阅读说明：本技术 具有脑靶向特性的aav突变体 (AAV mutants with brain targeting properties ) 是由 西江敏和 高岛扶有子 榎龙嗣 峰野纯一 田中佳典 于 2020-04-23 设计创作，主要内容包括：本发明提供编码腺相关病毒(AAV)衣壳蛋白突变体的核酸,所述突变体含有包含选自SEQ ID No.15-62的氨基酸序列的肽或者包含通过在选自SEQ ID No.15-62的氨基酸序列中置换、缺失、插入和/或添加一个或数个氨基酸残基产生的氨基酸序列的肽；包含该核酸的DNA；具有该DNA的细胞；以及产生该细胞的方法。(The present invention provides nucleic acids encoding mutants of adeno-associated virus (AAV) capsid proteins, said mutants comprising a peptide comprising an amino acid sequence selected from SEQ ID nos. 15-62 or a peptide comprising an amino acid sequence generated by substitution, deletion, insertion and/or addition of one or several amino acid residues in an amino acid sequence selected from SEQ ID nos. 15-62; DNA comprising the nucleic acid; a cell having the DNA; and a method for producing the cell.)

1. Nucleic acid encoding an adeno-associated virus (AAV) capsid protein mutant, said mutant comprising a peptide comprising an amino acid sequence selected from SEQ ID NOs 15-62 or a peptide comprising an amino acid sequence which differs from an amino acid sequence selected from SEQ ID NOs 15-62 by substitution, deletion, insertion and/or addition of one or several amino acids.

2. The nucleic acid according to claim 1, wherein the AAV capsid protein is derived from AAV 2.

3. The nucleic acid according to claim 2, wherein the peptide is placed at a position in VP1 of AAV2 between amino acid number 588 and amino acid number 589.

4. A recombinant DNA comprising a nucleic acid according to any one of claims 1 to 3.

5. A cell comprising a nucleic acid according to any one of claims 1 to 3 or a recombinant DNA according to claim 4.

6. An AAV particle comprising an AAV capsid protein mutant, said mutant comprising a peptide comprising an amino acid sequence selected from SEQ ID NOs 15-62 or a peptide comprising an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NOs 15-62 by substitution, deletion, insertion and/or addition of one or several amino acids.

7. The AAV particle according to claim 6, wherein the AAV capsid protein is derived from AAV 2.

8. The AAV particle of claim 7, wherein the peptide is disposed at a position in VP1 of AAV2 between amino acid number 588 and amino acid number 589.

9. A method for producing a gene-transduced cell, the method comprising the step of contacting an AAV particle comprising an AAV capsid protein mutant comprising a peptide comprising an amino acid sequence selected from SEQ ID NOs 15-62 or a peptide comprising an amino acid sequence which differs from an amino acid sequence selected from SEQ ID NOs 15-62 by substitution, deletion, insertion and/or addition of one or several amino acids with the cell.

10. The method according to claim 9, wherein the AAV capsid protein is derived from AAV 2.

11. The method according to claim 10, wherein the peptide is placed at a position in VP1 of AAV2 between amino acid number 588 and amino acid number 589.

Technical Field

The present invention relates to nucleic acids encoding adeno-associated virus (AAV) capsid protein mutants with brain tropism, AAV particles comprising the capsid protein variants, and methods of generating gene transduced cells by using the particles.

Background

AAV is a virus having a linear single-stranded DNA genome of 4.7 kb (an open reading frame comprising two genes, rep and cap). The Rep gene encodes four proteins necessary for genome replication (Rep78, Rep68, Rep52, and Rep 40). The cap gene expresses the three capsid proteins (VP1, VP2, VP3) that assemble to form the viral capsid, and the assembly-activating protein (AAP). Replication of AAV in nature is dependent on the presence of helper viruses (e.g., adenovirus or herpes virus). In the absence of helper virus, the AAV genome remains in episomes, or integrates into the host chromosome, so that the AAV is present in a latent state. More than one hundred serotypes and clades of AAV have been identified (non-patent document 1). In particular, the development of AAV 2-based gene delivery vectors has been advanced.

In 1989, a gene delivery vector system based on AAV2 was first developed. AAV-based vectors have been found to have a number of advantages. AAV-based vectors are considered to be very safe since wild-type AAV is non-pathogenic and has no etiologic relationship with any known disease. In addition, AAV has high gene transduction efficiency.

Administration of AAV particles achieves long-term and stable gene transduction into various target organs and target cells. Up to now, gene transduction has been reported with high efficiency into skeletal muscle, liver (liver cells), heart (heart muscle cells), nerve cells, pancreatic cells and pancreatic islet cells. In addition, AAV has been used in human clinical trials. On the other hand, attempts have been made to alter the cellular tropism of AAV by altering AAV capsid proteins, and to avoid removal of AAV particles by neutralizing antibodies. For example, AAV capsids having tropism for specific organs and cells such as glial cells, airway epithelial cells, coronary artery endothelial cells, and lungs, and AAV capsids having tropism for tumor cells such as glioblastoma cells, melanoma cells, lung cancer cells, and breast cancer cells have been produced (non-patent document 2).

CITATION LIST

Non-patent document

Non-patent document 1: gao et al, J. Virology, Vol. 78, pp. 6381-

Non-patent document 2: adachi K. et al, Gene ther. Regul, Vol.5, pp. 31-55, 2010

Summary of The Invention

Problems to be solved by the invention

The object of the present invention includes providing AAV capsid protein mutants having cerebral tropism, and providing a method for efficiently introducing genes into the brain.

Solution to this problem

The present inventors have made intensive efforts to solve the above problems and, as a result, have produced desired AAV particles comprising an AAV capsid protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 15-62. Thus, the present invention has been completed.

The present invention relates generally to:

[1] nucleic acid encoding an adeno-associated virus (AAV) capsid protein mutant, said mutant comprising a peptide comprising an amino acid sequence selected from SEQ ID NOs 15-62 or a peptide comprising an amino acid sequence which differs from an amino acid sequence selected from SEQ ID NOs 15-62 by substitution, deletion, insertion and/or addition of one or several amino acids;

[2] the nucleic acid according to [1], wherein the AAV capsid protein is derived from AAV 2;

[3] the nucleic acid according to [2], wherein the peptide is placed at a position between amino acid number 588 and amino acid number 589 in VP1 of AAV 2;

[4] a recombinant DNA comprising the nucleic acid according to any one of [1] to [3 ];

[5] a cell comprising the nucleic acid according to any one of [1] to [3] or the recombinant DNA according to [4 ];

[6] an AAV particle comprising an AAV capsid protein mutant comprising a peptide comprising an amino acid sequence selected from SEQ ID NOs 15-62 or a peptide comprising an amino acid sequence that differs from the amino acid sequence selected from SEQ ID NOs 15-62 by substitution, deletion, insertion and/or addition of one or several amino acids;

[7] the AAV particle according to [6], wherein the AAV capsid protein is derived from AAV 2;

[8] the AAV particle according to [7], wherein the peptide is placed at a position between amino acid number 588 and amino acid number 589 in VP1 of AAV 2;

[9] a method for producing a gene-transduced cell, the method comprising the step of contacting an AAV particle comprising an AAV capsid protein mutant comprising a peptide comprising an amino acid sequence selected from SEQ ID NOs 15-62 or a peptide comprising an amino acid sequence which differs from an amino acid sequence selected from SEQ ID NOs 15-62 by substitution, deletion, insertion and/or addition of one or several amino acids with the cell;

[10] the method according to [9], wherein the AAV capsid protein is derived from AAV 2; and

[11] the method according to [10], wherein the peptide is placed at a position between amino acid number 588 and amino acid number 589 in VP1 of AAV 2.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, a gene transduction system useful for gene transduction into the brain is provided. The AAV particles of the present invention have high cellular tropism for the brain, and genes transduced by the AAV particles can be strongly expressed.

Brief Description of Drawings

FIG. 1 illustrates a method for generating a nucleic acid construct for enabling a capsid protein to comprise a random peptide.

FIG. 2 shows the results of evaluation of tropism of AAV capsid protein mutants of the present invention.

Modes for carrying out the invention

As used herein, "adeno-associated virus" refers to a virus belonging to the genus dependovirus (Dependovirus) Of parvoviridae of family (a)Parvoviridae) And can infect primates, including humans, and other mammals. Hereinafter, adeno-associated virus is abbreviated AAV. AAV has a non-enveloped, regular icosahedral capsid (capsid) and linear single-stranded DNA within the capsid. As used herein, AAV encompasses wild-type viruses and derivatives thereof, and, unless specifically stated otherwise, encompasses all serotypes and clades of AAV.

As used herein, "vector" means a molecule or associated molecule for mediating delivery of a polynucleotide to a cell, and which comprises or is associated with a polynucleotide. Unless otherwise specifically stated, examples of vectors include vector DNA (such as plasmid vectors and phage vectors), viral vector particles, liposomes, and other carriers for gene delivery.

As used herein, "capsid protein" means a protein encoded by a cap gene present in the AAV genome and constituting the AAV capsid. The wild-type AAV genome encodes three capsid proteins, present as VP1, VP2, and VP 3. As used herein, capsid proteins comprise VP1, VP2, and VP 3.

As used herein, the term "several" in the context of amino acid substitutions, deletions, insertions and/or additions means, depending on the length of the reference amino acid sequence, for example, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids.

(1) Nucleic acids encoding AAV capsid protein mutants

The nucleic acids of the invention encode mutants of AAV capsid proteins, comprising a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 15-62 or a peptide comprising an amino acid sequence that differs from an amino acid sequence selected from the group consisting of SEQ ID NO 15-62 by substitution, deletion, insertion and/or addition of one or several amino acids. Preferably, the nucleic acid of the invention encodes an AAV capsid protein mutant comprising a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to SEQ ID NO: 62. More preferably, the nucleic acid of the invention encodes an AAV capsid protein mutant comprising a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19 and SEQ ID NO: 20 or a peptide comprising an amino acid sequence that differs from a sequence selected from SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19 and SEQ ID NO: 20, or a peptide having an amino acid sequence different from that of the amino acid sequence of 20. A peptide comprising an amino acid sequence that differs from the amino acid sequence shown by SEQ ID NO as described above by substitution, deletion, insertion and/or addition of one or several amino acids, when comprised in an AAV capsid protein, retains the cellular tropism of an AAV capsid protein mutant comprising a peptide comprising the amino acid sequence shown by SEQ ID NO as described above. In other words, the number of amino acids that are substituted, deleted, inserted, and/or added in the amino acid sequence shown by the above-mentioned SEQ ID NO is not limited as long as the peptide containing the amino acid sequence shown by the above-mentioned SEQ ID NO is retained to confer cellular tropism to the AAV capsid protein mutant comprising the peptide. Substitutions, deletions and/or insertions are for example 1 to 5, preferably 1 to 4, more preferably 1, 2 or 3 amino acids. For example, 1 to 9, preferably 1 to 8, more preferably 1 to 7, still more preferably 1 to 6, still more preferably 1 to 5, still more preferably 1, 2, 3 or 4 amino acids may be added.

For example, the peptide to be included in the AAV capsid protein mutant can be a peptide comprising an amino acid sequence identical to a sequence selected from SEQ ID NOs: 15 to SEQ ID NO: 62 having an amino acid sequence of at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity. A peptide comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to the amino acid sequence set forth in SEQ ID NO above, when comprised in an AAV capsid protein, retains cellular tropism for AAV capsid protein mutants comprising a peptide comprising the amino acid sequence set forth in SEQ ID NO above.

For example, the peptide to be included in the AAV capsid protein mutant may be a peptide comprising an amino acid sequence represented by the formula:

formula I: x¹X²GX³GWV；

Formula II: x⁴X⁵X⁶X⁷GWV；

Formula III: x⁸X⁹GX¹⁰X¹¹WV；

Formula IV: x¹²X¹³GX¹⁴GX¹⁵V；

Formula V: x¹⁶X¹⁷GX¹⁸GWX¹⁹(ii) a Or

Formula VI: x²⁰X²¹GX²²REX²³，

Wherein X¹To X²³Each of (a) is an arbitrary amino acid residue, G represents glycine, W represents tryptophan, V represents valine, R represents arginine, and E represents glutamic acid. Preferably, in formula I (X)¹X²GX³GWV), X¹Is E (glutamic acid), G (glycine) or T (threonine), and X²Is R (arginine), T (threonine), S (serine), N (asparagine), E (glutamic acid) or D (aspartic acid), and X³Is V (valine), H (histidine), R, M (methionine) or L (leucine). Preferably, in formula II (X)⁴X⁵X⁶X⁷GWV), X⁴Is A (alanine) or E, X⁵Is D, G or A, X⁶Is K (lysine), Q (glutamine) or N, and X⁷Is V or L. Preferably, in formula III (X)⁸X⁹GX¹⁰X¹¹WV) in which X is⁸Is A, E or G, X⁹Is S, D, G or R, X¹⁰Is T, M, D or V, and X¹¹R, V, S or T. Preferably, in formula IV (X)¹²X¹³GX¹⁴GX¹⁵In V), X¹²Is D, E, G or R, X¹³Is A, G, D or V, X¹⁴Is I, H, D, F (phenylalanine), G or L, and X¹⁵Is Y (tyrosine), F, R, G or V. Preferably, in the formulaV(X¹⁶X¹⁷GX¹⁸GWX¹⁹) In, X¹⁶Is A, E or G, X¹⁷Is G, R or S, X¹⁸Is V, H or D, and X¹⁹T, G, K, I (isoleucine) or A. Preferably, in formula VI (X)²⁰X²¹GX²²REX²³) In, X²⁰Is E or A, X²¹Is Y or H, X²²Is F or Y, and X²³Is G or P (proline).

Examples of peptides containing the amino acid sequence shown in formula I include, but are not limited to: comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 18. 20, 21, 22, 46, 55, 59, and 60, and peptides comprising amino acid sequences that are identical to those of SEQ ID NOs: 18. 20, 21, 22, 46, 55, 59, and 60, respectively. Examples of peptides containing the amino acid sequence shown in formula II include, but are not limited to: comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 25. 29 and 32, and peptides comprising amino acid sequences that are identical to those of a sequence selected from SEQ ID NOs: 25. 29 and 32, respectively. Examples of peptides containing the amino acid sequence shown in formula III include, but are not limited to: comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 15. 24, 38, 48 and 54, and peptides comprising amino acid sequences that are identical to those of a sequence selected from the group consisting of SEQ ID NOs: 15. 24, 38, 48 and 54, respectively. Examples of peptides containing the amino acid sequence shown in formula IV include, but are not limited to: comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 19. 31, 35, 44, 56, and 58, and peptides comprising amino acid sequences that are identical to those of SEQ ID NOs: 19. 31, 35, 44, 56 and 58, which differ in amino acid sequence. Examples of peptides containing the amino acid sequence shown in formula V include, but are not limited to: comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 26. 39, 42, 43, 47 and 50, and peptides comprising amino acid sequences that are identical to those of SEQ ID NOs: 26. 39, 42, 43, 47 and 50, respectively. Examples of peptides containing the amino acid sequence shown in formula VI include, but are not limited to: comprises a polypeptide consisting of SEQ ID NO: 16 or 17, and a peptide comprising an amino acid sequence consisting of a substitution, deletion, insertion and/or addition of 1 to 4 amino acids with a sequence represented by SEQ ID NO: 16 or 17, or a peptide having an amino acid sequence different from that shown in seq id no.

An AAV capsid protein mutant encoded by the nucleic acid of the present invention can be prepared by inserting a peptide into an AAV capsid protein of any wild type AAV (e.g., AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3A, AAV3B, etc.), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV), or by substituting the peptide for a part of the amino acid sequence of the AAV capsid protein (in other words, by allowing the AAV capsid protein to comprise the peptide). In the present invention, the capsid protein of AAV2 is preferably used.

The mutant AAV capsid proteins encoded by the nucleic acids of the invention may be proteins comprising one or more or several amino acid substitutions, deletions, insertions and/or additions in the wild type AAV capsid protein as well as the peptides described above. The "protein comprising one or more or several amino acid substitutions, deletions, insertions and/or additions and the above peptides" retains the properties of the original protein, for example, the cellular tropism of the AAV capsid protein mutant conferred by the above peptides, the capsid-forming ability, the function of the capsid protein (e.g., protection of the viral genome, uncoating after entry into a host cell), and the like.

Further, spacer sequences may be added to the N-terminus and/or C-terminus of the peptides contained in the AAV capsid proteins. The spacer sequence preferably consists of 1 to 5 amino acid residues. The amino acid residues constituting the spacer sequence are particularly limited. For example, the spacer sequence may comprise an amino acid selected from glycine, alanine and serine.

AAV VP1, VP2, or VP3 can be used as AAV capsid proteins comprising peptides. Only any one of VP1, VP2, and VP3 may be made to include a peptide, or VP1, VP2, and VP3 may all be made to include a peptide. In addition, two capsid proteins (e.g., VP1 and VP2, VP2 and VP3, or VP1 and VP3) can be made to comprise peptides. VP1 through VP3 are encoded in the AAV genome by the cap gene region. In one embodiment of the invention, the region shared by VP1 through VP3 is made to comprise a peptide, such that mutations can be introduced into all three of VP1 through VP 3. In another embodiment of the present invention, a gene encoding VP1, VP2, or VP3 is prepared separately from the cap gene region of AAV, and a mutation is introduced into the gene. In this case, a treatment may be performed which suppresses the expression of a wild-type capsid protein corresponding to a capsid protein encoded by a gene into which a mutation has been introduced from the cap gene region of AAV.

In the case of AAV2 VP1, the AAV capsid protein mutant encoded by the nucleic acid of the invention preferably comprises a peptide at a position between amino acid number 588 and amino acid number 589. Amino acid number 588 of AAV2 VP1 is arginine. AAV2 VP1 has amino acid number 589 of glutamine. Amino acid number 588 of AAV2 VP1 corresponds to amino acid number 451 of AAV2 VP2 and amino acid number 386 of AAV2 VP 3. In the case of using AAV serotypes other than AAV2 and capsid proteins of clades as AAV capsid proteins, the AAV capsid proteins are made to comprise peptides between amino acids corresponding to amino acid numbers 588 and 589 of AAV2 VP 1. One skilled in the art can readily determine the amino acids of the capsid proteins of AAV serotypes and clades other than AAV2 that correspond to amino acid number 588 of AAV2 VP 1. See, for example, alignment of the VP1 amino acid sequences shown in Gao et al, Proc. Natl. Acad. Sci. USA, Vol.99, No.18, pp. 11854-11859, 2002. For example, amino acid number 588 of AAV2 VP1 corresponds to amino acid number 589 of AAV1, amino acid number 590 of AAV7, and amino acid number 591 of AAV 8.

The nucleic acids of the invention may be operably linked to suitable control sequences. Examples of control sequences include promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, Internal Ribosome Entry Sites (IRES), and enhancers. Examples of promoter sequences include inducible promoter sequences and constitutive promoter sequences. The control sequences may be endogenous or exogenous to the AAV from which the capsid protein is derived, natural sequences, or synthetic sequences. The invention also includes such recombinant DNA capable of expressing AAV capsid protein mutants.

The recombinant DNA of the invention is useful for delivering the nucleic acid of the invention to cells in vitro, ex vivo, or in vivo, as well as conferring the ability to express AAV capsid protein mutants to cells. The cells to which the nucleic acids of the invention are delivered are then useful for the production of AAV particles. Recombinant DNA is particularly useful for delivering or introducing the nucleic acid of the invention into animal cells, preferably mammalian cells.

In the present invention, the recombinant DNA of the present invention can be prepared by retaining the nucleic acid of the present invention using the DNA as a vector. For example, plasmid DNA, phage DNA, transposon, cosmid DNA, episomal DNA, or viral genome can be used.

(2) Cells containing the nucleic acids of the invention

The present invention also provides a host cell, e.g., an isolated host cell, comprising a nucleic acid of the present invention, in particular, a recombinant DNA as described in (1) above. For example, the isolated cell is a cell line maintained in vitro. As set forth below, the host cells of the invention are useful for the production of AAV particles of the invention. When using the host cells of the invention to produce AAV particles, the host cells may be referred to as "packaging cells" or "producer cells. The host cell of the invention may comprise the recombinant DNA of the invention as described in (1) above integrated into the genome, or the recombinant DNA may be retained in the cell for transient expression of the AAV capsid protein mutant.

Introduction of the recombinant DNA of the present invention into a host cell can be carried out by a known method. For example, electroporation, calcium phosphate precipitation, direct cell-entry microinjection, liposome-mediated gene transfection, or nucleic acid delivery using a high-speed particle gun can be used. When a viral vector is used, an infection method suitable for the vector can be selected. By using such established techniques, the recombinant DNA of the present invention is stably introduced into the chromosome of the host cell or transiently introduced into the cytoplasm of the host cell. For stable transformation, a selection marker, for example, a well-known selection marker such as a neomycin resistance gene (encoding neomycin phosphotransferase) or a hygromycin B resistance gene (encoding Aminoglycoside Phosphotransferase (APH)), may be linked to the recombinant DNA of the present invention.

Various cells, such as mammalian cells including mouse cells and primate cells (e.g., human cells) or insect cells, can be used as host cells. Examples of suitable mammalian cells include, but are not limited to, primary cells and cell lines. Examples of suitable cell lines include 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO cells and cells derived therefrom.

(3) AAV particles comprising an AAV capsid protein comprising an amino acid sequence encoded by a nucleic acid of the invention

The AAV particle of the present invention is an AAV particle comprising an AAV capsid protein mutant comprising the peptide as described in (1) above. The AAV particle of the present invention can be produced from the host cell as described in (2) above. The AAV particle of the present invention has tropism for the brain, and is useful for introducing a gene into the brain. The brain includes brain cells such as neuronal cells and glial cells (microglia, oligodendrocytes, astrocytes). The gene introduced by the AAV particle of the present invention is strongly expressed in the above-mentioned tissues, organs and cells.

To produce AAV particles, cells comprising some of the elements necessary for production of AAV particles can be used as packaging cells. The first element is the vector genome (also referred to as an expression vector) of the recombinant AAV, which can replicate in a host cell and is packaged in an AAV particle. The recombinant AAV vector genome comprises a heterologous polynucleotide of interest and AAV Inverted Terminal Repeat (ITR) sequences located on each side (i.e., 5 '-and 3' -side) of the heterologous polynucleotide of interest. The heterologous polynucleotide of interest may have control sequences for expression. The nucleotide sequence of the ITR sequence is known. For example, see Human Gene Therapy, Vol.5, pp. 793-801, 1994 for AAV2-ITR sequences. An ITR sequence derived from any of various AAV serotypes including AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, and the like can be used as an AAV ITR sequence. The ITR sequences used in the present invention may be derived from wild-type AAV or may be altered by insertion, deletion or substitution of nucleotides. The ITR sequences enable the recombinant AAV vector genome to replicate in the presence of the Rep proteins and the recombinant AAV vector genome to be incorporated into capsid particles in the formation of AAV particles.

The size of the heterologous polynucleotide of interest that can be loaded into an AAV particle of the invention is typically less than about 5 kilobases (kb). For example, the heterologous polynucleotide of interest can be a gene encoding a protein of interest lacking or deleted from the receptor, a gene encoding a protein having a desired biological or therapeutic activity (e.g., antimicrobial, antiviral, or antitumor activity), a desired nucleotide sequence encoding an RNA that inhibits or reduces production of a deleterious or undesirable protein, or a nucleotide sequence encoding an antigenic protein. The heterologous polynucleotide of interest may be appropriately selected according to the purpose.

In one embodiment of the invention, the recombinant AAV vector genome lacks a cap gene region and/or a rep gene region. In this embodiment, the AAV particle (in which the recombinant AAV vector genome is packaged) does not replicate alone in infected cells to reform the AAV particle.

A second element necessary for production of AAV particles is a construct that provides the protein encoded in wild-type AAV. The constructs encode AAV-derived genes that provide AAV gene products required for AAV particle formation. In other words, the construct comprises one or both of the major AAV ORFs (the rep gene region and the coding region of the cap gene region). To produce the AAV particles of the invention, at least the nucleic acid encoding the AAV capsid protein mutant comprising a nucleotide sequence having a sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 62. The host cell of the present invention capable of expressing the mutant as described in (2) above can be used for production of AAV particles. AAV particles have a shell composed of many capsid proteins. All capsid proteins may be mutant, or some capsid proteins may be mutant and others may be wild-type capsid proteins. AAV particles of the invention can comprise one capsid protein mutant or multiple capsid protein mutants.

The Rep gene of AAV is contained in the coding region of the Rep gene, and it includes genes encoding replication proteins Rep78, Rep68, Rep52 and Rep 40. These Rep expression products have been shown to have a number of functions, including recognition, binding and nicking of the AAV genomic DNA origin of replication, DNA helicase activity, and regulation of transcription from AAV-derived promoters.

The third element necessary for production of AAV particles is the helper viral function (also referred to as accessory function) for AAV replication. Adenoviruses are commonly used to introduce helper functions. However, other viruses, such as herpes simplex virus type 1 or 2 and vaccinia virus, may also be used. When using viruses, the host cell is infected with the virus as a helper virus. For example, since packaging of AAV particles requires only expression of the adenovirus early genes, adenoviruses that do not exhibit expression of late genes may also be used. Adenovirus mutants lacking late gene expression (e.g., ts100K or ts149 adenovirus variants) may also be used. Nucleic acid constructs providing helper virus function can also be prepared by using nucleic acids isolated from helper virus necessary for helper virus function, which can then be introduced into host cells. Constructs providing helper virus function comprise nucleotide sequences providing one or more helper virus functions and are provided to a host cell in the form of a plasmid, phage, transposon, cosmid or other virus.

To produce an AAV particle, there are performed (a) a step of introducing a first element, i.e., a recombinant AAV vector genome, into a host cell, (b) a step of introducing a second element, i.e., a construct providing AAV helper functions, into a host cell, and (c) a step of introducing a third element, i.e., helper virus functions, into a host cell. These steps may be performed simultaneously, or may be performed sequentially. The order of steps (a) to (c) may be in any order. When the first to third elements are introduced into the host cell, the rep gene expression product excises and replicates the recombinant vector genome. The expressed capsid proteins form a capsid, and the recombinant vector genome is packaged in the capsid to produce AAV particles. When the AAV capsid protein mutant is expressed by the host cell, the coat of AAV particles produced comprises the AAV capsid protein mutant.

AAV particles can be isolated and purified from culture supernatants or lysates of host cells by various purification methods, such as CaCl density gradient centrifugation. For example, when viruses are used in step (c) above, a step of separating AAV particles from helper virus based on their size may be added. AAV particles can also be isolated from helper viruses based on their different affinities for heparin. In addition, the remaining helper virus can be inactivated by known methods. For example, the adenovirus may be inactivated by heating at about 60 ℃, e.g., for 20 minutes or more. Since AAV particles are very stable to heat, the above treatment is effective for selectively removing adenovirus used as a helper virus.

(4) Method for producing Gene-transduced cells of the present invention

The AAV particle of the present invention obtained by (3) above is used to deliver a heterologous polynucleotide of interest to a cell for gene therapy purposes or other purposes. AAV particles are typically introduced into cells in vivo or in vitro. For in vitro introduction, AAV particles are contacted with cells obtained from a living body. Then, the cells can also be transplanted into a living body. For introducing cells into a living body, the cells are formulated as pharmaceutical compositions, and may be administered using various techniques such as intramuscular, intravenous, subcutaneous, and intraperitoneal. For in vivo transduction, AAV particles are formulated as pharmaceutical compositions, and they are typically administered parenterally (e.g., by intramuscular, subcutaneous, intratumoral, transdermal, or intraspinal routes). Pharmaceutical compositions comprising AAV particles may contain a pharmaceutically acceptable carrier, and if necessary other agents, drugs, stabilizers, carriers, adjuvants, diluents, and the like.

Examples

Hereinafter, the present invention will be described with reference to examples, but the present invention is not particularly limited to the examples.

Example 1: preparation of AAV2 random peptide plasmid library

From a distributed host E.coli (Escherichia coli) Plasmid vector pAV1 carrying AAV2 genome was extracted from HB101 (ATCC no: 37215). Genomic DNA (about 4.7 kb) of AAV2 was excised from the extracted plasmid using restriction enzyme BgIII (manufactured by TAKARA BIO Inc.). The genomic DNA was inserted into pUC118 BamHI/BAP (manufactured by TAKARA BIO Inc.). The plasmid DNA thus obtained was designated AAV2WG/pUC 118.

AAV2WG/pUC118 was digested with restriction enzyme ScaI (produced by TAKARA BIO Inc.) to obtain an about 0.8 kb fragment containing nucleotides 1190 to 2017 of the cap gene. This fragment was inserted into pUC118 HincII/BAP (manufactured by TAKARA BIO Inc.). The plasmid DNA thus obtained was designated Cap-ScaI/pUC 118. Then, PCR was performed on Cap-ScaI/pUC118 to perform a series of changes in which the nucleotide sequence AAC (587N) consisting of nucleotides 1759 to 1761 of the Cap gene in Cap-ScaI/pUC118 was converted into CAG (587Q), 10 nucleotides consisting of GGC as a spacer, CAAG as a filler, and GCC as a spacer were inserted between the nucleotides 1764 and 1765, and the nucleotide sequence CAA (589Q) GCA (590A) GCT (591A) consisting of nucleotides 1765 to 1773 was converted into CAG (589Q) GCG (590A) GCC (591A), where the letters in the parentheses show the amino acid numbers and the encoded amino acids. Thus, two recognition sites for the restriction enzyme SfiI and a spacer, a filler and a spacer between the SfiI recognition sites were inserted. FIG. 1 shows the nucleotide sequences of Cap gene before and after nucleotide 1756 to 1773 conversion. The nucleotide sequence before conversion is shown in SEQ ID NO: 1. the converted nucleotide sequence is shown in SEQ ID NO: 2. the plasmid DNA containing the converted nucleotide sequence was designated Cap-ScaI-S4/pUC 118. For in-fusion cloning, the part of the Cap gene in Cap-ScaI-S4/pUC118 was amplified by PCR to obtain a fragment of about 0.8 kb. This fragment was used as insert DNA.

AAV2WG/pUC118 was subjected to PCR to introduce mutations into the recognition site for the restriction enzyme ScaI in the ampicillin resistance gene and the recognition site for the restriction enzyme SfiI in the Rep gene so that these recognition sites were converted into sequences not recognized by the restriction enzymes. For the ScaI recognition site, the nucleotide sequence GAG (E) consisting of nucleotides 304 to 306 of the ampicillin resistance gene was converted into GAA (E). The sequence before conversion is shown in SEQ ID NO: 3 and the converted sequence is shown in SEQ ID NO: 4. for the SfiI recognition site, the nucleotide sequence GCC (A) consisting of nucleotides 217 to 219 of Rep gene is converted into GCA (A). The sequence before conversion is shown in SEQ ID NO: 5, and the converted sequence is shown in SEQ ID NO: 6. the plasmid DNA thus obtained was digested with ScaI (produced by TAKARA BIO Inc.) to obtain a linear vector of about 0.8 kb lacking as a part of the Cap gene. This was used as a linear vector for in-fusion cloning.

The insert DNA was inserted into a linear vector using an In-Fusion (registered trademark) HD cloning kit (manufactured by Clontech Laboratories, inc.) and a cloning enhancer (manufactured by Clontech Laboratories, inc.), and thereby directional cloning was performed. The plasmid DNA thus obtained was designated AAV2WG-Cap-ScaI-S4/pUC118 Sx.

An oligo DNA (SEQ ID NO: 7) comprising a nucleotide sequence encoding a random peptide of 7 amino acids was produced by artificial synthesis. Double-stranded DNA was prepared from the oligomeric DNA by reaction with a primer (SEQ ID NO: 8) and Klenow fragment (produced by TAKARA BIO Inc.) at 37 ℃ for 3 hours. The double-stranded DNA was purified using a Nucleotide removal kit (manufactured by QIAGEN), and then digested with a restriction enzyme BglI (manufactured by TAKARA BIO Inc.). This DNA was inserted into AAV2WG-Cap-ScaI-S4/pUC118Sx digested with SfiI using DNA ligation kit "Mighty Mix" (manufactured by TAKARA BIO Inc.). The plasmid thus obtained was designated AAV2WG-RPL/pUC118Sx, and used as AAV2 random peptide plasmid library.

Example 2: preparation of AAV2 random peptide Virus library

(1) Inoculation of AAV293 cells

Cultured AAV293 cells (produced by Stratagene Corp.) were collected and then subjected to culturing in DMEM (produced by Sigma) containing 10% FBS and 2 mM sodium L-glutamate at 5X 10⁴Individual cells/mL were suspended. 40 mL of the suspension containing AAV293 cells was placed in T225 cm for cell culture²In a bottle (manufactured by Corning Incorporated), and then in CO₂The cells were incubated at 37 ℃ for 72 hours in an incubator.

(2) Introduction of plasmids into AAV293 cells

AAV293 CELLs were transfected by a general calcium phosphate method using 400 ng of AAV2WG-RPL/pUC118Sx obtained in example 1 and 40. mu.g of pHELP (CELL BIOLABS, manufactured by Inc.). Six hours after transfection, the medium was completely removed. After addition of 40 mL of DMEM containing 2% FBS and 2 mM sodium L-glutamate, cells were placed in CO₂The cells were incubated at 37 ℃ for 48 hours in an incubator.

(3) Collection of AAV2 random peptide Virus library

0.5 mL of 0.5M EDTA was added to the reaction vesselIncubated T225 cm²In a bottle, then left to stand for a few minutes. AAV293 cells were then stripped by pipetting and collected into 50 mL tubes and centrifuged at 300 x g for 10 minutes. The supernatant was then removed. Cells were resuspended in 2 mL TBS (Tris buffered saline) per vial and then subjected to three consecutive treatments consisting of freezing with ethanol/dry ice for 15 minutes, thawing in a 37 ℃ water bath for 15 minutes, and vortexing for 1 minute to collect cell lysates containing the AAV random peptide virus library. To cell lysate 5. mu.L of 1M MgCl was added per 1 mL TBS₂And a Benzonase (registered trademark) nuclease (manufactured by Merck KGaA) was added to the reaction mixture at a final concentration of 200U/mL, followed by reaction at 37 ℃ for 30 minutes. Then, the reaction was stopped by adding 6.5. mu.L of 0.5M EDTA per 1 mL of TBS. The cell lysate was centrifuged at 10000 rpm at 4 ℃ for 10 minutes, and then the supernatant was collected as an AAV vector solution.

(4) Quantification of AAV vector solution Titers by real-time PCR

mu.L of 10 XDNaseI buffer, 15.2. mu.L of water for injection (Otsuka Pharmaceutical Co., Ltd.) and 0.8. mu.L of DNaseI (TAKARA BIO Inc.) were added to 2. mu.L of AAV vector solution, and the mixture was incubated at 37 ℃ for 1 hour to remove free genomic DNA and plasmid DNA. To inactivate the DNaseI, the mixture was heated at 99 ℃ for 10 minutes. Then, 15. mu.L of water for injection, 4. mu.L of 10 XPROK buffer (0.1M Tris-HCl (pH 7.8), 0.1M EDTA, 5% SDS) and 1. mu.L of proteinase K (manufactured by TAKARA BIO Inc.) were added, and the mixture was incubated at 55 ℃ for 1 hour. Then, to inactivate proteinase K, the mixture was heated at 95 ℃ for 10 minutes. Quantification of AAV titer was carried out on this sample using SYBR (registered trademark) Premix ExTaq2 (manufactured by TAKARA BIO Inc.) and primers (SEQ ID NO: 9 and SEQ ID NO: 10) according to the instructions attached to the kit. The sample was diluted 50-fold with water for injection, and 2 μ L of the diluted solution was used for titer quantification. Linear DNA obtained by digesting pAV1 with a restriction enzyme was used as a standard.

Example 3: purification of AAV random peptide virus libraries

(1) Purification by cesium chloride density gradient centrifugation 1

In a 40PA tube (HITACHI-KOKI co., ltd., product) for ultracentrifugation, 4 mL of a cesium chloride solution having a density adjusted to 1.5, 4 mL of a cesium chloride solution having a density adjusted to 1.25, and 28 mL of the AAV vector solution prepared in example 2- (4) were put in this order from the bottom part layer. The tube was centrifuged at 25000 rpm for 3 hours at 16 ℃ with an ultracentrifuge HIMAC (HITACHI-KOKI Co., Ltd.). After centrifugation, 28 mL of solution was removed from the upper portion of the tube, and then 0.7 mL aliquots of the solution were successively collected from the upper portion into 1.5 mL tubes. In the same manner as in example 2- (4), the titer of AAV vector contained in each of the collected solutions was quantified.

(2) Purification by cesium chloride density gradient centrifugation 2

Cesium chloride solution adjusted to a density of 1.39 was added to several fractions with high titer shown in example 3- (1) to reach a total volume of 10.5 mL. The thus-obtained solution was put into a 13PA tube (HITACHI-KOKI co., ltd., product) for ultracentrifugation, and then centrifuged at 38000 rpm for 16 hours at 18 ℃. After centrifugation, 0.7 mL aliquots of the solution were collected from the upper portion of the tube one after the other. In the same manner as in example 2- (4), the titer of AAV vector contained in each of the collected solutions was quantified.

(3) Desalting by dialysis

The fractions shown to have high titers in example 3- (2) were mixed and then added to Slide-A-lyzer dialysis card (manufactured by Pierce). The purified AAV solution was desalted by dialysis against 1L Phosphate Buffered Saline (PBS) twice at 4 ℃ for 3 hours and against 500 mL PBS/5% sorbitol solution overnight at 4 ℃. The solution was then collected, sterilized with a 0.22 μm filter (manufactured by Millipore) and stored at-80 ℃ until just before use. The titer of each purified AAV particle was quantified in the same manner as in example 2- (4).

Example 4: AAV2 random peptide library screening

(1) Intravenous administration to the mouse tail

The purified AAV particle obtained in example 3- (3) was purified at 1.5X 10¹⁴Viral Genome (VG)/kg was administered to BALB/c mice via tail vein. After 72 hours of administration, brains were collected by using NucleoSpin (registered trademark) tissue (MACHEREY-NAGEL GmbH)&Raw material of Co and KGProduct) extraction of genomic DNA (round 1).

(2) Re-cloning of random peptide sequences by PCR

A DNA encoding a random peptide sequence was amplified using the genomic DNA extracted in example 4- (1) as a template and PrimeSTAR (registered trademark) GXL DNA polymerase (manufactured by TAKARA BIO inc.). As primers, forward primer 1(SEQ ID NO: 11) and reverse primer 1(SEQ ID NO: 12) were used. PCR was performed by repeating 30 cycles, and each cycle of PCR consisted of 98 ℃ for 10 seconds, 55 ℃ for 15 seconds, and 68 ℃ for 40 seconds. One twenty-fifth of the PCR reaction solution, forward primer 2(SEQ ID NO: 13) and reverse primer 2(SEQ ID NO: 14) were then used to prepare the same amount of reaction mixture as before. The reaction mixture was subjected to 30 cycles of PCR, where each cycle consisted of 98 ℃ for 10 seconds, 55 ℃ for 15 seconds, and 68 ℃ for 15 seconds. The DNA was purified from the thus-obtained reaction solution by using Nucleospin extract II (produced by MACHEREY-NAGEL GmbH & Co. KG), and digested with the restriction enzyme BglI. After electrophoresis, the digested product was purified by using Nucleospin extract II and re-cloned into AAV2WG-Cap-ScaI-S4/pUC118Sx prepared in example 1 by using DNA ligation kit "Mighty Mix".

(3) Production and purification of AAV2 random peptide virus library

AAV2 random peptide virus library production and purification of AAV particles were carried out in the same methods as those described in example 2 and example 3 using the plasmid obtained in example 4- (2).

(4) Screening

Screening (administration of AAV particles to mice and collection of brains) and extraction of genomic DNA (round 2) were performed in the same manner as in example 4- (1). Furthermore, using the extracted genomic DNA, re-cloning, library generation and purification, and screening were performed again, and genomic DNA was extracted (round 3).

(5) Random peptide sequencing

At each screening stage (rounds 1 to 3), sequencing was performed on the AAV random peptide plasmid library. Peptide sequences encoded by clones accumulated in the brain in rounds 2 and 3 and the frequency of appearance are shown in tables 1 and 2.

[ Table 1]

[ Table 2]

As shown in tables 1 and 2, AAV having a capsid comprising a particular peptide sequence accumulates in the brain. In particular, it was suggested that the peptide sequences GSGVTLV (SEQ ID NO: 15), AHGYREP (SEQ ID NO: 16), EYGFREG (SEQ ID NO: 17) and ETGHGWV (SEQ ID NO: 18) observed in round 3 were prone to infect the brain.

Furthermore, SEQ ID NO: 63 to SEQ ID NO: 110, which is a sequence comprising the sequence observed in round 2 and the spacer. In particular GGSGVTWVA (SEQ ID NO: 63), GAHGYREPA (SEQ ID NO: 64), GEYGFREGA (SEQ ID NO: 65) and GETGHGWVA (SEQ ID NO: 66), which are sequences comprising the sequence observed in round 3 and a spacer, are prone to infect the brain.

Example 5: evaluation of tropism of AAV vectors with acquired peptide sequences

(1) Construction of pRC-GDDGTRG having the obtained peptide sequence

AAV2WG-Cap-ScaI-S4/pUC118Sx clone having the peptide sequence (SEQ ID NO: 15-20) as obtained in example 4- (5) was digested with restriction enzymes SnaBI (produced by TAKARA BIO Inc.) and HindIII (produced by TAKARA BIO Inc.) to obtain a fragment. This fragment was ligated to a vector fragment obtained by digesting pAAVRC2 vector (produced by CELL BIOLABS, Inc.) with SnaBI and HindIII by a DNA ligation kit "Mighty Mix" (produced by TAKARA BIO Inc.) to obtain helper plasmids pRC-GSGVTRV, pRC-AHGYREP, pRC-EYGFREG, pRC-ETGHGWV, pRC-GGGIGYV and pRC-ERGVGLV.

(2) Production and purification of AAV2-LacZ capsid mutants

pAAV-LacZ (manufactured by TAKARA BIO Inc.), pHELP, and pRC helper plasmids (pRC-GSGVTEV, pRC-AHGYREP, pRC-EYGFREG, pRC-ETGHG) prepared in example 5- (1) were usedWV, pRC-GGGIGYV or pRC-ERGVGLV) in T255 cm²293T cells in the flask were transfected with PEIpro (produced by Polyplus Transfection). As a control, transfection was performed with pRC2 vector carrying wild-type capsid instead of the pRC helper plasmid with peptide sequence. In CO₂The transfected 293T cells were cultured in an incubator at 37 ℃ for 72 hours. From T255 cm²The supernatant containing AAV was collected in a flask, and then subjected to affinity purification using AVB agarose gel (produced by GE healthcare). Then, AAV was concentrated and purified by ultrafiltration to prepare a purified AAV solution. Then, the titer of AAV vector was quantified by the method described in example 2- (4).

(3) Administration of purified AAV solution to mice

The purified AAV solution obtained in example 5- (2) was filtered using a 0.22 μm filter, then passed through the tail vein at 0.5 x 10¹¹VG/mouse was administered to mice.

(4) Preparation of genomic DNA from brain and other tissues and quantification of AAV genome

Mice (to which AAV was administered in example 5- (3)) were euthanized 4 weeks after administration, and each tissue was collected. Genomic DNA was extracted from each tissue by using NucleoSpin tissue (produced by MACHEREY-NAGEL GmbH & Co. KG). The extracted genomic DNA was used as a sample to perform real-time PCR to determine the amount of AAV vector genome contained in each tissue. FIG. 2 shows the number of AAV genomic DNA molecules per 1. mu.g of total genomic DNA in each tissue.

As can be seen in figure 2, AAV vectors comprising capsids with the peptide sequences GSGVTWV, AHGYREP, EYGFREG, ETGHGWV, GGGIGYV, and erggwv tend to translocate to the brain. Further, some AAV vectors have tropism for the lung as well as the brain.

Industrial applicability

According to the present invention, AAV capsid protein mutants having cerebral tropism by systemic administration and their amino acid sequences are provided, and thus a method for efficiently introducing genes into the brain is provided.

Sequence Listing free text

SEQ ID NO: 1: AAV2 capsid 586-591 coding sequence

SEQ ID NO: 2: transformed AAV2 capsid coding sequences

SEQ ID NO: 3: ampicillin resistance gene before conversion

SEQ ID NO: 4: ampicillin resistance gene after conversion

SEQ ID NO: 5: AAV2 rep gene before conversion

SEQ ID NO: 6: transformed AAV2 rep gene

SEQ ID NO: 7: DNA sequence encoding a random peptide

SEQ ID NO: 8: primer for synthesizing double-stranded DNA

SEQ ID NO: 9: forward primers for quantification of AAV titers

SEQ ID NO: 10: reverse primer for quantification of AAV titers

SEQ ID NO: 11: forward primer 1 for amplification of random peptide coding region

SEQ ID NO: 12: reverse primer 1 for amplification of random peptide coding region

SEQ ID NO: 13: forward primer 2 for amplification of random peptide coding region

SEQ ID NO: 14: reverse primer 2 for amplification of random peptide coding region

SEQ ID NO: 15: peptide sequence GSGVTRV of AAV capsid protein mutant

SEQ ID NO: 16: peptide sequence AHGYREP of AAV capsid protein mutant

SEQ ID NO: 17: peptide sequence EYGFREG of AAV capsid protein mutant

SEQ ID NO: 18: peptide sequence ETGHGWV of AAV capsid protein mutant

SEQ ID NO: 19: peptide sequence GGGIGYV of AAV capsid protein mutant

SEQ ID NO: 20: peptide sequence ERGVGLV of AAV capsid protein mutant

SEQ ID NO: 21: peptide sequence of AAV capsid protein mutant ENGVGLV

SEQ ID NO: 22: peptide sequence GSGVGWV of AAV capsid protein mutant

SEQ ID NO: 23: peptide sequence ADGITWG of AAV capsid protein mutants

SEQ ID NO: 24: peptide sequence ADGTRWV of AAV capsid protein mutant

SEQ ID NO: 25: peptide sequence ADKVGWV of AAV capsid protein mutant

SEQ ID NO: 26: peptide sequence AGGVGWT of AAV capsid protein mutants

SEQ ID NO: 27: peptide sequence AGGVTDV of AAV capsid protein mutant

SEQ ID NO: 28: peptide sequence AGNAGGM of AAV capsid protein mutant

SEQ ID NO: 29: peptide sequence AGQLGWV of AAV capsid protein mutant

SEQ ID NO: 30: peptide sequence ARGTEWE of AAV capsid protein mutant

SEQ ID NO: 31: peptide sequence DAGGFV of AAV capsid protein mutant

SEQ ID NO: 32: peptide sequence of AAV capsid protein mutant EANVGWV

SEQ ID NO: 33: peptide sequence of AAV capsid protein mutant ECGLGEG

SEQ ID NO: 34: peptide sequence EGEVTWL of AAV capsid protein mutant

SEQ ID NO: 35: peptide sequence EGGDGRV of AAV capsid protein mutant

SEQ ID NO: 36: peptide sequence EGGFGEA of AAV capsid protein mutant

SEQ ID NO: 37: peptide sequence EGGG of AAV capsid protein mutant

SEQ ID NO: 38: peptide sequence EGGMVWV of AAV capsid protein mutant

SEQ ID NO: 39: peptide sequence of AAV capsid protein mutant EGGVGWT

SEQ ID NO: 40: peptide sequence EGGMWL of AAV capsid protein mutant

SEQ ID NO: 41: peptide sequence EGQVTWL of AAV capsid protein mutant

SEQ ID NO: 42: peptide sequence ERGHGWG of AAV capsid protein mutant

SEQ ID NO: 43: peptide sequence ESGVGWK of AAV capsid protein mutant

SEQ ID NO: 44: peptide sequence GDGFGGV of AAV capsid protein mutant

SEQ ID NO: 45: peptide sequence GDGVTRA of AAV capsid protein mutant

SEQ ID NO: 46: peptide sequence GEGRGWV of AAV capsid protein mutant

SEQ ID NO: 47: peptide sequence GGGDGWI of AAV capsid protein mutant

SEQ ID NO: 48: peptide sequence GGGDSWV of AAV capsid protein mutant

SEQ ID NO: 49: peptide sequence GGGIAWVAQAAL of AAV capsid protein mutant

SEQ ID NO: 50: peptide sequence GGGVGWA of AAV capsid protein mutant

SEQ ID NO: 51: peptide sequence GKGQVME of AAV capsid protein mutant

SEQ ID NO: 52: peptide sequence GNGTGGG of AAV capsid protein mutant

SEQ ID NO: 53: peptide sequence GQGGHME of AAV capsid protein mutant

SEQ ID NO: 54: peptide sequence GRGVTRV of AAV capsid protein mutant

SEQ ID NO: 55: peptide sequence GSGMGWV of AAV capsid protein mutant

SEQ ID NO: 56: peptide sequence GVGGGGVG of AAV capsid protein mutant

SEQ ID NO: 57: peptide sequence NDVRGRV of AAV capsid protein mutant

SEQ ID NO: 58: peptide sequence RDGLGFV of AAV capsid protein mutant

SEQ ID NO: 59: peptide sequence TDGLGWV of AAV capsid protein mutant

SEQ ID NO: 60: peptide sequence of AAV capsid protein mutant TEGHGWV

SEQ ID NO: 61: peptide sequence VAERLYG of AAV capsid protein mutant

SEQ ID NO: 62: peptide sequence VARGAGE of AAV capsid protein mutant

SEQ ID NO: 63: peptide sequence GGSGVTWVA of AAV capsid protein mutant

SEQ ID NO: 64: peptide sequence GAHGYREPA of AAV capsid protein mutant

SEQ ID NO: 65: peptide sequence GEYGFREGA of AAV capsid protein mutant

SEQ ID NO: 66: peptide sequence GETGHGWVA of AAV capsid protein mutant

SEQ ID NO: 67: peptide sequence GGGGIGYVA of AAV capsid protein mutant

SEQ ID NO: 68: peptide sequence GERGVGWVA of AAV capsid protein mutant

SEQ ID NO: 69: peptide sequence GENGVGWVA of AAV capsid protein mutant

SEQ ID NO: 70: peptide sequence GGSGVGWVA of AAV capsid protein mutant

SEQ ID NO: 71: peptide sequence GADGITWGA of AAV capsid protein mutant

SEQ ID NO: 72: peptide sequence GADGTRWVA of AAV capsid protein mutant

SEQ ID NO: 73: peptide sequence GADKVGWVA of AAV capsid protein mutant

SEQ ID NO: 74: peptide sequence GAGGVGWTA of AAV capsid protein mutant

SEQ ID NO: 75: peptide sequence GAGGVTGVA of AAV capsid protein mutant

SEQ ID NO: 76: peptide sequence GAGNAGGMA of AAV capsid protein mutant

SEQ ID NO: 77: peptide sequence GAGQLGWVA of AAV capsid protein mutant

SEQ ID NO: 78: peptide sequence GARGTEWEA of AAV capsid protein mutant

SEQ ID NO: 79: peptide sequence GDAGHGFVA of AAV capsid protein mutant

SEQ ID NO: 80: peptide sequence GEANVGWVA of AAV capsid protein mutant

SEQ ID NO: 81: peptide sequence GECGLGEGA of AAV capsid protein mutant

SEQ ID NO: 82: peptide sequence GEGEVTWLA of AAV capsid protein mutant

SEQ ID NO: 83: peptide sequence GEGGDGRVA of AAV capsid protein mutant

SEQ ID NO: 84: peptide sequence GEGGFGEAA of AAV capsid protein mutant

SEQ ID NO: 85: peptide sequence GEGGGA of AAV capsid protein mutant

SEQ ID NO: 86: peptide sequence GEGGMVWVA of AAV capsid protein mutant

SEQ ID NO: 87: peptide sequence GEGGVGWTA of AAV capsid protein mutant

SEQ ID NO: 88: peptide sequence GEGGVMWLA of AAV capsid protein mutant

SEQ ID NO: 89: peptide sequence GEGQVTWLA of AAV capsid protein mutant

SEQ ID NO: 90: peptide sequence GERGHGWGA of AAV capsid protein mutant

SEQ ID NO: 91: peptide sequence GESGVGWKA of AAV capsid protein mutant

SEQ ID NO: 92: peptide sequence GGDGFGGVA of AAV capsid protein mutant

SEQ ID NO: 93: peptide sequence GGDGVTWAA of AAV capsid protein mutant

SEQ ID NO: 94: peptide sequence GGEGRGWVA of AAV capsid protein mutant

SEQ ID NO: 95: peptide sequence GGGGDGWIA of AAV capsid protein mutant

SEQ ID NO: 96: peptide sequence GGGGDSWVA of AAV capsid protein mutant

SEQ ID NO: 97: peptide sequence GGGGIAWVAQAALA of AAV capsid protein mutant

SEQ ID NO: 98: peptide sequence GGGGVGWAA of AAV capsid protein mutant

SEQ ID NO: 99: peptide sequence GGKGQVMEA of AAV capsid protein mutant

SEQ ID NO: 100: peptide sequence GGNGTGGGA of AAV capsid protein mutant

SEQ ID NO: 101: peptide sequence GGQGGHMEA of AAV capsid protein mutant

SEQ ID NO: 102: peptide sequence GGRGVTWVA of AAV capsid protein mutant

SEQ ID NO: 103: peptide sequence GGSGMGWVA of AAV capsid protein mutant

SEQ ID NO: 104: peptide sequence GGVGGGVVA of AAV capsid protein mutant

SEQ ID NO: 105: peptide sequence GNDVRGRVA of AAV capsid protein mutant

SEQ ID NO: 106: peptide sequence GRDGLGFVA of AAV capsid protein mutant

SEQ ID NO: 107: peptide sequence GTDGLGWVA of AAV capsid protein mutant

SEQ ID NO: 108: peptide sequence GTEGHGWVA of AAV capsid protein mutant

SEQ ID NO: 109: peptide sequence GVAERLYGA of AAV capsid protein mutant

SEQ ID NO: 110: peptide sequence GVARGAGEA of AAV capsid protein mutant

SEQ ID NO: 111: peptide sequences represented by formula I

SEQ ID NO: 112: peptide sequences represented by formula II

SEQ ID NO: 113: peptide sequences represented by formula III

SEQ ID NO: 114: peptide sequences represented by formula IV

SEQ ID NO: 115: a peptide sequence represented by formula V

SEQ ID NO: 116: a peptide sequence represented by formula VI.