WO2015062516A1 - 定点突变和定点修饰的腺相关病毒、其制备方法及应用 - Google Patents

定点突变和定点修饰的腺相关病毒、其制备方法及应用 Download PDF

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WO2015062516A1
WO2015062516A1 PCT/CN2014/089880 CN2014089880W WO2015062516A1 WO 2015062516 A1 WO2015062516 A1 WO 2015062516A1 CN 2014089880 W CN2014089880 W CN 2014089880W WO 2015062516 A1 WO2015062516 A1 WO 2015062516A1
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amino acid
adeno
associated virus
fragment
site
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French (fr)
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周德敏
张传领
肖苏龙
姚天卓
郑永祥
俞飞
司龙龙
张礼和
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北京大学
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Priority to EP14857377.7A priority Critical patent/EP3070095B1/en
Priority to US15/033,723 priority patent/US10087217B2/en
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Definitions

  • the present invention relates to site-directed mutagenesis and site-directed modification of adeno-associated viruses.
  • the present invention relates to adeno-associated viruses utilizing non-natural amino acid site-directed mutagenesis and site-directed modification, and adeno-associated virus capsid protein VP1 or a fragment thereof.
  • the present invention also relates to a method and use for the preparation of the site-directed mutagenesis and site-directed modified adeno-associated virus.
  • Adeno-associated viruses belong to the family of microviridae [1] , a family member of the genus Viral genus, have no envelope, and belong to a single-stranded DNA virus. It has not been reported so far that AAV is associated with any known disease in humans and is therefore a promising gene transfer vector [2] .
  • AAV must rely on co-transfection with a helper virus such as adenovirus, herpes virus or papilloma virus to complete the life cycle [3] . Because AAV can infect dividing cells and non-dividing cells, it can establish long-term expression in vivo and does not cause known pathological infections, so it has long been considered as a promising human gene therapy vector [4-7] .
  • Type II AAV (AAV2) nanoparticles are the earliest cloned adeno-associated viruses, which are used for cystic fibrosis [8] , retinal degenerative disorders [9-11] and hemophilia B [12, 13 ] clinical gene transfer therapy, and achieved good results.
  • unnatural amino acids can be included (including Affinity tags and photoisomerized amino acids, carbonyl amino acids and glycosylated amino acids) are introduced into proteins (L. Wang et al., (2001), SCIENCE 292: 498-500; JW Chin et al., 2002, Journal of the American Chemical Society 124: 9026-9027; JW Chin, & P. G. Schultz, 2002, ChemBioChem 11: 1135-1137).
  • chemical functional groups for example, specific chemical groups such as carbonyl, alkynyl, and azide groups, which are generally effective and selective.
  • the formation of stable covalent bonds is more conducive to site-specific modification of proteins and improves the properties of proteins.
  • VP1 protein expression vector In one embodiment of the present invention, according to the structural analysis of the adeno-associated virus capsid protein VP1, 14 mutation sites were selected, and the codons of the amino acids corresponding to the 14 sites were mutated to TAG, and 14 was constructed. VP1 protein expression vector.
  • the 14 vectors are co-transfected with a vector encoding an orthogonal amber mutant repressor aminoacyl-tRNA synthetase/tRNA CUA gene, and non-naturally added to the cell culture medium.
  • the amino acid NAEK experimentally demonstrated that the non-natural amino acid NAEK can be inserted at the sites R447, G453, S578, N587, N587+1 and S662, respectively, but the non-natural amino acid NAEK cannot be inserted at other sites.
  • the principle of the mutation system is that the mutant tRNA Pyl and PylRS satisfy the following relationship: (1): tRNA Pyl cannot utilize the lysyl tRNA of the host cell and can only be acylated by the mutant PylRS; (2): mutation The type of PylRS can only acylate tRNAPyl and cannot acylate other tRNAs. Therefore, the relationship between the mutant tRNAPyl and PylRS is orthogonal. This orthogonality enzyme is the only one that can acylate an unnatural amino acid to such an orthogonal tRNA and can only acylate such a tRNA without acylating other tRNAs.
  • the obtained orthogonal lysyl tRNA synthase/tRNA system allows NAEK or DiZPK of 20 common amino acids to correspond to the amber codon TAG, thereby introducing the non-natural amino acid into the adenovirus capsid protein.
  • an adeno-associated virus having a non-natural amino acid inserted at a specific site is obtained.
  • the site-directed murine adeno-associated virus is comparable to the wild-type virus in terms of virus production and ability to transduce cells.
  • the site-directed murine adeno-associated virus is co-incubated with a fluorescently labeled molecule, and the non-natural amino acid is coupled to the fluorescently labeled molecule by a click reaction.
  • the trajectory of a single virus can be observed under a confocal microscope by a fluorescently labeled molecule coupled to a site-directed murine adeno-associated virus.
  • targeting of a site-directed murine adeno-associated virus to a targeting molecule by a click reaction can increase the targeting of a site-directed murine adeno-associated virus to a cell.
  • the site-directed mutant adeno-associated virus may further express a functional protein or nucleic acid to exert a functional protein or nucleic acid activity in the infected cell.
  • the present invention relates to the following aspects:
  • a first aspect of the invention relates to a site-directed mutagenized adeno-associated virus capsid protein VP1 or a fragment thereof, which is mutated to an unnatural amino acid at a specific position of a wild-type adeno-associated virus capsid protein VP1 or a fragment thereof, said specific The site is selected from at least one of positions R447, G453, S578, N587, N587+1, S662 of VP1 or a fragment thereof.
  • amino acid sequence of VP1 is represented by SEQ ID NO: 1
  • nucleotide sequence of VP1 is represented by SEQ ID NO: 2.
  • the adeno-associated virus capsid protein VP1 or a fragment thereof according to any one of the first aspects of the present invention, wherein the non-natural amino acid is, for example, an azide non-natural amino acid, a photocrosslinked non-natural amino acid, a keto-based unnatural amino acid, an alkyne Non-natural amino acids, acetyl unnatural amino acids, phosphate-based non-natural amino acids, methyl unnatural amino acids.
  • the non-natural amino acid is, for example, an azide non-natural amino acid, a photocrosslinked non-natural amino acid, a keto-based unnatural amino acid, an alkyne Non-natural amino acids, acetyl unnatural amino acids, phosphate-based non-natural amino acids, methyl unnatural amino acids.
  • the unnatural amino acid is an azide group-containing unnatural amino acid, such as N ⁇ -2-azidoethoxycarbonyl-L-lysine (NAEK),
  • the non-natural amino acid is an unnatural amino acid similar in structure to the azide-containing non-natural amino acid described above, such as DiZPK.
  • an amino acid at a specific site of the wild-type adeno-associated virus capsid protein VP1 or a fragment thereof is mutated to NAEK, the specific site being selected from the group R447 of VP1 or a fragment thereof, At least one of G453, S578, N587, N587+1, S662 bits Site.
  • an amino acid at a specific site of the wild-type adeno-associated virus capsid protein VP1 or a fragment thereof is mutated to DiZPK, and the specific site is position 587 of VP1 or a fragment thereof.
  • adeno-associated virus capsid protein VP1 or a fragment thereof according to any one of the first aspects of the present invention, wherein the NAEK is linked to the amino acid sequence of VP1 or a fragment thereof according to Formula I, wherein:
  • R1 to R2 The direction from R1 to R2 is the N-terminus to the C-terminal direction of the amino acid sequence, wherein the amino acid at the N-th position is selected from one of the R447, G453, S578, N587, N587+1, and S662 amino acids.
  • R1 is the amino acid residues 1 to N-1 of the amino acid sequence of VP1 or a fragment thereof
  • R2 is the amino acid residue from the N+1th to the C-terminus of the amino acid sequence of VP1 or a fragment thereof.
  • adeno-associated virus capsid protein VP1 or a fragment thereof according to any one of the first aspects of the present invention, wherein the DiZPK is linked to the amino acid sequence of VP1 or a fragment thereof as shown in Formula II:
  • R1 to R2 The direction from R1 to R2 is from the N-terminus to the C-terminus of the amino acid sequence, wherein the amino acid at the N-th position is the amino acid at position N587, and R1 is the amino acid residues 1 to N-1 of the amino acid sequence of the VP1 protein or a fragment thereof. , R2 is the amino acid residue from the N+1th to the C-terminus of the amino acid sequence of the VP1 protein or a fragment thereof,
  • adeno-associated virus capsid protein VP1 or a fragment thereof according to any one of the first aspects of the invention, wherein the adeno-associated virus is adeno-associated virus type II (AAV2).
  • AAV2 adeno-associated virus type II
  • adeno-associated virus capsid protein VP1 or a fragment thereof according to any one of the first aspects of the present invention, wherein the non-natural amino acid is further linked to a labeling group, such as a fluorescent labeling group, or, for example, to an azide group.
  • a labeling group such as a fluorescent labeling group, or, for example, to an azide group.
  • the labeling group is an Alexa fluorophore, such as Alexa 488 or Alexa 555.
  • the labeling group is DIBO-Alexa 488 or DIBO-Alexa 555.
  • a labeled molecule containing a DIBO group is attached to a non-natural amino acid containing an azide group by a click chemical reaction, particularly a copper-free click chemistry.
  • adeno-associated virus capsid protein VP1 or a fragment thereof according to any one of the first aspects of the present invention, wherein the non-natural amino acid is further linked to other functional molecules, such as a targeting molecule, preferably, the targeting molecule is further A group capable of undergoing a click chemical reaction with an azide group, such as DIBO (dibenzocyclooctyne), cyclooctyne, alkynyl group, is attached.
  • DIBO dibenzocyclooctyne
  • a second aspect of the invention relates to a site-directed mutagenized adeno-associated virus capsid protein comprising the adeno-associated virus capsid protein VP1 of any one of the first aspects of the invention or a fragment thereof.
  • a third aspect of the invention relates to a site-directed mutagenized adeno-associated virus comprising the adeno-associated virus capsid protein VP1 of any one of the first aspect of the invention, or a fragment thereof, or the adeno-associated virus capsid protein of any of the second aspect .
  • an adeno-associated virus according to any one of the third aspects of the present invention, wherein the non-natural amino acid is further linked to a labeling group, such as a fluorescent labeling group, or, for example, a label capable of undergoing a click chemical reaction with an azide group. Group.
  • a labeling group such as a fluorescent labeling group, or, for example, a label capable of undergoing a click chemical reaction with an azide group. Group.
  • the labeling group is an Alexa fluorophore, such as Alexa 488 or Alexa 555.
  • the labeling group is DIBO-Alexa 488 or DIBO-Alexa 555.
  • a labeled molecule containing a DIBO group is attached to a non-natural amino acid containing an azide group by a click chemical reaction, particularly a copper-free click chemistry.
  • an adeno-associated virus according to any one of the third aspects of the present invention, wherein the non-natural amino acid is further linked to other functional molecules, such as a targeting molecule, preferably, the targeting molecule is further linked to an azide group.
  • a targeting molecule preferably, the targeting molecule is further linked to an azide group.
  • a group in which a click chemical reaction occurs such as DIBO (dibenzocyclooctyne), cyclooctyne, alkynyl.
  • the targeting molecule includes any molecule known in the art that can serve as a target, and refers to a substance capable of targeting cells, tissues or organs, for example, capable of being associated with one or a specific type.
  • Protein or nucleic acid molecules eg, epidermal growth factor receptor, epidermal growth factor receptor tyrosine kinase, vascular endothelial growth factor
  • the targeting molecule can additionally facilitate the entry of a moiety associated therewith into a targeted cell, tissue or organ.
  • the targeting molecule refers to a molecule capable of targeting a tumor cell surface protein, such as a molecule capable of binding to integrin on the surface of a tumor cell, such as RGD, particularly a circular RGD.
  • An adeno-associated virus according to any one of the third aspects of the invention, which carries a nucleic acid fragment of a functional nucleic acid fragment or a labeled molecule.
  • the functional nucleic acid fragment can exert an active action in the form of a functional protein or nucleic acid into a cell, tissue or organ, which is known to the cell, tissue or organ as is known in the art.
  • the active protein is, for example, a cytotoxin, a tumor necrosis factor, a pro-apoptotic protein, a growth hormone, an interferon, a neurotrophic factor or the like
  • the functional nucleic acid is an active nucleic acid molecule known in the art, such as an RNA molecule.
  • RNA molecule such as small interfering RNA, microRNA, etc.
  • the labeling molecule is a molecule having a labeling function known in the art, such as a fluorescent molecule, a polypeptide, an antibody, an enzyme, a polysaccharide, a functional small molecule compound, and the like.
  • the functional nucleic acid fragment refers to a nucleic acid fragment encoding a thymidine kinase or a tumor necrosis factor-related apoptosis ligand.
  • the nucleic acid fragment of the marker molecule refers to a nucleic acid fragment encoding GFP.
  • a fourth aspect of the invention relates to a nucleic acid molecule encoding the adeno-associated virus capsid protein VP1 or a fragment thereof according to any one of the first aspects of the invention, wherein the nucleic acid molecule differs from the nucleic acid molecule encoding a wild-type adeno-associated virus capsid protein in Wherein the codon encoding the amino acid of the particular site of the unnatural amino acid is TAG.
  • the codon encoding one of the amino acids at positions R447, G453, S578, N587, N587+1, S662 of the wild type adeno-associated virus capsid protein VP1 or a fragment thereof is mutated For TAG.
  • the codon encoding the amino acid number N587 of the wild type adeno-associated virus capsid protein VP1 or a fragment thereof is mutated to TAG.
  • a fifth aspect of the invention relates to a nucleic acid vector operably linked to the nucleic acid molecule of any of the fourth aspects of the invention.
  • a nucleic acid vector according to any one of the fifth aspects of the invention which is a eukaryotic cell expression vector or an adeno-associated virus vector.
  • the vector pCMV-VP1-Flag is operably linked to the nucleic acid molecule of any of the fourth aspects of the invention.
  • the invention also relates to a host cell comprising the nucleic acid vector of any of the fifth aspects of the invention.
  • the host cell according to any of the present invention, further comprising a vector encoding an orthogonal amber mutant suppressor aminoacyl-tRNA synthetase/tRNA CUA gene.
  • the vector encoding the orthogonal amber mutant suppressor aminoacyl-tRNA synthetase/tRNA CUA gene is plasmid pACYC-tRNA/PylRS, which is deposited from June 14, 2011, Obtained in Escherichia coli pACYC-tRNA/PylRS with accession number CGMCC No:4951.
  • the host cell according to any of the invention, further comprising a pHelper vector and a pAAV-GFP vector.
  • the host cell is a mammalian cell, such as a human, monkey, mouse, cow, horse, sheep, and the like.
  • the host cell is an AAV-293 cell.
  • the host cell is a HeLa cell.
  • the host cell is a U87 cell.
  • the invention also relates to a method of preparing a site-directed murine adeno-associated virus capsid protein VP1, VP2 or VP3 comprising the steps of:
  • the specific amino acid position is selected from one of the positions R447, G453, S578, N587, N587+1, S662 of VP1 or a fragment thereof.
  • the specific amino acid position is position 587 of VP1 or a fragment thereof.
  • the suitable vector is a eukaryotic expression vector, such as the vector pCMV-VP1-flag.
  • the mutant sequence expression vector is pCMV-VP1-flag-R447, pCMV-VP1-flag-G453, pCMV-VP1-flag-S578, pCMV-VP1-flag-N587, pCMV-VP1 -flag-N587+1 or pCMV-VP1-flag-S662.
  • the vector encoding the orthogonal amber mutant suppressor aminoacyl-tRNA synthetase/tRNA CUA gene is plasmid pACYC-tRNA/PylRS, which is deposited from June 14, 2011, Obtained in Escherichia coli pACYC-tRNA/PylRS with accession number CGMCC No:4951.
  • the vector simultaneously co-transfected in step (4) further comprises the vectors pHelper and pAAV-GFP.
  • the suitable host cell in step (4) is an AAV-293 packaging cell.
  • the unnatural amino acid is an azide group-containing unnatural amino acid, such as N ⁇ -2-azidoethoxycarbonyl-L-lysine (NAEK), or the non-natural
  • the amino acid is an unnatural amino acid similar in structure to the above-described azide-containing non-natural amino acid, such as DiZPK.
  • host cells after successful transfection are cultured for 48 hours in a medium containing 1 mM of unnatural amino acid.
  • the invention also relates to a method of preparing a site-directed mutant adeno-associated virus comprising the steps of:
  • the plasmid pAAV-RC for viral packaging which contains the coding gene for the capsid protein VP1, VP2 or VP3
  • a plurality of specific amino acid positions preferably, the specific amino acid position is selected from at least one of positions R447, G453, S578, N587, N587+1, S662 of VP1 or a fragment thereof;
  • a codon encoding an amino acid corresponding to VP1 or a fragment thereof at the selected site in the step (1) is mutated to a codon TAG by a genetic engineering method to obtain a fixed point.
  • the specific amino acid position is selected from one of the positions R447, G453, S578, N587, N587+1, S662 of VP1 or a fragment thereof.
  • the specific amino acid position is position 587 of VP1 or a fragment thereof.
  • the mutant sequence expression vector is pAAV-RC-R447, pAAV-RC-G453, pAAV-RC-S578, pAAV-RC-N587, pAAV-RC-N587+1 or pAAV-RC -S662.
  • the vector encoding the orthogonal amber mutant suppressor aminoacyl-tRNA synthetase/tRNA CUA gene is plasmid pACYC-tRNA/PylRS, which is deposited from June 14, 2011, Obtained in Escherichia coli pACYC-tRNA/PylRS with accession number CGMCC No:4951.
  • the vector simultaneously co-transfected in step (4) further comprises the vectors pHelper and pAAV-GFP.
  • the suitable host cell in step (4) is an AAV-293 packaging cell.
  • the unnatural amino acid is NAEK or DiZPK.
  • host cells after successful transfection are cultured for 72 hours in a medium containing 1 mM of unnatural amino acid.
  • the invention also relates to a composition (e.g., a pharmaceutical composition) or kit comprising the invention
  • a composition e.g., a pharmaceutical composition
  • kit comprising the invention
  • the invention also relates to a genetic vaccine comprising the adeno-associated virus of any of the third aspects of the invention, or the nucleic acid molecule of any of the fourth aspect, or the nucleic acid vector of any of the fifth aspect.
  • the invention also relates to the use of the adeno-associated virus of any one of the third aspects of the invention, or the nucleic acid molecule of any of the fourth aspect, or the nucleic acid vector of any of the fifth aspect, for the preparation of a preparation for obtaining an adeno-associated virus-binding protein , or in the preparation of a gene therapy drug, or for the preparation of a DNA vaccine.
  • the invention further relates to the use of an adeno-associated virus according to any of the third aspects of the invention as a tool adeno-associated virus.
  • the adeno-associated virus can be used as a tool viral vector, carrying a corresponding functional gene according to specific needs or coupling a functional molecule on its surface for use in basic research, gene therapy or DNA vaccine preparation.
  • the invention also relates to a method of gene therapy comprising administering to a subject in need thereof an effective amount of the adeno-associated virus of any of the third aspects of the invention or the nucleic acid molecule of any of the fourth aspects or the fifth aspect A nucleic acid vector of any of the following.
  • a targeting molecule is coupled to the surface of the site-directed mutagenesis and the modified adeno-associated virus, and the adeno-associated virus further expresses a functional protein, and the targeting molecule is used to target the adeno-associated virus to a specific cell.
  • the functional protein acts on the specific cell for the purpose of gene therapy.
  • a targeted molecular circular RGD is coupled to the surface of the site-directed mutagenesis and the modified adeno-associated virus, and the adeno-associated virus also expresses a functional protein tumor necrosis factor-related apoptosis ligand or thymidine.
  • the kinase uses a targeting molecule to target the adeno-associated virus to a tumor cell that expresses integrin highly, and the functional protein acts on the tumor cell to achieve the purpose of gene therapy.
  • the adeno-associated virus may be an adeno-associated virus of various serotypes, and may be, for example, AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9.
  • the adeno-associated virus is AAV2.
  • a plasmid containing the corresponding capsid protein coding sequence can be selected based on the viral capsid protein.
  • the adeno-associated virus has the same meaning as the adeno-associated virus vector.
  • the position of the mutated amino acid on the VP1 is based on VP1 of the AAV2-type adeno-associated virus standard strain (the amino acid sequence of which is represented by SEQ ID NO: 1); if other types of adenovirus are used,
  • One skilled in the art can derive sites corresponding to the mutation sites R447, G453, S578, N587, N587+1, S662 of the present invention based on the amino acid sequences of other types of adenovirus VP1.
  • the VP1 fragment refers to a protein formed by a partial sequence of VP1, particularly a protein formed by a C-terminal sequence thereof, for example, a capsid protein VP2 or a capsid protein VP3; wherein VP2 is a C-terminal 598 of VP1.
  • a protein formed by an amino acid, VP3 is a protein formed by the C-terminal 533 amino acids of VP1.
  • the manner in which the amino acid site on the VP1 fragment is described is determined with reference to VP1; for example, the amino acid R447 of VP2 described in the present invention corresponds to the position R310 of the actual amino acid position in the VP2 protein.
  • the expression "amino acid at position R447 of VP1 or a fragment thereof” means the amino acid residue at position R447 of the polypeptide of SEQ ID NO: 1.
  • mutations or mutations including but not limited to, substitutions, deletions and/or additions, such as different serotypes or chimeric serotypes
  • the adeno-associated virus VP1 or a fragment thereof or a mutant thereof thereof does not affect its biological function.
  • the term "VP1 or a fragment thereof” shall include all such sequences, including, for example, the sequence set forth in SEQ ID NO: 1 and natural or artificial variants thereof. Also, when describing a sequence fragment of VP1, it includes not only the sequence fragment of SEQ ID NO: 1, but also the corresponding sequence fragment in its natural or artificial variant.
  • amino acid at position R447 of VP1 refers to the amino acid residue at position R447 of SEQ ID NO: 1, and the corresponding amino acid residue in a variant thereof (natural or artificial).
  • the expression "corresponding site” means a site located at an equivalent position in the sequence to be compared when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage identity.
  • BLAST and BLAST 2.0 algorithms for determining sequence identity and percent sequence similarity are, for example, BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25: 3389-3402 and Altschul et al. 1990) J. Mol. Biol. 215: 403-410.
  • BLAST and BLAST 2.0 can be used to determine this, for example, as described in the literature or by default parameters.
  • Software for performing BLAST analyses is available to the public through the National Center for Biotechnology Information.
  • the amino acid sequence having at least 90% sequence identity to the amino acid sequence comprises a polypeptide sequence substantially identical to the amino acid sequence, for example when using the methods described herein (eg, BLAST analysis using standard parameters) Having the sequence identity of at least 90% sequence identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher compared to the polypeptide sequence of the invention Those sequences of sex.
  • the AAV2-type adeno-associated virus refers to an AAV2-type adeno-associated virus standard strain, and the amino acid sequence of VP1 is as shown in SEQ ID NO: 1.
  • the adeno-associated virus capsid protein is encoded by an adeno-associated virus cap gene, encoding three structural proteins VP1, VP2, and VP3, respectively, having molecular weights of 87, 73, and 61 kDa, respectively.
  • the non-natural amino acid may be any unnatural amino acid known in the art, for example, may be selected from the group consisting of an azide non-natural amino acid, a photocrosslinked non-natural amino acid, a keto-based unnatural amino acid, an alkynyl unnatural amino acid, Acetyl unnatural amino acids, phosphate-based unnatural amino acids, methyl unnatural amino acids, the meanings of which can be found in references [21], [22].
  • the gene therapy refers to the insertion of a foreign gene into an appropriate target cell by a gene transfer technique, so that the product produced by the foreign gene can treat a certain disease.
  • gene therapy can also include measures and techniques for treating certain diseases taken at the DNA level.
  • the DNA vaccine also referred to as a nucleic acid vaccine or a genetic vaccine, refers to a eukaryotic expression plasmid DNA (sometimes also an RNA) or a viral expression vector (eg, an adeno-associated virus) encoding an immunogen or a protein associated with an immunogen.
  • the vector enters the animal through a certain route, is taken up by the host cell, and then transcribed and translated to express an immunogenic protein or a protein associated with the immunogen, which can stimulate the body to produce a non-specific and/or specific immune response, thereby To immune protection.
  • the N587+1 refers to the insertion of a non-natural amino acid between N587 and R588; when constructing the expression plasmid, a TAG is inserted between the codons of N587 and R588; N450+1, N385+1 The meaning is like this.
  • the present invention obtains an adeno-associated virus which has undergone site-directed mutagenesis and modification of a non-natural amino acid, which is comparable to a wild-type adeno-associated virus in production, transfection and mobile transport ability, and has been verified to further pass unnatural amino acids and other functional molecules.
  • targeting molecule coupling to improve the transduction efficiency of targeted cells while also carrying functional genes normally, indicating that the site-directed murine adeno-associated virus can be used as a tool for adeno-associated virus, in the search for adeno-associated virus-binding proteins or as Various aspects related to adeno-associated viruses, such as targeted gene therapy vectors, are applied.
  • Figure 1 shows the adeno-associated virus modification process based on the click chemical reaction
  • A indicates that the AAV2 particle can be site-modified by the genetically encoded azide-containing amino acid NAEK, and B indicates that the fluorescent molecule can be labeled by bioorthogonal reaction.
  • Figure 2 shows that NAEK can be efficiently genetically inserted into the AAV capsid protein VP1.
  • A, NAEK is inserted into the VP1 site selection.
  • Figure 3 shows azimuth tag pointing markers that AAV2 particles can be genetically encoded
  • A is a model diagram of AAV2 major capsid protein and viral capsid obtained according to the atomic structure of Xie et al. (from the protein database), in which the inserted Arg447 site (red) is indicated, and B is transfected with HT-1080 cells.
  • B is transfected with HT-1080 cells.
  • C is transfected with serial dilution of rAAVr HT-1080 cells were then counted for virus titers, the error bars represent the standard deviation of the three experiments, and the ordinate is the functional titer (x10 7 virions/ml).
  • Figure 4 shows the successful insertion of NAEK at different sites of the AAV2 capsid
  • the mutant virus dependent on NAEK was co-cultured with HT-1080 cells for 48 hours. If the mutant virus was successfully transferred into HT-1080 cells, the reporter gene GFP was expressed, indicating the viral infectivity of R447-AAV2 and S568-AAV2 with wild-type virus. Basically equivalent.
  • Figure 5 shows that NAEK cannot be inserted into the AAV2 capsid at certain sites.
  • Figure 6 shows that AAV2-GFP virions can be genetically encoded by NAEK tags.
  • a pattern of A, B, AAV2 major capsid protein and AAV2 virus particles can be successfully mutated by the amino acid azide compound (NAEK) at positions R447, G453, S578, N587, S662.
  • NAEK amino acid azide compound
  • AAV2-GFP cannot be mutated at positions S261, N381, Y444, S458, S492, Y500, F534, T573.
  • HT-1080 cells were transduced with serially diluted AAV2-GFP and virus titers were calculated. Error bars represent the standard deviation of three replicate experiments.
  • Figure 7 shows that Alexa 488 was successfully modified on the surface of the virus
  • A is fluorescently labeled NAEK; wild type virus (WT AAV2), NAEK-labeled virus (R447-AAV2) particles and S578-AAV2 virions were co-cultured with Hela cells with or without DIBO-Alexa488 (green) for 30 min at 37 °C. , then fixed, permeabilized, and immunostained (red) with a murine monoclonal antibody (A20 clone) against intact AAV2.
  • the labeled azide label (green) is co-localized with the R447-AAV2 particle (red).
  • the overlapping green and red signals appear as yellow in the fused image.
  • the first row of AAV2 was not co-cultured with the A20 antibody as a negative control.
  • the scale is equivalent to 10 ⁇ m.
  • B, C is AAV which was reacted with or reacted with DIBO-Alexa 488 by SDS-PAGE, wherein Alexa 488 (C) was detected by 488 nm transmitted light, and then the gel was stained with Coomassie brilliant blue (B).
  • Figure 8 shows the results of confocal microscopy of R447 and S578 in the capsid protein, which Alexa 555 also successfully linked to AAV2 by NAEK.
  • Figure 9 shows a quantitative analysis of intracellular Alexa 488-labeled AAV2 virus movement.
  • A, B, and C represent typical trajectories of Alexa 488-labeled AAV2 in Hela cells; Hela cells were co-cultured with Alexa 488-labeled AAV2 at 4 °C for 30 min, and then confocal real-time imaging was recorded.
  • A is a fluorescence map
  • B is a white light map
  • C is a photo after AB superposition
  • D is a typical motion selected therein, enlarged display.
  • D is a typical trajectory of Alexa 488-labeled AAV2, where 1 is fast and directional, 2 is fast and non-directional, 3 is slow and non-directional, and E, F, G are time trajectories of viral velocity.
  • the track for H is Alexa 488-AAV2 is classified as "slow undirected”, “fast undirected”, and “fast directed”, where slow undirected means slow and undirected, fast undirected means fast and undirected, fast directed Fast and directional.
  • Error bars represent the standard deviation of the three experiments (a total of 195 trajectories). The scale is equivalent to 10 ⁇ m.
  • Figure 10 shows the real-time monitoring of the endocytosis of Alexa488-AAV2 through the clathrin envelope.
  • FIG. 11 shows cRGD-modified AAV2-GFP vector-mediated gene transduction
  • E, F, vectors labeled with cRGD have higher gene transduction capacity for Hela and U87 cells, respectively.
  • the same number of vectors without RAD/RGD markers, RAD/RGD-labeled vectors were incubated for 2 h at 4 °C. Unbound virus was removed, then fresh medium was added and expression of GFP was analyzed by FACS 48 h later. Data represent the percentage of cells expressing the eGFP transgene and the data are expressed as mean and standard deviation of three replicate experiments.
  • Figure 12 shows that RAD/RGD was successfully labeled on the surface of AAV2-GFP capsid protein by a "click" chemical reaction between NAEK and DIBO.
  • A.AAV2 N587+1/azido (AAV2 labeled NAEK between N587 and R588) at a dose of 300 genome copies per cell with RAD, RGD, DIBO, RAD-DIBO, RGD-DIBO or a combination thereof at 4 Incubate for 2 h at °C. Excess unreacted molecules were removed using a 100 kD Millipore Amicon Ultra-100. The diafiltered virus was incubated with U87 cells for 2 h at 4 °C. Unbound virus was removed, then fresh medium was added and expression of GFP was analyzed by FACS 48 h later.
  • AAV2 N587+1/azido + RGD-mediated competition for integrin-targeted transduction U87 cells were incubated with AAV2 N587+1/azido + RGD (RGD modified AAV2) or AAV2 N587+1/azido (RGD unmodified AAV2) for 2 h at 4 ° C at a dose of 700 genomic copies per cell. Unbound virus was removed, then fresh medium was added and expression of GFP was analyzed by FACS 48 h later. For competition experiments, the virus was tested for binding to cells at 400 ⁇ g/ml RAD or RGD peptide, control or LM609 antibody (1:100 dilution), or the combination of RGD peptide and LM609 antibody.
  • Figure 13 shows the transduction efficiency of different viruses against Hela and U87 tumor cells.
  • AAV2 wild type AAV2
  • AAV2 N587+1/azido insert NAEK tag at positions 587 and 588 of AAV2 capsid protein
  • AAV2 N587+RAD insert at positions 587 and 588 of AAV2 capsid protein surface
  • RAD peptide insert at positions 587 and 588 of AAV2 capsid protein surface
  • AAV2 N587+RGD insert RGD peptide at positions 587 and 588 of AAV2 capsid protein
  • AAV2 N587+1/azido + RAD AAV2 is chemically linked to cRAD by NAEK and DIBO
  • AAV2 N587+ 1/azido +RGD AAV2 chemically linked to cRGD via NAEK and DIBO
  • Figure 14 shows the binding of carrier particles to Hela and U87 cells.
  • Figure 15 shows the killing effect of differently modified AAV2-TK (thymidine kinase) on Hela and U87 cells.
  • AAV2-GFP wild-type AAV2 contains GFP reporter gene
  • AAV2-TK wild-type AAV2 contains thymidine kinase gene
  • AAV2 N587+1/azido- TK AAV2-TK integrates NAEK at site N587+1)
  • AAV2 N587+1/azido +RAD-TK AAV2-TK is chemically coupled with cRAD by NAEK and DIBO
  • AAV2 N587+1/azido +RGD-TK AAV2-TK is chemically coupled with NARG and DIBO by cRGD)
  • AAV2 N587+RAD- TK AAV2-TK fused with RAD peptide between its capsid protein surface sites 587 and 588
  • AAV2 N587+RGD- TK AAV2-TK at its capsid protein surface site 587)
  • AAV2-GFP, AAV2-TK, and AAV2 N587+1/azido- TK, AAV2 N587+1/azido +RAD-TK, and AAV2 N587+1/azido +RGD-TK, AAV2 N587+RAD -TK , AAV2 N587 +RGD- TK was incubated with U87 cells at a dose of 500 genomic copies/cell. These cells were incubated with different doses of GCV for 48 h and cell viability was quantified using cell Titer-Glo (promega). MEM medium was used as a negative control.
  • Figure 16 shows the killing effect of differently modified AAV2-TRAIL on Hela and U87 cells.
  • AAV2 vectors containing the TRAIL (TNF-related apoptosis-inducing ligand) gene were incubated with HeLa (A) and U87 cells (B) at a dose of 500 genomic copies per cell. After 48 h, cell viability was quantified using cell Titer-Glo (promega). MEM medium was used as a negative control.
  • TRAIL TNF-related apoptosis-inducing ligand
  • Figure 17 shows the imaging results of insertion of DiZPK at different sites of the AAV2 capsid.
  • the medium for AAV-293, HT-1080 and Hela cells is DMEM medium containing 10% fetal calf serum (PAA, Austria) and 2 mM L-glutamine (Zhongkemaichen Beijing Technology Co., Ltd.) Morning Beijing Technology Co., Ltd.), cultured under 5% CO 2 conditions.
  • Murine monoclonal antibody (A20 clone) against intact AAV2 was obtained from ARP (American Research Products, Belmont, MA).
  • DIBO-Alexa 488, DIBO-Alexa 555 dye was purchased from Invitrogen.
  • N ⁇ -2-azidoethyloxycarbonyl-L-lysine N ⁇ -2-azidoethoxycarbonyl-L-lysine, Synthesis of NAEK
  • the above product 2 was dissolved in pyridine, and 11 g of TsCl was added with stirring at 0 ° C overnight. After the reaction was completed, the reaction mixture was poured into a mixture of concentrated hydrochloric acid and ice water, and extracted with diethyl ether. The ether layer was washed with 1N hydrochloric acid and 1N NaOH. The organic phase dried column gave 11.8 g of a colorless viscous liquid product.
  • AAV-293 cells represent human embryonic kidney cells stably transfected with adenovirus type 5 DNA, and have the adenoviral e1 gene required for in vitro preparation of rAAV.
  • AAV-293 cells were cultured in DMEM medium, and 10% fetal calf serum, 4 mM L-glutamine and 4.5 g/L glucose were added under the culture conditions of 37 ° C, 5% CO 2 . The cells were cultured to confluence 60-70% and then the triple co-transfection step of the AAV plasmid was initiated.
  • the expression plasmid vectors pAAV-RC, pHelper and pAAV-GFP were used in this experiment.
  • the construct contains AAV and is required for the preparation of infectious AAV particles.
  • the adenovirus gene is required.
  • pAAV-RC provides rep and cap genes encoding AAV replication and capsid proteins, respectively.
  • the pHelper vector contains the adenovirus E2A, E4 and VA genes, and the pAAV-GFP contains the GFP reporter gene.
  • the reporter vector represents a plasmid containing an ITR with a cmv promoter.
  • the pAAV-RC-R447 plasmid was prepared using the Quik Change Lightning Site-Directed Mutagenesis Kit (Agilent), in which the genetic code of the arginine residue at position 447 of the AAV capsid protein was mutated to TAG, and the other mutant plasmids were constructed in the same manner.
  • the pACYC-tRNA/PylRS vector containing the orthogonal amber mutant suppressor aminoacyl-tRNA synthetase/tRNA CUA gene was kindly provided by Professor Chen Peng from Peking University School of Chemistry (Duy P. Nguyen, Hrvoje Lusic, Heinz Neumann, Prashant B. Kapadnis, Alexander Deiters, and Jason W. Chin. Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNA CUA Pair and Click Chemistry. J.AM.CHEM.SOC.2009,131,8720 -8721).
  • Azide-labeled AAV2 infected particles (e.g., R447-AAV2 vector) were prepared in AAV Helper-Free System without the use of helper adenovirus or herpes virus.
  • AAV2 plasmid vector pAAV-RC, pHelper, pAAV-GFP and vector pACYC-tRNA/PylRS (molar ratio 1: 1: 1: 2) were transiently co-transfected into AAV-293 packaging cells by calcium phosphate precipitation. Six hours after transfection, the cell culture medium was replaced with fresh medium containing 1 mM NAEK. Infected cells were collected after 72 hours.
  • the infected cells are lysed by freeze-thaw.
  • the separation and purification steps refer to the operation of Ping Guo et al. (Guo P, El-Gohary Y, Prasadan K, Shiota C, Xiao X, Wiersch J, Paredes J, Tulachan S, Gittes GK: Rapid and simplified purification of recombinant adeno-associated virus .J Virol Methods, 183(2): 139-146).
  • AAV-HT1080 cells were cultured in a six-well tissue culture plate at 2 ml DMEM medium per well at a cell density of 3 ⁇ 10 5 /well. Incubate overnight at 37 °C. The cells need to be cultured to cover about 50%. 10 times diluted virus stock solution. Five-fold serial dilutions were made in a 5 ml volume on a 10-fold dilution ranging from 2 x 10 -2 to 8 x 10 -4 . 1 ml of each dilution was added to each well of a six-well plate, and three holes were added to each titer. At the same time, the well without the virus stock solution was used as a negative control. Incubate at 37 ° C for 1-2 hours.
  • the plate was vortexed gently every 30 min during incubation. Then, 1 ml of pre-warmed H-DMEM was added to each well, and cultured at 37 ° C for 40-48 hours. The pAAV-hrGFP AAV-infected cells were detected by FACS.
  • the genomic copy of the vector was quantified using an Agilent Mx3000P real-time PCR instrument (Agilent Technologies, La Jolla, CA, USA), and the specific primer pair for the GFP gene was: 5'-AAGCAGCACGACTTCTTCAAGTC-3' (SEQ ID NO: 31) ( Forward) and 5'-TCGCCCTCGAACTTCACCTC-3' (SEQ ID NO: 32) (reverse). See the published operating guide [20] for details.
  • AAV2-azide For fluorescent labeling of AAV2-azide, purified virions were incubated with Alexa488-DIBO or Alexa 555 (500 ⁇ M) for 2 hours at room temperature under pH 7.0. Unreacted dye was removed using a 100 kD Millipore Amicon Ultra-100.
  • the method of attaching a targeting molecule to the CRGD is like attaching a fluorescent probe.
  • Alexa488-labeled AAV2 was co-cultured with HeLa cells in a glass-bottomed dish at 37 ° C for 30 min, and then the cells were fixed with phosphate buffer (pH 7.0) (PBS) containing 4% paraformaldehyde for 15 min. The cells were then permeabilized for 10 min in PBS containing 0.5% Triton X-100 and blocked with 3% bovine serum albumin (BSA) in PBS for 60 min. The cells were then incubated with a murine monoclonal antibody (A20) against intact AAV2 at 4 °C overnight, followed by incubation with a second antibody (life tecnology) ligated with Alexa594 for 1 hour at room temperature. The nuclei were stained with DAPI (Sigma). Imaging under confocal laser-scanning microscopy (SP8 Series, Leica, Germany).
  • Alexa488-AAV2 was seeded in glass bottom culture dishes and cultured overnight at 37 °C. Alexa488-labeled AAV2 was then co-cultured with HeLa cells for 4 min at 4 °C, and then confocal real-time imaging was recorded using a live cell imaging system (PerkinElmer, MA, USA).
  • a mutation site as shown in Table 1 was selected on the adeno-associated virus capsid protein VP1.
  • the amino acid sequence of the VP1 protein is:
  • the nucleotide sequence of the VP1 protein is:
  • the position described in Table 1 is the position in the VP1 protein.
  • mutant primers shown in Table 2 were designed (which can also be used as sequencing primers).
  • #1 is a forward primer and #2 is a reverse primer.
  • 587+1 indicates insertion of a non-natural amino acid after position 587, ie insertion of an unnatural amino acid between positions 587 and 588.
  • the VP1 gene was cloned into the vector pCMV-FLAG (FLAG tag at the C-terminus) to obtain the VP1 coding vector pCMV-VP1-FLAG, using the Quik Change Lightning Site-Directed Mutagenesis Kit (Agilent), according to its instructions for use, pCMV-VP1 -FLAG plasmid as a template, using the mutation primers listed in Table 2, the vector encoding S261, N381, Y444, R447, G453, S458, S492, Y500, F534, T573, S578, N587, N587+1, S662
  • the codons of the base were mutated to the amber stop codon (TAG), respectively, and the sequencing results showed that each mutation was successfully introduced.
  • the plasmid obtained after the mutation of R447 was named pCMV-VP1-FLAG-R447, which indicated that the genetic code of the arginine residue of VP1447 of AAV capsid protein was mutated to TAG, and the plasmids obtained after other mutations were named and expressed. This type of push.
  • the present invention utilizes the genetic code extension technique to site-specifically label AAV2 by introducing the azide-containing amino acid NAEK into the viral capsid.
  • each mutation was completed using the pAAV-RC plasmid as a template and the mutant primers listed in Table 2, and the sequencing results showed that each mutation was successfully introduced.
  • the plasmid obtained after the mutation of R447 was named pAAV-RC-R447, which indicated that the AAV capsid protein VP1 447 arginine
  • the genetic code of the residue is mutated to TAG, and the plasmid naming and meaning obtained after other mutations are analogous.
  • the present invention utilizes a genetic approach to insert an azide non-natural amino acid at the 447 amino acid position of the cap gene located in the VP1/VP2/VP3 region.
  • the genetic code of the AAV2 capsid protein encoding plasmid pAAV-RC was mutated and mutated to the amber stop codon TAG.
  • the mutant plasmid pAAV-RC was mutated by co-expression of a NAEK-specific orthogonal tRNA/aaRS pair (pACYC-tRNA/PylRS vector), pHelper vector and pAAV-GFP vector in AAV-293 packaging cells.
  • R447-AAV2 AAV2 particles with azide
  • cell lysates were collected, and after 10-fold dilution, they were added to HT-1080 cells, and a fluorescent microscope was used to detect whether or not the mutant virus was produced.
  • the NAEK-containing cell lysate obtained after transfection with the mutant plasmid was transfected into HT-1080 cells. After transfecting HT-1080 cells for 48 hours, we found strong green fluorescence in HT-1080 cells, almost in the wild. The type is comparable, but significantly different from no NAEK (see Figures 3B, 3C).
  • AAV was successfully packaged using the mutated AAV2 capsid due to the presence of NAEK.
  • modified or unlabeled AAV2 carrying the GFP reporter gene was used to infect HT-1080 cells to detect viral titers, while qPCR was used to detect viral genome titers.
  • the functional titer size (transfection unit/mL) of R447-AAV2 and S578-AAV2 is the same as that of WT-AAV2 (see Figure 6E, Figure 6F), indicating the effect of the introduction of azide label on the production and transfection ability of AAV2 particles. Very small.
  • Example 3 we obtained NAEK site-labeled AAV2, and the azide group in the NAEK tag on AAV2 can be coupled to the surface of the virion by a copper-free click chemical reaction with a DIBO-fluorescent molecule. 1).
  • the copper-free click chemical reaction is a click reaction achieved by introducing a cyclooctyne to maintain cell activity, wherein the tension of the eight-membered ring allows reaction with azide in the absence of a catalyst.
  • One of these agents consists of a so-called DIBO compound [18] .
  • Azide-modified macromolecules can now be labeled without a metal catalyst, which not only allows for the study of living cells, but also prevents damage to proteins.
  • NAEK has been site-specifically displayed on mutant viruses, and fluorescent molecules have indeed been linked to mutant viruses via NAEK. Not only R447-AAV2, but also S578-AAV2 was successfully coupled with Alexa 488 (see Figures 7A, 7B, 7C).
  • Another fluorophore Alexa 555 can also be linked to AAV2 via NAEK (see Figure 8), as described above.
  • the above results indicate that the azide tag is capable of covalently linking the fluorescent probe to the AAV2 particle in a site-specific manner.
  • the bioorthogonal reaction of the virus with Alexa488 and 555 has little effect on the infectivity of fluorescently labeled viruses due to mild reaction conditions (2 h incubation at 25 °C) and NAEK-DIBO-Alexa488 or 555 probes.
  • the size is small. According to Chem 3D software, the size of the probe is approximately 2.6868 nm, which is approximately 1% of the size of a single AAV2.
  • the present invention verifies whether such a marker can be used on a single virus tracer.
  • the virus carrying the 447th mutation of Alexa 488 was added to HeLa cells, incubated at 4 ° C for 30 min, the binding was synchronized, and then the virus was monitored under real-time imaging by confocal microscopy.
  • confocal Focusing For the specific experimental method, see "Confocal Focusing" in the Experimental Materials and Methods section. Imaging”.
  • Figure 9 shows Alexa 488-labeled AAV2 in HeLa cells. Representative trajectory (Figs. 9A, 9B, 9C).
  • Figures 9D, 9E, and 9F are time trajectories of virus speed.
  • the maximum velocity of the trajectory is ⁇ 0.002 ⁇ m/s
  • the motion with a single direction in more than 5 consecutive frames is defined as directional transport, fast ( ⁇ 0.002 ⁇ m/s) but non-directional motion definition
  • fast non-directional transport slow ( ⁇ 0.002 ⁇ m/s) and non-directional motion is defined as slow non-directional transport.
  • Alexa 488-labeled virus was co-incubated with Hela cells expressing clathrin fused with red fluorescent protein, using time-lapse spinning. Confocal microscopy) for live cell imaging. Representative trajectories and photographs of Alexa 488-AAV2 are shown in Figure 10A.
  • the viral particles green
  • the viral particles were initially co-localized with the clathrin signal (red). This colocalization lasted for about 10 s and then the clathrin signal disappeared rapidly indicating that the virus dissociated from the uncoated clathrin vesicles (Fig. 10B).
  • Example 6 Enhancement of cell transduction by coupling of AAV2 to a targeting ligand
  • a tumor targeting motif (cyclic RGD) to the AAV2 capsid protein to target the delivery of the gene.
  • Integrins are receptors for RGD peptides that are highly expressed in a variety of tumors. Therefore, RGD may be useful for coating AAV vectors to increase delivery efficiency and to target integrin ⁇ v ⁇ 3 (high expression in tumor cells).
  • RAD is a mutant motif in which the glycine in RGD is mutated to alanine and can be used as a negative control.
  • connection of RAD instead of RGD to the AAV vector had little effect on the transduction efficiency of all of these sites, indicating that RGD plays a key role in the improvement of transduction efficiency. Therefore, ligation of RGD at a suitable position of the AAV vector, but not at any position, is an effective way to increase the transduction efficiency of difficult-to-transform cells.
  • the infection efficiency of coupling RGD at position 587+1 of the AAV vector was only about 20% increased, probably due to low expression of cell surface integrin ⁇ v ⁇ 3 (Fig. 11C). The results indicate that site-selective coupling of RGD in this manner is an effective way to adjust the tropism of the AAV vector and increase the efficiency of gene delivery.
  • the GFP reporter gene in the viral capsid. If the GFP gene is replaced by a therapeutic gene, can the virus exhibit the above characteristics?
  • HSV-tk herpes simplex virus thymidine kinase
  • TRAIL tumor necrosis factor-related apoptosis ligand
  • AAV N587+1/azido +RGD-TK/TRAIL virus was significantly better than WT-AAV and RGD fusion modified AAV N587+RGD- TK/TRAIL virus.
  • the difference in killing effect on HeLa cells with low expression of integrin is not so obvious. This indicates that the chemical free modification of cRGD not only enhances the killing effect of the virus, but also enhances the targeting of the virus (Fig. 15, 16).
  • the photocross-linked unnatural amino acid (DiZPK) is introduced into the surface of the AAV2 capsid, and the virus will cross-link to capture new interacting proteins during the process of entering the cell, thereby discovering the virus. New receptors.
  • the S261, R447, F534, T573, and N587 sites have been tried, and it has been found that DiZPK can be successfully introduced at the N587 site.
  • the R447 site was able to introduce NAEK, but not the DiZPK, and the two unnatural amino acids were able to be inserted at the N587 site. This may be because the spatial structure of each amino acid residue site of the protein molecule is different, and the structural requirements for the unnatural amino acid that can be introduced are also different.

Abstract

提供了一种定点突变和定点修饰的腺相关病毒，其制备方法为，利用遗传密码扩展技术，将非天然氨基酸引入腺相关病毒衣壳蛋白VP1或其片段，进而得到利用非天然氨基酸定点突变的腺相关病毒。该定点突变的腺相关病毒在生产、转导和移动能力等方面与野生型病毒相当，可与其它功能分子例如靶向分子偶联，还可以正常携带功能基因，作为工具腺相关病毒。

Description

定点突变和定点修饰的腺相关病毒、其制备方法及应用 技术领域
本发明涉及定点突变和定点修饰的腺相关病毒，具体地，本发明涉及利用非天然氨基酸定点突变和定点修饰的腺相关病毒以及腺相关病毒衣壳蛋白VP1或其片段。本发明还涉及所述定点突变和定点修饰的腺相关病毒的制备方法和用途。
背景技术
腺相关病毒(AAVs)属微小病毒科[1]、依赖病毒属的家庭成员之一，无包膜，属于单链DNA病毒。至今未报道AAV与任何人类已知疾病相关，因此是很有潜力的基因转移载体[2]。AAV必须依赖于与辅助病毒例如腺病毒、疱疹病毒或乳头瘤病毒共转染才能完成生命周期[3]。由于AAV能感染分裂细胞和非分裂细胞，在体内能够建立长期表达且不会引起已知的病理性感染后果，因此一直以来被认为是有希望的人类基因治疗载体[4-7]。II型AAV(AAV2)纳米粒是最早克隆的腺相关病毒，将这种纳米载体用于囊性纤维化[8]、视网膜退行性紊乱[9-11]和B型血友病[12，13]的临床基因转移治疗，取得了很好的效果。
近年来，许多实验室致力于通过修饰这些粒子的细胞结合特性来制备靶向AAV。最基本的策略是将能够靶向特异细胞类型的多肽基序通过基因修饰的方法插入AAV的衣壳蛋白。这种方法已成功地用于使AAV靶向动脉内皮细胞[14]、横纹肌[15]和脑血管系统[16]。但是，这种操作最重要的技术问题包括产量低、病毒滴度大幅下降或者DNA包装效率明显降低[17]。对病毒衣壳蛋白的大范围遗传修饰可能使病毒的感染性消失，甚至改变病毒和宿主细胞的相互作用。因此，需要开发出新的具有位点特异性且不具有破坏性的技术来修饰腺相关病毒。
经过数年的研究，人们对原核生物核糖体的翻译机制已有较全面的理解，多种核糖体不同功能状态的晶体和电镜结构已得到解析，大多数氨酰tRNA合成酶的结构也已获得。基于这些研究成果，近年来发展起来了遗传密码扩展的技术-利用琥珀终止密码子(TAG)来编码多种非天然氨基酸并在生物活体内将其定点插入。到目前为止，这一技术已经将几十种非天然氨基酸成功地定点表达在活细胞的蛋白质当中，赋予了这些蛋白质新颖的物理、化学和生理性质。使用这一方法，可以将非天然氨基酸(包括 亲和标记和光致异构化的氨基酸、羰基氨基酸和糖基化氨基酸)引入蛋白质中(L.Wang等人，(2001)，SCIENCE 292：498-500；J.W.Chin等，2002，Journal of the American Chemical Society 124：9026-9027；J.W.Chin，&P.G.Schultz，2002，ChemBioChem 11：1135-1137)。这些研究表明，有可能且有选择性且常规地引入化学官能基团到蛋白质中，例如，羰基、炔基、和叠氮基团等特殊化学基团，这些基团一般能够有效且选择性地形成稳定的共价键，更加有利于蛋白质的定点特异修饰，改善蛋白质的性质。
但目前尚没有研究将该技术应用于腺相关病毒的定点修饰中。
发明内容
发明人经过大量实验研究和反复摸索，令人惊奇地发现，利用密码子扩展技术，可以将非天然氨基酸引入腺相关病毒衣壳蛋白VP1或其片段的一些特定位点，进而表达在腺相关病毒上，由此完成了本发明。
在本发明的一个实施方案中，根据对腺相关病毒衣壳蛋白VP1的结构分析，选取了14个突变位点，将这14个位点所对应氨基酸的密码子突变为TAG，并构建了14个VP1蛋白表达载体。
在本发明的一个实施方案中，分别将这14个载体与编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体共转染细胞，并在细胞培养基中加入非天然氨基酸NAEK，实验证明在位点R447，G453，S578，N587，N587+1和S662，可分别插入非天然氨基酸NAEK，但在其它位点不能插入非天然氨基酸NAEK。在本发明的另一个实施方案中，通过实验证明了在位点N587可以插入非天然氨基酸DiZPK。
该突变系统的原理在于：突变型的tRNAPyl，PylRS满足下列关系：(1)：tRNAPyl不能利用宿主细胞的赖氨酰tRNA酶，只能被突变型的PylRS酰化；(2)：突变型的PylRS只能酰化tRNAPyl，不能酰化其它tRNA，因此，突变性tRNAPyl和PylRS之间的关系是正交性的。这种正交性的酶并且是只有这种酶可以把非天然氨基酸酰化到这种正交的tRNA上，并且只能酰化这种tRNA，而不能酰化其它的tRNA。获得的正交赖氨酰tRNA合酶/tRNA系统，使非20种常见氨基酸的NAEK或DiZPK等与琥珀密码子TAG相对应，从而将非天然氨基酸定点引入到腺病毒衣壳蛋白中。
在本发明的一个实施方案中，获得了在特定位点插入非天然氨基酸的腺相关病毒。
在本发明的实施方案中，定点突变的腺相关病毒在病毒生产和对细胞的转导能力方面，与野生型病毒相当。
在本发明的实施方案中，将定点突变的腺相关病毒与荧光标记分子共孵育，通过click反应将非天然氨基酸与荧光标记分子偶联起来。
在本发明的一个实施方案中，通过与定点突变的腺相关病毒偶联的荧光标记分子可以在共聚焦显微镜下观察单个病毒的运动轨迹。
在本发明的一个实施方案中，将定点突变的腺相关病毒与靶向分子通过click反应偶联，可以提高定点突变的腺相关病毒对细胞的靶向性。
在本发明的一个实施方案中，定点突变的腺相关病毒还可以进一步表达功能性蛋白或核酸，以在感染细胞中发挥功能性蛋白或核酸的活性作用。
更为具体地，本发明涉及以下几个方面：
本发明第一方面涉及定点突变的腺相关病毒衣壳蛋白VP1或其片段，其在野生型腺相关病毒衣壳蛋白VP1或其片段的特定位点的氨基酸被突变为非天然氨基酸，所述特定位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的至少一个位点。
在本发明的实施方案中，所述VP1的氨基酸序列为SEQ ID NO：1所示，VP1的核苷酸序列为SEQ ID NO：2所示。
根据本发明第一方面任一项的腺相关病毒衣壳蛋白VP1或其片段，其中所述非天然氨基酸例如可以为叠氮非天然氨基酸、光交联非天然氨基酸、酮基非天然氨基酸、炔基非天然氨基酸、乙酰基非天然氨基酸、磷酸基非天然氨基酸、甲基非天然氨基酸。在本发明的实施方案中，所述非天然氨基酸为含有叠氮基团的非天然氨基酸，例如为Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)、
Figure PCTCN2014089880-appb-000001
或者所述非天然氨基酸为与上述含有叠氮基团的非天然氨基酸结构相似的非天然氨基酸，例如DiZPK。
在本发明的一个实施方案中，其在野生型腺相关病毒衣壳蛋白VP1或其片段的特定位点的一个氨基酸被突变为NAEK，所述特定位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的至少一个 位点。
在本发明的另一个实施方案中，其在野生型腺相关病毒衣壳蛋白VP1或其片段的特定位点的一个氨基酸被突变为DiZPK，所述特定位点为VP1或其片段的第N587位。
根据本发明第一方面任一项的腺相关病毒衣壳蛋白VP1或其片段，其中所述NAEK与VP1或其片段氨基酸序列的连接方式如式I所示，其中：
Figure PCTCN2014089880-appb-000002
由R1到R2的方向为氨基酸序列的N末端到C末端方向，其中第N位的氨基酸选自第R447位、G453位、S578位、N587位、N587+1位、S662位氨基酸中的一个，R1为VP1或其片段氨基酸序列的第1至第N-1位氨基酸残基，R2为VP1或其片段氨基酸序列的第N+1位至C末端的氨基酸残基。
根据本发明第一方面任一项的腺相关病毒衣壳蛋白VP1或其片段，其中所述DiZPK与VP1或其片段氨基酸序列的连接方式如式II所示：
Figure PCTCN2014089880-appb-000003
由R1到R2的方向为氨基酸序列的N末端到C末端方向，其中第N位的氨基酸为第N587位氨基酸，R1为VP1蛋白或其片段氨基酸序列的第1至第N-1位氨基酸残基，R2为VP1蛋白或其片段氨基酸序列的第N+1位至C末端的氨基酸残基，
R3为
Figure PCTCN2014089880-appb-000004
根据本发明第一方面任一项的腺相关病毒衣壳蛋白VP1或其片段，其中所述腺相关病毒为腺相关病毒II型(AAV2)。
根据本发明第一方面任一项的腺相关病毒衣壳蛋白VP1或其片段，其中所述非天然氨基酸上还连接有标记基团，例如为荧光标记基团，或者例如为能与叠氮基团发生click化学反应的标记基团。
在本发明的实施方案中，所述标记基团为Alexa荧光基团，例如为Alexa 488或者Alexa 555。在本发明的具体实施方案中，所述标记基团为DIBO-Alexa 488或DIBO-Alexa 555。
在本发明的实施方案中，通过click化学反应，特别是无铜的click化学反应将含有DIBO基团的标记分子与含有叠氮基团的非天然氨基酸相连接。
根据本发明第一方面任一项的腺相关病毒衣壳蛋白VP1或其片段，其中所述非天然氨基酸上还连接有其它功能分子，例如靶向分子，优选地，所述靶向分子上还连接有能与叠氮基团发生click化学反应的基团，例如DIBO(二苯并环辛炔)、环辛炔、炔基。
本发明第二方面涉及定点突变的腺相关病毒衣壳蛋白，其含有本发明第一方面任一项所述的腺相关病毒衣壳蛋白VP1或其片段。
本发明第三方面涉及定点突变的腺相关病毒，其含有本发明第一方面任一项所述的腺相关病毒衣壳蛋白VP1或其片段或者第二方面任一项的腺相关病毒衣壳蛋白。
根据本发明第三方面任一项的腺相关病毒，其中所述非天然氨基酸上还连接有标记基团，例如为荧光标记基团，或者例如为能与叠氮基团发生click化学反应的标记基团。
在本发明的实施方案中，所述标记基团为Alexa荧光基团，例如为Alexa 488或者Alexa 555。在本发明的具体实施方案中，所述标记基团为DIBO-Alexa 488或DIBO-Alexa 555。
在本发明的实施方案中，通过click化学反应，特别是无铜的click化学反应将含有DIBO基团的标记分子与含有叠氮基团的非天然氨基酸相连接。
根据本发明第三方面任一项的腺相关病毒，其中所述非天然氨基酸上还连接有其它功能分子，例如靶向分子，优选地，所述靶向分子上还连接有能与叠氮基团发生click化学反应的基团，例如DIBO(二苯并环辛炔)、环辛炔、炔基。
在本发明中，所述靶向分子包括本领域公知的可以起到靶向作用的任何分子，是指能够靶向细胞、组织或器官的物质，例如是指能够与一种或一类特定的细胞(例如肿瘤细胞、免疫细胞)表面或细胞内部(如特定的亚细胞位置或细胞器)的蛋白或核酸分子(例如表皮生长因子受体、表皮生长因子受体酪氨酸激酶、血管内皮生长因子受体、白细胞分化抗原、整合素、乙酰胆碱受体、叶酸受体)特异性结合的物质，所述靶向分子例如为抗体、配体、信号肽、毒素、核酸、多糖、叶酸等。靶向分子可以额外地促进与其相连的部分进入靶定的细胞、组织或器官。
在本发明的实施方案中，所述靶向分子是指能够靶向肿瘤细胞表面蛋白的分子，例如能够与肿瘤细胞表面的整合素结合的分子，例如为RGD，特别是环状RGD。
根据本发明第三方面任一项的腺相关病毒，其携带有功能性核酸片段或标记分子的核酸片段。
在本发明中，所述功能性核酸片段在进入细胞、组织或器官内可以以功能性蛋白或核酸的形式发挥活性作用，所述功能性蛋白为本领域所公知的对细胞、组织或器官具有活性作用的蛋白，例如为细胞毒素、肿瘤坏死因子、促凋亡蛋白、生长激素、干扰素、神经营养因子等，所述功能性核酸为本领域公知的具有活性作用的核酸分子，例如RNA分子，如小干扰RNA，微小RNA等。
在本发明中，所述标记分子为本领域公知的具有标记功能的分子，例如为荧光分子、多肽、抗体、酶、多糖、功能性小分子化合物等。
在本发明的实施方案中，所述功能性核酸片段是指编码胸苷激酶或肿瘤坏死因子相关凋亡配体的核酸片段。
在本发明的实施方案中，所述标记分子的核酸片段是指编码GFP的核酸片段。
本发明第四方面涉及编码本发明第一方面任一项的腺相关病毒衣壳蛋白VP1或其片段的核酸分子，所述核酸分子与编码野生型腺相关病毒衣壳蛋白的核酸分子的区别在于，其中编码非天然氨基酸的特定位点氨基酸的密码子为TAG。
在本发明的一个实施方案中，其中编码野生型腺相关病毒衣壳蛋白VP1或其片段的第R447、G453、S578、N587、N587+1、S662位的氨基酸中的一个氨基酸的密码子被突变为TAG。
在本发明的另一个实施方案中，其中编码野生型腺相关病毒衣壳蛋白VP1或其片段的第N587位氨基酸的密码子被突变为TAG。
本发明第五方面涉及核酸载体，其可操作地连接有本发明第四方面任一项的核酸分子。
根据本发明第五方面任一项的核酸载体，所述载体为真核细胞表达载体或腺相关病毒载体。
在本发明的一个实施方案中，其为载体pCMV-VP1-Flag可操作地与本发明第四方面任一项的核酸分子连接。
在本发明的一个实施方案中，其为载体pAAV-RC中编码野生型腺相关病毒衣壳蛋白VP1或其片段的第R447、G453、S578、N587、N587+1、S662位的氨基酸中的一个氨基酸的密码子被突变为TAG。
本发明还涉及宿主细胞，其中含有本发明第五方面任一项的核酸载体。
根据本发明任一项所述的宿主细胞，其中还含有编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体。
在本发明的实施方案中，所述编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体为质粒pACYC-tRNA/PylRS，其从保藏日为2011年6月14日、保藏号为CGMCC No：4951的大肠埃希氏菌pACYC-tRNA/PylRS中获取。
根据本发明任一项所述的宿主细胞，其中还含有pHelper载体和pAAV-GFP载体。
在本发明的实施方案中，所述宿主细胞为哺乳动物细胞，所述哺乳动物例如为人、猴、鼠、牛、马、羊等。
在本发明的实施方案中，所述宿主细胞为AAV-293细胞。
在本发明的另外的实施方案中，所述宿主细胞为HeLa细胞。
在本发明的另外的实施方案中，所述宿主细胞为U87细胞。
本发明还涉及制备定点突变的腺相关病毒衣壳蛋白VP1、VP2或VP3的方法，其包括以下步骤：
(1)将野生型VP1蛋白的基因克隆进合适的表达载体中，得到重组表达载体；
(2)在野生型腺相关病毒衣壳蛋白VP1或其片段的氨基酸序列中选择 期望突变的一个或多个特定氨基酸位点，优选地，所述特定氨基酸位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的至少一个位点；
(3)将重组表达载体中编码对应于步骤(2)中选择位点的VP1或其片段的氨基酸的密码子用基因工程方法突变为密码子TAG，得到定点突变的VP1或其片段的突变序列表达载体；
(4)将步骤(3)获得的突变序列表达载体与编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体共同转染合适的宿主细胞，将转染成功后的宿主细胞在含有非天然氨基酸的培养基中培养，并在合适的条件下诱导表达，得到定点突变的腺相关病毒衣壳蛋白VP1或其片段。
在本发明的一个实施方案中，所述特定氨基酸位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的一个位点。
在本发明的另一个实施方案中，所述特定氨基酸位点为VP1或其片段的第N587位。
在本发明的实施方案中，所述合适的载体为真核细胞表达载体，例如为载体pCMV-VP1-flag。
在本发明的实施方案中，所述突变序列表达载体为pCMV-VP1-flag-R447，pCMV-VP1-flag-G453，pCMV-VP1-flag-S578，pCMV-VP1-flag-N587，pCMV-VP1-flag-N587+1或pCMV-VP1-flag-S662。
在本发明的实施方案中，所述编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体为质粒pACYC-tRNA/PylRS，其从保藏日为2011年6月14日、保藏号为CGMCC No：4951的大肠埃希氏菌pACYC-tRNA/PylRS中获取。
在本发明的实施方案中，步骤(4)中同时共转染的载体还包括载体pHelper和pAAV-GFP。
在本发明的实施方案中，步骤(4)中所述合适的宿主细胞是AAV-293包装细胞。
在本发明的实施方案中，所述非天然氨基酸为含有叠氮基团的非天然氨基酸，例如为Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)，或者所述非天然氨基酸为与上述含有叠氮基团的非天然氨基酸结构相似的非天然氨基酸，例如DiZPK。
在本发明的实施方案中，将转染成功后的宿主细胞在含有1mM的非天然氨基酸的培养基中培养48小时。
本发明还涉及制备定点突变的腺相关病毒的方法，其包括以下步骤：
(1)取病毒包装用质粒pAAV-RC(其含有衣壳蛋白VP1、VP2或VP3的编码基因)，在野生型腺相关病毒衣壳蛋白VP1或其片段的氨基酸序列中选择期望突变的一个或多个特定氨基酸位点，优选地，所述特定氨基酸位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的至少一个位点；
(2)以(1)中的质粒pAAV-RC为模板，将编码对应于步骤(1)中选择位点的VP1或其片段的氨基酸的密码子用基因工程方法突变为密码子TAG，得到定点突变的病毒包装质粒；
(3)将步骤(3)获得的突变序列表达载体与编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体共同转染合适的宿主细胞，将转染成功后的宿主细胞在含有非天然氨基酸的培养基中培养，并在合适的条件下诱导表达，即可得到定点突变的腺相关病毒；
在本发明的一个实施方案中，所述特定氨基酸位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的一个位点。
在本发明的另一个实施方案中，所述特定氨基酸位点为VP1或其片段的第N587位。
在本发明的实施方案中，所述突变序列表达载体为pAAV-RC-R447，pAAV-RC-G453，pAAV-RC-S578，pAAV-RC-N587，pAAV-RC-N587+1或pAAV-RC-S662。
在本发明的实施方案中，所述编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体为质粒pACYC-tRNA/PylRS，其从保藏日为2011年6月14日、保藏号为CGMCC No：4951的大肠埃希氏菌pACYC-tRNA/PylRS中获取。
在本发明的实施方案中，步骤(4)中同时共转染的载体还包括载体pHelper和pAAV-GFP。
在本发明的实施方案中，步骤(4)中所述合适的宿主细胞是AAV-293包装细胞。
在本发明的实施方案中，所述非天然氨基酸为NAEK或DiZPK。
在本发明的实施方案中，将转染成功后的宿主细胞在含有1mM的非天然氨基酸的培养基中培养72小时。
本发明还涉及组合物(例如药物组合物)或试剂盒，其中含有本发明第 三方面任一项的腺相关病毒或第四方面任一项的核酸分子或第五方面任一项的核酸载体，以及任选的药学上可接受的辅料。
本发明还涉及基因疫苗，其含有本发明第三方面任一项的腺相关病毒或第四方面任一项的核酸分子或第五方面任一项的核酸载体。
本发明还涉及本发明第三方面任一项的腺相关病毒或第四方面任一项的核酸分子或第五方面任一项的核酸载体在制备用于获得腺相关病毒结合蛋白的制剂的用途，或在制备基因治疗的药物中的用途，或用于制备DNA疫苗的用途。
本发明还涉及本发明第三方面任一项的腺相关病毒作为工具腺相关病毒的用途。
在本发明中，所述腺相关病毒可以作为工具病毒载体，根据具体需要携带相应的功能基因或在其表面偶联功能分子，以用于基础研究、基因治疗或DNA疫苗的制备。
本发明还涉及一种基因治疗方法，所述方法包括给予有需要的受试者有效量的本发明第三方面任一项的腺相关病毒或第四方面任一项的核酸分子或第五方面任一项的核酸载体。
在本发明的实施方案中，在定点突变和修饰的腺相关病毒表面偶联有靶向分子，同时该腺相关病毒还表达功能性蛋白，利用靶向分子将该腺相关病毒靶向特定细胞，该功能性蛋白针对该特定细胞发挥活性作用，以达到基因治疗的目的。
在本发明的具体实施方案中，在定点突变和修饰的腺相关病毒表面偶联有靶向分子环状RGD，同时该腺相关病毒还表达功能性蛋白肿瘤坏死因子相关凋亡配体或胸苷激酶，利用靶向分子将该腺相关病毒靶向高表达整合素的肿瘤细胞，该功能性蛋白针对该肿瘤细胞发挥活性作用，以达到基因治疗的目的。
在本发明中，所述腺相关病毒可以为各种血清型的腺相关病毒，例如可以为AAV1、AAV2、AAV5、AAV6、AAV7、AAV8、AAV9。在本发明的实施方案中，所述腺相关病毒为AAV2。当选用其它血清型的腺相关病 毒时，本领域技术人员知晓可以根据病毒衣壳蛋白的不同选择含有相应衣壳蛋白编码序列的质粒。
在本发明中，所述腺相关病毒与腺相关病毒载体具有同一含义。
在本发明中，所述VP1上突变氨基酸的位置都是以AAV2型腺相关病毒标准株的VP1(其氨基酸序列如SEQ ID NO：1所示)为基准；如果采用其它类型的腺病毒，本领域技术人员可以根据其它类型腺病毒VP1的氨基酸序列得出与本发明突变位点R447、G453、S578、N587、N587+1、S662位相应的位点。
在本发明中，所述VP1片段是指VP1的部分序列形成的蛋白，特别是其C端序列形成的蛋白，例如是指衣壳蛋白VP2或者衣壳蛋白VP3；其中VP2是VP1的C端598个氨基酸形成的蛋白，VP3是VP1的C端533个氨基酸形成的蛋白。
在本发明中，所述VP1片段上氨基酸位点的描述方式参照VP1确定；例如本发明中所述的VP2的第R447位氨基酸，其对应于VP2蛋白中实际氨基酸位置的第R310位。
在本发明中，当提及腺相关病毒VP1或其片段时，其使用SEQ ID NO：1所示的序列来进行描述。例如，表述“VP1或其片段的第R447位氨基酸”是指，SEQ ID NO：1所示的多肽的第R447位氨基酸残基。然而，本领域技术人员理解，在腺相关病毒VP1的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同血清型或嵌合血清型的腺相关病毒VP1或其片段或其突变体)，而不影响其生物学功能。因此，在本发明中，术语“VP1或其片段”应包括所有此类序列，包括例如SEQ ID NO：1所示的序列以及其天然或人工的变体。并且，当描述VP1的序列片段时，其不仅包括SEQ ID NO：1的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“VP1的第R447位氨基酸”是指，SEQ ID NO：1的第R447位氨基酸残基，以及其变体(天然或人工)中的相应氨基酸残基。根据本发明，表述“相应位点”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的位点。
在本发明中，用于确定序列同一性和序列相似性百分数的算法是例如BLAST和BLAST 2.0算法，它们分别描述在Altschul等(1977)Nucl.Acid.Res.25：3389-3402和Altschul等(1990)J.Mol.Biol.215：403-410。采用例如文献中所述或者默认参数，BLAST和BLAST 2.0可以用于确定本 发明的氨基酸序列同一性百分数。执行BLAST分析的软件可以通过国立生物技术信息中心为公众所获得。
在本发明中，所述与氨基酸序列具有至少90％的序列同一性的氨基酸序列包括与所述氨基酸序列基本同一的多肽序列，例如当采用本文所述方法(例如采用标准参数的BLAST分析)时，与本发明多肽序列相比含有至少90％序列同一性、优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的序列同一性的那些序列。
在本发明的实施方案中，所述AAV2型腺相关病毒是指AAV2型腺相关病毒标准株，其VP1的氨基酸序列如SEQ ID NO：1所示。
在本发明中，所述腺相关病毒衣壳蛋白由腺相关病毒cap基因编码，分别编码三个结构蛋白VP1、VP2和VP3，分子量分别为87、73、61kDa。
在本发明中，所述非天然氨基酸可以为本领域公知的任何非天然氨基酸，例如可以选自叠氮非天然氨基酸、光交联非天然氨基酸、酮基非天然氨基酸、炔基非天然氨基酸、乙酰基非天然氨基酸、磷酸基非天然氨基酸、甲基非天然氨基酸，其含义可见参考文献[21]、[22]。
在本发明中，所述基因治疗(gene therapy)是指将外源基因通过基因转移技术将其插入适当的靶细胞中，使外源基因制造的产物能治疗某种疾病。从广义说，基因治疗还可包括从DNA水平采取的治疗某些疾病的措施和技术。
在本发明中，所述DNA疫苗又称核酸疫苗或基因疫苗，是指编码免疫原或与免疫原相关的蛋白的真核表达质粒DNA(有时也可是RNA)或病毒表达载体(例如腺相关病毒载体)，经一定途径进入动物体内，被宿主细胞摄取后转录和翻译表达出免疫原蛋白或与免疫原相关的蛋白，此蛋白能刺激机体产生非特异性和/或特异性免疫应答反应，从而起到免疫保护作用。
在本发明中，所述N587+1是指在N587和R588之间插入非天然氨基酸；在构建该表达质粒时，在N587和R588的密码子之间插入TAG；N450+1、N385+1的含义以此类推。
本发明获得了经过非天然氨基酸定点突变和修饰的腺相关病毒，其在生产、转染和移动运输能力上与野生型腺相关病毒相当，并验证了其可以进一步通过非天然氨基酸与其它功能分子例如靶向分子偶联，以提高对靶向细胞的转导效率，同时还可以正常携带功能基因，表明该定点突变的腺相关病毒可以作为工具腺相关病毒，在寻找腺相关病毒结合蛋白或作为靶向基因治疗载体等与腺相关病毒相关的各个方面得到应用。
附图说明
图1显示了以click化学反应为基础的腺相关病毒修饰流程；
A表示AAV2粒子可以被遗传编码的含有叠氮的氨基酸NAEK进行定点修饰，B表示接下来可以通过生物正交反应标记上荧光分子。
图2显示了将NAEK可以有效地遗传插入到AAV衣壳蛋白VP1中。
A，NAEK插入VP1的位点选择。
B，分别在14个位点带有琥珀终止密码子的NAEK依赖的VP1的表达。
C，DIBO-Alexa488标记的VP1蛋白的考马斯亮蓝染色和胶内荧光染色。偶联依赖于NAEK呈现在VP1表面。
D，纯化的胰酶消化的VP1G453NAEK的MS/MS片段谱。NAEK的位置由g标记。
图3显示了AAV2粒子可以被遗传编码的叠氮标签定点标记；
A为根据谢等的原子结构(来自蛋白数据库)得到的AAV2主要衣壳蛋白和病毒衣壳的模型图，其中标出了插入的Arg447位点(红色)，B为转染HT-1080细胞后加入(+)或不加(-)NAEK培养的AAV-293包装细胞中的病毒提取物，病毒转染后48小时检测的GFP表达情况，比例尺相当于100μm，C为转染有系列稀释的rAAVr HT-1080细胞，然后计算病毒滴度，误差线表示三次实验平均的标准差，纵坐标为功能滴度(×107病毒颗粒/ml)。
图4显示了AAV2衣壳的不同位点上成功地插入了NAEK；
依赖NAEK生产的突变病毒与HT-1080细胞共培养48小时，如果突变病毒成功转入HT-1080细胞，则报告基因GFP表达，结果表明R447-AAV2和S568-AAV2与野生型病毒的病毒感染性基本相当。
图5显示了在某些位点不能将NAEK插入AAV2衣壳。
图6显示了AAV2-GFP病毒粒子可以被基因编码的NAEK标签定点标记。
A，B，AAV2主要衣壳蛋白和AAV2病毒颗粒的模式图。AAV2-GFP可以成功地被携带有氨基酸的叠氮化合物(NAEK)在位点R447，G453，S578，N587，S662突变。
C，D，但AAV2-GFP在位点S261，N381，Y444，S458，S492，Y500，F534，T573不能突变。
E，用系列稀释的AAV2-GFP转导HT-1080细胞，计算病毒滴度。误差棒代表三次重复实验的标准差。
F，在不同位点整合有NAEK的AAV2-GFP的基因组滴度通过Q-PCR对GFP进行定量。
图7显示了Alexa 488成功地修饰在病毒表面；
A为荧光标记的NAEK；野生型病毒(WT AAV2)，NAEK标记的病毒(R447-AAV2)粒子和S578-AAV2病毒粒子与加入或不加入DIBO-Alexa488(绿色)的Hela细胞37℃共培养30min，然后固定，透化，并用针对完整AAV2的鼠单克隆抗体(A20克隆)免疫染色(红色)。标记的叠氮标签(绿色)与R447-AAV2粒子(红色)共定位。重叠的绿色和红色信号显示为融合影像的黄色。第一行的AAV2没有与A20抗体共培养，作为阴性对照。比例尺相当于10μm。
B，C为用SDS-PAGE检测与DIBO-Alexa 488反应或未反应的AAV，其中利用488nm透射光检测Alexa 488(C)，然后凝胶用考马斯亮蓝染色(B)。
图8显示了在衣壳蛋白的R447和S578，Alexa 555也通过NAEK成功地与AAV2连接的共聚焦显微镜结果。
图9显示了对细胞内Alexa 488标记的AAV2病毒运动的定量分析。
A，B，C代表了Alexa 488标记的AAV2在Hela细胞中运动的典型轨迹；Hela细胞与Alexa 488标记的AAV2 4℃共培养30min，然后记录共聚焦实时成像。其中A是荧光图，B是白光图，C是AB叠加后照片，D是选取其中典型运动，放大显示。
D为Alexa 488标记的AAV2的典型轨迹，其中1为快速且定向的，2为快速且无定向的，3为慢速且无定向的，E，F，G为病毒速度的时间轨迹。
H为Alexa 488-AAV2的轨迹被分类为“slow undirected”，“fast undirected”，和“fast directed”，其中slow undirected表示慢速且无定向的，fast undirected表示快速且无定向的，fast directed表示快速且定向的。误差线表示三次实验平均的标准差(共195个轨迹)。比例尺相当于10μm。
图10显示了实时监测Alexa488-AAV2通过网格蛋白被膜小窝的内吞过程。
转染后24h，细胞与Alexa488标记的AAV2(绿色)在4℃共孵育30min，然后加温至37℃启动病毒内吞。接着记录共聚焦延时成像。图中显示了在表达有mRFP-clathrin(A)的Hela细胞中Alexa488-AAV2的代表性轨迹，以及选择出的实时成像框(B)。C，单个AAV2在表达有mRFP-clathrin的Hela细胞中的三维轨迹(绿色)。
图11显示了cRGD修饰的AAV2-GFP载体介导的基因转导
A，B，RAD-DIBO和RGD-DIBO的化学分子式。
C，D，细胞流式技术(FACS)分析整合素分别在Hela(左)和U87(右)细胞的表达。αvβ3整合素的表达由抗体LM609确定。利用Alexa488-标记的抗鼠抗体作为第二抗体用FACS检测(黑线)。阴影线为阴性对照的结果。
E，F，用cRGD标记的载体分别对Hela和U87细胞具有更高的基因转导能力。数量相同的没有RAD/RGD标记的载体、RAD/RGD标记的载体在4℃分别孵育细胞2h。除去未结合的病毒，然后加入新鲜培养基，48h后利用FACS分析GFP的表达。数据代表了表达有eGFP转基因的细胞百分数，数据用平均值和三次重复实验的标准差表示。
图12显示了RAD/RGD通过NAEK和DIBO之间的“click”化学反应成功地在AAV2-GFP衣壳蛋白表面进行了标记。
A.AAV2N587+1/azido(AAV2在N587和R588之间标记有NAEK)在300基因组拷贝/细胞的剂量下分别与RAD，RGD，DIBO，RAD-DIBO，RGD-DIBO或它们的组合在4℃共孵育2h。利用100kD Millipore Amicon Ultra-100除去多余的未反应的分子。透析后的病毒与U87细胞在4℃孵育2h。除去未结合的病毒，然后加入新鲜培养基，48h后利用FACS分析GFP的表达。
B，AAV2N587+1/azido+RGD介导的整合素靶向转导的竞争作用。U87细胞与AAV2N587+1/azido+RGD(RGD修饰的AAV2)或者AAV2N587+1/azido(RGD未修饰的AAV2)在4℃下共孵育2h，剂量为700基因组拷贝/细胞。除去未结合的病毒，然后加入新鲜培养基，48h后利用FACS分析GFP的表达。对于竞争实验，检测病毒在400μg/ml RAD或RGD肽，对照或LM609抗体(1∶100稀释)，或者RGD肽与LM609抗体的联合的条件下与细胞的结合。
图13显示了不同病毒对Hela和U87肿瘤细胞的转导效率比较
WT AAV2(野生型AAV2)，AAV2N587+1/azido(在AAV2衣壳蛋白表面的587和588位点插入NAEK标签)，AAV2N587+RAD(在AAV2衣壳蛋白表面的587和588位点插入RAD肽)，AAV2N587+RGD(在AAV2衣壳蛋白表面的587和588位点插入RGD肽)，AAV2N587+1/azido+RAD(AAV2通过NAEK和DIBO与cRAD化学连接)，and AAV2N587+1/azido+RGD(AAV2通过NAEK和DIBO与cRGD化学连接)与Hela(A)或U87细胞(B)在4℃ 孵育2h，剂量为700基因组拷贝/细胞。除去未结合的病毒，然后加入新鲜培养基，48h后利用荧光显微镜和FACS分析GFP的表达。所有的病毒载体包含eGFP基因。
图14显示了载体颗粒对Hela和U87细胞的结合力分析
相同数量的病毒载体与细胞在4℃下孵育2h，然后用PBS洗去未结合的载体颗粒。与Hela(A)或U87细胞(B)结合的载体颗粒通过抗AAV单抗A20和FACS检测。
图15显示了不同修饰的AAV2-TK(thymidine kinase，胸苷激酶)对Hela和U87细胞的杀伤作用。
A，C，E，G环氧鸟苷(GCV)对Hela/TK细胞在体外的剂量依赖性细胞毒性。AAV2-GFP(野生型AAV2含有GFP报告基因)，AAV2-TK(野生型AAV2含有胸苷激酶基因)，和AAV2N587+1/azido-TK(AAV2-TK在位点N587+1整合有NAEK)，AAV2N587+1/azido+RAD-TK(AAV2-TK通过NAEK和DIBO化学偶联有cRAD)，和AAV2N587+1/azido+RGD-TK(AAV2-TK通过NAEK和DIBO化学偶联有cRGD)，AAV2N587+RAD-TK(AAV2-TK在其衣壳蛋白表面位点587和588之间融合有RAD肽)，AAV2N587+RGD-TK(AAV2-TK在其衣壳蛋白表面位点587和588之间融合有RGD肽)与U87细胞孵育，剂量为500基因组拷贝/细胞。这些细胞与不同剂量的GCV孵育48h，然后利用cell Titer-Glo(promega)定量细胞存活率。MEM培养基作为阴性对照。
B，D，F，G GCV在体外对U87/TK细胞的毒性及剂量依赖性。AAV2-GFP，AAV2-TK，和AAV2N587+1/azido-TK，AAV2N587+1/azido+RAD-TK，和AAV2N587+1/azido+RGD-TK，AAV2N587+RAD-TK，AAV2N587+RGD-TK与U87细胞孵育，剂量为500基因组拷贝/细胞。这些细胞与不同剂量的GCV孵育48h，然后利用cell Titer-Glo(promega)定量细胞存活率。MEM培养基作为阴性对照。
图16显示了不同修饰的AAV2-TRAIL对Hela和U87细胞的杀伤作用。
含有TRAIL(TNF-related apoptosis-inducing ligand，肿瘤坏死因子相关凋亡配体)基因的不同AAV2载体与Hela(A)和U87细胞(B)孵育，剂量为500基因组拷贝/细胞。48h后，利用cell Titer-Glo(promega)定量细胞存活率。MEM培养基作为阴性对照。
图17显示了在AAV2衣壳的不同位点插入DiZPK的成像结果。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实验材料和方法：
细胞株，抗体和试剂
AAV-293、HT-1080和Hela细胞的培养基为含有10％胎牛血清(PAA，奥地利)和2mM L-谷氨酰胺(中科迈晨北京科技有限公司)的DMEM培养基(中科迈晨北京科技有限公司)，在5％CO2条件下培养。抗完整AAV2的鼠单克隆抗体(A20克隆)获自ARP公司(American Research Products，Belmont，MA)。DIBO-Alexa 488、DIBO-Alexa 555染料购自Invitrogen。
Nε-2-azidoethyloxycarbonyl-L-lysine(Nε-2-叠氮乙氧羰基-L-赖氨酸， NAEK)的合成：
Figure PCTCN2014089880-appb-000005
将2-溴乙醇(8g，64mmol)和叠氮钠(6.24g，96mmol)室温下加入丙酮(60ml)和水(30ml)中。反应混合液60℃回流10h，冷却至室温，真空蒸发除去丙酮。残留物用二乙基乙醚提取。有机层用盐水洗两次，然后用Na2SO4干燥，过滤，蒸发，得到2-叠氮乙醇(化合物2)，产率99％(5.5g，63.2mmol)，不用进一步纯化。
化合物2(5.5g，63.2mmol)溶于二氯甲烷(120ml)得到的溶液在-3℃条件下缓慢加入溶于二氯甲烷(55ml)的N，N’-羰基二咪唑(15.36g，94.8mmol)悬浮液中。搅拌条件下反应12小时。然后加入200mL水，有机层用盐水先后洗两次，然后用Na2SO4干燥，过滤并在真空条件下浓缩。残留物进一步用二 氧化硅凝胶层析纯化，PE/EtOAc(1∶1)洗脱，得到呈无色油状物的化合物3(10.7g，59mmol)，产率93％。
化合物3(10.7g，59mmol)溶于二氯甲烷(100ml)得到的溶液在室温下加入溶于1M NaOH(50ml)水溶液的Boc-Lys-OH(12.2g，49.2mmol)溶液中。接着加入TBAB(0.16g，0.01eq)。反应混合液在搅拌条件下反应12小时，冷却至0℃，然后用冰浴的1M HCl水溶液调pH值至2-3。水相用DCM提取，有机层用盐水先后洗两次。然后有机层用Na2SO4干燥，过滤并在真空条件下浓缩。残留物进一步用二氧化硅凝胶层析纯化，PE/EtOAc/HAc(100∶100∶1)洗脱，得到呈无色油状物的化合物4(15.1g，41.94mmol)，产率85％。
化合物4(15.1g，41.94mmol)溶于二氯甲烷(80ml)中，然后缓慢加入三氟乙酸(20ml)。反应液在室温下搅拌反应0.5小时，然后在真空条件下蒸发除去溶剂。残留物重溶于甲醇(5ml)中，并在乙醚中沉淀。收集沉淀物在真空条件下干燥，得到呈白色固体的化合物5(6.63g，25.58mmol)，即NAEK，产率61％。
非天然氨基酸DiZPK的合成和鉴定：
非天然氨基酸DiZPK的化学合成反应式如下：
Figure PCTCN2014089880-appb-000006
如上式所示，将原料1(5-羟基-2-戊酮)15mL与液氨40mL在-40℃下搅拌反应5h，之后降温至-60℃，缓慢滴加NH2OSO3H(20g)的甲醇溶液，加毕升至室温，反应过夜。滤除沉淀，向上清液中加入三乙胺，冰浴 条件下缓慢加入I2，至反应液颜色变深，不再产生气泡为止。反应完全后蒸除溶剂，经乙醚萃取后干燥。蒸除乙醚，剩余液体减压蒸馏获得25.4g无色粘稠液体产物2。
将上述产物2用吡啶溶解，0℃搅拌下加入11g TsCl，反应过夜。待反应完全后将反应液倒入浓盐酸与冰水的混合液中，乙醚萃取，醚层分别用1N盐酸和1N NaOH洗涤。有机相干燥柱分得到11.8g无色粘稠液体产物3。
将上述产物3用DMF溶解，加入NaN3室温反应隔夜至反应完全，加入大量水，乙醚萃取。蒸除乙醚，剩余产物用THF∶水(9∶1)混溶，加入三苯基磷，室温反应。反应完后加1N HCl混匀，旋干THF，二氯甲烷把未反应的原料，PPh3和O＝PPh3洗掉，液相加1N NaOH调pH到12，二氯甲烷萃取出4.0g产物4。
将5.2g原料5(Boc-Lys-OMe)与羰基二咪唑反应，制备出5.9g化合物6。之后化合物6与上述产物4(4.0g)偶联得到化合物7，最后经过两步脱保护，将Boc和甲酯脱除，得到目标4.5g产物8，即DiZPK。经谱学验证，结果为：
1H NMR(400MHz，D2O)：δ3.10(1H，t，J＝6.3Hz)，2.96(4H，m)，1.25(10H，m)，0.90(3H，s)；13C NMR(100MHz，D2O)：183.63，160.66，56.00，39.80，39.30，34.49，30.84，29.20，26.75，23.92，22.43，18.80；HREIMS m/z 308.16937[M+1]+(calcd for C12H22N5NaO3，308.16931)，证明所得到的DiZPK结构正确。
包装细胞培养
用AAV-293细胞(stratagene)生产重组感染性AAV颗粒。本发明中仅制备并使用AAV2血清型。AAV-293细胞代表稳定转染有5型腺病毒DNA的人胚胎肾细胞，并且具有rAAV体外制备所需的腺病毒e1基因。为了制备rAAV，AAV-293细胞在DMEM培养基中培养，并添加10％胎牛血清、4mM L-谷氨酰胺和4.5g/L葡萄糖，培养条件为37℃、5％CO2。细胞培养至铺满60-70％，然后开始AAV质粒的三重共转染步骤。
质粒构建
表达质粒载体pAAV-RC，pHelper和pAAV-GFP(安捷伦公司，SantaClara，CA)用于本实验。构建体含有AAV和制备感染性AAV颗粒所需 要的腺病毒基因。pAAV-RC提供分别编码AAV复制和衣壳蛋白的rep和cap基因。pHelper载体含有腺病毒E2A，E4和VA基因，pAAV-GFP含有GFP报告基因。该报告载体代表含有ITR的质粒，该质粒带有cmv启动子。pAAV-RC-R447质粒利用Quik Change Lightning Site-Directed Mutagenesis Kit(Agilent)制备，其中AAV衣壳蛋白VP1 447位精氨酸残基的遗传密码突变为TAG，其它突变质粒构建方法相同。
含有编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的pACYC-tRNA/PylRS载体由北京大学化学院陈鹏教授惠赠(Duy P.Nguyen，Hrvoje Lusic，Heinz Neumann，Prashant B.Kapadnis，Alexander Deiters，and Jason W.Chin.Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUAPair and Click Chemistry.J.AM.CHEM.SOC.2009，131，8720-8721)。
从保藏地：中国普通微生物菌种保藏管理中心 菌种保藏地址：地址：北京市朝阳区北辰西路1号院，中国科学院微生物研究所，保藏日为2011年6月14日、保藏号为CGMCC No：4951的分类命名为大肠埃希氏菌(Escherichia coli)的含有质粒pACYC-tRNA/PylRS的大肠埃希氏菌pACYC-tRNA/PylRS(由北京大学化学院陈鹏教授馈赠)中获取质粒pACYC-tRNA/PylRS，该质粒可以表达特异识别非天然氨基酸DiZPK和NAEK的tRNA和tRNA合成酶，该保藏信息已在公开号为CN102838663A的专利申请中公开。
AAV的制备和纯化
在AAV Helper-Free System中制备叠氮标记的AAV2感染颗粒(例如R447-AAV2载体)，感染时没有使用辅助腺病毒或疱疹病毒。用磷酸钙沉淀法将AAV2质粒载体pAAV-RC，pHelper，pAAV-GFP和载体pACYC-tRNA/PylRS(摩尔比1∶1∶1∶2)瞬时共转染AAV-293包装细胞。转染后6小时，将细胞培养基替换为含有1mM NAEK的新鲜培养基。72小时后收集感染细胞。为了释放rAAV病毒粒子，感染细胞用冻融法裂解。分离和纯化步骤参照Ping Guo等的操作(Guo P，El-Gohary Y，Prasadan K，Shiota C，Xiao X，Wiersch J，Paredes J，Tulachan S，Gittes GK：Rapid and simplified purification of recombinant adeno-associated virus.J Virol Methods，183(2)：139-146)。
病毒滴度测定
在六孔组织培养板中培养AAV-HT1080细胞，每孔2ml DMEM培养基，细胞密度为3×105/孔。37℃过夜培养。细胞需要培养至铺满约50％。10倍稀释病毒储存液。在10倍稀释的基础上，在5ml体积中进行5倍系列稀释，浓度范围从2×10-2至8×10-4。在六孔板的每孔中各加入1ml各稀释液，每个滴度各加三孔。同时以未加病毒储存液的孔做为阴性对照。37℃孵育1-2小时。孵育时每隔30min轻轻涡旋振荡培养板。然后在每孔中加入1ml事先预温的H-DMEM，37℃培养40-48小时。利用FACS检测pAAV-hrGFP AAV感染细胞。
利用实时定量PCR(qPCR)定量基因组拷贝
载体的基因组拷贝利用安捷伦公司Mx3000P实时PCR仪(Agilent Technologies，La Jolla，CA，USA)进行定量，针对GFP基因的特异性引物对为：5’-AAGCAGCACGACTTCTTCAAGTC-3’(SEQ ID NO：31)(forward)and 5’-TCGCCCTCGAACTTCACCTC-3’(SEQ ID NO：32)(reverse)。详细方法参见出版的操作指南[20]。
连接荧光探针
为了荧光标记AAV2-叠氮，取纯化的病毒粒子与Alexa488-DIBO或Alexa 555(500μM)在pH7.0条件下室温孵育2小时。利用100kD Millipore Amicon Ultra-100除去未反应的染料。
连接靶向分子cRGD方法如同连接荧光探针。
共聚焦成像
Alexa488标记的AAV2与Hela细胞在玻璃底培养皿中37℃共培养30min，然后细胞用含4％多聚甲醛的磷酸盐缓冲液(pH 7.0)(PBS)固定15min。接着细胞在含有0.5％Triton X-100的PBS溶液中透化10min，并用含3％牛血清白蛋白(BSA)的PBS溶液封闭60min。接下来细胞与抗完整AAV2的鼠单克隆抗体(A20)4℃孵育过夜，继而在室温下与连接有Alexa594的第二抗体(life tecnology)孵育1小时。细胞核用DAPI(Sigma)染色。共聚焦激光-扫描显微镜(SP8 Series，Leica，Germany)下成像。
活细胞成像
为了实时观察Alexa488-AAV2的运动，Hela细胞接种在玻璃底培养皿中，37℃培养过夜。接着Alexa488标记的AAV2与Hela细胞4℃共培养30min，然后利用活细胞成像系统(PerkinElmer，MA，USA)记录共聚焦实时成像。
实施例1腺相关病毒衣壳蛋白突变位点的选择和突变引物设计
(1)突变位点的选择
在腺相关病毒衣壳蛋白VP1上选择了如表1所示的突变位点。
其中VP1蛋白的氨基酸序列为：
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(SEQ ID NO：1)；
VP1蛋白的核苷酸序列为：
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGT ACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACA AGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA(SEQ ID NO：2)。
表1突变位点
Figure PCTCN2014089880-appb-000007
表1中所述的位置是在VP1蛋白中的位置。
(2)突变引物设计
为了定点突变表1中所述的位点，设计了表2所示的突变引物(同时可以用作测序引物)。
表2突变引物列表
Figure PCTCN2014089880-appb-000008
Figure PCTCN2014089880-appb-000009
表2中#1为正向引物，#2为反向引物。
587+1表示在587位点后插入非天然氨基酸，即在587位点和588位点之间插入非天然氨基酸。
实施例2腺相关病毒VP1突变蛋白的表达与检测
我们首先考察了正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA系统对AAV衣壳蛋白VP1表达中的相容性。将VP1基因克隆入载体pCMV-FLAG中(FLAG标签在C末端)，得到VP1编码载体pCMV-VP1-FLAG，利用Quik Change Lightning Site-Directed Mutagenesis Kit(Agilent)，根据其使用说明，以pCMV-VP1-FLAG质粒做为模板，使用表2所列突变引物，将该载体中编码S261，N381，Y444，R447，G453，S458，S492，Y500，F534，T573，S578，N587，N587+1，S662残基的密码子分别突变为琥珀终止密码子(TAG)，测序结果表明，已成功引入了各突变。对于R447位点突变后获得的质粒命名为pCMV-VP1-FLAG-R447，其表示将AAV衣壳蛋白VP1447位精氨酸残基的遗传密码突变为TAG，其它突变后获得的质粒命名和含义以此类推。
然后验证突变质粒是否能通过共表达NAEK特异的正交tRNA/aaRS载体在293T细胞中正确地展示NAEK。Western blotting的结果表明，当293T细胞在含有NAEK的培养中培养时，利用抗FLAG抗体可以检测到所有突变体的VP1蛋白；根据突变位点的不同，突变VP1的表达水平大约是野生型蛋白的10-100％(图2A、2B)。如果293T细胞在没有NAEK的条件下培养，则无法检测到VP1蛋白。进一步地，NAEK插入VP1的G453位点并且NAEK的展示通过MS/MS质谱测序证实(图2D)。
另外，通过其与DIBO-Alexa488(一种单个病毒的荧光标签)在温和条件下的正交反应也证实了NAEK已插入VP1蛋白中(图2C)，当突变VP1蛋白与荧光基团在4℃下共孵育1h，可以观察到明亮的绿色荧光。对于野生型VP1蛋白或者没有用荧光基团处理过的突变VP1蛋白，则观察不到绿色信号(图2C)。实验结果表明：NAEK被成功标记在VP1蛋白之上，并能够通过NAEK进一步的偶联其它分子。
实施例3定点突变的腺相关病毒及检测
本发明利用遗传密码扩展技术，通过将含有叠氮的氨基酸NAEK引入病毒衣壳来对AAV2进行定点标记。
利用Quik Change Lightning Site-Directed Mutagenesis Kit(Agilent)，根据其使用说明，以pAAV-RC质粒做为模板，使用表2所列突变引物，完成各突变，测序结果表明，已成功引入了各突变。对于R447位点突变后获得的质粒命名为pAAV-RC-R447，其表示将AAV衣壳蛋白VP1 447位精氨酸 残基的遗传密码突变为TAG，其它突变后获得的质粒命名和含义以此类推。
如图3A所示，本发明利用遗传学方法，在cap基因位于VP1/VP2/VP3区的第447氨基酸位点插入叠氮非天然氨基酸。相应地，AAV2衣壳蛋白编码质粒pAAV-RC的遗传密码进行了突变，将其突变为琥珀终止密码子TAG。然后，在NAEK存在的情况下，通过在AAV-293包装细胞中共表达NAEK特异的正交tRNA/aaRS对(pACYC-tRNA/PylRS载体)、pHelper载体和pAAV-GFP载体，突变的质粒pAAV-RC-R447用于带有叠氮的AAV2(R447-AAV2)粒子的传代(具体参见材料和方法部分)。72小时后，收集细胞裂解物，经过10倍稀释后加入HT-1080细胞中，利用荧光显微镜检测突变的病毒是否已生产。
利用突变质粒转染后得到的含有NAEK的细胞裂解物转染HT-1080细胞，在转染HT-1080细胞48小时后，我们在HT-1080细胞中发现了很强的绿色荧光，几乎与野生型相当，但是与没有NAEK的显著不同(参见图3B、3C)。
具体实验方法参照材料和方法部分的“AAV的制备和纯化”。
不仅在R447位点，并且在其它位点，G453，S578，N587，N587+1、S662都可以插入NAEK(参见图4，图6A，图6B)。但是，位点S261，N381，Y444，S458，S492，Y500，F534，T573不能用NAEK标记(参见图5，图6C，图6D)。具体实验方法同上。
这表明，由于NAEK的存在，利用突变的AAV2衣壳包装AAV确实是成功的。而且，携带有GFP报告基因的经过修饰或未经标记的AAV2都用于感染HT-1080细胞，来检测病毒滴度，同时用qPCR检测病毒基因组滴度。R447-AAV2和S578-AAV2的功能滴度大小(转染单位/mL)与WT-AAV2相同(参见图6E，图6F)，表明叠氮标签的引入对于AAV2粒子的生产和转染能力的影响很小。
实施例4NAEK定点标记AAV2的荧光修饰
实施例3中，我们获得NAEK定点标记的AAV2，AAV2上的NAEK标签内的叠氮基团可以与DIBO-荧光分子通过无铜click化学反应将荧光基团定点偶联在病毒粒子表面(见图1)。
无铜click化学反应是通过引入环辛炔来维持细胞活性而实现的click反应，其中八元环的张力允许在没有催化剂的条件下与叠氮发生反应。这 些试剂中的一种由所谓的DIBO化合物组成[18]。叠氮修饰的大分子现在可以在没有金属催化剂的条件下进行标记，这样不仅可用于活细胞的研究，而且阻止了对蛋白的损伤。
为了确定叠氮标签是否能在方便且温和的条件下(25℃，2h)与Alexa 488发生正交反应，制备了纯化的野生型AAV2(WT-AAV2)和叠氮标记的AAV2(R447-AAV2和S578-AAV2)颗粒，并且在室温下与DIBO-Alexa 488反应2h，利用100kD Millipore Amicon Ultra-100将反应缓冲液替换为PBS。接着，将含有病毒颗粒的溶液加在HeLa细胞上，并用特异性针对完整AAV2颗粒的抗体进行免疫染色。具体实验方法参见实验材料和方法部分的“连接荧光探针”和“共聚焦成像”。
大部分肼信号与AAV2信号共定位(参见图7A)，而没有叠氮标签的病毒颗粒没有观察到明显的肼信号(参见图7A)，表明叠氮基团已有效地表达在AAV2表面。标记有绿色荧光的AAV2衣壳蛋白VP1/VP2/VP3进一步用SDS-PAGE成像得到了验证，而在WT-AAV2没有发现信号(参见图7B，图7C)。
显然，NAEK已位点特异性地展示在突变病毒上，荧光分子也确实通过NAEK连接到了突变病毒上。不仅R447-AAV2，而且S578-AAV2也成功地偶联了Alexa 488(参见图7A、7B、7C)。
进一步地，另一种荧光基团Alexa 555也可以通过NAEK与AAV2相连(参见图8)，方法同上。上述结果表明，叠氮标签能够将荧光探针以位点特异的方式共价连接到AAV2颗粒上。
需要注意的是，病毒与Alexa488和555的生物正交反应对荧光标记病毒的感染性几乎没有什么影响，这是由于温和的反应条件(25℃孵育2h)和NAEK-DIBO-Alexa488或555探针的尺寸很小。根据Chem 3D软件，探针的大小约为2.6868nm，大约是单个AAV2大小的1％。
实施例5利用荧光标记的AAV2观察单个病毒的运动
在建立了用Alexa 488定点标记AAV2方法的基础上，本发明验证了这种标记是否能够用在单个病毒示踪上。
携带有Alexa 488的第447位突变的病毒加入Hela细胞中，4℃孵育30min，使结合同步，然后在共聚焦显微镜实时成像下监测这些病毒，具体实验方法参见实验材料和方法部分的“共聚焦成像”。成像中看到了病毒颗粒细胞内运动的多种形式，图9显示了Hela细胞中Alexa 488标记的AAV2的 代表性轨迹(图9A、9B、9C)。
以三维AAV2颗粒运动的二维数据分析结果为基础，我们发现许多颗粒表现出相对较慢的运动(例如图9D中的粉色轨迹)，但是有一些颗粒表现出快速的和定向的运输(例如图9D中的橙色轨迹)[19]。图9D、9E、9F为病毒速度的时间轨迹。
根据我们的分析，轨迹的最高速度≥0.002μm/s，并且将在超过5个连续的框中具有单一方向的运动定义为定向运输，快速(≥0.002μm/s)但是无方向性的运动定义为快速非定向运输，慢速(≤0.002μm/s)且没有方向性的运动定义为慢速非定向运输。
利用这些定义，16.3％Alexa-AAV2的轨迹是快速且定向的，24.7％的Alexa-AAV2的轨迹是快速但非定向的，而其余的是慢速非定向的(参见图9H)。
为了实时监测AAV2与网格蛋白(clathrin)结构的相互作用，Alexa 488标记的病毒与表达融合了红色荧光蛋白的网格蛋白的Hela细胞共孵育，利用延时旋转共聚焦显微镜(time-lapse spinning confocal microscopy)进行活细胞成像。Alexa 488-AAV2的代表性轨迹和照片如图10A所示。病毒颗粒(绿色)最初与网格蛋白信号(红色)共定位。这种共定位持续约10s，然后网格蛋白信号迅速消失，表明病毒与未包被的网格蛋白囊泡解离(图10B)。在病毒颗粒与网格蛋白信号共定位的最初10s，病毒颗粒的瞬时扩散系数(instantaneous diffusion coefficients)显著低于从网格蛋白信号解离后(图10C)，表明病毒颗粒通过网格蛋白被膜小窝(clathrin-coated pit)限定的区域进入细胞。
实施例6AAV2与靶向配体偶联对细胞转导的增强作用
在上述实验的基础上，接下来我们将肿瘤靶向基序(环状RGD)偶联到AAV2衣壳蛋白上以靶向递送基因。整合素是的RGD肽的受体，在多种肿瘤中高表达。因此，RGD可能可以用于包被AAV载体以提高递送效率和靶向整合素αvβ3(高表达于肿瘤细胞)的选择性。RAD是RGD中的甘氨酸突变为丙氨酸的突变基序，可以作为阴性对照。
如图11所示，用获得的载体转染U87细胞(整合素αvβ3阳性细胞)和Hela细胞(作为阴性对照)。我们发现在AAV载体的不同位点偶联RGD对U87的转导效率有不同的影响。在位点N587+1，可以观察到转导效率的显著提高，与携带RAD和含有叠氮化物的AAV载体(对照)相比，几乎提高了10倍，在位点447,587,662偶联RGD未观察到提高。在位点453，578偶联RGD观察 到显著的降低。作为对照，连接RAD而不是RGD到AAV载体上对所有这些位点的转导效率几乎都没有什么影响，表明在转导效率的提高中RGD起关键作用。因此，在AAV载体的合适位置而不是任意位置连接RGD是提高难转导细胞的转导效率的有效途径。对于Hela细胞，在AAV载体的位点587+1偶联RGD的感染效率仅有约20％的提高，可能是由于细胞表面整合素αvβ3的低表达(图11C)。结果表明，按照这种方式位点选择性的偶联RGD是调整AAV载体的趋向性和提高基因递送效率的有效方式。
为了进一步验证cRGD标记的AAV2衣壳蛋白是通过DIBO和NAEK之间的“click”反应，RAD、RGD、DIBO、RAD-DIBO、RGD-DIBO或者它们的组合分别与AAV2N587+1 azido孵育。如图12A所示，只有DIBO-RGD组表现出增强的病毒转导效率，而且，只有DIBO分子能够竞争性地抑制DIBO-RGD。这些结果表明，标记在AAV2衣壳蛋白上的RGD肽仅仅是通过NAEK和DIBO之间的“click”化学反应连接。
为了从另一方面验证上述结论，我们利用这些载体对竞争性抑制AAV介导的基因递送进行了实验。我们利用一种合成的RGD多肽和抗整合素抗体来看是否它们能够抑制转导。如图12B所示，由AAV2N587+1 azido+RGD载体介导的U87细胞内的GFP表达能够被合成的RGD多肽和抗整合素抗体显著抑制。这些结果显示了细胞表面RGD和整合素之间相互作用是特异的。
接下来我们想知道是否cRGD化学修饰的AAV2比RGD融合在AAV2表面的效果要更好。如图13所示，cRGD化学修饰的AAV2和RGD融合在AAV2表面与未被修饰的AAV2(野生型AAV2)相比，都能够显著提高病毒的转导效率。但是，cRGD化学修饰的AAV2是RGD融合在AAV2表面的病毒转导效率的1.5倍。这些结果表明，本发明中对AAV2进行应点特异性修饰的方法优于目前普遍用于病毒靶向的AAV2表面修饰方法。
为了研究为什么AAV2的cRGD化学修饰在病毒靶向方面优于AAV2的RGD表面融合修饰，我们对病毒和细胞的结合能力进行了分析。相同数量的不同病毒与U87细胞在4℃孵育2h，然后利用抗AAV单抗A20和FACS进行分析。如图14所示。与未修饰的AAV2或者线性RAD修饰的AAV2相比，cRGD化学修饰的AAV2和RGD融合修饰的AAV2都能够显著提高结合病毒结合病毒的能力。但是，cRGD化学修饰的AAV2仍是RGD融合修饰AAV2结合能力的1.5倍。结果表明，cRGD化学偶联的AAV2的细胞结合能力明显优于RGD融合修饰的AAV2，因而产生更好的病毒靶向作用。
上述工作中，为了研究方便我们在病毒衣壳内中包装的是GFP报告基 因。如果将GFP基因换成治疗用基因，病毒还能否展现上述特性？实验过程中，我们将具有细胞杀伤功能的单纯疱疹病毒胸苷激酶(HSV-tk)基因和肿瘤坏死因子相关凋亡配体(TRAIL)基因包装到NAEK标记的病毒中，并通过NAEK偶联cRGD分子，制备出RGD化学游离修饰的病毒AAVN587+1/azido+RGD-TK/TRAIL。实验结果显示，AAVN587+1/azido+RGD-TK/TRAIL病毒杀伤肿瘤细胞的效果明显优于WT-AAV和RGD融合修饰AAVN587+RGD-TK/TRAIL病毒。而对整合素低表达的HeLa细胞的杀伤效果的差异就没有那么明显。说明cRGD的化学游离修饰不但增强病毒的杀伤效果，而且提高了病毒的靶向性(图15，16)。
实施例7光交联非天然氨基酸(DiZPK)定点标记的AAV2的制备
利用与上述相同的方法，将光交联非天然氨基酸(DiZPK)引入定点到AAV2衣壳表面，将会在病毒进入细胞的过程中，通过光照交联，捕捉新的相互作用蛋白，从而发现病毒新的受体。如图17所示，已经尝试过S261、R447、F534、T573、N587位点，目前发现在N587位点可以成功引入DiZPK。
从研究结果看，R447位点能够引入NAEK，但是不能插入DiZPK，而在N587位点这两个非天然氨基酸都能够插入。这可能是因为蛋白分子的每个氨基酸残基位点的空间结构不同，对能够引入的非天然氨基酸的结构要求也是不同的。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
参考文献
1.Atchison RW，Casto BC，Hammon WM：Adenovirus-Associated Defective Virus Particles.Science 1965，149(3685)：754-756.
2.Gray SJ，Samulski RJ：Optimizing gene delivery vectors for the treatment of heart disease.Expert Opin Biol Ther 2008，8(7)：911-922.
3.Grimm D，Kay MA：From virus evolution to vector revolution：use of naturally occurring serotypes of adeno-associated virus(AAV)as novel vectors for human gene  therapy.Curr Gene Ther 2003，3(4)：281-304.
4.Mueller C，Flotte TR：Clinical gene therapy using recombinant adeno-associated virus vectors.Gene Ther 2008，15(11)：858-863.
5.Choi KJ，Kim JH，Lee YS，Kim J，Suh BS，Kim H，Cho S，Sohn JH，Kim GE，Yun CO：Concurrent delivery of GM-CSF and B7-1 using an oncolytic adenovirus elicits potent antitumor effect.Gene Ther 2006，13(13)：1010-1020.
6.Wu Z，Asokan A，Samulski RJ：Adeno-associated virus serotypes：vector toolkit for human gene therapy.Mol Ther 2006，14(3)：316-327.
7.Fisher KJ，Jooss K，Alston J，Yang Y，Haecker SE，High K，Pathak R，Raper SE，Wilson JM：Recombinant adeno-associated virus for muscle directed gene therapy.Nat Med 1997，3(3)：306-312.
8.Moss RB，Rodman D，Spencer LT，Aitken ML，Zeitlin PL，Waltz D，Milla C，Brody AS，Clancy JP，Ramsey B et al：Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis：a multicenter，double-blind，placebo-controlled trial.Chest 2004，125(2)：509-521.
9.Mingozzi F，High KA：Therapeutic in vivo gene transfer for genetic disease using AAV：progress and challenges.Nat Rev Genet，12(5)：341-355.
10.Bainbridge JW，Smith AJ，Barker SS，Robbie S，Henderson R，Balaggan K，Viswanathan A，Holder GE，Stockman A，Tyler N et al：Effect of gene therapy on visual function in Leber′s congenital amaurosis.N Engl J Med 2008，358(21)：2231-2239.
11.Hauswirth WW，Aleman TS，Kaushal S，Cideciyan AY，Schwartz SB，Wang L，Conlon TJ，Boye SL，Flotte TR，Byrne BJ et al：Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector：short-term results of a phase I trial.Hum Gene Ther 2008，19(10)：979-990.
12.Manno CS，Pierce GF，Arruda VR，Glader B，Ragni M，Rasko JJ，Ozelo MC，Hoots K，Blatt P，Konkle B et al：Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med 2006，12(3)：342-347.
13.Manno CS，Chew AJ，Hutchison S，Larson PJ，Herzog RW，Arruda VR，Tai SJ，Ragni MV，Thompson A，Ozelo M et al：AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B.Blood 2003，101(8)：2963-2972.
14.White SJ，Nicklin SA，Buning H，Brosnan MJ，Leike K，Papadakis ED，Hallek M， Baker AH：Targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-associated virus vectors.Circulation 2004，109(4)：513-519.
15.Yu CY，Yuan Z，Cao Z，Wang B，Qiao C，Li J，Xiao X：A muscle-targeting peptide displayed on AAV2 improves muscle tropism on systemic delivery.Gene Ther 2009，16(8)：953-962.
16.Work LM，Buning H，Hunt E，Nicklin SA，Denby L，Britton N，Leike K，Odenthal M，Drebber U，Hallek M et al：Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses.Mol Ther 2006，13(4)：683-693.
17.Douar AM，Poulard K，Danos O：Deleterious effect of peptide insertions in a permissive site of the AAV2 capsid.Virology 2003，309(2)：203-208.
18.Ning X，Guo J，Wolfert MA，Boons GJ：Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions.Angew Chem Iht Ed Engl 2008，47(12)：2253-2255.
19.Joo KI，Fang Y，Liu Y，Xiao L，Gu Z，Tai A，Lee CL，Tang Y，Wang P：Enhanced real-time monitoring of adeno-associated virus trafficking by virus-quantum dot conjugates.ACS Nano，5(5)：3523-3535.
20.Rohr，U.P.；Wulf，M.A.；Stahn，S.；Steidl，U.；Haas，R.；Kronenwett，R.J Virol Methods 2002，106，81-8.
21.Travis S.Young and Peter G.Schultz：Beyond the Canonical 20 Amino Acids：Expanding the Genetic Lexicon.J.Biol.Chem.2010，285：11039-11044.
22.Jianming Xie and Peter G Schultz：Adding amino acids to the genetic repertoire.Current Opinion in Chemical Biology 2005，9：548-554.

Claims (23)

  1. 定点突变的腺相关病毒衣壳蛋白VP1或其片段，其在野生型腺相关病毒衣壳蛋白VP1或其片段的特定位点的氨基酸被突变为非天然氨基酸，所述特定位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的至少一个位点。
  2. 权利要求1的腺相关病毒衣壳蛋白VP1或其片段，其中所述非天然氨基酸选自叠氮非天然氨基酸、光交联非天然氨基酸、酮基非天然氨基酸、炔基非天然氨基酸、乙酰基非天然氨基酸、磷酸基非天然氨基酸、甲基非天然氨基酸。
  3. 权利要求1的腺相关病毒衣壳蛋白VP1或其片段，其中所述非天然氨基酸为含有叠氮基团的非天然氨基酸，例如为Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)、
    Figure PCTCN2014089880-appb-100001
    或
    Figure PCTCN2014089880-appb-100002
    或者所述非天然氨基酸为与上述含有叠氮基团的非天然氨基酸结构相似的非天然氨基酸，例如DiZPK。
  4. 权利要求3的腺相关病毒衣壳蛋白VP1或其片段，其中所述NAEK与VP1或其片段氨基酸序列的连接方式如式I所示，其中：
    Figure PCTCN2014089880-appb-100003
    由R1到R2的方向为氨基酸序列的N末端到C末端方向，其中第N位的氨基酸选自第R447位、G453位、S578位、N587位、N587+1位、S662位氨基酸中的一个，R1为VP1蛋白或其片段氨基酸序列的第1至第N-1位氨基酸残基，R2为VP1蛋白或其片段氨基酸序列的第N+1位至C末端的氨基酸残基；
    其中所述DiZPK与VP1或其片段氨基酸序列的连接方式如式II所示，其中：
    Figure PCTCN2014089880-appb-100004
    由R1到R2的方向为氨基酸序列的N末端到C末端方向，其中第N位的氨基酸选自第R447位、G453位、S578位、N587位、N587+1位、S662位氨基酸中的一个，R1为VP1蛋白或其片段氨基酸序列的第1至第N-1位氨基酸残基，R2为VP1蛋白或其片段氨基酸序列的第N+1位至C末端的氨基酸残基，
    R3为
    Figure PCTCN2014089880-appb-100005
  5. 权利要求1的腺相关病毒衣壳蛋白VP1或其片段，其中所述腺相关 病毒为腺相关病毒II型。
  6. 权利要求1的腺相关病毒衣壳蛋白VP1或其片段，其中所述非天然氨基酸上还连接有标记基团，例如为荧光标记基团，或者例如为能与叠氮基团发生click化学反应的标记基团；或者所述非天然氨基酸上还连接有靶向分子，优选地，所述靶向分子上还连接有能与叠氮基团发生click化学反应的基团。
  7. 定点突变的腺相关病毒衣壳蛋白，其含有权利要求1-5任一项的腺相关病毒衣壳蛋白VP1或其片段。
  8. 定点突变的腺相关病毒，其含有权利要求1-5任一项的腺相关病毒衣壳蛋白VP1或其片段或者权利要求7的腺相关病毒衣壳蛋白。
  9. 权利要求8的腺相关病毒，其中所述非天然氨基酸上还连接有标记基团，例如为荧光标记基团，或者例如为能与叠氮基团发生click化学反应的标记基团。
  10. 权利要求8的腺相关病毒，其中所述非天然氨基酸上还连接有靶向分子，优选地，所述靶向分子上还连接有能与叠氮基团发生click化学反应的基团。
  11. 权利要求8的腺相关病毒，其携带有功能性核酸片段或标记分子的核酸片段。
  12. 编码权利要求1-5任一项的腺相关病毒衣壳蛋白VP1或其片段的核酸分子，所述核酸分子与编码野生型腺相关病毒衣壳蛋白的核酸分子的区别在于，其中编码非天然氨基酸的特定位点氨基酸的密码子为TAG。
  13. 核酸载体，其可操作地连接有权利要求12的核酸分子。
  14. 权利要求13的核酸载体，其为载体pAAV-RC，其中编码野生型腺相关病毒衣壳蛋白VP1或其片段的特定位点氨基酸的密码子被突变为TAG，所述特定位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的至少一个位点。
  15. 宿主细胞，其中含有权利要求13或14的核酸载体。
  16. 权利要求15的宿主细胞，其中还含有编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体，例如为质粒pACYC-tRNA/PylRS。
  17. 制备定点突变的腺相关病毒衣壳蛋白VP1或其片段的方法，其包括以下步骤：
    (1)将野生型VP1或其片段的基因克隆进合适的表达载体中，得到重组表达载体；
    (2)在野生型腺相关病毒衣壳蛋白VP1或其片段的氨基酸序列中选择期望突变的一个或多个特定氨基酸位点，优选地，所述特定氨基酸位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的至少一个位点；
    (3)将重组表达载体中编码对应于步骤(1)中选择位点的VP1或其片段的氨基酸的密码子用基因工程方法突变为密码子TAG，得到定点突变的VP1或其片段的突变序列表达载体；
    (4)将步骤(3)获得的突变序列表达载体与编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体共同转染合适的宿主细胞，将转染成功后的宿主细胞在含有非天然氨基酸的培养基中培养，并在合适的条件下诱导表达，得到定点突变的腺相关病毒衣壳蛋白VP1或其片段；
    优选地，所述编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体为质粒pACYC-tRNA/PylRS，其从保藏日为2011年6月14日、保藏 号为CGMCC No：4951的大肠埃希氏菌pACYC-tRNA/PylRS中获取；
    优选地，所述非天然氨基酸为含有叠氮基团的非天然氨基酸，例如为Nε-2-叠氮乙氧羰基-L-赖氨酸(NAEK)、
    Figure PCTCN2014089880-appb-100006
    或
    Figure PCTCN2014089880-appb-100007
    或者所述非天然氨基酸为与上述含有叠氮基团的非天然氨基酸结构相似的非天然氨基酸，例如DiZPK。
  18. 制备定点突变的腺相关病毒的方法，其包括以下步骤：
    (1)取病毒包装用质粒pAAV-RC，在野生型腺相关病毒衣壳蛋白VP1或其片段的氨基酸序列中选择期望突变的一个或多个特定氨基酸位点，优选地，所述特定氨基酸位点选自VP1或其片段的第R447、G453、S578、N587、N587+1、S662位中的至少一个位点；
    (2)以(1)中的质粒pAAV-RC为模板，将编码对应于步骤(1)中选择位点的VP1或其片段的氨基酸的密码子用基因工程方法突变为密码子TAG，得到定点突变的病毒包装质粒；
    (3)将步骤(3)获得的突变序列表达载体与编码正交琥珀突变型抑制子氨酰-tRNA合成酶/tRNACUA基因的载体共同转染合适的宿主细胞，将转染成功后的宿主细胞在含有非天然氨基酸的培养基中培养，并在合适的条件下诱导表达，即可得到定点突变的腺相关病毒；
    优选地，步骤(3)中还同时与载体pHelper和pAAV-GFP共同转染合适的宿主细胞，步骤(3)中所述合适的宿主细胞是AAV-293包装细胞。
  19. 组合物或试剂盒，其中含有权利要求8-11任一项的腺相关病毒或权利要求12的核酸分子或权利要求13或14的核酸载体。
  20. 基因疫苗，其含有权利要求8-11任一项的腺相关病毒或权利要求12的核酸分子或权利要求13或14的核酸载体。
  21. 权利要求8-11任一项的腺相关病毒或权利要求12的核酸分子或权利要求13或14任一项的核酸载体在制备用于获得腺相关病毒结合蛋白的制剂的用途，或在制备基因治疗的药物中的用途，或用于制备DNA疫苗的用途。
  22. 权利要求8-11任一项的腺相关病毒作为工具腺相关病毒的用途。
  23. 一种基因治疗方法，所述方法包括给予有需要的受试者有效量的权利要求8-11任一项的腺相关病毒或权利要求12的核酸分子或权利要求13或14的核酸载体。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017212019A1 (en) 2016-06-09 2017-12-14 Centre National De La Recherche Scientifique (Cnrs) Raav with chemically modified capsid
WO2018035503A1 (en) * 2016-08-18 2018-02-22 The Regents Of The University Of California Crispr-cas genome engineering via a modular aav delivery system
US10758623B2 (en) 2013-12-09 2020-09-01 Durect Corporation Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same
WO2021005210A1 (en) 2019-07-11 2021-01-14 Centre National De La Recherche Scientifique Chemically-modified adeno-associated virus
US20210230231A1 (en) * 2016-06-15 2021-07-29 Howard Hughes Medical Institute Variant adeno-associated viruses and methods of using
US11382988B2 (en) 2019-11-08 2022-07-12 Coave Therapeutics Modified adeno-associated virus vectors and delivery thereof into the central nervous system
RU2793112C2 (ru) * 2017-10-16 2023-03-29 ФиГенерон ГмбХ Векторы aav
WO2023198652A1 (en) 2022-04-11 2023-10-19 Centre National De La Recherche Scientifique Chemically-modified adeno-associated viruses

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Publication number Priority date Publication date Assignee Title
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CN106928093B (zh) * 2015-12-31 2019-02-01 北京大学 氰基非天然氨基酸的制备及其在生物正交拉曼检测上的应用
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WO2019006418A2 (en) * 2017-06-30 2019-01-03 Intima Bioscience, Inc. ADENO-ASSOCIATED VIRAL VECTORS FOR GENE THERAPY
WO2019014924A1 (zh) * 2017-07-21 2019-01-24 中国科学院深圳先进技术研究院 一种病毒转染增效剂和基于点击化学的病毒转染应用
CN109897881A (zh) * 2019-03-21 2019-06-18 中国科学院深圳先进技术研究院 非囊膜病毒的代谢修饰与原位生物正交标记方法及应用
CN113727992A (zh) * 2019-03-21 2021-11-30 斯特里迪比奥公司 重组腺相关病毒载体
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004027019A2 (en) * 2002-05-01 2004-04-01 University Of Florida Research Foundation, Inc. Improved raav expression systems for genetic modification of specific capsid proteins
CN102618605A (zh) * 2003-06-18 2012-08-01 斯克利普斯研究院 非天然活性氨基酸遗传密码增加
CN102838663A (zh) 2011-06-23 2012-12-26 北京大学 定点突变和定点修饰的病毒膜蛋白、其制备方法及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090215879A1 (en) 2008-02-26 2009-08-27 University Of North Carolina At Chapel Hill Methods and compositions for adeno-associated virus (aav) with hi loop mutations
US20140140962A1 (en) * 2011-04-29 2014-05-22 The Research Foundation Of State University Of New York Viruses modified with unnatural moieties and methods of use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004027019A2 (en) * 2002-05-01 2004-04-01 University Of Florida Research Foundation, Inc. Improved raav expression systems for genetic modification of specific capsid proteins
CN102618605A (zh) * 2003-06-18 2012-08-01 斯克利普斯研究院 非天然活性氨基酸遗传密码增加
CN102838663A (zh) 2011-06-23 2012-12-26 北京大学 定点突变和定点修饰的病毒膜蛋白、其制备方法及其应用

Non-Patent Citations (30)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCL. ACID. RES, vol. 25, 1977, pages 3389 - 3402
ATCHISON RW; CASTO BC; HAMMON WM: "Adenovirus-Associated Defective Virus Particles", SCIENCE, vol. 149, no. 3685, 1965, pages 754 - 756
BAINBRIDGE JW; SMITH AJ; BARKER SS; ROBBIE S; HENDERSON R; BALAGGAN K; VISWANATHAN A; HOLDER GE; STOCKMAN A; TYLER N ET AL.: "Effect of gene therapy on visual function in Leber's congenital amaurosis", N ENGL J MED, vol. 358, no. 21, 2008, pages 2231 - 2239
CHOI KJ; KIM JH; LEE YS; KIM J; SUH BS; KIM H; CHO S; SOHN JH; KIM GE; YUN CO: "Concurrent delivery of GM-CSF and B7-1 using an oncolytic adenovirus elicits potent antitumor effect", GENE THER, vol. 13, no. 13, 2006, pages 1010 - 1020
DOUAR AM; POULARD K; DANOS O: "Deleterious effect of peptide insertions in a permissive site of the AAV2 capsid", VIROLOGY, vol. 309, no. 2, 2003, pages 203 - 208
DUY P. NGUYEN; HRVOJE LUSIC; HEINZ NEUMANN; PRASHANT B. KAPADNIS; ALEXANDER DEITERS; JASON W. CHIN: "Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNA Pair and Click Chemistry", J. AM. CHEM. SOC, vol. 131, 2009, pages 8720 - 8721
FISHER KJ; JOOSS K; ALSTON J; YANG Y; HAECKER SE; HIGH K; PATHAK R; RAPER SE; WILSON JM: "Recombinant adeno-associated virus for muscle directed gene therapy", NAT MED, vol. 3, no. 3, 1997, pages 306 - 312
GRAY SJ; SAMULSKI RJ: "Optimizing gene delivery vectors for the treatment of heart disease", EXPERT OPIN BIOL THER, vol. 8, no. 7, 2008, pages 911 - 922
GRIMM D; KAY MA: "From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy", CURR GENE THER, vol. 3, no. 4, 2003, pages 281 - 304
GUO P; EL-GOHARY Y; PRASADAN K; SHIOTA C; XIAO X; WIERSCH J; PAREDES J; TULACHAN S; GITTES GK: "Rapid and simplified purification of recombinant adeno-associated virus", J VIROL METHODS, vol. 183, no. 2, pages 139 - 146
HAUSWIRTH WW; ALEMAN TS; KAUSHAL S; CIDECIYAN AV; SCHWARTZ SB; WANG L; CONLON TJ; BOYE SL; FLOTTE TR; BYRNE BJ ET AL.: "Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial.", HUM GENE THER, vol. 19, no. 10, 2008, pages 979 - 990
J. W. CHIN ET AL., JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 124, 2002, pages 9026 - 9027
J. W. CHIN; P. G. SCHULTZ, CHEMBIOCHEM, vol. 11, 2002, pages 1135 - 1137
JIANMING XIE; PETER G SCHULTZ: "Adding amino acids to the genetic repertoire", CURRENT OPINION IN CHEMICAL BIOLOGY, vol. 9, 2005, pages 548 - 554
JOO KI; FANG Y; LIU Y; XIAO L; GU Z; TAI A; LEE CL; TANG Y; WANG P: "Enhanced real-time monitoring of adeno-associated virus trafficking by virus-quantum dot conjugates", ACS NANO, vol. 5, no. 5, pages 3523 - 3535
L. WANG ET AL., SCIENCE, vol. 292, 2001, pages 498 - 500
MANNO CS; CHEW AJ; HUTCHISON S; LARSON PJ; HERZOG RW; ARRUDA VR; TAI SJ; RAGNI MV; THOMPSON A; OZELO M ET AL.: "AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B.", BLOOD, vol. 101, no. 8, 2003, pages 2963 - 2972
MANNO CS; PIERCE GF; ARRUDA VR; GLADER B; RAGNI M; RASKO JJ; OZELO MC; HOOTS K; BLATT P; KONKLE B ET AL.: "Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.", NAT MED, vol. 12, no. 3, 2006, pages 342 - 347
MINGOZZI F; HIGH KA: "Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges.", NAT REV GENET, vol. 12, no. 5, pages 341 - 355
MOSS RB; RODMAN D; SPENCER LT; AITKEN ML; ZEITLIN PL; WALTZ D; MILLA C; BRODY AS; CLANCY JP; RAMSEY B ET AL.: "Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial", CHEST, vol. 125, no. 2, 2004, pages 509 - 521
MUELLER C; FLOTTE TR: "Clinical gene therapy using recombinant adeno-associated virus vectors", GENE THER, vol. 15, no. 11, 2008, pages 858 - 863
NING X; GUO J; WOLFERT MA; BOONS GJ: "Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions", ANGEW CHEM INT ED ENGL, vol. 47, no. 12, 2008, pages 2253 - 2255
ROHR, U. P.; WULF, M. A.; STAHN, S.; STEIDL, U.; HAAS, R.; KRONENWETT, R., J VIROL METHODS, vol. 106, 2002, pages 81 - 8
See also references of EP3070095A4 *
TRAVIS S.: "Young and Peter G. Schultz: Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon", J BIOL. CHEM., vol. 285, 2010, pages 11039 - 11044
WHITE SJ; NICKLIN SA; BUNING H; BROSNAN MJ; LEIKE K; PAPADAKIS ED; HALLEK M; BAKER AH: "Targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-associated virus vectors", CIRCULATION, vol. 109, no. 4, 2004, pages 513 - 519
WORK LM; BUNING H; HUNT E; NICKLIN SA; DENBY L; BRITTON N; LEIKE K; ODENTHAL M; DREBBER U; HALLEK M ET AL.: "Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses", MOL THER, vol. 13, no. 4, 2006, pages 683 - 693
WU Z; ASOKAN A; SAMULSKI RJ: "Adeno-associated virus serotypes: vector toolkit for human gene therapy", MOL THER, vol. 14, no. 3, 2006, pages 316 - 327
YU CY; YUAN Z; CAO Z; WANG B; QIAO C; LI J; XIAO X: "A muscle-targeting peptide displayed on AAV2 improves muscle tropism on systemic delivery", GENE THER, vol. 16, no. 8, 2009, pages 953 - 962

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US20210230231A1 (en) * 2016-06-15 2021-07-29 Howard Hughes Medical Institute Variant adeno-associated viruses and methods of using
US11939355B2 (en) * 2016-06-15 2024-03-26 Howard Hughes Medical Institute Variant adeno-associated viruses and methods of using
CN109996880A (zh) * 2016-08-18 2019-07-09 加利福尼亚大学董事会 基于模块化aav递送系统的crispr-cas基因组工程
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RU2793112C2 (ru) * 2017-10-16 2023-03-29 ФиГенерон ГмбХ Векторы aav
WO2021005210A1 (en) 2019-07-11 2021-01-14 Centre National De La Recherche Scientifique Chemically-modified adeno-associated virus
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US11981914B2 (en) 2023-09-25 2024-05-14 Ginkgo Bioworks, Inc. Recombinant adeno-associated virus vectors

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