associated virus, comprising: (a) a capsid, wherein the capsid comprises the recombinant adeno-associated virus capsid protein of claim 1; and (b) a recombinant adeno-associated virus genome.

10. The recombinant adeno-associated virus according to claim 9, wherein the recombinant adeno-associated virus genome comprises a transgene, wherein the transgene preferably encodes a polypeptide or a non-coding RNA; Optionally, the recombinant adeno-associated virus is a self-complementary AAV.

11. A method for delivering a transgene to a cell, the method comprising contacting the cell with the recombinant adeno-associated virus of claim 9 or 10 under conditions that transduce the cell and express the transgene.

12. The method according to claim 11, wherein the cell is a hepatocyte.

13. A pharmaceutical composition comprising the recombinant adeno-associated virus according to claim 9 or 10.

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