Nucleic acid combination and use thereof

Abstract

The present invention relates to a nucleic acid combination, comprising a first nucleic acid molecule and a second nucleic acid molecule. The first nucleic acid molecule and the second nucleic acid molecule respectively express one or two of VP1, VP2, and VP3. The types of VP1, VP2, and VP3 expressed by the first nucleic acid molecule and the second nucleic acid molecule are different, and the nucleic acid combination can express all VP1, VP2, and VP3 proteins. The nucleic acid combination may further comprise a third nucleic acid molecule. In addition, the present invention also relates to a vector comprising the nucleic acid combination, a vector combination, a recombinant AAV virion, a cell, and / or a pharmaceutical composition.

Description

Nucleic acid combination and use thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a nucleic acid combination encoding an adeno-associated virus (AAV) capsid protein. BACKGROUND

[0002] Adeno-associated virus (AAV) vectors are extremely potential gene therapy vectors, with non-pathogenicity, low immunogenicity, stable physical properties and long-term stable expression of exogenous genes, and are the preferred viral vectors in current gene therapy development, and are widely used in clinical experiments of gene therapy. However, the existing AAV vectors still have the following defects: 1, off-target effect, that is, in addition to the target organ, it can also be taken up by various tissue organs; 2, naturally occurring neutralizing antibodies in patients. The presence of off-target effects and neutralizing antibodies reduces the biological effectiveness of AAV vectors, resulting in the need to use high-dose viruses for effective gene delivery, thereby bringing greater safety risks. In clinical trials and monitoring of marketed drugs, high-dose AAV has been clearly identified as being associated with an increase in patient mortality. Therefore, the development of new AAV vectors, enhancing the ability of organ-targeted infection, reducing off-target effects, and escaping naturally occurring neutralizing antibodies, can theoretically effectively reduce the working dose of AAV and improve the safety of human drug use.

[0003] AAV is a non-enveloped DNA virus, whose genome is wrapped by a positive icosahedral capsid protein layer composed of about 60 capsid proteins, with a size of about 20 nm. The capsid protein determines the infection ability of AAV to the target organ, and is also the epitope of neutralizing antibodies. Therefore, in order to improve the targeting of AAV and reduce the neutralizing ability of neutralizing antibodies, the capsid protein needs to be engineered. There are currently various strategies for engineering AAV capsid proteins: 1, finding new AAV viruses from nature and using them as vectors; 2, using DNA shuffling to randomly combine existing AAV capsid protein sequences, and obtaining new AAV capsid proteins through appropriate screening methods; 3, using error-prone PCR to randomly mutate the capsid protein sequence and screening new mutants; 4, rational design, that is, by mutating or inserting affinity polypeptides or sequences with targeting ability in specific regions of the AAV capsid protein sequence, to achieve the ability of targeted infection; 5, through computer-aided design or artificial intelligence technology, based on a large number of different types of AAV capsid protein structures and functions, to design new AAV vectors with better infection or lower neutralizing antibody neutralization ability.

[0004] The random sequences obtained by DNA shuffling or error-prone mutation need large-scale library construction and subsequent screening. However, due to the great difference between species, the results of screening using a mouse model may be difficult to achieve similar effects in humans. The use of computer-aided design is limited by the lack of accurate data on the infection ability of different capsid proteins of AAV in different tissues and organs, resulting in low calculation accuracy. Rational design of AAV capsid proteins has the characteristics of less workload and direct targeting of human tissue and organ target proteins, and can be used as an important way to engineer AAV capsid.

[0005] However, the AAV capsid contains VP1, VP2 and VP3 proteins, which are translated from different transcription splice bodies of the same gene, and the ratio of VP1:VP2:VP3 is about 1:1:10. Therefore, engineering the common part of VP1, VP2 and VP3 on the original capsid gene will result in modification of all three capsid proteins, which will affect the conformation of the capsid protein and cause the assembly of the virus particle to fail.

[0006] Therefore, there is an urgent need for a modification technology that can accurately manipulate the engineered capsid protein to avoid the adverse effects of the engineered capsid protein on AAV particle packaging. SUMMARY

[0007] To solve the above problems, the present application constructs a series of AAV virus capsid protein expression vectors, which can effectively express VP1, VP2 or VP3 in AAV alone, or express two capsid proteins (VP1 and VP2, VP1 and VP3, VP2 and VP3) at the same time. Through this technology, the present application can realize the separation of VP1, VP2 and VP3 into two or even three expression frames (or open reading frames) for expression, so as to realize the modification of only one kind of capsid protein without affecting the functional structure of other capsid proteins, and minimize the impact of modification on the overall AAV virus body.

[0008] Further, the present application tests the efficiency of different capsid protein expression plasmids for packaging AAV, and screens the plasmid combination capable of viral packaging. Finally, the present application tests one or more VP inserted with large fragment protein, and performs viral packaging and infection test, and finds that: 1) the large fragment protein can be integrated into the capsid protein and packaged into AAV particles; 2) the integration efficiency of the capsid protein-large fragment protein fusion protein into the AAV particles is dependent on the site of the large fragment protein insertion, and the integration efficiency is high at some sites and low at some sites; 3) the engineered capsid protein virus particles containing the large fragment protein insertion not only can improve the infection efficiency of cells expressing affinity ligands, but also can reduce the non-specific infection ability of the virus particles. The present application proves the rationality and effectiveness of the capsid protein split expression system for packaging AAV particles containing one or two large fragment insertion protein sequences. In addition, the nucleic acid molecule of the present application can also contain a sequence encoding a functional protein / peptide.

[0009] On this basis, the present application provides different nucleic acid combinations, which comprise two or three nucleic acid molecules.

[0010] In the first aspect, the nucleic acid combination comprises two nucleic acid molecules, which are a first nucleic acid molecule and a second nucleic acid molecule. The first nucleic acid molecule and the second nucleic acid molecule each independently comprises one or two of the nucleotide sequences encoding VP1, VP2 and VP3.

[0011] In a preferred embodiment, the first nucleic acid molecule and the second nucleic acid molecule each independently comprises a genetic mutation.

[0012] For example, both the first nucleic acid molecule and the second nucleic acid molecule comprise a genetic mutation.

[0013] In a more preferred embodiment, the genetic mutation occurs in the nucleotide sequence expressing the capsid protein on the first nucleic acid molecule and the second nucleic acid molecule. Specifically, the genetic mutation inhibits the expression of one or two capsid proteins, so that the first nucleic acid molecule and the second nucleic acid molecule express (or encode) two or one capsid proteins of VP1, VP2 and VP3. In some embodiments, the nucleic acid combination is just capable of expressing three capsid proteins VP1, VP2 and VP3.

[0014] For example, the expression of VP2 and VP3 is inhibited on the first nucleic acid molecule, so that the first nucleic acid molecule only expresses VP1; the expression of VP1 is inhibited on the second nucleic acid molecule, so that the second nucleic acid molecule only expresses VP2 and / or VP3.

[0015] For example, expression of VP1 and VP3 is inhibited on the first nucleic acid molecule, such that the first nucleic acid molecule only expresses VP2; expression of VP2 is inhibited on the second nucleic acid molecule, such that the second nucleic acid molecule only expresses VP1 and / or VP3.

[0016] For example, expression of VP1 and VP2 is inhibited on the first nucleic acid molecule, such that the first nucleic acid molecule only expresses VP3; expression of VP3 is inhibited on the second nucleic acid molecule, such that the second nucleic acid molecule only expresses VP1 and / or VP2.

[0017] The first nucleic acid molecule and the second nucleic acid molecule can express a capsid protein subunit of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh8, AAVrh8, AAV-DJ, AAV-LK03, AAV-PHP.B / PHP.eB / PHP.S, Anc80L65, etc.

[0018] In a first embodiment of the first aspect, the VP1, VP2 and VP3 expressed by the nucleic acid combination are VP1, VP2 and VP3 of AAV6.

[0019] In a first embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that inhibits expression of VP1 of AAV6. Meanwhile, the genetic mutation that inhibits expression of VP1 of AAV6 does not affect expression of VP2 of AAV6 and VP3 of AAV6.

[0020] In a preferred embodiment, the genetic mutation that inhibits expression of VP1 of AAV6 is a genetic mutation that results in RNA splicing cancellation. Specifically, the genetic mutation occurs at Splicing acceptor Al.

[0021] In a preferred embodiment, the genetic mutation that inhibits expression of VP1 of AAV6 is a genetic mutation that mutates the start codon on the nucleotide sequence encoding VP1 of AAV6 into a non-start codon. Specifically, the genetic mutation results in mutation of the codon corresponding to M1 site on VP1 protein of AAV6, preferably into a non-start codon, more preferably into a codon corresponding to M1L.

[0022] In a preferred embodiment, the genetic mutation that inhibits expression of VP1 of AAV6 is a genetic mutation that mutates a certain codon between the start codon on the nucleotide sequence encoding VP1 of AAV6 and the start codon on the nucleotide sequence encoding VP2 of AAV6 into a stop codon. Specifically, the genetic mutation results in mutation of the codon corresponding to Y6 and / or E12 site on VP1 protein of AAV6, preferably into a stop codon.

[0023] In a second embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP2 of AAV6.

[0024] In a preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV6 is a genetic mutation that results in the abrogation of RNA splicing. Specifically, the genetic mutation occurs at Splicing acceptor A2.

[0025] In a preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV6 is a genetic mutation that results in the abrogation of RNA splicing. Specifically, the genetic mutation occurs at Splicing acceptor A2.

[0026] In a third embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP3 of AAV6.

[0027] In a preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV6 is a genetic mutation that results in the abrogation of RNA splicing. Specifically, the genetic mutation occurs at Splicing acceptor A2.

[0028] In a preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV6 is a genetic mutation that results in the abrogation of RNA splicing. Specifically, the genetic mutation occurs at Splicing acceptor A2.

[0029] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP1 protein of AAV6 and the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP2 protein of AAV6.

[0030] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP1 protein of AAV6 and the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP3 protein of AAV6.

[0031] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP3 protein of AAV6 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP2 protein of AAV6.

[0032] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP1 protein of AAV6 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP2 protein of AAV6 and the VP3 protein of AAV6.

[0033] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP2 protein of AAV6 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP1 protein of AAV6 and the VP3 protein of AAV6.

[0034] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP3 protein of AAV6 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP1 protein of AAV6 and the VP2 protein of AAV6.

[0035] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M203L, M211L, and L235L (CTG-CTC) and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L, Y6*, and E12* (* is a stop codon).

[0036] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M203L, M211L, and L235L (CTG-CTC) and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138T (ACG-ACC).

[0037] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L and T138A and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138M, M203L, M211L, and L235L (CTG-CTC).

[0038] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M203L, M211L, and L235L (CTG-CTC) and the second nucleic acid molecule comprises a genetic mutation selected from the group consisting of a genetic mutation that occurs at Splicing acceptor Al.

[0039] In one specific example, the amino acid sequence of VP1 of AAV6 is set forth in SEQ ID NO: 4, the amino acid sequence of VP2 of AAV6 is set forth in SEQ ID NO: 5, and the amino acid sequence of VP3 of AAV6 is set forth in SEQ ID NO: 6. In one specific example, the nucleic acid sequence encoding VP1 of AAV6 is set forth in SEQ ID NO: 20, the nucleic acid sequence encoding VP2 of AAV6 is set forth in SEQ ID NO: 21, and the nucleic acid sequence encoding VP3 of AAV6 is set forth in SEQ ID NO: 22. When referring to the nucleic acid combination expressing AAV6 capsid proteins, the above sequences can be referred to. Since the mRNAs of VP1, VP2 and VP3 are identical in reading frame, the different proteins are mainly produced by translational initiation regulation, thus, the first nucleic acid molecule and the second nucleic acid molecule can be obtained by the genetic mutation to the nucleic acid molecules as set forth below: (1) a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 20 or 21, or (2) a nucleic acid molecule comprising a sequence having at least 90% or more (e.g., 92% or more, 95% or more, 98% or more, 99% or more) sequence identity to the sequence set forth in SEQ ID NO: 20 or 21, as long as the expressed VP1, VP2 and VP3 proteins are as described above.

[0040] In a second embodiment of the first aspect, the VP1, VP2 and VP3 expressed by the nucleic acid combination are VP1, VP2 and VP3 of AAV2.

[0041] In a first specific embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP1 of AAV2.

[0042] In a preferred embodiment, the genetic mutation that suppresses the expression of VP1 of AAV2 is a genetic mutation that results in the abrogation of RNA splicing. Specifically, the genetic mutation occurs at Splicing acceptor Al.

[0043] In a preferred embodiment, the genetic mutation that suppresses the expression of VP1 of AAV2 is a genetic mutation that mutates the start codon in the nucleotide sequence encoding VP1 of AAV2 to a non-start codon. Specifically, the genetic mutation results in the mutation of the codon corresponding to the M1 site in the VP1 protein of AAV2, preferably to a non-start codon, more preferably to the codon corresponding to M1L.

[0044] In a preferred embodiment, the genetic mutation that suppresses the expression of VP1 of AAV2 is a mutation of a codon between the start codon and the start codon of VP2 of AAV2 to a stop codon in the VP1-encoding nucleotide sequence of AAV2. In particular, the genetic mutation results in a mutation of the codon corresponding to the Y6 and / or E12 site of the VP1 protein of AAV2, preferably to a stop codon.

[0045] In a second particular embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP2 of AAV2.

[0046] In a preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV2 is a genetic mutation that results in the cancellation of RNA splicing. In particular, the genetic mutation occurs at Splicing acceptor A2.

[0047] In a preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV2 is a mutation of the start codon to a non-start codon in the VP2-encoding nucleotide sequence of AAV2. In particular, the genetic mutation results in a mutation of the codon corresponding to the T138 site of the VP2 protein of AAV2, preferably to a non-start codon. In a more preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV2 comprises a mutation of the T138 codon from ACG to ACC, in the form of a T138A codon mutation.

[0048] In a third particular embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP3 of AAV2.

[0049] In a preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV2 is a genetic mutation that results in the cancellation of RNA splicing. In particular, the genetic mutation occurs at Splicing acceptor A2.

[0050] In a preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV2 is a mutation of the start codon to a non-start codon in the VP3-encoding nucleotide sequence of AAV2. In particular, the genetic mutation results in a mutation of the codon corresponding to one or more of the T138, M203, M211, V221 and M235 sites of the VP3 protein of AAV2, preferably to a non-start codon. In a more preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV2 comprises a T138M, M203L, M211L and / or M235L codon mutation, and a mutation of the V221 codon from GTG to GTC.

[0051] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP1 protein of AAV2 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP2 protein of AAV2.

[0052] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP1 protein of AAV2 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP3 protein of AAV2.

[0053] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP3 protein of AAV2 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP2 protein of AAV2.

[0054] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP1 protein of AAV2 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP2 protein and a VP3 protein of AAV2.

[0055] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP2 protein of AAV2 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP1 protein and a VP3 protein of AAV2.

[0056] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP3 protein of AAV2 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP1 protein and a VP2 protein of AAV2.

[0057] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M203L, M211L, and M235L and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L, Y6*, and E12* (* is a stop codon).

[0058] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L, M203L, M211L, and M235L and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, T138T (ACG-ACC).

[0059] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L and T138A and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138M, M203L, M211L, V221V (GTG-GTC), and M235L.

[0060] In a specific embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M203L, M211L, and M235L, and the second nucleic acid molecule comprises a genetic mutation selected from the group consisting of a genetic mutation that occurs at Splicing acceptor Al.

[0061] In one specific example, the amino acid sequence of VP1 of AAV2 is set forth in SEQ ID NO: 7, the amino acid sequence of VP2 of AAV2 is set forth in SEQ ID NO: 8, and the amino acid sequence of VP3 of AAV2 is set forth in SEQ ID NO: 9. In one specific example, the nucleic acid sequence encoding VP1 of AAV2 is set forth in SEQ ID NO: 23, the nucleic acid sequence encoding VP2 of AAV2 is set forth in SEQ ID NO: 24, and the nucleic acid sequence encoding VP3 of AAV2 is set forth in SEQ ID NO: 25. When referring to a nucleic acid combination that expresses AAV2 capsid proteins, the above sequences can be referred to. Since the mRNAs of VP1, VP2, and VP3 are identical in reading frame, the different proteins are produced mainly by translational initiation regulation, and thus the first nucleic acid molecule and the second nucleic acid molecule can be modified from the nucleic acid molecules set forth below: (1) a nucleic acid molecule comprising a sequence set forth in SEQ ID NO: 23 or 24, or (2) a nucleic acid molecule comprising a sequence having at least 90% or more (e.g., 92% or more, 95% or more, 98% or more, 99% or more) sequence identity to a sequence set forth in SEQ ID NO: 23 or 24, as long as the expressed VP1, VP2, and VP3 proteins are as described above.

[0062] In a third embodiment of the first aspect, the nucleic acid combination expresses VP1, VP2, and VP3 of AAV5.

[0063] In a first specific embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP1 of AAV5.

[0064] In a preferred embodiment, the genetic mutation that suppresses the expression of VP1 of AAV5 is a genetic mutation that results in RNA splicing cancellation. Specifically, the genetic mutation occurs at Splicing acceptor Al.

[0065] In a preferred embodiment, the genetic mutation that suppresses the expression of VP1 of AAV5 is a mutation of the start codon in the VP1-encoding nucleotide sequence of AAV5 to a non-start codon. In particular, the genetic mutation results in a mutation of the codon corresponding to the Ml site in the VP1 protein of AAV5, preferably to a non-start codon, more preferably to MlL.

[0066] In a preferred embodiment, the genetic mutation that suppresses the expression of VP1 of AAV5 is a mutation of a codon between the N-terminal of the start codon and the start codon of VP2 of AAV5 in the VP1-encoding nucleotide sequence of AAV5 to a stop codon. In particular, the genetic mutation results in a mutation of the codon corresponding to the V4 and / or E13 site in the VP1 protein of AAV5, preferably to a stop codon.

[0067] In a second particular embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP2 of AAV5.

[0068] In a preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV5 is a genetic mutation that results in the abrogation of RNA splicing. In particular, the genetic mutation occurs at Splicing acceptor A2.

[0069] In a preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV5 is a mutation of the start codon in the VP2-encoding nucleotide sequence of AAV5 to a non-start codon. In particular, the genetic mutation results in a mutation of the codon corresponding to the T137 site in the VP2 protein of AAV5, preferably to a non-start codon. In a more preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV5 comprises a mutation of the T137 codon from ACG to ACC, T137A.

[0070] In a third particular embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP3 of AAV5.

[0071] In a preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV5 is a genetic mutation that results in the abrogation of RNA splicing. In particular, the genetic mutation occurs at Splicing acceptor A2.

[0072] In preferred embodiments, the genetic mutation that suppresses expression of the VP3 protein of AAV5 is a mutation of the start codon in the VP3-encoding nucleotide sequence of AAV5 to a non-start codon. Specifically, the genetic mutation results in a mutation of the codon corresponding to one or more of positions T137, M193, and M225 in the VP3 protein of AAV5, preferably to a non-start codon. In more preferred embodiments, the genetic mutation that suppresses expression of the VP3 protein of AAV5 comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T137M, M193L, and M225L.

[0073] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP1 protein of AAV5 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP2 protein of AAV5.

[0074] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP1 protein of AAV5 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP3 protein of AAV5.

[0075] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP3 protein of AAV5 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP2 protein of AAV5.

[0076] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP1 protein of AAV5 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP2 protein and the VP3 protein of AAV5.

[0077] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP2 protein of AAV5 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP1 protein and the VP3 protein of AAV5.

[0078] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP3 protein of AAV5 and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of the VP1 protein and the VP2 protein of AAV5.

[0079] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T137A, M193L, and M225L and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L, V4*, and E13* (* is a stop codon).

[0080] In a specific embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L, M193L, and M225L, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T137A, T137T (ACG-ACC).

[0081] In a specific embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L and T137A, and the second nucleic acid molecule comprises an amino acid mutation selected from the group consisting of T137M, M193L, and M225L.

[0082] In a specific embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T137A, M193L, and M225L, and the second nucleic acid molecule comprises a genetic mutation selected from the group consisting of a genetic mutation that occurs at Splicing acceptor A1.

[0083] In a specific embodiment, the first nucleic acid molecule comprises a genetic mutation selected from the group consisting of a genetic mutation that occurs at Splicing acceptor A2, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L, V4*, and E13* (* is a stop codon).

[0084] In a specific example, the amino acid sequence of VP1 of AAV5 is shown in SEQ ID NO: 10, the amino acid sequence of VP2 of AAV5 is shown in SEQ ID NO: 11, and the amino acid sequence of VP3 of AAV5 is shown in SEQ ID NO: 12. In a specific example, the nucleic acid sequence encoding VP1 of AAV5 is shown in SEQ ID NO: 26, the nucleic acid sequence encoding VP2 of AAV5 is shown in SEQ ID NO: 27, and the nucleic acid sequence encoding VP3 of AAV5 is shown in SEQ ID NO: 28. In referring to a combination of nucleic acids that express AAV5 capsid proteins, the above sequences can be referred to. Since the mRNAs of VP1, VP2, and VP3 are identical in reading frame, different proteins are produced mainly by translational initiation regulation, and thus the first nucleic acid molecule and the second nucleic acid molecule can be modified on the basis of the nucleic acid molecules shown below: (1) a nucleic acid molecule comprising a sequence shown in SEQ ID NO: 26 or 27, or (2) a nucleic acid molecule comprising a sequence having at least 90% or more (e.g., 92% or more, 95% or more, 98% or more, 99% or more) sequence identity to a sequence shown in SEQ ID NO: 26 or 27, as long as the expressed VP1, VP2, and VP3 proteins are as described above.

[0085] In a fourth embodiment of the first aspect, the nucleic acid combination expresses AAV9 VP1, VP2 and VP3 of AAV9 VP1, VP2 and VP3.

[0086] In a first embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of AAV9 VP1.

[0087] In a preferred embodiment, the genetic mutation that suppresses the expression of AAV9 VP1 is a genetic mutation that results in the abrogation of RNA splicing. In particular, the genetic mutation occurs at Splicing acceptor Al.

[0088] In a preferred embodiment, the genetic mutation that suppresses the expression of AAV9 VP1 is a genetic mutation that mutates the start codon in the AAV9 VP1-encoding nucleotide sequence into a non-start codon. In particular, the genetic mutation results in the mutation of the codon corresponding to the Ml site in the AAV9 VP1 protein, preferably into a non-start codon, more preferably M1L.

[0089] In a preferred embodiment, the genetic mutation that suppresses the expression of AAV9 VP1 is a genetic mutation that mutates a codon between the N-terminal of the start codon in the AAV9 VP1-encoding nucleotide sequence and the start codon of AAV9 VP2 into a stop codon. In particular, the genetic mutation results in the mutation of the codon corresponding to the Y6 and / or E12 site in the AAV9 VP1 protein, preferably into a stop codon.

[0090] In a second embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of AAV9 VP2.

[0091] In a preferred embodiment, the genetic mutation that suppresses the expression of AAV9 VP2 is a genetic mutation that results in the abrogation of RNA splicing. In particular, the genetic mutation occurs at Splicing acceptor A2.

[0092] In a preferred embodiment, the genetic mutation that suppresses the expression of AAV9 VP2 is a genetic mutation that mutates the start codon in the AAV9 VP2-encoding nucleotide sequence into a non-start codon. In particular, the genetic mutation results in the mutation of the codon corresponding to the T138 site in the AAV9 VP2 protein, preferably into a non-start codon. In a more preferred embodiment, the genetic mutation that suppresses the expression of AAV9 VP2 comprises a genetic mutation that mutates the T138 codon from ACG to ACC, resulting in T138A.

[0093] In a third embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP3 of AAV9.

[0094] In a preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV9 is a genetic mutation that results in an abrogation of RNA splicing. In particular, the genetic mutation occurs at Splicing acceptor A2.

[0095] In a preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV9 is a genetic mutation that mutates the start codon on the VP3-encoding nucleotide sequence of AAV9 to a non-start codon. In particular, the genetic mutation results in a mutation of one or more of the codons corresponding to T138, M203, L235 and L249 on the VP3 protein of AAV9, preferably to a non-start codon. In a more preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV9 comprises a mutation of the T138 codon to M, the M203 codon to L, the L235 codon from CTG to CTC, and the L249 codon from CTG to CTC.

[0096] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP1 protein of AAV9 and the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP2 protein of AAV9.

[0097] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP1 protein of AAV9 and the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP3 protein of AAV9.

[0098] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP3 protein of AAV9 and the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP2 protein of AAV9.

[0099] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP1 protein of AAV9 and the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP2 protein and the VP3 protein of AAV9.

[0100] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP2 protein of AAV9 and the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP1 protein and the VP3 protein of AAV9.

[0101] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP3 protein of AAV9 and the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP1 protein and the VP2 protein of AAV9.

[0102] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation leading to an amino acid mutation selected from the group consisting of T138A, M203L, L235L (CTG-CTC) and L249L (CTG-CTC), and the second nucleic acid molecule comprises a genetic mutation leading to an amino acid mutation selected from the group consisting of M1L, Y6* and E12* (* is a stop codon).

[0103] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation leading to an amino acid mutation selected from the group consisting of M1L, M203L, L235L (CTG-CTC) and L249L (CTG-CTC), and the second nucleic acid molecule comprises a genetic mutation leading to an amino acid mutation selected from the group consisting of T138A, T138T (ACG-ACC).

[0104] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation leading to an amino acid mutation selected from the group consisting of M1L and T138A, and the second nucleic acid molecule comprises a genetic mutation leading to an amino acid mutation selected from the group consisting of T138M, M203L, L235L (CTG-CTC) and L249L (CTG-CTC).

[0105] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation leading to an amino acid mutation selected from the group consisting of T138A, M203L, L235L (CTG-CTC) and L249L (CTG-CTC), and the second nucleic acid molecule comprises a genetic mutation selected from the group consisting of a genetic mutation occurring on Splicing acceptor A1.

[0106] In one specific example, the amino acid sequence of VP1 of AAV9 is set forth in SEQ ID NO: 13, the amino acid sequence of VP2 of AAV9 is set forth in SEQ ID NO: 14, and the amino acid sequence of VP3 of AAV9 is set forth in SEQ ID NO: 15. In one specific example, the nucleic acid sequence encoding VP1 of AAV9 is set forth in SEQ ID NO: 29, the nucleic acid sequence encoding VP2 of AAV9 is set forth in SEQ ID NO: 30, and the nucleic acid sequence encoding VP3 of AAV9 is set forth in SEQ ID NO: 31. When referring to the nucleic acid combination expressing AAV9 capsid proteins, the above sequences can be referred to. Since the mRNAs of VP1, VP2 and VP3 are identical in reading frame, the different proteins are mainly produced by translational initiation regulation, thus the first nucleic acid molecule and the second nucleic acid molecule can be modified based on the nucleic acid molecules as set forth below: (1) a nucleic acid molecule comprising a sequence as set forth in SEQ ID NO: 29 or 30, or (2) a nucleic acid molecule comprising a sequence having at least 90% or more (e.g., 92% or more, 95% or more, 98% or more, 99% or more) sequence identity to the sequence as set forth in SEQ ID NO: 29 or 30, as long as the expressed VP1, VP2 and VP3 proteins are as described above.

[0107] In a fifth embodiment of the first aspect, the nucleic acid combination expresses VP1, VP2 and VP3 of AAV8 as VP1, VP2 and VP3 of AAV8.

[0108] In a first specific embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP1 of AAV8.

[0109] In a preferred embodiment, the genetic mutation that suppresses the expression of VP1 of AAV8 is a genetic mutation that results in abrogation of RNA splicing. Specifically, the genetic mutation occurs at Splicing acceptor Al.

[0110] In a preferred embodiment, the genetic mutation that suppresses the expression of VP1 of AAV8 is a genetic mutation that mutates the start codon on the nucleotide sequence encoding VP1 of AAV8 to a non-start codon. Specifically, the genetic mutation results in mutation of the codon corresponding to the M1 site on the VP1 protein of AAV8, preferably to a non-start codon, more preferably to M1L.

[0111] In a preferred embodiment, the genetic mutation that suppresses the expression of VP1 of AAV8 is a mutation of a codon from the start codon N-terminally to the VP2 start codon of AAV8 to a stop codon in the VP1-encoding nucleotide sequence of AAV8. In particular, the genetic mutation results in a mutation of the codon corresponding to the Y6 and / or E12 site of the VP1 protein of AAV8, preferably to a stop codon.

[0112] In a second particular embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP2 of AAV8.

[0113] In a preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV8 is a genetic mutation that results in the abrogation of RNA splicing. In particular, the genetic mutation occurs at Splicing acceptor A2.

[0114] In a preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV8 is a mutation of the start codon in the VP2-encoding nucleotide sequence of AAV8 to a non-start codon. In particular, the genetic mutation results in a mutation of the codon corresponding to the T138 site of the VP2 protein of AAV8, preferably to a non-start codon. In a more preferred embodiment, the genetic mutation that suppresses the expression of VP2 of AAV8 comprises a mutation of the T138 codon from ACG to ACC, T138A.

[0115] In a third particular embodiment, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of VP3 of AAV8.

[0116] In a preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV8 is a genetic mutation that results in the abrogation of RNA splicing. In particular, the genetic mutation occurs at Splicing acceptor A2.

[0117] In a preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV8 is a mutation of the start codon in the VP3-encoding nucleotide sequence of AAV8 to a non-start codon. In particular, the genetic mutation results in a mutation of the codon corresponding to one or more of the T138, M204 and M212 sites of the VP3 protein of AAV8, preferably to a non-start codon. In a more preferred embodiment, the genetic mutation that suppresses the expression of VP3 of AAV8 comprises T138M, M204L and M212L.

[0118] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP1 protein of AAV8 and the second nucleic acid molecule comprises a genetic mutation that suppresses the expression of the VP2 protein of AAV8.

[0119] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP1 protein of AAV8 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP3 protein of AAV8.

[0120] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP3 protein of AAV8 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP2 protein of AAV8.

[0121] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP1 protein of AAV8 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP2 protein of AAV8 and a VP3 protein of AAV8.

[0122] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP2 protein of AAV8 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP1 protein of AAV8 and a VP3 protein of AAV8.

[0123] In certain embodiments, the first nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP3 protein of AAV8 and the second nucleic acid molecule comprises a genetic mutation that inhibits expression of a VP1 protein of AAV8 and a VP2 protein of AAV8.

[0124] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L, and M212L, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L (ATG-CTA), M204L, and M212L. In specific embodiments, the nucleic acid combination further comprises a third nucleic acid molecule, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L and T138A.

[0125] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L, and M212L, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of Y6* (TAA).

[0126] In specific embodiments, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L, and M212L, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of E12* (TAG).

[0127] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L and M212L, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L.

[0128] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L and M212L, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L.

[0129] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L and M212L, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L.

[0130] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M204L and M212L (ATG-CTC), and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138T.

[0131] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M204L and M212L (ATG-CTC), and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138M.

[0132] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L and T138A, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of T138M.

[0133] In a particular embodiment, the first nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L and T138A, and the second nucleic acid molecule comprises a genetic mutation that results in an amino acid mutation selected from the group consisting of M204L and M212L.

[0134] In one specific example, the amino acid sequence of VP1 of AAV8 is set forth in SEQ ID NO: 37, the amino acid sequence of VP2 of AAV8 is set forth in SEQ ID NO: 38, and the amino acid sequence of VP3 of AAV8 is set forth in SEQ ID NO: 39. In one specific example, the nucleic acid sequence encoding VP1 of AAV8 is set forth in SEQ ID NO: 40, the nucleic acid sequence encoding VP2 of AAV8 is set forth in SEQ ID NO: 41, and the nucleic acid sequence encoding VP3 of AAV8 is set forth in SEQ ID NO: 42. When referring to the nucleic acid combination expressing AAV8 capsid proteins, the above sequences can be referred to. Since the mRNAs of VP1, VP2 and VP3 are identical in reading frame, the different proteins are mainly produced by translational initiation regulation, therefore, the first nucleic acid molecule and the second nucleic acid molecule can be modified based on the nucleic acid molecules as set forth below: (1) a nucleic acid molecule comprising a sequence as set forth in SEQ ID NO: 41 or 42, or (2) a nucleic acid molecule comprising a sequence having at least 90% or more (e.g., 92% or more, 95% or more, 98% or more, 99% or more) sequence identity to the sequence as set forth in SEQ ID NO: 41 or 42, as long as the expressed VP1, VP2 and VP3 proteins are as described above.

[0135] In a further embodiment of the first aspect, the nucleic acid combination, the first nucleic acid molecule and / or the second nucleic acid molecule further comprises a gene of interest, which encodes a protein of interest.

[0136] In a preferred embodiment, the protein of interest has a molecular weight of about 1 KD to about 100 KD.

[0137] In a specific embodiment, the insertion site of the protein of interest is located in a region selected from the group consisting of: loop between βΒ and βΟ, variable region I, variable region II, variable region IV, variable region V, variable region VI, variable region VII, variable region VIII, HI loop, and variable region IX.

[0138] In a specific embodiment, the insertion site of the protein of interest is located in a region selected from the group consisting of: loop between βΒ and βΟ, variable region I, variable region II, variable region IV, variable region V, variable region VI, variable region VII, variable region VIII, HI loop, and variable region IX.

[0139] In more particular embodiments, the insertion site for the protein of interest is selected from one or more of the following: between N254 and H255, between S264 and G266, between D328 and G329, between G454 and S455, between N498 and S499, between D530 and K531, between A548 and G549, between S588 and D590, between S708 and A709, and, between N717 and N718.

[0140] In more particular embodiments, the insertion site for the protein of interest is selected from one or more of the following: between N254 and H255, between S264 and G266, between D328 and G329, between G454 and S455, between N498 and S499, between D530 and K531, between A548 and G549, between S588 and D590, between S663 and A664, between S708 and A709, and, between N717 and N718.

[0141] In particular embodiments, the insertion site for the protein of interest is selected from one or more of the following: between T492 and N493, between Q574 and S575, and between A581 and T582, using the VP protein of AAV5 as an example.

[0142] In particular embodiments, the insertion site for the protein of interest is selected from one or more of the following: between G453 and S454, between A589 and Q590, between N498 and S499, and between G530 and E531, using the VP protein of AAV9 as an example.

[0143] In certain embodiments, the protein of interest comprises one or more proteins selected from the group consisting of: a reporter protein, a therapeutic protein, and a prophylactic protein.

[0144] In certain embodiments, the protein of interest is a protein that alters AAV targeting. In certain embodiments, the protein of interest comprises an antigen binding protein. In certain embodiments, the antigen binding protein comprises an antibody or an antigen binding fragment thereof. In certain embodiments, the antibody comprises a nanobody, e.g., a TfRl nanobody.

[0145] In certain embodiments, the N-terminus and / or C-terminus of the protein of interest is connected to the VP protein via a linker. Exemplary linkers can be those shown in SEQ ID NO: 1, 2, 43, 44, or 45. For example, the linker for the N-terminus of the protein of interest is the linker shown in SEQ ID NO: 43, the linker for the C-terminus is the linker shown in SEQ ID NO: 44 and the linker shown in SEQ ID NO: 2. For example, the linker for the N-terminus of the protein of interest is the linker shown in SEQ ID NO: 43, the linker for the C-terminus is the linker shown in SEQ ID NO: 2. For example, the linker for the N-terminus of the protein of interest is the linker shown in SEQ ID NO: 44, the linker for the C-terminus is the linker shown in SEQ ID NO: 2. For example, the linker for the N-terminus of the protein of interest is the linker shown in SEQ ID NO: 45, the linker for the C-terminus is the linker shown in SEQ ID NO: 2.

[0146] For example, in the first nucleic acid molecule or the second nucleic acid molecule, the sequence encoding the protein of interest is inserted via the linker in the sequence encoding the VP protein.

[0147] In certain embodiments, the protein of interest can be an endosomal escape peptide.

[0148] In certain embodiments, two or more proteins of interest are inserted. In certain embodiments, the two or more proteins of interest are inserted into the first nucleic acid molecule. In certain embodiments, one of the two or more proteins of interest is inserted into the first nucleic acid molecule and the other protein(s) is inserted into the second nucleic acid molecule.

[0149] In further embodiments, the first nucleic acid molecule and / or the second nucleic acid molecule comprises a promoter, preferably a strong promoter, more preferably a CMV promoter.

[0150] In further embodiments, the nucleic acid combination further comprises a nucleic acid molecule encoding an assembly-activated protein (AAP).

[0151] In further embodiments, the first nucleic acid molecule and / or the second nucleic acid molecule further comprises other genetic mutations, preferably genetic mutations that alter the targeting.

[0152] In specific embodiments, the first nucleic acid molecule and the second nucleic acid molecule are located in different expression cassettes.

[0153] In specific embodiments, the first nucleic acid molecule and the second nucleic acid molecule are located in the same molecule in a tandem form.

[0154] In another specific embodiment, the first nucleic acid molecule and the second nucleic acid molecule are located on different molecules.

[0155] In another further embodiment of the first aspect, the nucleic acid combination comprises three nucleic acid molecules, a first nucleic acid molecule, a second nucleic acid molecule and a third nucleic acid molecule, respectively. The third nucleic acid molecule comprises a nucleotide sequence capable of expressing one or two proteins selected from the group consisting of VP1, VP2 and VP3, and the third nucleic acid molecule and the first nucleic acid molecule and the second nucleic acid molecule express different VP1, VP2 and VP3 protein species.

[0156] In a second aspect, the present application provides a vector comprising any of the aforementioned first nucleic acid molecule and / or any of the aforementioned second nucleic acid molecule.

[0157] In certain embodiments, the vector is an expression vector.

[0158] In a specific embodiment, the vector is a plasmid.

[0159] In another specific embodiment, the vector is an adeno-associated virus (AAV).

[0160] In a third aspect, the present application provides a vector combination comprising a first vector and a second vector. The first vector comprises any of the aforementioned first nucleic acid molecule, and the second vector comprises any of the aforementioned second nucleic acid molecule. The first nucleic acid molecule and the second nucleic acid molecule are capable of expressing three capsid proteins VP1, VP2 and VP3 of AAV. In certain embodiments, the vector combination further comprises a third vector, which comprises the aforementioned third nucleic acid molecule.

[0161] In certain embodiments, the first vector or the second vector is an expression vector.

[0162] In a specific embodiment, the first vector or the second vector is a plasmid.

[0163] In another specific embodiment, the first vector or the second vector is an adeno-associated virus (AAV).

[0164] In a fourth aspect, the present application provides a recombinant AAV virion expressed packaged from any of the aforementioned first nucleic acid molecule and any of the aforementioned second nucleic acid molecule. The first nucleic acid molecule and the second nucleic acid molecule are capable of expressing three capsid proteins VP1, VP2 and VP3 of AAV. In some embodiments, the recombinant AAV virion is expressed packaged from any of the aforementioned first nucleic acid molecule, any of the aforementioned second nucleic acid molecule, and any of the aforementioned third nucleic acid molecule. The first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule are capable of expressing three capsid proteins VP1, VP2 and VP3 of AAV.

[0165] In a fifth aspect, the present application provides a cell comprising any of the aforementioned nucleic acid combination, any of the aforementioned vector, and / or any of the aforementioned vector combination.

[0166] In a sixth aspect, the present application provides a pharmaceutical composition comprising any of the aforementioned nucleic acid combination, any of the aforementioned vector, any of the aforementioned vector combination, any of the aforementioned recombinant AAV virion, and / or, optionally, a pharmaceutically acceptable carrier.

[0167] In a seventh aspect, the present application provides a method of preventing, alleviating, and / or treating a disease, comprising administering to a subject in need thereof an effective amount of any of the aforementioned nucleic acid combination, any of the aforementioned vector, any of the aforementioned vector combination, any of the aforementioned recombinant AAV virion, and / or any of the aforementioned pharmaceutical composition.

[0168] In an eighth aspect, the present application also provides use of any of the aforementioned nucleic acid combination, any of the aforementioned vector, any of the aforementioned vector combination, any of the aforementioned recombinant AAV virion, and / or any of the aforementioned pharmaceutical composition in preventing, alleviating, and / or treating a disease.

[0169] In a ninth aspect, the present application also provides use of any of the aforementioned nucleic acid combination, any of the aforementioned vector, any of the aforementioned vector combination, any of the aforementioned recombinant AAV virion, and / or any of the aforementioned pharmaceutical composition in the manufacture of a medicament for preventing, alleviating, and / or treating a disease.

[0170] Other aspects and advantages of the present application will become apparent from the following detailed description, taken in conjunction with the accompanying drawings. Only examples of the present application are described in the detailed description. Changes can be made to the examples described, by one skilled in the art, without departing from the spirit and scope of the application as recited in the following claims. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature, and not as restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0171] The specific features of the invention involved in the present application are shown in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the detailed description of exemplary embodiments and the attached drawings. The drawings are briefly described as follows:

[0172] Figure 1 shows the expression identification results of AAV6 capsid protein split plasmid.

[0173] Figure 2 shows the schematic diagram of AAV6 capsid protein monomer structure (taking VP3 as an example) and flexible region where foreign proteins can be inserted.

[0174] Figure 3 shows the expression identification of fusion protein plasmid after AAV6 capsid protein-inserting large fragments, and the right side is the transfection plasmid number and corresponding name.

[0175] Figures 4 and 5 show the efficiency identification of capsid protein integration into AAV virus particles after inserting large fragment proteins at different sites, and the lower side is the transfection plasmid number and corresponding name, and the integrated plasmid combination. Figure 6 shows the identification of the infection ability of AAV particles carrying large fragment inserted proteins on target cells, and the lower side is the transfection plasmid number and corresponding name, and the integrated plasmid combination.

[0176] Figure 7 shows the expression identification of fusion protein plasmid after capsid protein-inserting large fragments, and the lower side is the transfection plasmid number and corresponding name.

[0177] Figure 8 shows the expression identification results of AAV2 capsid protein split plasmid.

[0178] Figure 9 shows the expression identification results of AAV5 capsid protein split plasmid.

[0179] Figure 10 shows the expression identification results of AAV9 capsid protein split plasmid.

[0180] Figure 11 shows the liver aggregation detection results of AAV6 inserted with large fragments by tail vein injection. The upper figure is the AAV6 packaging capsid protein expression plasmid. The lower figure is the liver infection immunofluorescence detection results after AAV6 tail vein injection for 4 weeks.

[0181] Figure 12 shows the AAV5 capsid protein VP2 expression plasmid inserted with large fragments at different sites and the expression identification results. The upper figure is the AAV5 capsid protein VP2 expression plasmid inserted with large fragments. The lower figure is the plasmid expression identification.

[0182] Figure 13 shows the packaging and infection effect test results of AAV5 capsid protein VP2 inserting large fragments at different sites. The upper panel is the capsid expression plasmid of AAV5 capsid protein VP2 inserting large fragments at different sites. The middle panel is the packaging and infection effect of AAV5 capsid protein VP2 inserting large fragments at different sites. The lower panel is the immunoblot detection after affinity chromatography purification of virus particles, the VP1 of AAV5 virus particles with capsid protein VP2 inserting TFR-3 coincides with the VP2-TFR-3 band.

[0183] Figure 14 shows the capsid protein expression plasmid of AAV5 capsid inserting endosome escape peptide and expression identification results. The upper panel is the capsid protein expression plasmid inserting endosome escape peptide. The lower panel is the immunoblot identification of capsid protein expression after plasmid transfection.

[0184] Figure 15 shows the packaging effect of AAV5 capsid protein VP1 and VP3 inserting endosome escape peptide. The upper left panel is the capsid expression plasmid of AAV5 capsid protein VP1 and VP3 inserting endosome escape peptide. The upper right panel is the combination of capsid expression plasmid and packaging titer. The lower panel is the immunoblot detection after iodixanol density gradient centrifugation purification of virus particles.

[0185] Figure 16 shows the packaging effect of AAV5 capsid protein inserting endosome escape peptide and large fragments. The upper panel is the capsid expression plasmid of AAV5 capsid protein VP1 and VP3 inserting endosome escape peptide, and capsid protein VP2 inserting large fragments. The lower left panel is the combination of capsid expression plasmid and packaging titer. The lower right panel is the immunoblot detection after affinity chromatography purification of virus particles, the VP1 of AAV5 virus particles with capsid protein VP2 inserting TFR-3 coincides with the VP2-TFR-3 band, and the VP1-peptide of AAV5 virus particles with capsid protein VP2 inserting TFR-3 and capsid protein VP1 and VP3 inserting polypeptide coincides with the VP2-TFR-3 band.

[0186] Figure 17 shows the capsid protein VP2 expression plasmid of AAV9 inserting large fragments at different sites and expression identification results. The upper panel is the capsid protein VP2 expression plasmid of AAV9 inserting large fragments at different sites. The lower panel is the plasmid expression identification.

[0187] Figure 18 shows the packaging and infection effect test results of AAV9 capsid protein VP2 inserting large fragments at different sites. The upper panel is the capsid expression plasmid of AAV9 capsid protein VP2 inserting large fragments at different sites. The middle panel is the packaging and infection effect of AAV9 capsid protein VP2 inserting large fragments at different sites. The lower panel is the immunoblot detection after affinity chromatography purification of virus particles, the VP1 of AAV9 virus particles with capsid protein VP2 inserting TFR-3 coincides with the VP2-TFR-3 band.

[0188] Figure 19 shows the results of the detection of the trans-blood brain barrier effect of the TFR-3 AAV9 inserted with the capsid protein VP2 by tail vein injection. The upper panel is the capsid protein expression plasmid for AAV9 packaging. The lower panel is the brain infection in vivo imaging detection after AAV9 tail vein injection for 3 weeks.

[0189] Figure 20 shows the results of the expression plasmid and expression identification of the endosome escape peptide inserted capsid protein of AAV9 capsid. The upper panel is the endosome escape peptide inserted capsid protein expression plasmid. The lower panel is the immunoblot identification of the capsid protein expression after plasmid transfection.

[0190] Figure 21 shows the packaging effect of the endosome escape peptide inserted AAV9 capsid protein VP1 and VP3. The upper left panel is the capsid expression plasmid of the endosome escape peptide inserted AAV5 capsid protein VP1 and VP3. The upper right panel is the combination of the capsid expression plasmid and the packaging titer. The lower panel is the immunoblot detection after iodixanol density gradient centrifugation purification of the virus particles.

[0191] Figure 22 shows the packaging effect of the endosome escape peptide and large fragment inserted AAV9 capsid protein. The upper panel is the capsid expression plasmid of the endosome escape peptide inserted AAV9 capsid protein VP1 and VP3, and the capsid protein VP2 inserted large fragment. The lower left panel is the combination of the capsid expression plasmid and the packaging titer. The lower right panel is the immunoblot detection of the capsid protein VP2 inserted TFR-3 AAV9 virus particles after affinity chromatography purification of the virus particles. The VP1 and VP2-TFR-3 bands of the capsid protein VP2 inserted TFR-3 AAV9 virus particles coincide. The VP1-peptide and VP2-TFR-3 bands of the capsid protein VP2 inserted TFR-3 and the capsid protein VP1 and VP3 inserted polypeptide AAV9 virus particles coincide.

[0192] Figure 23 shows the results of the D26 capsid protein VP2 expression plasmid inserted at different sites of AAV9 and expression identification. The upper panel is the D26 capsid protein VP2 expression plasmid inserted at different sites. The lower panel is the plasmid expression identification.

[0193] Figure 24 shows the results of the packaging and infection effect test of the large fragment inserted at different sites of AAV9 capsid protein VP2. The upper panel is the D26 inserted at different sites of AAV9 capsid protein VP2 expression plasmid. The middle panel is the packaging and infection effect of the D26 inserted at different sites of AAV9 capsid protein VP2. The lower panel is the immunoblot detection after affinity chromatography purification of the virus particles. The VP1 and VP2-D26 bands of the D26 inserted AAV9 virus particles coincide.

[0194] Figure 25 shows the results of the immunoblot detection of the split expression of AAV8 capsid protein. DETAILED DESCRIPTION

[0195] The following illustrates the embodiments of the present application by specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of the present application.

[0196] Definitions of terms

[0197] In the present application, the term "gene mutation leading to the cancellation of post-transcriptional RNA splicing" generally refers to a gene mutation caused by the failure of the post-transcriptional RNA splicing step. Since the three capsid proteins VP1, VP2 and VP3 of AAV are translated from different transcriptional splice bodies of the same gene, they are three structural proteins with overlapping parts. Specifically, VP2 contains the part of VP3, and VP1 contains the part of VP2. Therefore, interfering with post-transcriptional RNA splicing can affect the formation of VP1, VP2 and VP3 transcriptional splice bodies, thereby affecting the expression of capsid proteins. Specifically, the "gene mutation leading to the cancellation of post-transcriptional RNA splicing" can be a gene site mutation targeting splice acceptor A1 or A2.

[0198] In the present application, the terms "first nucleic acid molecule", "second nucleic acid molecule" and "third nucleic acid molecule" refer to one of the nucleic acid molecules expressing the three capsid proteins VP1, VP2 and VP3 of AAV. Among them, the third nucleic acid molecule can exist or not exist. The first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule can be on the same nucleotide chain or on different nucleotide chains, but must have different expression frames, such as having insulators between each other, having elements that terminate or inhibit transcription between each other.

[0199] In the present application, the term "initiation codon" refers to the codon that specifies the beginning of protein synthesis on a messenger RNA (mRNA). The term "codon" refers to an oligonucleotide consisting of three nucleotides that encode a defined amino acid. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon. In particular to AAV capsid proteins, the initiation codon of VP1 refers to the codon that specifies the beginning of VP1 protein synthesis on the mRNA encoding VP proteins, the initiation codon of VP1 is usually at a specific site, i.e., M1, and usually includes AUG, not excluding the case that a codon at other site or other sequence can initiate translation of VP1 protein; the initiation codon of VP2 refers to the codon that specifies the beginning of VP2 protein synthesis on the mRNA encoding VP proteins, the initiation codon of VP2 is usually at a fixed site, i.e., T138 or T137 (depending on different serotypes), and usually includes ACG; the initiation codon of VP3 refers to the codon that specifies the beginning of VP3 protein synthesis on the mRNA encoding VP proteins, usually M203 or M193 (depending on different serotypes), unlike VP1 or VP2, VP3 can usually be initiated by a codon at a different position, such as M211, V221, M235, L235, or M225 (depending on different serotypes), when the initiation codon at one position is mutated to be unable to initiate translation, the translation of VP3 can be initiated by a codon at another position. For a specific serotype of AAV, the positions and sequences of the initiation codons of VP1, VP2, and VP3 are known to those skilled in the art, and exemplary initiation codons of VP1, VP2, and VP3 are provided in the examples for the purpose of implementing the application.

[0200] In the present application, the term "non-initiation codon" refers to a codon that cannot specify the beginning of protein synthesis on an mRNA. The non-initiation codon can be a stop codon, or can be a codon other than the initiation codon and the stop codon. Mutating the initiation codon in a coding gene to a non-initiation codon can make the gene that is originally expressed at this position unable to be expressed at this position, and can only continue to move downstream until the next initiation codon appears to start expression. Mutating the initiation codon to a non-initiation codon usually results in no expression or truncated expression of the protein.

[0201] In the present application, the term "nucleic acid combination" refers to a composition comprising two or more nucleic acid molecules. Specifically, the nucleic acid combination can comprise a first nucleic acid molecule and a second nucleic acid molecule, or a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule. Further, the nucleic acid combination can exist in any possible form, such as a plasmid, a colony, a solution, etc.

[0202] In the present application, when referring to codon mutation, various ways of expression are used interchangeably, such as "CTG-CTC", "CTG to CTC" or "mutation from CTG to CTC" all mean the same, i.e. the codon CTG is mutated to codon CTC.

[0203] In the present application, the term "site" refers to mutation site, insertion site and other possible forms on amino acid sequence or nucleotide sequence.

[0204] In some embodiments of the present application, the site is a relative concept rather than an absolute position. For example, since VP1, VP2 and VP3 are translated from different transcript splicers of the same gene, the three capsid proteins have overlapping protein regions, and the expression of the site is not according to the actual amino acid sequence of each protein, but generally the full-length amino acid sequence of VP1 wild type is used as the expression of the site. Therefore, in the present application, under AAV6, unless otherwise specified, the position of the amino acid M encoded by the start codon of VP1 is taken as the first site of the amino acid sequence of VP1, VP2 and VP3 capsid proteins, i.e. M1; the position of the amino acid T encoded by the start codon of VP2 is taken as the 138th site of the amino acid sequence of VP1, VP2 and VP3 capsid proteins, i.e. T138, even though for the VP2 protein, T138 is the first amino acid of its actual sequence. Similarly, for nucleic acid sequence, under AAV6, the first base of the start codon of VP1 is taken as the first site of the nucleic acid sequence.

[0205] In certain embodiments, the numerical value of the site of the amino acid sequence and the nucleotide sequence is not affected by exogenous modification. For example, even if deletion or insertion of amino acid or nucleotide is made before the site by genetic engineering or other possible modification methods, so that the actual sequence of the site is changed, but for the purpose of uniformity of the text format, the site is still expressed as the 138th amino acid and the 220th nucleotide. In short, the description of the amino acid or nucleotide site in the present application is based on the unmodified protein or nucleic acid. For example, if one amino acid is inserted / deleted before the 138th amino acid T, so that the actual sequence of the amino acid T becomes the 137th or 139th, the site of the amino acid T is still called T138; for another example, even if the 138th amino acid is mutated from wild-type amino acid T to other amino acid, the site is still called T138.

[0206] That is, when referring to an amino acid position, the position of the first amino acid in the VP1 sequence of wild-type AAV6 (as shown in SEQ ID NO: 4) is referred to as the "M1" position, and the position of the 138th amino acid in the sequence is referred to as the "T138" position. Those skilled in the art will understand that the designation of the position is not affected by mutations in the amino acid. For example, when the amino acid at position 138 of the sequence is mutated from T to A, the position can still be referred to as "T138", and the mutation that occurs is "T138A", and for another example, when the amino acid before the 138th amino acid of the sequence is mutated by deletion, substitution, insertion, etc., for the mutated amino acid sequence, the position can not be at the absolute position of the 138th amino acid, but in the present application it is still referred to as "T138". Similarly, for other serotypes, such as AAV2, AAV5, AAV8 and AAV9, those skilled in the art can obtain the actual position represented by the respective corresponding amino acid positions according to common general knowledge.

[0207] In the present application, the term "recombinant AAV virion" refers to an AAV virion comprising a different AAV genome than a native AAV genome. The difference can be in the rep gene or in the cap gene. In the narrow sense of the present application, the difference refers to a genetic mutation in the nucleic acid sequence encoding the capsid proteins VP1, VP2 and / or VP3 in the cap gene.

[0208] In the present application, the term "heterologous" means derived from an entity that is genetically different from the remainder of the entity with which it is compared or the remainder of the entity into which it is introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0209] In the present application, the terms "VP1", "VP2" and "VP3" refer to the three capsid proteins of adeno-associated virus (AAV) or nucleic acid molecules encoding said VP1, VP2 and VP3, and whether the specific reference is to the capsid protein or the corresponding nucleic acid can be determined by the context. The VP1, VP2 and VP3 can each be wild-type (e.g., without change in the nucleotide and amino acid sequences) or each can be mutated (e.g., with mutations in the nucleotide and / or amino acid sequences), and the mutations can be mutations that change the amino acid sequence of the encoded protein or mutations that do not change the amino acid sequence of the encoded protein.

[0210] In the present application, the term "selected from" generally refers to include the selected objects and all combinations thereof. For example, "selected from A, B and C" means to include all combinations of A, B and C, e.g., A, B, C, A+B, A+C, B+C, or A+B+C.

[0211] In the present application, the term "about" generally refers to a variation within a range of 0.5-10% above or below the specified numerical value, e.g., within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0212] DETAILED DESCRIPTION

[0213] Nucleic acid combination

[0214] The present application provides a nucleic acid combination comprising nucleic acid molecules encoding AAV capsid proteins VP1, VP2 and VP3.

[0215] In one aspect, the nucleic acid combination can comprise two nucleic acid molecules, a first nucleic acid molecule and a second nucleic acid molecule, respectively. The first nucleic acid molecule and the second nucleic acid molecule can each independently comprise nucleotide sequences encoding VP1, VP2 and VP3.

[0216] In a preferred embodiment, the first nucleic acid molecule and the second nucleic acid molecule can each independently comprise a genetic mutation.

[0217] For example, both the first nucleic acid molecule and the second nucleic acid molecule comprise a genetic mutation.

[0218] For another example, the first nucleic acid molecule comprises a genetic mutation and the second nucleic acid molecule does not comprise a genetic mutation.

[0219] For another example, the first nucleic acid molecule does not comprise a genetic mutation and the second nucleic acid molecule comprises a genetic mutation.

[0220] In a more preferred embodiment, the genetic mutation can occur in the nucleotide sequences expressing capsid proteins on the first nucleic acid molecule and the second nucleic acid molecule.

[0221] In certain embodiments, the genetic mutation can inhibit expression of one or two of the capsid proteins, such that the first nucleic acid molecule and the second nucleic acid molecule express two or one of the capsid proteins. Meanwhile, the nucleic acid combination is just capable of expressing three capsid proteins VP1, VP2 and VP3. In certain embodiments, the inhibition of expression of the capsid protein can be a reduction of the expression level of the capsid protein (e.g. the expression level of the capsid protein is lower than before the genetic mutation, or the expression level of the capsid protein is 0). In certain embodiments, the inhibition of expression of the capsid protein can be a change of the amino acid sequence to express a protein that cannot normally function.

[0222] For example, the expression of VP2 and VP3 can be inhibited on the first nucleic acid molecule, such that the first nucleic acid molecule only expresses VP1; the expression of VP1 can be inhibited on the second nucleic acid molecule, such that the second nucleic acid molecule only expresses VP2 and / or VP3.

[0223] For example, the expression of VP2 and VP3 can be inhibited on the first nucleic acid molecule, such that the first nucleic acid molecule only expresses VP1; the expression of VP1 can be inhibited on the second nucleic acid molecule, such that the second nucleic acid molecule only expresses VP2 and / or VP3.

[0224] For example, the expression of VP2 and VP3 can be inhibited on the first nucleic acid molecule, such that the first nucleic acid molecule only expresses VP1; the expression of VP1 can be inhibited on the second nucleic acid molecule, such that the second nucleic acid molecule only expresses VP2 and / or VP3.

[0225] For example, the expression of VP2 and VP3 can be inhibited on the first nucleic acid molecule, such that the first nucleic acid molecule only expresses VP1; the expression of VP1 can be inhibited on the second nucleic acid molecule, such that the second nucleic acid molecule only expresses VP2 and / or VP3.

[0226] For example, the expression of VP2 and VP3 can be inhibited on the first nucleic acid molecule, such that the first nucleic acid molecule only expresses VP1; the expression of VP1 can be inhibited on the second nucleic acid molecule, such that the second nucleic acid molecule only expresses VP2 and / or VP3.

[0227] For example, the expression of VP2 and VP3 can be inhibited on the first nucleic acid molecule, such that the first nucleic acid molecule only expresses VP1; the expression of VP1 can be inhibited on the second nucleic acid molecule, such that the second nucleic acid molecule only expresses VP2 and / or VP3.

[0228] In certain embodiments, the first nucleic acid molecule and / or the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing one or two of the capsid proteins VP1, VP2 and VP3, while the nucleic acid combination is capable of expressing all three capsid proteins VP1, VP2 and VP3.

[0229] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP1 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP2 protein and / or the VP3 protein.

[0230] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP2 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP1 protein and / or the VP3 protein.

[0231] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP3 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP1 protein and / or the VP2 protein.

[0232] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP2 protein and / or the VP3 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP1 protein.

[0233] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP1 protein and / or the VP3 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP2 protein.

[0234] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP1 protein and / or the VP2 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing the VP3 protein.

[0235] In certain embodiments, the nucleic acid combination can comprise three nucleic acid molecules, namely a first nucleic acid molecule, a second nucleic acid molecule and a third nucleic acid molecule. Examples of the third nucleic acid molecule can be found in the above description of the first nucleic acid molecule and / or the second nucleic acid molecule.

[0236] In certain embodiments, the first nucleic acid molecule, the second nucleic acid molecule and / or the third nucleic acid molecule can comprise a nucleotide sequence capable of expressing one or two of the capsid proteins VP1, VP2 and VP3, while the nucleic acid combination is capable of expressing all three capsid proteins VP1, VP2 and VP3.

[0237] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the third nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein.

[0238] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the third nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein.

[0239] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the third nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein.

[0240] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the third nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein.

[0241] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the third nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein.

[0242] For example, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the third nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein.

[0243] In certain embodiments, the first nucleic acid molecule can further comprise a promoter. In certain embodiments, the second nucleic acid molecule can further comprise a promoter. In certain embodiments, the third nucleic acid molecule can further comprise a promoter. The promoter can be selected from one or more of the following: CMV, EF1a, SV40, PGK1, Ubc, CAG, TRE, UAS, Ac5, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, and pL. In certain embodiments, the promoter can be a strong promoter. For example, the strong promoter can be a CMV promoter.

[0244] In certain embodiments, the first nucleic acid molecule, the second nucleic acid molecule, and / or the third nucleic acid molecule can comprise other genetic mutations, preferably genetic mutations that alter targeting.

[0245] In certain embodiments, the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule can be in different expression cassettes. In certain embodiments, the different expression cassettes can be on different molecules, or can be on the same molecule. In certain embodiments, the different expression cassettes on the same molecule can be separated by one or more of the following elements: an insulator, an intron, and other elements that can serve to separate gene expression. In certain embodiments, the first nucleic acid molecule and the second nucleic acid molecule can be in tandem on the same molecule. In certain embodiments, the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule are in one vector.

[0246] In certain embodiments, the first nucleic acid molecule and the second nucleic acid molecule can be on different molecules.

[0247] In certain embodiments, the first nucleic acid molecule and the second nucleic acid molecule can be in tandem on the same molecule, and the third nucleic acid molecule can be on a different molecule from the first nucleic acid molecule and the second nucleic acid molecule.

[0248] In certain embodiments, the first nucleic acid molecule and the third nucleic acid molecule can be in tandem on the same molecule, and the second nucleic acid molecule can be on a different molecule from the first nucleic acid molecule and the third nucleic acid molecule.

[0249] In certain embodiments, the second nucleic acid molecule and the third nucleic acid molecule can be in tandem on the same molecule, and the first nucleic acid molecule can be on a different molecule from the second nucleic acid molecule and the third nucleic acid molecule.

[0250] In certain embodiments, the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule can be in tandem on the same molecule.

[0251] In certain embodiments, the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule can be on different molecules. In certain embodiments, the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule on different molecules can be in the same vector. In certain embodiments, the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule on different molecules can be in different vectors, respectively.

[0252] In some embodiments, the nucleic acid combination can further comprise a nucleic acid molecule encoding an assembly-activated protein (AAP). The nucleic acid molecule encoding the AAP can be located in the same molecule in tandem with the first nucleic acid molecule; can be located in the same molecule in tandem with the second nucleic acid molecule; or can be located on a different molecule from the first nucleic acid molecule and the second nucleic acid molecule. For example, the AAP can comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 16-19. For another example, the AAP can be encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOs: 32-35.

[0253] Mutated forms of capsid proteins

[0254] In the present application, a gene encoding an AAV capsid protein can be mutated to achieve the above-mentioned expression modes. Since VP1, VP2 and VP3 of AAV share the same mRNA and reading frame, different proteins are mainly produced by translation initiation regulation, therefore, a nucleic acid sequence encoding a VP1 protein can be mutated to obtain the above-mentioned first nucleic acid molecule, and the nucleic acid sequence encoding the VP1 protein can be mutated differently from the first nucleic acid molecule to obtain the above-mentioned second nucleic acid molecule. Further, the nucleic acid sequence encoding the VP1 protein can be mutated differently from the first nucleic acid molecule and the second nucleic acid molecule to obtain the above-mentioned third nucleic acid molecule. The present application provides specific embodiments for achieving the above-mentioned expression in the following.

[0255] Therefore, in another aspect, the present application relates to a gene mutation mode of capsid protein VP1 in an adeno-associated virus (AAV). Specifically, it can be a gene mutation that inhibits the expression of VP1. At the same time, the gene mutation that inhibits the expression of VP1 does not affect the expression of VP2 and VP3.

[0256] In some embodiments, the gene mutation that inhibits the expression of VP1 can be a mutation that changes the amino acid sequence of VP1, which causes a change in the secondary structure or tertiary structure of VP1, thereby causing VP1 to be unable to perform its function.

[0257] In some embodiments, the gene mutation that inhibits the expression of VP1 can be a mutation that reduces the expression amount of VP1. Specifically, the expression amount can be reduced to 0.

[0258] In some embodiments, the gene mutation that inhibits the expression of VP1 can be a gene mutation that causes RNA splicing to be cancelled. Specifically, the gene mutation that causes RNA splicing to be cancelled can be a gene mutation occurring at Splicing acceptor A1 or Splicing acceptor A2, preferably a gene mutation occurring at Splicing acceptor A1, and more preferably an AG mutation to TT.

[0259] In some embodiments, the gene mutation that suppresses the expression of VP1 can be a mutation that changes the start codon on the VP1-encoding nucleotide sequence to a non-start codon. Specifically, the gene mutation can result in a mutation of the codon corresponding to the M1 site on the VP1 protein, preferably to a non-start codon, and specifically can be a mutation of the start codon corresponding to the methionine at position 1 on the VP1 protein to a non-start codon corresponding to leucine (i.e., M1L).

[0260] In the AAV capsid protein, the start codon can generally be selected from one or more of the following: ACG, ATG, TTG, CTG, GTG, and other possible choices.

[0261] In some embodiments, the gene mutation that suppresses the expression of VP1 can be a mutation that changes a certain codon between the N-terminal of the start codon on the VP1-encoding nucleotide sequence and the start codon of VP2 to a stop codon. Specifically, the gene mutation can result in a mutation of the codon corresponding to one or more of the following amino acids on the VP1 protein: valine at position 4, tyrosine at position 6, glutamic acid at position 12, and glutamic acid at position 13, to a stop codon.

[0262] In another aspect, the present application relates to a gene mutation mode of the capsid protein VP2 in an adeno-associated virus (AAV). Specifically, it can be a gene mutation that suppresses the expression of VP2. At the same time, the gene mutation that suppresses the expression of VP2 does not affect the expression of VP1 and VP3.

[0263] In some embodiments, the gene mutation that suppresses the expression of VP2 can be a mutation that changes the VP2 amino acid sequence, which causes a change in the secondary structure or tertiary structure of VP2, thereby causing VP2 to be unable to perform its function.

[0264] In some embodiments, the gene mutation that suppresses the expression of VP2 can be a mutation that reduces the expression amount of VP2. Specifically, the expression amount can be reduced to 0.

[0265] In some embodiments, the gene mutation that suppresses the expression of VP2 can be a gene mutation that causes RNA splicing to be cancelled. Specifically, the gene mutation that causes RNA splicing to be cancelled can be a gene mutation that occurs at Splicing acceptor A1 or Splicing acceptor A2, preferably a gene mutation that occurs at Splicing acceptor A2, and more preferably an AG mutation to TT.

[0266] In some embodiments, the gene mutation that suppresses the expression of VP2 can be a mutation of the start codon on the VP2-encoding nucleotide sequence to a non-start codon. Specifically, the gene mutation can result in a mutation of the codon corresponding to the T137 and / or T138 site on the VP2 protein, preferably can be mutated to a non-start codon, specifically can be a mutation of the start codon corresponding to the threonine at position 137 and / or 138 on the VP2 protein to a non-start codon corresponding to alanine (i.e., T137A and / or T138A), or can be a mutation of the codon corresponding to the threonine at position 137 and / or 138 on the VP2 protein to a non-start codon (such as ACC).

[0267] In the AAV capsid protein, the start codon can generally be selected from one or more of the following: ACG, ATG, TTG, CTG, GTG, and other possible choices.

[0268] In another aspect, the present application relates to a gene mutation mode of the capsid protein VP3 in adeno-associated virus (AAV). Specifically, it can be a gene mutation that suppresses the expression of VP3. At the same time, the gene mutation that suppresses the expression of VP3 does not affect the expression of VP1 and VP2.

[0269] In some embodiments, the gene mutation that suppresses the expression of VP3 can be a mutation that changes the VP3 amino acid sequence, which causes a change in the secondary structure or tertiary structure of VP3, thereby resulting in VP3 being unable to perform its function.

[0270] In some embodiments, the gene mutation that suppresses the expression of VP3 can be a mutation that reduces the expression amount of VP3. Specifically, the expression amount can be reduced to 0.

[0271] In some embodiments, the gene mutation that suppresses the expression of VP3 can be a gene mutation that results in the cancellation of RNA splicing. Specifically, the gene mutation that results in the cancellation of RNA splicing can be a gene mutation that occurs at Splicing acceptor A1 or Splicing acceptor A2, preferably can be a gene mutation that occurs at Splicing acceptor A2, more preferably is a mutation of AG to TT.

[0272] In certain embodiments, the gene mutation that suppresses the expression of VP3 can be a mutation of the start codon in the VP3-encoding nucleotide sequence to a non-start codon. Specifically, the gene mutation can be a mutation of the codon corresponding to one or more of the following amino acid positions in the VP3 protein: threonine at position 137, threonine at position 138, methionine at position 193, methionine at position 203, methionine at position 211, valine at position 221, methionine at position 225, leucine at position 235, and leucine at position 249, to a non-start codon.

[0273] In specific embodiments, the gene mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to a specific amino acid position in the VP3 protein to a non-start codon corresponding to another amino acid.

[0274] For example, the gene mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to threonine at position 137 in the VP3 protein to a non-start codon corresponding to methionine (i.e., T137M).

[0275] For another example, the gene mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to threonine at position 138 in the VP3 protein to a non-start codon corresponding to methionine (i.e., T138M).

[0276] For another example, the gene mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to methionine at position 193 in the VP3 protein to a non-start codon corresponding to leucine (i.e., M193L).

[0277] For another example, the gene mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to methionine at position 203 in the VP3 protein to a non-start codon corresponding to leucine (i.e., M203L).

[0278] For another example, the gene mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to methionine at position 211 in the VP3 protein to a non-start codon corresponding to leucine (i.e., M211L).

[0279] For another example, the gene mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to methionine at position 225 in the VP3 protein to a non-start codon corresponding to leucine (i.e., M225L).

[0280] In specific embodiments, the gene mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to a specific amino acid position in the VP3 protein to a non-start codon corresponding to another amino acid.

[0281] For example, the genetic mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to valine at position 221 of the VP3 protein to a non-start codon corresponding to valine (such as GTC).

[0282] For another example, the genetic mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to leucine at position 235 of the VP3 protein to a non-start codon corresponding to leucine (such as CTC).

[0283] For another example, the genetic mutation that suppresses the expression of VP3 can be a mutation of the start codon corresponding to leucine at position 249 of the VP3 protein to a non-start codon corresponding to leucine (such as CTC).

[0284] In the AAV capsid protein, the start codon can generally be selected from one or more of the following: ACG, ATG, TTG, CTG, GTG, and other possible choices.

[0285] Mutated forms of capsid proteins on different AAVs

[0286] The present application also relates to the use of the nucleic acid combination in a specific serotype of adeno-associated virus (AAV). Specifically, the use can involve specific mutated forms of VP1, VP2 and VP3 in the specific serotype in the nucleic acid combination.

[0287] In certain embodiments, the specific serotype of the AAV can be selected from one or more of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrh10, AAV-PHP.eB, AAV-PSP.S, AAV-DJ, AAVDJ / 8, AAVDJ / 9, AAV1.1, AAV2.5, AAV6.1, AAV6.2, AAV6.3.1, and other naturally occurring or artificially engineered serotypes.

[0288] In a first aspect, the specific serotype can be AAV6.

[0289] In certain embodiments, the VP1 can comprise an amino acid sequence as shown in SEQ ID NO: 4. More specifically, the VP1 can be formed by encoding a nucleotide sequence as shown in SEQ ID NO: 20.

[0290] In certain embodiments, the VP2 can comprise an amino acid sequence as shown in SEQ ID NO: 5. More specifically, the VP1 can be formed by encoding a nucleotide sequence as shown in SEQ ID NO: 21.

[0291] In certain embodiments, the VP3 can comprise an amino acid sequence as set forth in SEQ ID NO: 6. More particularly, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 22.

[0292] In the following description of mutations, the above-mentioned AAV6 sequence is taken as the reference sequence.

[0293] In specific embodiments, the genetic mutation that suppresses the expression of VP1 can be selected from one or more of the following: a genetic mutation that occurs at Splicing acceptor Al, a mutation of the start codon corresponding to the methionine at position 1 of the VP1 protein to a non-start codon corresponding to leucine (i.e., M1L), a mutation of the codon corresponding to tyrosine at position 6 to a stop codon, and a mutation of the codon corresponding to glutamic acid at position 12 to a stop codon.

[0294] In specific embodiments, the genetic mutation that suppresses the expression of VP2 can be selected from one or more of the following: a genetic mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to the threonine at position 138 of the VP2 protein to a non-start codon corresponding to alanine (i.e., T138A), and a mutation of the codon corresponding to the threonine at position 138 to a non-start codon (such as ACC).

[0295] In specific embodiments, the genetic mutation that suppresses the expression of VP3 can be selected from one or more of the following: a genetic mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to the threonine at position 138 of the VP3 protein to a non-start codon corresponding to methionine (i.e., T138M), a mutation of the start codon corresponding to the methionine at position 203 to a non-start codon corresponding to leucine (i.e., M203L), a mutation of the start codon corresponding to the methionine at position 211 to a non-start codon corresponding to leucine (i.e., M211L), and a mutation of the start codon corresponding to the leucine at position 235 to a non-start codon corresponding to leucine (such as CTC).

[0296] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein and / or a VP3 protein.

[0297] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP3 protein.

[0298] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP2 protein.

[0299] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138A, M203L, M211L, and L235L (CTG-CTC), and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L, Y6*, and E12* (* is a stop codon).

[0300] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M203L, M211L, and L235L (CTG-CTC), and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138T (ACG-ACC).

[0301] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L and T138A, and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138M, M203L, M211L, and L235L (CTG-CTC).

[0302] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138A, M203L, M211L, and L235L (CTG-CTC), and the second nucleic acid molecule can comprise a genetic mutation selected from the group consisting of a genetic mutation occurring at Splicing acceptor A1.

[0303] In a second aspect, the specific serotype can be AAV2.

[0304] In certain embodiments, the VP1 can comprise an amino acid sequence as set forth in SEQ ID NO: 7. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 23.

[0305] In certain embodiments, the VP2 can comprise an amino acid sequence as set forth in SEQ ID NO: 8. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 24.

[0306] In certain embodiments, the VP3 can comprise an amino acid sequence as set forth in SEQ ID NO: 9. More particularly, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 25.

[0307] In the following description of mutations, the above-mentioned AAV2 sequence is taken as the reference sequence.

[0308] In specific embodiments, the gene mutation that suppresses the expression of VP1 can be selected from one or more of the following: a gene mutation that occurs at Splicing acceptor Al, a mutation of the start codon corresponding to the methionine at position 1 of the VP1 protein to a non-start codon corresponding to leucine (i.e., M1L), a mutation of the codon corresponding to tyrosine at position 6 to a stop codon, and a mutation of the codon corresponding to glutamic acid at position 12 to a stop codon.

[0309] In specific embodiments, the gene mutation that suppresses the expression of VP2 can be selected from one or more of the following: a gene mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to the threonine at position 138 of the VP2 protein to a non-start codon corresponding to alanine (i.e., T138A), and a mutation of the codon corresponding to the threonine at position 138 to a non-start codon (such as ACC).

[0310] In specific embodiments, the gene mutation that suppresses the expression of VP3 can be selected from one or more of the following: a gene mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to the threonine at position 138 of the VP3 protein to a non-start codon corresponding to methionine (i.e., T138M), a mutation of the start codon corresponding to the methionine at position 203 to a non-start codon corresponding to leucine (i.e., M203L), a mutation of the start codon corresponding to the methionine at position 211 to a non-start codon corresponding to leucine (i.e., M211L), a mutation of the start codon corresponding to the methionine at position 235 to a non-start codon corresponding to leucine, and a mutation of the start codon corresponding to the valine at position 221 to a non-start codon corresponding to valine (such as GTC).

[0311] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein and / or a VP3 protein.

[0312] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP3 protein.

[0313] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP2 protein.

[0314] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138A, M203L, M211L, and M235L, and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L, Y6*, and E12* (* is a stop codon).

[0315] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L, M203L, M211L, and M235L, and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138A, T138T (ACG-ACC).

[0316] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L and T138A, and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138M, M203L, M211L, V221V (GTG-GTC), and M235L.

[0317] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138A, M203L, M211L, and M235L, and the second nucleic acid molecule can comprise a genetic mutation selected from the group consisting of a genetic mutation occurring at Splicing acceptor A1.

[0318] In a third aspect, the specific serotype can be AAV5.

[0319] In certain embodiments, the VP1 can comprise an amino acid sequence as set forth in SEQ ID NO: 10. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 26.

[0320] In certain embodiments, the VP1 can comprise an amino acid sequence as set forth in SEQ ID NO: 11. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 27.

[0321] In certain embodiments, the VP1 can comprise an amino acid sequence as set forth in SEQ ID NO: 12. More particularly, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 28.

[0322] In the following description of mutations, the above-mentioned AAV5 sequence is taken as the reference sequence.

[0323] In specific embodiments, the gene mutation that suppresses the expression of VP1 can be selected from one or more of the following: a gene mutation that occurs at Splicing acceptor Al, a mutation of the start codon corresponding to the methionine at position 1 of the VP1 protein to a non-start codon corresponding to leucine (i.e., M1L), a mutation of the codon corresponding to valine at position 4 to a stop codon, and a mutation of the codon corresponding to glutamic acid at position 13 to a stop codon.

[0324] In specific embodiments, the gene mutation that suppresses the expression of VP2 can be selected from one or more of the following: a gene mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to the threonine at position 137 of the VP2 protein to a non-start codon corresponding to alanine (i.e., T138A), and a mutation of the codon corresponding to threonine at position 138 to a non-start codon (such as ACC).

[0325] In specific embodiments, the gene mutation that suppresses the expression of VP3 can be selected from one or more of the following: a gene mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to the threonine at position 137 of the VP3 protein to a non-start codon corresponding to methionine (i.e., T138M), a mutation of the start codon corresponding to the methionine at position 193 to a non-start codon corresponding to leucine (i.e., M203L), and a mutation of the start codon corresponding to the methionine at position 225 to a non-start codon corresponding to leucine.

[0326] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein and / or a VP3 protein.

[0327] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP3 protein.

[0328] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP2 protein.

[0329] In particular embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T137A, M193L and M225L, and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L, V4* and E13* (* is a stop codon).

[0330] In particular embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L, M193L and M225L, and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T137A, T137T (ACG-ACC).

[0331] In particular embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L and T137A, and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T137M, M193L and M225L.

[0332] In particular embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T137A, M193L and M225L, and the second nucleic acid molecule can comprise a genetic mutation selected from the group consisting of a genetic mutation occurring at Splicing acceptor A1.

[0333] In particular embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of a genetic mutation occurring at Splicing acceptor A2, and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L, V4* and E13* (* is a stop codon).

[0334] In particular embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T137A, M193L and M225L, the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L, M193L and M225L, and the third nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L and T137A.

[0335] In a fourth aspect, the particular serotype can be AAV9.

[0336] In certain embodiments, the VP1 can comprise an amino acid sequence as set forth in SEQ ID NO: 13. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 29.

[0337] In certain embodiments, the VP2 can comprise an amino acid sequence as set forth in SEQ ID NO: 14. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 30.

[0338] In certain embodiments, the VP3 can comprise an amino acid sequence as set forth in SEQ ID NO: 15. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 31.

[0339] In the following description of mutations, the above-mentioned AAV9 sequence is taken as the reference sequence.

[0340] In specific embodiments, the genetic mutation that suppresses the expression of VP1 can be selected from one or more of the following: a genetic mutation that occurs at Splicing acceptor Al, a mutation of the start codon corresponding to the first methionine of the VP1 protein to a non-start codon corresponding to leucine (i.e., M1L), a mutation of the codon corresponding to the sixth tyrosine to a stop codon, and a mutation of the codon corresponding to the twelfth glutamic acid to a stop codon.

[0341] In specific embodiments, the genetic mutation that suppresses the expression of VP2 can be selected from one or more of the following: a genetic mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to the 138th threonine of the VP2 protein to a non-start codon corresponding to alanine (i.e., T138A), and a mutation of the codon corresponding to the 138th threonine to a non-start codon (such as ACC).

[0342] In specific embodiments, the genetic mutation that suppresses the expression of VP3 can be selected from one or more of the following: a genetic mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to the 138th threonine of the VP3 protein to a non-start codon corresponding to methionine (i.e., T138M), a mutation of the start codon corresponding to the 203rd methionine to a non-start codon corresponding to leucine (i.e., M203L), a mutation of the start codon corresponding to the 235th leucine to a non-start codon corresponding to leucine (such as CTC), and a mutation of the start codon corresponding to the 249th leucine to a non-start codon corresponding to leucine (such as CTC).

[0343] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein and / or a VP3 protein.

[0344] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP3 protein.

[0345] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP2 protein.

[0346] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138A, M203L, L235L (CTG-CTC), and L249L (CTG-CTC), and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L, Y6* and E12* (* is a stop codon).

[0347] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L, M203L, L235L (CTG-CTC), and L249L (CTG-CTC), and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138A, T138T (ACG-ACC).

[0348] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L and T138A, and the second nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138M, M203L, L235L (CTG-CTC), and L249L (CTG-CTC).

[0349] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138A, M203L, L235L (CTG-CTC), and L249L (CTG-CTC), and the second nucleic acid molecule can comprise a genetic mutation selected from the group consisting of a genetic mutation occurring at Splicing acceptor Al.

[0350] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A and M203L, the second nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L and M203L, and the third nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L and T138A.

[0351] In a fourth aspect, the specific serotype can be AAV8.

[0352] In certain embodiments, the VP1 can comprise an amino acid sequence as set forth in SEQ ID NO: 37. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 40.

[0353] In certain embodiments, the VP2 can comprise an amino acid sequence as set forth in SEQ ID NO: 38. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 41.

[0354] In certain embodiments, the VP3 can comprise an amino acid sequence as set forth in SEQ ID NO: 39. More specifically, the VP1 can be encoded by a nucleotide sequence as set forth in SEQ ID NO: 42.

[0355] In the following description of mutations, the AAV8 sequence described above is used as the reference sequence.

[0356] In specific embodiments, the genetic mutation that suppresses the expression of VP1 can be selected from one or more of the following: a genetic mutation that occurs at Splicing acceptor A1, a mutation of the start codon corresponding to the methionine at position 1 of the VP1 protein to a non-start codon corresponding to leucine (i.e., M1L), a mutation of the codon corresponding to the tyrosine at position 6 to a stop codon, and a mutation of the codon corresponding to the glutamic acid at position 12 to a stop codon.

[0357] In specific embodiments, the genetic mutation that suppresses the expression of VP2 can be selected from one or more of the following: a genetic mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to the threonine at position 138 of the VP2 protein to a non-start codon corresponding to alanine (i.e., T138A), and a mutation of the codon corresponding to the threonine at position 138 to a non-start codon (such as ACC).

[0358] In specific embodiments, the genetic mutation that suppresses expression of VP3 can be selected from one or more of the following: a genetic mutation that occurs at Splicing acceptor A2, a mutation of the start codon corresponding to threonine at position 138 of the VP3 protein to a non-start codon corresponding to methionine (i.e., T138M), a mutation of the start codon corresponding to methionine at position 204 to a non-start codon corresponding to leucine (i.e., M204L), a mutation of the start codon corresponding to methionine at position 212 to a non-start codon corresponding to leucine (i.e., M212L).

[0359] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein and / or a VP3 protein.

[0360] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP2 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP3 protein.

[0361] In certain embodiments, the first nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP3 protein, and the second nucleic acid molecule can comprise a nucleotide sequence capable of expressing a VP1 protein and / or a VP2 protein.

[0362] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L, and M212L, and the second nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L, M204L, and M212L. In certain embodiments, the nucleic acid combination further comprises a third nucleic acid molecule, which can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L and T138A.

[0363] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L, and M212L, and the second nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of Y6* (e.g., mutated to TAA).

[0364] In specific embodiments, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L, and M212L, and the second nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of E12* (e.g., mutated to TAG).

[0365] In a particular embodiment, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L and M212L, and the second nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L.

[0366] In a particular embodiment, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L and M212L, and the second nucleic acid molecule can comprise a genetic mutation selected from the group consisting of a genetic mutation that occurs at Splicing acceptor Al.

[0367] In a particular embodiment, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L and M212L, and the second nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L.

[0368] In a particular embodiment, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L and M212L, and the second nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L.

[0369] In a particular embodiment, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L and M212L, and the second nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L.

[0370] In a particular embodiment, the first nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of T138A, M204L and M212L, and the second nucleic acid molecule can comprise a genetic mutation that results in an amino acid mutation selected from the group consisting of M1L.

[0371] Tables A to D list mutations of each AAV that affect VP protein expression. A person skilled in the art can select appropriate mutations to engineer the gene encoding VP1 or VP2 to suppress the expression of one or more of VP1, VP2 and VP3.

[0372] Protein of interest and other mutant forms

[0373] In another aspect, in the nucleic acid combination, the first nucleic acid molecule, the second nucleic acid molecule and / or the third nucleic acid molecule can further comprise a gene of interest, the gene of interest encoding a protein of interest.

[0374] In certain embodiments, the insertion site of the protein of interest can be located in one or more regions selected from the group consisting of: the loop between βΒ and βc, variable region I, variable region II, variable region IV, variable region V, variable region VI, variable region VII, variable region VIII, HI loop, and variable region IX.

[0375] In certain embodiments, the insertion site of the protein of interest can be located in one or more regions selected from the group consisting of: the loop between βΒ and βc, variable region I, variable region II, variable region IV, variable region V, variable region VI, variable region VII, variable region VIII, and variable region IX.

[0376] In certain embodiments, the insertion site of the protein of interest can be located in the loop between βΒ and βc.

[0377] In certain embodiments, the insertion site of the protein of interest can be located in variable region I.

[0378] In certain embodiments, the insertion site of the protein of interest can be located in variable region II.

[0379] In certain embodiments, the insertion site of the protein of interest can be located in variable region IV.

[0380] In certain embodiments, the insertion site of the protein of interest can be located in variable region V.

[0381] In certain embodiments, the insertion site of the protein of interest can be located in variable region VI.

[0382] In certain embodiments, the insertion site of the protein of interest can be located in variable region VII.

[0383] In certain embodiments, the insertion site of the protein of interest can be located in variable region VIII.

[0384] In certain embodiments, the insertion site of the protein of interest can be located in the HI loop.

[0385] In certain embodiments, the insertion site of the protein of interest can be located in variable region IX. In certain embodiments, the molecular weight of the protein of interest can be between about 1 KD and about 100 KD.

[0386] In certain embodiments, the molecular weight of the protein of interest can be between about 1 KD and about 2 KD; the molecular weight of the protein of interest can be between about 2 KD and about 3 KD; the molecular weight of the protein of interest can be between about 3 KD and about 4 KD; the molecular weight of the protein of interest can be between about 4 KD and about 5 KD; the molecular weight of the protein of interest can be between about 5 KD and about 6 KD; the molecular weight of the protein of interest can be between about 6 KD and about 7 KD; the molecular weight of the protein of interest can be between about 7 KD and about 8 KD; the molecular weight of the protein of interest can be between about 8 KD and about 9 KD; the molecular weight of the protein of interest can be between about 9 KD and about 10 KD; the molecular weight of the protein of interest can be between about 10 KD and about 20 KD; the molecular weight of the protein of interest can be between about 20 KD and about 30 KD; the molecular weight of the protein of interest can be between about 30 KD and about 40 KD; the molecular weight of the protein of interest can be between about 40 KD and about 50 KD; the molecular weight of the protein of interest can be between about 50 KD and about 60 KD; the molecular weight of the protein of interest can be between about 60 KD and about 70 KD; the molecular weight of the protein of interest can be between about 70 KD and about 80 KD; the molecular weight of the protein of interest can be between about 80 KD and about 90 KD; the molecular weight of the protein of interest can be between about 90 KD and about 100 KD.

[0387] In certain embodiments, the molecular weight of the protein of interest can be about 1 KD; the molecular weight of the protein of interest can be about 2 KD; the molecular weight of the protein of interest can be about 3 KD; the molecular weight of the protein of interest can be about 4 KD; the molecular weight of the protein of interest can be about 5 KD; the molecular weight of the protein of interest can be about 6 KD; the molecular weight of the protein of interest can be about 7 KD; the molecular weight of the protein of interest can be about 8 KD; the molecular weight of the protein of interest can be about 9 KD; the molecular weight of the protein of interest can be about 10 KD; the molecular weight of the protein of interest can be about 20 KD; the molecular weight of the protein of interest can be about 30 KD; the molecular weight of the protein of interest can be about 40 KD; the molecular weight of the protein of interest can be about 50 KD; the molecular weight of the protein of interest can be about 60 KD; the molecular weight of the protein of interest can be about 70 KD; the molecular weight of the protein of interest can be about 80 KD; the molecular weight of the protein of interest can be about 90 KD; the molecular weight of the protein of interest can be about 100 KD.

[0388] In certain embodiments, the protein of interest can be selected from one or more proteins of the group consisting of: a reporter protein, a therapeutic protein, and a prophylactic protein.

[0389] In certain embodiments, the protein of interest can be a reporter protein, which can be selected from one or more of the following: green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), luciferase, β-galactosidase (LacZ), chloramphenicol acetyltransferase (CAT), secreted alkaline phosphatase (SEAP), variants of yellow fluorescent protein (such as Citrine and mVenus), mCherry, mNeonGreen, HaloTag, and SNAPTag.

[0390] In certain embodiments, the protein of interest can be a therapeutic protein, which can be selected from one or more of the following: an antibody, a recombinant enzyme, a hormone, a growth factor, a coagulation factor, a cytokine, and a therapeutic vaccine.

[0391] For example, the antibody can be a neutralizing antibody for a virus or a bacterium, or an antibody drug. The neutralizing antibody can be a neutralizing antibody for a virus. The virus can be selected from one or more of the following: human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza virus. The target of the antibody drug can comprise an immune checkpoint and / or a cytokine. The immune checkpoint can be selected from one or more of the following: PD-1, PD-L1, PD-L2, CTLA4, VISTA, CD155, TIGIT, CD28, ICOS, GITR, GITRL, TIM-3, CD137, and LAG-3. The cytokine can be selected from one or more of the following: IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-a, and IFN-g.

[0392] For another example, the recombinant enzyme can be a digestive enzyme or a tool enzyme. The digestive enzyme can be selected from one or more of the following: amylase, protease, lipase, lactase, maltase, sucrase, phosphatase, nuclease, cellulase, peptidase, carboxypeptidase, aminopeptidase, and transglutaminase. The tool enzyme can be selected from one or more of the following: Cre recombinase, Flp recombinase, Dre recombinase, restriction endonuclease, DNA ligase, polymerase, reverse transcriptase, phosphatase, kinase, alkaline phosphatase, exonuclease, endonuclease, methylase, demethylase, and transposase.

[0393] For another example, the hormone can be selected from one or more of the following: insulin, glucagon, growth hormone, thyroid-stimulating hormone, gonadotropin, adrenocortical hormone, gastrin, angiotensin, antidiuretic hormone, erythropoietin, and thrombopoietin.

[0394] For another example, the growth factor can be selected from one or more of platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and bone morphogenetic protein (BMP).

[0395] For another example, the coagulation factor can be selected from one or more of coagulation factor I (fibrinogen), coagulation factor II (prostacyclin), coagulation factor III (tissue factor), coagulation factor V (a factor that accelerates the clotting process), coagulation factor VIII (anti-hemophilic factor A), coagulation factor IX (anti-hemophilic factor B), coagulation factor X (Stuart factor), coagulation factor XI (platelet factor 4), coagulation factor XII (Hageman factor), and coagulation factor XIII (fibrin-stabilizing factor).

[0396] For another example, the cytokine can be selected from one or more of IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-a, and IFN-g.

[0397] For another example, the therapeutic vaccine can be a vaccine for treating cancer (such as a DC vaccine), or a vaccine for treating autoimmune diseases.

[0398] In certain embodiments, the protein of interest can be a therapeutic protein, which can be a fusion protein, meaning a protein that combines the properties of two or more proteins, the composition of which can be referred to above.

[0399] In certain embodiments, the protein of interest can be a prophylactic protein, which can be a prophylactic vaccine.

[0400] In certain embodiments, the protein of interest can be an antigen-binding protein. In certain embodiments, the antigen-binding protein can comprise an antibody or an antigen-binding fragment thereof. The antibody or binding fragment thereof can be selected from one or more of a monospecific antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a Fab, a Fv, a F(ab’)2, and a single-chain Fv (scFv). In certain embodiments, the antibody can be a nanobody.

[0401] In a first embodiment, the insertion site of the protein of interest can be located on the AAV6 capsid protein. In particular, it can be located in one or more regions selected from the group consisting of: the loop between βB and βC, variable region I, variable region II, variable region IV, variable region V, variable region VI, variable region VII, variable region VIII, HI loop and variable region IX derived from AAV6.

[0402] In a particular embodiment, the loop between βB and βC can be located between positions 250 to 258 of the VP1, VP2 and / or VP3 capsid protein.

[0403] In a particular embodiment, the variable region I can be located between positions 259 to 273 of the VP1, VP2 and / or VP3 capsid protein, more particularly between positions 261 to 273 of the VP1, VP2 and / or VP3 capsid protein.

[0404] In a particular embodiment, the variable region II can be located between positions 325 to 331 of the VP1, VP2 and / or VP3 capsid protein, more particularly between positions 327 to 330 of the VP1, VP2 and / or VP3 capsid protein.

[0405] In a particular embodiment, the variable region IV can be located between positions 443 to 476 of the VP1, VP2 and / or VP3 capsid protein, more particularly between positions 452 to 458 of the VP1, VP2 and / or VP3 capsid protein.

[0406] In a particular embodiment, the variable region V can be located between positions 488 to 506 of the VP1, VP2 and / or VP3 capsid protein, more particularly between positions 496 to 499 of the VP1, VP2 and / or VP3 capsid protein.

[0407] In a particular embodiment, the variable region VI can be located between positions 525 to 544 of the VP1, VP2 and / or VP3 capsid protein, more particularly between positions 528 to 535 of the VP1, VP2 and / or VP3 capsid protein.

[0408] In a particular embodiment, the variable region VII can be located between positions 545 to 560 of the VP1, VP2 and / or VP3 capsid protein, more particularly between positions 546 to 554 of the VP1, VP2 and / or VP3 capsid protein.

[0409] In specific embodiments, the variable region VIII can be located between positions 580 to 595 of the VP1, VP2 and / or VP3 capsid protein, more specifically between positions 581 to 593 of the VP1, VP2 and / or VP3 capsid protein.

[0410] In specific embodiments, the HI loop can be located between positions 653 to 674 of the VP1, VP2 and / or VP3 capsid protein, more specifically between positions 660 to 672 of the VP1, VP2 and / or VP3 capsid protein.

[0411] In specific embodiments, the variable region IX can be located between positions 702 to 719 of the VP1, VP2 and / or VP3 capsid protein.

[0412] In specific embodiments, the insertion site of the protein of interest can be selected from one or more of the following: between N254 and H255, between S264 and G266, between D328 and G329, between G454 and S455, between N498 and S499, between D530 and K531, between A548 and G549, between S588 and D590, between S663 and A664, between S708 and A709, and, between N717 and N718. The N254 refers to the asparagine at position 254 of the VP1, VP2 and / or VP3 capsid protein, the H255 refers to the histidine at position 255 of the VP1, VP2 and / or VP3 capsid protein, and so on.

[0413] In another aspect, the nucleic acid combination can further comprise other genetic mutations on VP1, VP2 and / or VP3.

[0414] In certain embodiments, the other mutations can not affect the normal expression of VP1, VP2 and / or VP3.

[0415] In certain embodiments, the other mutations can be mutations that affect the assembly ability of AAV. Specifically, the other mutations can be mutations that enhance the assembly ability of AAV, or mutations that weaken the assembly ability of AAV.

[0416] In certain embodiments, the other mutations can be mutations that improve the targeting ability of AAV. For example, the other mutations can improve the targeting ability of AAV to specific tissues and / or specific organs (e.g., heart, brain, etc.).

[0417] Vectors and vector combinations

[0418] In another aspect, the present application provides a vector. The vector can comprise one or more of: any of the foregoing first nucleic acid molecules, any of the foregoing second nucleic acid molecules, and any of the foregoing third nucleic acid molecules.

[0419] In certain embodiments, the vector can be an expression vector.

[0420] In certain embodiments, the vector can be DNA and / or RNA.

[0421] In certain embodiments, the vector can be selected from one or more of: a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector.

[0422] For example, the RNA vector can be selected from one or more of: a messenger RNA, a transfer RNA, a ribosomal RNA.

[0423] For another example, the DNA vector can be a plasmid.

[0424] In another aspect, the present application provides a vector combination. The vector combination can comprise one or more vectors: a first vector, a second vector, and a third vector. The vector combination can express the three capsid proteins VP1, VP2, and VP3 of AAV.

[0425] In certain embodiments, the first vector can comprise one or more of: any of the foregoing first nucleic acid molecules, any of the foregoing second nucleic acid molecules, and any of the foregoing third nucleic acid molecules.

[0426] In certain embodiments, the second vector can comprise one or more of: any of the foregoing first nucleic acid molecules, any of the foregoing second nucleic acid molecules, and any of the foregoing third nucleic acid molecules.

[0427] In certain embodiments, the third vector can comprise one or more of: any of the foregoing first nucleic acid molecules, any of the foregoing second nucleic acid molecules, and any of the foregoing third nucleic acid molecules.

[0428] In certain embodiments, the first vector, the second vector, and the third vector can be expression vectors, respectively.

[0429] In certain embodiments, the first vector, the second vector, and the third vector can be DNA and / or RNA, respectively.

[0430] In certain embodiments, the first vector, the second vector, and the third vector can be selected from one or more of: a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector, respectively.

[0431] For example, the RNA vector can be selected from one or more of the following: messenger RNA, transfer RNA, ribosomal RNA.

[0432] For another example, the DNA vector can be a plasmid.

[0433] Recombinant AAV virions and cells

[0434] In another aspect, the present application provides a recombinant AAV virion, which can be packaged from two or three nucleic acid molecules selected from the group consisting of any of the first nucleic acid molecules, any of the second nucleic acid molecules, and any of the third nucleic acid molecules.

[0435] In some embodiments, the recombinant AAV virion can be packaged by a triple plasmid system. The triple plasmid system can be a viral packaging system that produces high titer virus by co-transfecting three plasmids (i.e., a vector plasmid, a packaging plasmid, and a helper plasmid) associated with AAV packaging into producer cells (e.g., HEK293 cells) for a period of time, and then purifying a virus-containing composition, which can be cell lysate and / or cell culture medium.

[0436] In some embodiments, the vector plasmid can comprise a gene of interest. The gene of interest can be exogenous or endogenous. The gene of interest can be a gene that alters the nature of AAV itself, or a gene that encodes another drug carried by AAV. The gene that alters the nature of AAV itself can be a gene that increases the expression efficiency of AAV, increases the therapeutic efficiency of AAV, enhances the targeting ability of AAV, or changes other functions.

[0437] For example, the gene that alters the nature of AAV itself can be directed to an endogenous gene of AAV. The gene that alters the nature of AAV itself can be a mutant of an endogenous gene of AAV, can be an overexpression vector of an endogenous gene of AAV, can be a knockdown vector of an endogenous gene of AAV, or can be a knockout vector of an endogenous gene of AAV.

[0438] For another example, the gene that alters the nature of AAV itself can be directed to an exogenous gene. The gene that alters the nature of AAV itself can be an overexpression vector of an exogenous gene, or can be a mutant of an exogenous gene.

[0439] In some embodiments, the vector plasmid can comprise a promoter for gene expression. For example, the promoter for gene expression can be CMV.

[0440] In some embodiments, the vector plasmid can comprise an ITR. The gene of interest can be located within two ITRs.

[0441] In some embodiments, the vector plasmid can comprise a gene of interest, a promoter for gene expression, and an ITR. The gene of interest can be any of the aforementioned genes of interest. The promoter can be CMV.

[0442] In some embodiments, the method of inserting a gene of interest sequence can be accomplished by conventional genetic engineering methods in the art. For example, a plasmid containing a nucleotide sequence of interest, such as a gene of interest, can be used as a template to insert the gene of interest into a predetermined position by conventional molecular cloning methods such as homologous recombination. With the development of technology, it is also contemplated that the entire plasmid can be synthesized using synthetic technology after designing the structure of the plasmid, although in this case the step of "insertion" can not be included, but the plasmid obtained by this method should also be considered as part of the present application.

[0443] In some embodiments, the gene of interest can be a coding sequence or a non-coding sequence. For example, the transgene can be a therapeutic gene and / or a reporter gene. The therapeutic gene can encode a product having therapeutic activity. For example, the protein encoded by the therapeutic gene can be used in replacement therapy to treat diseases caused by defects in the protein. When it is a coding sequence, it can encode one or more target products. When more than one target product is encoded, they can be connected by a cleavable linker, such as a 2A peptide, such as E2A, P2A, T2A, F2A. There is no special limitation on the length of the transgene, which can be those suitable in the art. For example, the length of the transgene is preferably consistent with the packaging capacity of AAV.

[0444] In some embodiments, the packaging plasmid and the helper plasmid can use any suitable packaging plasmid and helper plasmid.

[0445] In some embodiments, the packaging plasmid can comprise a Rep gene of AAV. The Rep gene can be derived from different serotypes of AAV.

[0446] In some embodiments, the packaging plasmid can comprise a Cap gene of AAV. The Cap gene can be a Cap gene encoding a capsid protein of any AAV serotype. Specifically, the capsid protein can be VP1, VP2, and VP3 as described herein.

[0447] In some embodiments, the packaging plasmid can comprise a Cap and Rep gene of AAV.

[0448] In some embodiments, the helper plasmid can be a plasmid comprising a helper virus genome. The helper virus can be a virus that rescues AAV from a dormant state. For example, the helper virus can be an adenovirus. For another example, the helper virus can be a herpes simplex virus.

[0449] In certain embodiments, the helper plasmid can generally comprise one or more of E1A / B, E2A / B, E4 orf 6, VA genes of adenovirus. As commonly used adenovirus, Ad5 and Ad2 can be listed, preferably Ad5. The genes comprised in the helper plasmid can depend on the type of production cell. For example, when HEK293 cells are used as production cells, since HEK293 cells comprise E1A and E1B, a helper plasmid containing only E2A, E4 orf 6 and VA genes can be used.

[0450] In certain embodiments, the helper plasmid can assist the expression of Rep and Cap genes.

[0451] In another aspect, the present application provides a cell.

[0452] In certain embodiments, the cell can comprise any one of the aforementioned nucleic acid combinations.

[0453] In certain embodiments, the cell can comprise any one of the aforementioned vectors.

[0454] In certain embodiments, the cell can comprise any one of the aforementioned vector combinations.

[0455] In certain embodiments, the cell can comprise any one of the aforementioned recombinant AAV virions.

[0456] In certain embodiments, the cell can comprise a host cell.

[0457] In certain embodiments, the cell can comprise an immune cell and / or an inflammatory cell.

[0458] In certain embodiments, the cell can comprise a tumor cell.

[0459] In certain embodiments, the cell can exist in vivo.

[0460] For example, the cell can be a cell mass of cells gathered in a certain organ and / or tissue in vivo. For another example, the cell can be a single or multiple cells dispersed in a certain organ and / or tissue in vivo. For yet another example, the cell can be a circulating cell in the circulatory system in vivo. The circulatory system can comprise blood vessels and / or lymphatic vessels.

[0461] In certain embodiments, the cell can comprise a cell line isolated and cultured in vitro.

[0462] For example, the cell can be a primary cell line isolated from a tissue in vivo. For another example, the cell can be a passaged cell line. The passaged cell line can be commercialized or non-commercialized.

[0463] In some embodiments, the cell can be from any organism.

[0464] For example, the cell can be of human origin or of non-human origin. The non-human origin can be of mammalian origin or of non-mammalian origin. Examples of the mammalian origin can be selected from one or more of the following: mouse, rat, guinea pig, rabbit, and monkey.

[0465] Pharmaceutical compositions and therapeutic uses

[0466] In another aspect, the present application provides a pharmaceutical composition. It can comprise one or more of the following: any of the aforementioned nucleic acid combinations, any of the aforementioned vectors, any of the aforementioned vector combinations, any of the aforementioned recombinant AAV virions, any of the aforementioned cells, and optionally a pharmaceutically acceptable carrier.

[0467] In some embodiments, the optional pharmaceutically acceptable carrier can comprise saline, buffer, glucose, water, glycerol, ethanol, and any combination thereof. The pharmaceutical formulation should be matched with the mode of administration.

[0468] In some embodiments, the pharmaceutical composition can be used alone to treat a disease.

[0469] In some embodiments, the pharmaceutical composition can be used in combination with other drugs to treat a disease.

[0470] In some embodiments, the combination of the pharmaceutical composition with other drugs can be to link a nucleic acid molecule encoding other drugs to the nucleic acid molecules in the nucleic acid combination. For example, the nucleic acid molecule encoding other drugs can be linked outside the capsid protein coding sequence. For another example, the nucleic acid molecule encoding other drugs can be linked in the expression frame of the capsid protein coding sequence.

[0471] In some embodiments, the combination of the pharmaceutical composition with other drugs can be to link a nucleic acid molecule encoding other drugs to the nucleic acid molecules in the nucleic acid combination, preferably the nucleic acid molecule encoding other drugs can be linked in the expression frame of the capsid protein coding sequence. For example, the other drugs are expressed on the outer surface of the recombinant AAV virion. For another example, the other drugs are expressed on the inner surface of the recombinant AAV virion.

[0472] In some embodiments, the combination of the pharmaceutical composition with other drugs can be to use the recombinant AAV packaged using the nucleic acid combination, vector, or vector combination provided by the present application and the recombinant AAV virion provided by the present application to deliver other drugs. Specifically, it can be to put the coding sequence of the other drugs into the vector plasmid.

[0473] In another aspect, the present application provides a method of treating a disease. It can comprise use of one or more of the following: any of the aforementioned nucleic acid combinations, any of the aforementioned vectors, any of the aforementioned vector combinations, any of the aforementioned recombinant AAV virions, any of the aforementioned cells, and any of the aforementioned pharmaceutical compositions.

[0474] In certain embodiments, the disease can be selected from one or more of the following: a cardiac disease, a brain disease, an ophthalmologic disease, a tumor, an autoimmune disease, and a blood disease.

[0475] For example, the cardiac disease can be selected from one or more of the following: coronary heart disease, cardiomyopathy, valvular heart disease, congenital heart disease, and inflammatory heart disease.

[0476] For another example, the brain disease can be selected from one or more of the following: a neurodevelopmental disorder, a neurodegenerative disease, a traumatic brain injury, and a stroke.

[0477] For another example, the ophthalmologic disease can be selected from one or more of the following: deafness, a retinal disease, an ocular fundus disease, and a corneal conjunctival disease.

[0478] For another example, the tumor can be selected from one or more of the following: B-cell lymphoma, T-cell lymphoma, leukemia, Hodgkin's lymphoma, myeloma, plasmacytoma, adrenocortical carcinoma, bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioma, head and neck squamous carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, stomach cancer, thymic carcinoma, and endometrial cancer.

[0479] For another example, the autoimmune disease can be selected from one or more of the following: systemic lupus erythematosus, lupus nephritis, type 1 diabetes, systemic sclerosis, idiopathic inflammatory myopathy, Sjogren's syndrome, systemic vasculitis, rheumatoid arthritis, primary membranous nephropathy, limbic encephalitis, scleroderma myopathy, anti-phospholipid syndrome, autoimmune hemolytic anemia, IgG4-related disease, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, Takayasu's arteritis, chronic autoimmune hepatitis, primary biliary cirrhosis, myasthenia gravis, neuromyelitis optica spectrum disorder, multiple sclerosis, Lambert-Eaton syndrome, Hashimoto's thyroiditis, Graves' disease, and paraneoplastic cerebellar syndrome.

[0480] For another example, the blood disease can be selected from one or more of the following: hemophilia, aplastic anemia, thrombocytopenia, fibrosis of the bone, and thalassemia.

[0481] Table A Mutations in AAV6 capsid protein expression

[0482] Table B Mutations in AAV2 capsid protein expression

[0483] Table C Mutations in AAV5 capsid protein expression

[0484] Table D Mutations in AAV9 capsid protein expression

[0485] Table E Mutations in AAV8 capsid protein expression

[0486] Without wishing to be bound by any theory, the examples below are merely intended to illustrate the fusion proteins, methods of making, and uses of the present application, and are not intended to limit the scope of the present application.

[0487] Examples

[0488] Materials and methods

[0489] 1. Plasmid construction

[0490] For point mutation plasmids:

[0491] Primers were designed according to the mutation site, which contained about 20 bp sequences on both sides of the mutation site, and the mutation site used the sequence after mutation. High-fidelity DNA synthesis enzyme was used for PCR. After amplification, Dpnl endonuclease was added to remove the template plasmid, and then E. coli was transformed. After picking clones, sequencing was performed for identification.

[0492] For fragments that need to replace the expression vector backbone:

[0493] First, primers were designed according to the target fragment and the target backbone plasmid, which contained about 20 bp of the target fragment sequence and about 20 bp of the sequence next to the insertion site of the backbone plasmid. The desired target fragment was amplified using the designed primers, and then purified and cloned into the backbone plasmid using the HB-infusion seamless cloning kit. After transforming E. coli, single colonies were picked for sequencing identification.

[0494] 2. Protein expression identification

[0495] The constructed plasmid was transfected into 293T, and the cells were collected 48 hours after transfection. The cells were lysed with cell lysis solution (Tris 40 mM, NaCl 150 mM, Triton X-100 1%, EDTA 1 mM, SDS 0.1%, pH = 7.4, and protease inhibitors were added before use), and the supernatant was collected after centrifugation at 20000g for 5 minutes. The protein concentration was determined by BCA method and was uniformed to the same concentration. The protein loading buffer was added at a ratio of 3:1, and the protein was denatured at 95 degrees for 5 minutes. Protein separation was performed using 10% SDS-PAGE, and the protein loading amount was 20 ug / sample. Anti-AAV VP1 / VP2 / VP3 mouse monoclonal, B1 antibody was used to detect the expression of VP1, VP2, and VP3; Anti-Flag antibody was used to detect the expression of the inserted fragment in the capsid protein.

[0496] 3. Recombinant AAV virus particle packaging and titer identification

[0497] The 293T was used to package the virus. The capsid protein expression plasmid, pHelper, and pITR-CMV-ZsGreen-Luciferase-ITR plasmid were co-transfected into 293T cells, and the cell body was collected after 72 hours. PBS was added for resuspension, and the cell debris was removed by centrifugation after freezing-thawing 3 times. The supernatant was collected, Benzonase was added to remove nucleic acids in the sample, and then AAV particles were purified by iodixanol density gradient ultracentrifugation. Fluorescent quantitative PCR was used to identify the virus genome, and the virus genome (vg) titer was obtained. After uniforming according to the virus genome titer, 1*10 10 vg, 20ul 2x Loading buffer was added, and the virus protein was denatured at 95 degrees for 5 minutes. Then the expression of the capsid protein and the integration of the inserted fragment in the virus capsid were detected according to the expression part of 1.2.

[0498] 4. Recombinant AAV virus particle infection ability test

[0499] The purified virus was used to infect 293T at MOI = 100000, and the virus infection and gene expression efficiency were tested by detecting the proportion of fluorescent protein expression or Luciferase activity by flow cytometry 3 days after infection. For AAV virus particles with nanobodies inserted into the capsid protein, 293T cells overexpressing target membrane antigens were infected at MOI = 1000, 10000, 100000, and the fluorescent protein expression efficiency was detected 3 days after infection.

[0500] Example 1: Plasmid construction and expression detection

[0501] The plasmid was designed according to the following rules:

[0502] 1. M1 site is the start codon of VP1, which is mutated to a non-M codon, which is expected to inhibit the expression of VP1;

[0503] 2. T138 site, codon ACG, is the start codon of VP2, which is mutated to other non-ATG codons, which is expected to inhibit the expression of VP2, and is mutated to M, which is expected to inhibit the expression of VP3;

[0504] 3. The start codon of VP3 is M203, but there are other possible start codons in the C-terminal direction of the M203 site, such as M211, L235, etc., which need to be mutated to multiple possible classic or non-classic start codons, which are expected to inhibit the expression of VP3;

[0505] 4. It is expected that the inhibition of VP1 can also be achieved by mutating the N-terminal codon of VP1 to a stop codon;

[0506] 5. In the case of insufficient expression of capsid proteins after mutation of RC6 plasmid (also referred to as "R2C6" herein), the corresponding capsid proteins need to be constructed into a vector with a strong promoter such as CMV promoter, which is expected to increase the expression of capsid proteins;

[0507] 6. AAP protein is very important for AAV6 packaging, and its gene is located inside the capsid protein expression frame. In order to avoid the influence of various mutations on AAP expression and further affect the packaging of virus particles, it is necessary to separately split out the AAP expression plasmid.

[0508] Table 1 is a summary of the plasmids for split expression of capsid proteins. Among them, the plasmids starting with R2C6 represent the plasmids constructed based on R2C6 (Hanheng Biotechnology (Shanghai) Co., Ltd.) as the backbone plasmid, and then according to the point mutations (T138A, M203L, etc.) described above; The plasmid starting with pCMV represents the construction of the corresponding capsid protein into the expression plasmid of pcDNA3.1 (Hanheng Biotechnology (Shanghai) Co., Ltd.) etc. backbone; T138A, M203L, M211L, L235L, M1L, etc. represent the amino acid mutations of the corresponding VP proteins; * in the plasmid represents the mutation of the site to a stop codon, for example, R2C6-Y6* and R2C6-E12*; Mut-SA1 or Mut-SA-2 in the plasmid represents the mutation of the splice acceptor site in the AAV genome, which inhibits the expression of specific capsid proteins by canceling RNA splicing, for example, R2C6-Mut-SA1 and R2C6-Mut-SA2.

[0509] After the plasmid is constructed, 293T cells are transfected, and cell lysates are collected to identify the split expression of capsid proteins. Table 2 and Figure 1 are the actual expression of the capsid protein expression plasmid detected by immunoblotting. Figure 1 shows the results of the identification of capsid protein expression, wherein groups 1 to 17 correspond to the sequence numbers in Table 2, and the results show that the expression of the capsid expression plasmid meets the expectations.

[0510] Table 1. Capsid protein split expression plasmid construction table

[0511] Table 2. Capsid protein expression plasmid and expression results

[0512] Example 2 Split plasmid packaging combination test

[0513] The capsid protein coding gene in the AAV genome has a fusion gene phenomenon, in which, in addition to the three capsid proteins, at least two proteins AAP and MAAP are included, which are important auxiliary proteins in viral particle packaging. The split expression plasmid of capsid protein constructed by point mutation strategy may affect the expression of fusion gene, leading to failure of viral particle packaging, so it is necessary to test different combinations of split expression plasmid of capsid protein.

[0514] According to the plasmid combinations in Table 3 and Table 4, Luciferase expression virus is packaged, and 293T cells are infected. 72 hours later, the cells are collected to determine the Luciferase activity, and the effects of different plasmid combinations on virus packaging are compared.

[0515] From the combination of capsid plasmids, it can be seen that 1) AAP significantly improves the packaging efficiency of AAV6; 2) Even different expression plasmids of the same capsid protein have a large difference in the packaging efficiency of AAV6 virus particles. Plasmid A and plasmid B, plasmid G and plasmid B, plasmid H and plasmid D, plasmid E and plasmid F, plasmid J and plasmid L, etc. Combination still maintains good packaging efficiency, and plasmid O expressing AAP protein of AAV6 can be added to further improve the packaging efficiency.

[0516] Table 3. Test plasmid list

[0517] Table 4. Test plasmid combination and packaging efficiency

[0518] Example 3 Construction of AAV6 capsid protein expression plasmid with large fragment protein inserted based on split expression plasmid of capsid

[0519] The advantage of the capsid split expression plasmid is that one or two capsid proteins can be engineered while retaining the other wild-type capsid proteins, reducing the decline in packaging efficiency due to engineering. To achieve this purpose, we selected to insert a nanobody of about 20KD in a specific region of the capsid protein for testing. The selection strategy for the insertion region is: 1) exposed on the surface of AAV6 particles; 2) the insertion region should be a flexible region without secondary structure. Based on the above two criteria, there are 10 regions in the AAV6 capsid protein that can be inserted with exogenous fragments as shown in Table 5. They are all flexible loop structures exposed on the surface of AAV particles, which are: the loop between βB and βC, variable region I, variable region II, variable region IV, variable region V, variable region VI, variable region VII, variable region VIII, HI loop and variable region IX. The positions of the sites in the capsid protein are shown in Figure 2, where A is the VP3 structure and the optional insertion site position, and B is the AAV6 virus particle structure (part) and the insertable site position. Different colors represent different insertion sites (right side).

[0520] Table 5. Selected exogenous protein insertion sites (position information is ordered by VP1 of AAV6) in capsid protein

[0521] Variable region I, variable region IV, variable region VIII and HI loop in Table 5 were selected for large fragment protein insertion testing, where the variable region I insertion site is between S264 and G266, the variable region IV insertion site is between G454 and S455, the variable region VIII insertion site is between S588 and D590, and the HI loop insertion site is between S663 and A664. The inserted fragment is selected from the nanobody (nanobody) A0194009G09 sequence in the WO2015193452A1 patent, represented by "G09". G09 is connected to VP on the left through (GGGGS)5 and connected to VP on the right through GGGGA, and has three Flag tags. The plasmid is constructed as described in Table 6. Among them, the amino acid sequence GGGGS has the sequence number SEQ ID NO: 1 in this application, and the amino acid sequence GGGGA has the sequence number SEQ ID NO: 2 in this application.

[0522] To verify whether the capsid protein carrying large fragments can be expressed, the above plasmids were transfected into 293T cells alone, and 48 hours after transfection, the cell lysates were collected for immunoblotting to identify the expression of capsid protein and inserted protein. As shown in FIG. 3, in the collected cell lysates, the capsid protein and the inserted protein can be detected at the same immunoblotting band, and the molecular weight of the fusion protein is significantly higher than that of the wild-type capsid protein, indicating that the above plasmids can express the fusion protein of capsid protein-inserted protein. In FIG. 3A, the expression of plasmids 4-11 was normal; in FIG. 3B, no expression of VP2-nanobody fusion protein was detected in plasmid 8, and the expression of other plasmids was normal.

[0523] Further tests were conducted on the insertion of large fragment proteins in other loop regions in Table 5, wherein the loop insertion site between βB and βC is between N254 and H255, the variable region II insertion site is between D328 and G329, the variable region V insertion site is between N498 and S499, the variable region VI insertion site is between D530 and K531, the variable region VII insertion site is between A548 and G549, the variable region IX insertion site is between S708 and A709, and the N717 and N718 insertion site. The inserted fragment is selected from the A0194009G09 sequence (SEQ ID NO: 3) in the WO2015193452A1 patent, and the plasmids as described in Table 7 were constructed.

[0524] To verify whether the capsid protein carrying large fragments can be expressed, the above plasmids were transfected into 293T cells alone, and 48 hours after transfection, the cell lysates were collected for immunoblotting to identify the expression of capsid protein and inserted protein. As shown in FIG. 7, in the collected cell lysates, the capsid protein and the inserted protein can be detected at the same immunoblotting band, and the molecular weight of the fusion protein is significantly higher than that of the wild-type capsid protein, indicating that the above plasmids can express the fusion protein of capsid protein-inserted protein.

[0525] Table 6. AAV6 capsid protein expression plasmid containing an inserted fragment

[0526] Table 7. AAV6 capsid protein expression plasmid containing an inserted fragment

[0527] Example 4. Identification of the efficiency of AAV6 capsid protein integrated into AAV virus particles after insertion of large fragment proteins at different sites

[0528] To further identify whether the capsid protein-nanobody fusion protein can be integrated into the AAV virus particles, we used 293T co-transfected capsid protein expression plasmid, helper plasmid, AAP expression plasmid and core plasmid (ITR-CMV-ZsGreen-ITR), and collected the packaged virus particles by iodixanol density gradient centrifugation for immunoblotting detection. As shown in Figure 4, the capsid proteins expressed by plasmids 8, 9, 10, 12, 13, and 14, i.e., the large fragment protein insertion region at variable region I, variable region IV, and variable region VIII, can be integrated into the AAV virus particles, while no large fragment protein insertion site at H1-loop capsid protein (lanes F and K of Figure 4) was detected. The above results show that: 1) the large fragment protein inserted at variable region I, variable region IV, and variable region VIII can be integrated into the packaged AAV particles; 2) the large fragment protein inserted at H1-loop cannot be integrated or integrated into the packaged AAV particles with very low efficiency.

[0529] The immunoblotting experiment proved that after inserting the large fragment protein into the 11 flexible regions in VP1 and VP2, the VP-fusion protein can be normally expressed. Further, we used 293T to test its packaging and infection effect. As shown in Figure 5, the immunoblotting detection experiment after iodixanol density gradient purification of virus particles showed that the VP-fusion protein inserted at the loop site between βB and βC (inserted between N254-H255) and the H1 loop region (inserted between A663-A664) cannot be integrated into the AAV virus particles, and the insertion of variable region IX (inserted between N717-N718) greatly affects the integration of VP-fusion protein into virus particles. Among the insertion sites shown in Table 5, except for the HI loop and the loop between βB and βC, the large fragment protein inserted in other regions can be integrated into the packaged AAV particles.

[0530] Example 5 Identification of the Infection Ability of AAV6 Particles Carrying Large Fragment Insertion Proteins on Target Cells

[0531] To verify whether the AAV particles integrated with Nanobody can improve the infection ability to cells expressing the antigen of interest, we infected 293T cells expressing KCNA3 with the virus particles obtained in Figure 4, with a multiplicity of infection (MOI) of 10000, and collected the cells 72 hours after infection for flow cytometry to identify the efficiency of ZsGreen expression. As shown in Figure 6, the virus particles containing nanobody inserted at the positions of variable region I, variable region IV, and variable region VIII had significantly higher infection efficiency on 293-Pos (antigen-expressing positive cells) than on 293-Neg (antigen-expressing negative cells) (groups C, D, E, H, I, and J of Figure 6); while the virus particles containing nanobody inserted at the H1 loop region had no significant improvement in the infection ability on 293-Pos (groups F and K of Figure 6). Moreover, the infection efficiency of the VP1-nanobody fusion protein group (groups C, D, and E of Figure 6) on 293-Neg was significantly lower than that of the VP1 wild-type control group (group B of Figure 6), indicating that the fusion of VP1 with nanobody shielded the interaction between the original capsid protein and the receptor cells, indicating that the virus particles integrated with large fragments of proteins not only can improve the infection ability on target cells, but also can reduce the non-specific infection ability of the virus particles.

[0532] Further, HEK293T cells overexpressing the target of G09 antibody were used as target cells for infection test. Different capsid-packaged viruses carried the same Luciferase gene overexpression sequence, and the infection effect was characterized by the Luciferase activity of the cells after infection.

[0533] Table 8. Capsid expression plasmids of AAV6 capsid proteins with large fragments inserted at different positions

[0534] As shown in Table 9, the final virus packaging efficiency and infection ability are related to the site of large fragment insertion. The virus yield after inserting large fragment protein at the loop site between βΒ and βΟ (inserted between N254-H255 in the example) can maintain 50% packaging efficiency relative to wild type, the virus yield after inserting large fragment protein at variable region IV (inserted between G454-S455 in the example) can maintain 70% packaging efficiency relative to wild type, and the rest of the combinations have less relative impact. Comparing the infection ability after inserting large fragment protein at different sites, the infection effect is significantly improved by 1.7-3.2 times after inserting large fragment at variable region IV (inserted between G454-S455 in the example), variable region V (inserted between N498-S499 in the example), variable region VI (inserted between D530-K531 in the example), variable region VII (inserted between A548-G549 in the example), and variable region VIII (inserted between S588-D590 in the example) sites; the infection effect is significantly improved by about 1.3 times after inserting large fragment at variable region I (inserted between S264-G266 in the example) and variable region IX (inserted between S708-A709 in the example) sites.

[0535] Table 9. Infection effect test after inserting large fragment at different sites of AAV6 capsid protein

[0536] Example 6 Split expression and packaging combination of AAV2 capsid protein

[0537] The above examples are based on AAV6 for split expression. In this example, AAV2 capsid protein is used for split expression. The plasmids are constructed according to the same method, 293T cells are transfected, and cell lysates are collected to identify the split expression of capsid protein. The constructed plasmids are shown in Table 10, and the AAV2 capsid protein expression results are shown in Table 11 and Figure 8.

[0538] Table 10. Split expression plasmid construction table of AAV2 capsid protein

[0539] Table 11. Summary of split expression of AAV2 capsid protein

[0540] According to the plasmid expression in Table 11, the plasmids in Table 12 are selected for further AAV packaging verification. Luciferase expression virus is packaged, and 293T cells are infected for 72 hours to collect cells to determine Luciferase activity, and the effects of different plasmid combinations on virus packaging are compared.

[0541] The results are shown in Table 13. From the combinations of capsid plasmids, it can be seen that 1) AAP significantly improves the packaging efficiency of AAV2; 2) even different expression plasmids of the same capsid protein have a large difference in the packaging efficiency of AAV6 virus particles. The combinations of plasmid I and plasmid C, plasmid I and plasmid D, etc. still maintain good packaging efficiency, and the plasmid M expressing the AAP protein of AAV2 can be added to further improve the packaging efficiency.

[0542] Table 12. AAV2 capsid protein split plasmid packaging test list

[0543] Table 13. Test plasmid combinations and packaging efficiency

[0544] Example 7. Split expression and packaging combination of AAV5 capsid protein

[0545] This example uses AAV5 capsid protein for split expression. After the plasmids are constructed in the same way, 293T cells are transfected, and cell lysates are collected to identify the split expression of capsid protein. The constructed plasmids are shown in Table 14, the results of immunoblotting are shown in Figure 9, and the statistics are shown in Table 15.

[0546] Table 14. AAV5 capsid protein split expression plasmid construction table

[0547] Table 15. AAV5 capsid protein split expression plasmid construction table

[0548] According to the plasmid expression, the plasmids in Table 16 are selected for further AAV packaging verification. By packaging Luciferase expression virus, 293T cells are infected, and 72 hours later, the cells are collected to determine the Luciferase activity, and the effects of different plasmid combinations on virus packaging are compared.

[0549] The results are shown in Table 17. From the combinations of capsid plasmids, it can be seen that 1) AAP significantly improves the packaging efficiency of AAV5; 2) even different expression plasmids of the same capsid protein have a large difference in the packaging efficiency of AAV5 virus particles. The combinations of plasmid D and plasmid E, plasmid N and plasmid H, plasmid N and plasmid J, or plasmid M and plasmid G still maintain good packaging efficiency, and the plasmid C expressing the AAP protein of AAV5 can be added to further improve the packaging efficiency.

[0550] Table 16. AAV5 capsid protein split plasmid packaging test list

[0551] Table 17. Test plasmid combinations and packaging efficiency

[0552] Example 8 Split expression and packaging combination of AAV9 capsid protein

[0553] This example uses AAV9 capsid protein for split expression. After the plasmids are constructed in the same way, 293T cells are transfected, and cell lysates are collected to identify the split expression of capsid protein. The constructed plasmids are shown in Table 18, the results of immunoblotting are shown in Figure 10, and the statistics are shown in Table 19.

[0554] Table 18. Split expression plasmid construction table of AAV9 capsid protein

[0555] Table 19. Split expression plasmid construction table of AAV9 capsid protein

[0556] According to the plasmid expression in Table 19, the plasmids in Table 20 are selected for further AAV packaging verification. By packaging Luciferase expression virus, 293T cells are infected, and the Luciferase activity is measured 72 hours after cell collection to compare the effects of different plasmid combinations on virus packaging.

[0557] The results are shown in Table 21. From the capsid plasmid combination, it can be seen that 1) AAP significantly improves the packaging efficiency of AAV9; 2) Even different expression plasmids of the same capsid protein, there is a large difference in the packaging efficiency of AAV9 virus particles. Plasmid D and plasmid E, plasmid D and plasmid F, plasmid D, plasmid G and plasmid H, plasmid I and plasmid J, plasmid K and plasmid J, plasmid N and plasmid I, plasmid N and plasmid K, etc. Combination still maintains good packaging efficiency, while adding plasmid C expressing AAV9 AAP protein can further improve the packaging efficiency.

[0558] Table 20. AAV9 capsid protein split plasmid packaging test list

[0559] Table 21. Test plasmid combination and packaging efficiency

[0560] Example 9 AAV6 capsid protein insertion large fragment after hepatotropism test

[0561] To verify whether the AAV particles integrated with nanobodies can reduce the AAV liver aggregation caused by intravenous injection, this embodiment inserts a large fragment of AAV6 at the variable region VI (between the sites D530-K531) to infect mice through tail vein injection, and the virus is used at a dose of 1E+12 vg. Four weeks after injection, the mouse liver is sampled and frozen sectioned, and the number of EGFP positive cells in the liver is detected by full slide scanner. As shown in Figure 11, the number of EGFP positive cells in the liver of the mouse injected with AAV6 inserted with a large fragment at the variable region VI (between the sites D530-K531) is significantly less than that of the mouse injected with wild-type AAV6, indicating that the AAV particles integrated with nanobodies can reduce the liver tropism and reduce the AAV liver aggregation caused by intravenous injection.

[0562] The nanobody of this embodiment is the same as that of Example 5.

[0563] Example 10 Construction of an expression plasmid for inserting a large fragment of protein into AAV5 capsid protein based on capsid split expression system

[0564] Based on the capsid split expression plasmid, the nanobody is successfully inserted into AAV6 capsid protein VP2, and the AAV6 inserted with the nanobody enhances the infection ability of the cell expressing the target antigen. To verify whether this engineering method is applicable to other types of AAV, further selection is made to test the large fragment insertion into AAV5 capsid protein VP2. The large fragment insertion sites of AAV5 capsid protein are determined based on the packaging and infection effects of large fragment insertion into different regions of AAV6 and the high-frequency capsid modification sites used in the literature. The finally selected sites (the position information is arranged according to VP1 of AAV5) are: between T492 and N493, between Q574 and S575, and between A581 and T582. The insertion fragment selects the (VHH) sequence targeting TfR1, denoted as “TFR-3”, which is connected to VP on the left through (GGGGS)4 and connected to VP on the right through GGGGA, with or without (GGGGS)2, to construct the plasmid as shown in the upper panel of Figure 12.

[0565] To verify whether the capsid protein carrying a large fragment can be expressed, the above plasmid is used to transfect 293T cells alone, and the cells are collected 72 hours after transfection. After lysis, the expression of the capsid protein inserted with a large fragment of protein is identified by immunoblotting. As shown in the lower panel of Figure 12, in the collected cell lysate, the molecular weight of the capsid protein VP2 inserted with a large fragment of protein is significantly higher than that of the wild-type capsid protein VP2, indicating that the above plasmid can express the fusion protein of capsid protein VP2-inserted protein, i.e., the above three sites of AAV5 can be used to express the fusion protein of capsid protein-inserted protein.

[0566] Example 11 AAV5 virus packaging of capsid protein carrying large fragment insertion and identification of infection ability to target cells

[0567] To further identify whether the capsid protein-nanobody fusion protein can be integrated into the AAV5 virus particles, the 293T co-transfected capsid protein expression plasmid, helper plasmid and core plasmid (ITR-CMV-Luc-ZsGreen-ITR) were used, and the purified AAV virus particles after packaging were collected and subjected to immunoblotting detection. As shown in the lower panel of FIG. 13, the VP2-nanobody fusion protein inserted between T492 and N493, between Q574 and S575, and between A581 and T582 can be integrated into the packaged AAV5 particles.

[0568] To verify whether the AAV5 particles integrated with Nanobody can improve the infection ability to cells expressing target antigens, the titer of the purified virus was detected by qPCR, the purified virus particles were used to infect 293T cells expressing TfRl at a MOI of 1E+5, and the cells were collected 72 hours after infection for Luciferase activity detection. The Luciferase activity represents the infection effect.

[0569] As shown in the middle panel of FIG. 13, the packaging efficiency and infection ability of the capsid insertion large fragment protein virus are affected by the insertion site and the fusion protein connection mode. The virus yield after inserting the large fragment protein between Q574 and S575 through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection form is increased by about 80% relative to the original packaging system; the virus yield after inserting the large fragment protein between A581 and T582 through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection form is relatively less affected, and the virus yield after inserting the large fragment protein through the (GGGGS)4-TFR-3-GGGGA connection form can still maintain 70% packaging efficiency; the virus yield after inserting the large fragment protein between T492 and N493 through the two connection modes is about 70% relative to the original VP plasmid. After comparing the infection abilities after inserting the large fragment protein at different sites, the infection effect is significantly improved by about 14-fold after inserting the large fragment protein between T492 and N493 through the (GGGGS)4-TFR-3-GGGGA connection form, and the infection effect is significantly improved by about 3.8-fold after inserting the large fragment protein through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection form; the infection effect is significantly improved by about 1.8-2.5-fold after inserting the large fragment protein between Q574 and S575 and between A581 and T582.

[0570] Example 12 Construction of expression plasmid for inserting endosome escape peptide into AAV5 capsid protein based on split expression system of capsid

[0571] AAV capsid protein VP1 is essential for viral infection, and VP3 is essential for viral packaging. In order to verify whether the capsid proteins VP1 and VP3 can be functionally inserted with polypeptides based on the split expression system of capsid we designed, we selected to test the insertion of endosome escape peptide into AAV5 capsid protein VP1 and VP3. The insertion sites of endosome escape peptide on capsid protein VP1 and VP3 were selected from the commonly used sites of AAV5 capsid modification (the position information is ordered according to VP1 of AAV5): between Q574 and S575 and between A581 and T582. The endosome escape peptides selected were Aurein1.2 in 26465072 (PMID number) and GALA3 sequence in 32002589 (PMID number), represented as “Ayrein 1.2” and “GALA3” respectively, which were directly connected on the left and right sides, and the plasmids were constructed as shown in the upper panel of FIG. 14.

[0572] In order to verify whether the capsid proteins carrying the endosome escape peptide can be expressed, the above plasmids were transfected into 293T cells alone, and the cells were collected 72 hours after transfection. After lysis, the expression of capsid proteins inserted with endosome escape peptide was identified by immunoblotting. As shown in the lower panel of FIG. 14, in the collected cell lysates, the molecular weight of capsid protein VP3 inserted with endosome escape peptide was slightly higher than that of wild-type capsid protein VP3, indicating that the above plasmids can all express fusion proteins of capsid protein VP3-endosome escape peptide. At the same time, the split expression plasmid for expressing capsid protein VP1 and VP3 based on the expression plasmid for inserting endosome escape peptide into capsid protein VP1 and VP3 was constructed, so the above plasmids also expressed fusion proteins of capsid protein VP1-endosome escape peptide, i.e. both of the above two sites of AAV5 can be used to express fusion proteins of capsid protein-endosome escape peptide.

[0573] Example 13 AAV5 virus packaging carrying endosome escape peptide inserted protein

[0574] To further identify whether the capsid protein-endosome escape peptide fusion protein can be integrated into the AAV5 virus particles, 293T co-transfected capsid protein expression plasmid, helper plasmid and core plasmid (ITR-CMV-Luc-ZsGreen-ITR) were used to collect and purify the packaged AAV virus particles by iodixanol density gradient centrifugation, and Western blot detection was performed. As shown in the lower panel of FIG. 15, the molecular position of VP1-endosome escape peptide, VP3-endosome escape peptide fusion protein inserted between Q574 and S575 and between A581 and T582 is slightly higher than that of wild-type VP1 and VP3, indicating that the VP1-endosome escape peptide, VP3-endosome escape peptide fusion protein can be integrated into the packaged AAV5 particles.

[0575] Example 14 AAV5 virus packaging of proteins carrying both endosome escape peptide and large fragment insertion

[0576] Further, to verify whether the capsid protein VP1 / VP3 containing functional polypeptide insertion can be packaged into complete virus particles with the capsid protein VP2 containing large fragment insertion, we tested the combination of endosome escape peptide and large fragment insertion for AAV5 packaging. We selected the two highest infection efficiency from the tested capsid protein VP2 large fragment insertion sites and methods, and selected the highest packaging titer from the tested capsid protein VP1 and VP3 endosome escape peptide insertion design, and tested the combination of endosome escape peptide and large fragment insertion for packaging. We used 293T co-transfected capsid protein expression plasmid, helper plasmid and core plasmid (ITR-CMV-Luc-ZsGreen-ITR) to collect and purify the packaged AAV virus particles by affinity chromatography, and Western blot detection was performed. As shown in the lower right panel of FIG. 16, plasmid combination Code C, D corresponding to lanes C, D shows that the capsid protein VP2-large fragment fusion protein is integrated into the AAV virus particles; plasmid combination Code E corresponding to lane E shows that the capsid protein VP1-endosome escape peptide, VP3-endosome escape peptide fusion protein is integrated into the AAV virus particles; plasmid combination Code F, G corresponding to lanes F, G shows that the capsid protein VP2-large fragment fusion protein, capsid protein VP1-endosome escape peptide, VP3-endosome escape peptide fusion protein is integrated into the AAV virus particles.

[0577] Example 15 Construction of expression plasmid for inserting large fragment protein into AAV9 capsid protein based on capsid split expression plasmid

[0578] This embodiment tests large fragment insertion of AAV9 capsid protein VP2. The large fragment insertion site of AAV9 capsid protein is determined based on the large fragment insertion packaging and infection effect of different regions of AAV6 and the high-frequency capsid modification site used in the literature. The finally selected site (position information is ordered according to VP1 of AAV9) is: between G453 and S454, between A589 and Q590, between N498 and S499, and between G530 and E531. The insertion fragment is selected as TFR-3, which is connected to VP on the left through (GGGGS)4 and connected to VP on the right through GGGGA with or without (GGGGS)2, and the plasmid is constructed as shown in the upper panel of FIG. 17.

[0579] To verify whether the capsid protein carrying the large fragment can be expressed, the above plasmid is transfected into 293T cells alone, and the cells are collected 72 hours after transfection. The expression of the capsid protein inserted with the large fragment protein is identified by immunoblotting after lysis. As shown in the lower panel of FIG. 17, in the collected cell lysate, the molecular weight of the capsid protein VP2 inserted with the large fragment protein is significantly higher than that of the wild-type capsid protein VP2, indicating that the above plasmid can express the fusion protein of capsid protein VP2-inserted protein, i.e., the above four sites of AAV9 can be used to express the fusion protein of capsid protein-inserted protein.

[0580] Example 16 AAV9 virus packaging carrying large fragment inserted protein and identification of infection ability on target cells

[0581] To further identify whether the capsid protein-nanobody fusion protein can be integrated into the AAV9 virus particle, we use 293T to co-transfect the capsid protein expression plasmid, the helper plasmid and the core plasmid (ITR-CMV-Luc-ZsGreen-ITR), and collect the purified AAV virus particles after AAV purification affinity chromatography column for immunoblotting detection. As shown in the lower panel of FIG. 18, the VP2-nanobody fusion protein inserted between G453 and S454, between A589 and Q590, between N498 and S499, and between G530 and E531 can be integrated into the packaged AAV9 particles.

[0582] To verify whether the AAV9 particles integrated with Nanobody can improve the infection ability on cells expressing target antigens, we detect the titer of the purified virus by qPCR, infect the 293T cells expressing TfR1 with the purified virus particles, with MOI of 1E+5, collect the cells 72 hours after infection for Luciferase activity detection, and the Luciferase activity represents the infection effect.

[0583] As shown in the graph in FIG. 18, the packaging efficiency and infection ability of the capsid inserted large fragment protein virus are affected by the insertion site and the fusion protein connection mode. The virus yield after inserting the large fragment protein through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection mode between G453 and S454 has relatively small influence; the virus yield after inserting the large fragment protein through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection mode between A589 and Q590 has relatively small influence, the virus yield after inserting the large fragment protein through the (GGGGS)4-TFR-3-GGGGA connection mode is about 50% higher than that of the original packaging system; the virus yield after inserting the large fragment protein through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection mode between N498 and S499 is about 70% higher than that of the original packaging system; the virus yield after inserting the large fragment protein through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection mode between G530 and E531 is about 20% higher than that of the original packaging system. Comparing the infection abilities after inserting the large fragment protein at different sites, the infection effect after inserting the large fragment protein through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection mode between G530 and E531 is significantly improved by about 6.6 times; the infection effect after inserting the large fragment protein through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection mode between N498 and S499 is significantly improved by about 4.9 times; the infection effect after inserting the large fragment protein through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection mode between G453 and S454 is significantly improved by about 2.7 times; the infection effect after inserting the large fragment protein through the (GGGGS)4-TFR-3-(GGGGS)2-GGGGA connection mode between A589 and Q590 is significantly improved by about 2 times, and the infection effect after inserting the large fragment protein through the (GGGGS)4-TFR-3-GGGGA connection mode is improved by about 1.4 times.

[0584] Example 17 Test of the Trans-Blood-Brain-Barrier Effect of AAV9 Carrying Large Fragment Inserted Protein

[0585] To verify whether AAV particles integrated with TfR1-binding TFR-3 can improve the effect of AAV crossing the blood-brain barrier, we selected AAV9 with TFR-3 inserted between G530-E531 sites for mouse infection by tail vein injection, and the virus was used at a dose of 1E+12 vg. Three weeks after injection, the mice were subjected to in vivo imaging to detect the infection of AAV9 in the brain of the mice. As shown in FIG. 19, the luciferase signal in the brain of the mice injected with AAV9 with TFR-3 inserted between G530-E531 sites was about 2.4-2.8 times that of the mice injected with split wild-type AAV9 expressing capsid protein, indicating that AAV9 particles integrated with TFR-3 have stronger brain infection ability and better blood-brain barrier crossing effect.

[0586] Example 18 Construction of an expression plasmid for inserting an endosome escape peptide into AAV9 capsid protein based on a split expression plasmid for capsid

[0587] For this example, we selected to test the insertion of an endosome escape peptide into AAV9 capsid proteins VP1 and VP3 to verify the split expression system for capsid, whether the functional polypeptide insertion into capsid proteins VP1 and VP3 is suitable for other types of AAV. The insertion sites of the endosome escape peptide into capsid proteins VP1 and VP3 were selected from the insertion sites used in the large fragment of AAV9 capsid protein VP2 (the position information is arranged according to VP1 of AAV9): between N498 and S499 and between G530 and E531. The endosome escape peptide was selected as INF7 in PMID: 17973492 and GALA3 sequence in PMID: 32002589, represented as “INF7” and “GALA3”, respectively, and directly connected on the left and right sides to construct the plasmid as shown in the upper panel of FIG. 20.

[0588] To verify whether the capsid protein carrying the endosome escape peptide can be expressed, the above plasmids were individually transfected into 293T cells, and the cells were collected 72 hours after transfection. The cell lysates were subjected to immunoblotting to identify the expression of the capsid protein with the inserted endosome escape peptide. As shown in the lower panel of FIG. 20, the molecular weight of the capsid protein VP3 with the inserted endosome escape peptide was slightly higher than that of the wild-type capsid protein VP3 in the collected cell lysates, indicating that the above plasmids can all express the fusion protein of capsid protein VP3-endosome escape peptide. At the same time, the split expression plasmid for expressing capsid proteins VP1 and VP3 based on the plasmid for expressing capsid proteins VP1 and VP3 with the inserted endosome escape peptide, so the above plasmids also express the fusion protein of capsid protein VP1-endosome escape peptide, i.e., both of the above sites of AAV9 can be used to express the fusion protein of capsid protein-endosome escape peptide.

[0589] Example 19 AAV9 virus packaging with inserted endosome escape peptide

[0590] To further identify whether the capsid protein-endosome escape peptide fusion protein can be integrated into the AAV9 virus particle, we used 293T co-transfection of capsid protein expression plasmid, helper plasmid and core plasmid (ITR-CMV-Luc-ZsGreen-ITR), and collected the packaged AAV virus particles after iodixanol density gradient centrifugation for immunoblotting detection. As shown in the lower panel of FIG. 21, the molecular position of VP1-endosome escape peptide, VP3-endosome escape peptide fusion protein after insertion between Q574 and S575 and between A581 and T582 is slightly higher than that of wild-type VP1 and VP3, indicating that the VP1-endosome escape peptide, VP3-endosome escape peptide fusion protein can be integrated into the packaged AAV9 particles.

[0591] Example 20 AAV9 virus packaging carrying endosome escape peptide and large fragment insertion protein

[0592] The advantage of the VP split expression system is that different VP proteins can be modified separately to obtain multifunctional AAV particles. The combination of endosome escape peptide and large fragment insertion packaging test was performed on AAV9 to verify whether the capsid split expression system based on the present application, the functional polypeptide insertion of capsid proteins VP1 and VP3, and the large fragment insertion of capsid protein VP2, can be completed in one AAV9 virus particle. We selected the two highest infection efficiency from the tested capsid protein VP2 large fragment insertion sites and methods, and selected one with higher packaging titer from the tested capsid protein VP1 and VP3 endosome escape peptide insertion design, to perform the combination of endosome escape peptide and large fragment insertion packaging test. We used 293T co-transfection of capsid protein expression plasmid, helper plasmid and core plasmid (ITR-CMV-Luc-ZsGreen-ITR), and collected the packaged AAV virus particles after affinity chromatography for immunoblotting detection. As shown in the lower right panel of FIG. 22, plasmid combination Code C, D corresponds to lanes C, D showing that the capsid protein VP2-large fragment fusion protein is integrated into the AAV virus particle; plasmid combination Code E corresponds to lane E showing that the capsid protein VP1-endosome escape peptide, VP3-endosome escape peptide fusion protein is integrated into the AAV virus particle; plasmid combination Code F, G corresponds to lanes F, G showing that the capsid protein VP2-large fragment fusion protein, capsid protein VP1-endosome escape peptide, VP3-endosome escape peptide fusion protein is integrated into the AAV virus particle.

[0593] Example 21 AAV9 virus packaging carrying large fragment insertion protein

[0594] AAV has low infection efficiency on primary T cells. The improvement of infection efficiency on primary human T cells by using AAV strategy mainly through inserting large fragments or polypeptides binding to T cell surface antigens into AAV capsid. The AAV9 capsid protein VP2 was selected for large fragment insertion test to further verify whether the capsid split expression system based on the application can improve the infection efficiency of AAV on T cells. The insertion site of large fragments of AAV9 capsid protein VP2 is consistent with the insertion site of TFR-3 (the position information is arranged according to VP1 of AAV9): between G453 and S454, between A589 and Q590, between N498 and S499, and between G530 and E531. The insertion fragment is selected from the R2HCD26 sequence in the WO2017134306A1 patent, and the humanized modification is made on the basis of the sequence, represented as "D26". The left side of D26 is connected to VP through (GGGGS)4, and the right side is connected to VP through GGGGA, with or without (GGGGS)2. The plasmid is constructed as shown in the upper panel of FIG. 23.

[0595] To verify whether the capsid protein carrying large fragments can be expressed, the above plasmids were transfected into 293T cells alone, and the cells were collected 72 hours after transfection. The expression of capsid protein inserted with large fragment protein was identified by immunoblotting after lysis. As shown in the lower panel of FIG. 23, in the collected cell lysate, the molecular weight of capsid protein VP2 inserted with large fragment protein is significantly higher than that of wild-type capsid protein VP2, indicating that the above plasmids can express fusion proteins of capsid protein VP2-inserted protein, i.e., the above four sites of AAV9 can be used to express capsid protein-D26 fusion proteins.

[0596] Example 22 Infection ability of AAV9 virus carrying large fragment inserted protein on CD8 positive T cells

[0597] To further identify whether the capsid protein-D26 fusion protein can be integrated into the AAV9 virus particle, we used 293T co-transfected capsid protein expression plasmid, helper plasmid and core plasmid (ITR-CMV-Luc-ZsGreen-ITR), and collected the packaged AAV virus particles after AAV purification affinity chromatography column for immunoblotting detection. As shown in the lower panel of FIG. 24, the VP2-nanobody fusion protein inserted at four sites of G453 and S454, A589 and Q590, N498 and S499, and G530 and E531 can be integrated into the packaged AAV9 particles.

[0598] To verify whether the AAV9 particles integrated with D26 can improve the infection ability to cells expressing the target antigen, we detected the titer of the purified virus by qPCR, infected the purified virus particles to human peripheral blood mononuclear cells (hPBMCs) treated by T cell activation, with MOI of 1E+6, and collected the cells 72 hours after infection to identify the proportion of ZsGreen positive cells in CD8 positive T cells by flow cytometry.

[0599] As shown in the graph in FIG. 24, the packaging efficiency and infection ability of the capsid inserted large fragment protein virus are affected by the insertion site and the connection mode of the fusion protein.

[0600] The virus yield after inserting the large fragment protein between G453 and S454 through (GGGGS)4-D26-(GGGGS)2-GGGGA connection mode can maintain 51% of the packaging efficiency relative to the wild type, and the virus yield after inserting the large fragment protein through (GGGGS)4-D26-GGGGA connection mode can be increased by about 12% relative to the original packaging system; the virus yield after inserting the large fragment protein between A589 and Q590 through (GGGGS)4-D26-(GGGGS)2-GGGGA connection mode can maintain 56% of the packaging efficiency relative to the wild type; the virus yield after inserting the large fragment protein between N498 and S499 through (GGGGS)4-D26-(GGGGS)2-GGGGA connection mode can maintain 72% of the packaging efficiency relative to the wild type, and the virus yield after inserting the large fragment protein through (GGGGS)4-D26-GGGGA connection mode has no significant effect on the packaging efficiency; the virus yield after inserting the large fragment protein between G530 and E531 through (GGGGS)4-D26-(GGGGS)2-GGGGA connection mode can maintain 44% of the packaging efficiency relative to the wild type, and the virus yield after inserting the large fragment protein through (GGGGS)4-D26-GGGGA connection mode can maintain 52% of the packaging efficiency relative to the wild type.

[0601] The infection ability of CD8 positive cells after inserting D26 at different sites was compared, wherein the infection effect was significantly increased by about 1.8 times after inserting D26 between N498 and S499 through two connection modes; the infection effect was significantly increased by about 1.7 times after inserting D26 between G530 and E531 through two connection modes; the infection effect was significantly increased by about 1.5 times after inserting the large fragment protein between A589 and Q590 through (GGGGS)4-D26-(GGGGS)2-GGGGA connection mode; the infection effect was increased by about 1.3-1.7 times after inserting D26 between G453 and S454 through two connection modes.

[0602] Example 23 Split expression of AAV8 capsid protein and packaging combination

[0603] To verify whether AAV8 capsid protein can be split expressed as AAV6, 2, 5, 9 mentioned above, we followed the same strategy to test. After the plasmids were constructed in the same way, 293T cells were transfected, and cell lysates were collected to identify the split expression of capsid protein. The constructed plasmids are shown in Table 22, the results of immunoblotting are shown in Figure 25, and the statistics are shown in Table 23.

[0604] According to the plasmid expression in Table 23, the plasmids in Table 24 were selected for further AAV packaging verification. By packaging Luciferase expression virus, 293T cells were infected, and 72 hours later, the cells were collected to determine Luciferase activity, and the effects of different plasmid combinations on virus packaging were compared.

[0605] The results are shown in Table 25. From the combination of capsid plasmids, it can be seen that even different expression plasmids of the same capsid protein have a large difference in the packaging efficiency of AAV8 virus particles. Plasmid B and plasmid O, plasmid B and plasmid P, plasmid B and plasmid Q, plasmid B and plasmid R, plasmid E and plasmid L, plasmid E and plasmid M, plasmid F and plasmid I, etc. The combination still maintains good packaging efficiency.

[0606] Table 22 AAV8 capsid protein split expression plasmid construction table

[0607] Table 23 AAV8 capsid protein split expression summary

[0608] Table 24 AAV8 capsid protein split plasmid packaging test list

[0609] Table 25 Test AAV8 capsid protein split plasmid combination and packaging efficiency

Claims

1. A nucleic acid combination comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein, The first nucleic acid molecule comprises a nucleotide sequence capable of expressing one or two proteins selected from the group consisting of capsid proteins VP1, VP2 and VP3, the second nucleic acid molecule comprises a nucleotide sequence capable of expressing one or two proteins selected from the group consisting of VP1, VP2 and VP3, and the first and second nucleic acid molecules express different VP1, VP2 and VP3 protein species.

2. The nucleic acid combination of claim 1, wherein the first and second nucleic acid molecules each independently comprise a genetic mutation that is a mutation that inhibits expression of one or two of VP1, VP2 and VP3 proteins.

3. The nucleic acid combination of any one of claims 1-2, wherein the first and / or second nucleic acid molecule comprises a genetic mutation that inhibits expression of VP1.

4. The nucleic acid combination of claim 3, wherein the genetic mutation that inhibits expression of VP1 comprises: a) a genetic mutation that results in the VP1 start codon being mutated to a non-start codon; b) a genetic mutation that results in any one of the codons between the VP1 start codon and the VP2 start codon being mutated to a stop codon; and / or c) a genetic mutation that results in the abrogation of RNA splicing.

5. The nucleic acid combination of any one of claims 3-4, wherein the genetic mutation that inhibits expression of VP1 comprises: a) a genetic mutation that results in one or more amino acid mutations at one or more sites selected from the group consisting of M1, Y6 and E12; and / or b) a mutation at Splicing acceptor A1.

6. The nucleic acid combination of any one of claims 3-5, wherein the genetic mutation that inhibits expression of VP1 comprises: a) a genetic mutation that results in one or more amino acid mutations selected from the group consisting of M1 mutated to a non-M amino acid, Y6 codon mutated to a stop codon, E12 codon mutated to a stop codon; and / or b) a mutation at Splicing acceptor A1.

7. The nucleic acid combination of any one of claims 1-6, wherein the first and / or second nucleic acid molecule comprises a genetic mutation that inhibits expression of VP2.

8. The nucleic acid combination of claim 7, wherein the genetic mutation that inhibits expression of VP2 comprises: a) a genetic mutation that results in the start codon of VP2 being mutated to a non-start codon, and / or b) a genetic mutation that results in the abrogation of RNA splicing.

9. The nucleic acid combination of any one of claims 7-8, wherein the start codon of VP2 is T137 or T138.

10. The nucleic acid combination of any one of claims 7-9, wherein the genetic mutation that inhibits expression of VP2 comprises: a) T138 codon mutated from ACG to ACC, T138A, T137 codon mutated from ACG to ACC, or T137A; and / or b) a genetic mutation at Splicing acceptor A2. ​ 11. The nucleic acid combination according to any one of claims 1 to 10, wherein the first nucleic acid molecule and / or the second nucleic acid molecule comprises a genetic mutation that suppresses VP3 expression.

12. The nucleic acid combination according to claim 11, wherein the genetic mutation that suppresses VP3 expression comprises: a) a genetic mutation that results in a mutation of the start codon of VP3 to a non- start codon, b) a mutation of the start codon of VP2 to M; and / or c) a genetic mutation that results in an abrogation of RNA splicing.

13. The nucleic acid combination according to any one of claims 11 to 12, wherein the genetic mutation that suppresses VP3 expression comprises: a) a genetic mutation that results in an amino acid mutation at one or more positions selected from any one of groups (1) to (4): (1) T138, M203, M211, and L235, (2) T138, M203, M211, V221, and M235, (3) T137, M193, and M225, (4) T138, M203, L235, and L249, (5) T138, M204, M212, and / or b) a mutation at Splicing acceptor A2.

14. The nucleic acid combination according to any one of claims 11 to 13, wherein the genetic mutation that suppresses VP3 expression comprises: a) a genetic mutation that results in an amino acid mutation at one or more positions selected from any one of groups (1) to (4): (1) T138M, M203L, M211L, and L235L (CTG-CTC), (2) T138M, M203L, M211L, M235L, and V221V (GTG-GTC), (3) T137M, M193L, and M225L, and (4) T138M, M203L, L235L (CTG-CTC), and L249L (CTG-CTC), (5) T138M, M204L, M212L, and / or b) a genetic mutation at Splicing acceptor A2.

15. The nucleic acid combination according to any one of claims 1 to 14, wherein the first nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP1 protein and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP2 protein.

16. The nucleic acid combination according to any one of claims 1 to 14, wherein the first nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP1 protein and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP3 protein.

17. The nucleic acid combination according to any one of claims 1 to 14, wherein the first nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP3 protein and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP2 protein.

18. The nucleic acid combination according to any one of claims 1 to 14, wherein the first nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP1 protein and the second nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP2 protein and a VP3 protein. ​ 19. The nucleic acid combination of any one of claims 1-14, wherein the first nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP2 protein, and the second nucleic acid molecule comprises genetic mutations that suppress expression of a VP1 protein and a VP3 protein.

20. The nucleic acid combination of any one of claims 1-14, wherein the first nucleic acid molecule comprises a genetic mutation that suppresses expression of a VP3 protein, and the second nucleic acid molecule comprises genetic mutations that suppress expression of a VP1 protein and a VP2 protein.

21. The nucleic acid combination of any one of claims 1-20, wherein the genetic mutations comprised by the first nucleic acid molecule and the second nucleic acid molecule are selected from the group consisting of: (1) first nucleic acid molecule: T138A and M203L, second nucleic acid molecule: M1L, (2) first nucleic acid molecule: T138A and M203L, second nucleic acid molecule: Y6*, (3) first nucleic acid molecule: T138A and M203L, second nucleic acid molecule: M1L and M203L, or M1L and T138A, (4) first nucleic acid molecule: T138A, second nucleic acid molecule: is a nucleic acid molecule encoding VP2 and does not comprise a genetic mutation that suppresses expression of a VP, (5) first nucleic acid molecule: T138T, second nucleic acid molecule: is a nucleic acid molecule encoding VP2 and does not comprise a genetic mutation that suppresses expression of a VP, (6) first nucleic acid molecule: M203L, L235L (CTG-CTC), and L249L (CTG-CTC), second nucleic acid molecule: T138A, and (7) first nucleic acid molecule: M203L, L235L (CTG-CTC), and L249L (CTG-CTC), second nucleic acid molecule: T138T.

22. The nucleic acid combination of any one of claims 1-21, wherein the first nucleic acid molecule and the second nucleic acid molecule comprise the genetic mutations on the basis of the nucleotide sequence set forth in any one of SEQ ID NOs: 20-31 and 40-42.

23. The nucleic acid combination of any one of claims 1-22, wherein the VP1 protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 4, 7, 10, 13, and 37.

24. The nucleic acid combination of any one of claims 1-23, wherein the VP2 protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5, 8, 11, and 38.

25. The nucleic acid combination of any one of claims 1-24, wherein the VP3 protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 6, 9, 12, and 39.

26. The nucleic acid combination of any one of claims 1-25, wherein the first nucleic acid molecule and / or the second nucleic acid molecule further comprises a gene of interest, which encodes a protein of interest.

27. The nucleic acid combination of claim 26, wherein the protein of interest has a molecular weight of between about 1 KD and about 100 KD.

28. The nucleic acid combination of any one of claims 26-27, wherein the insertion site of the protein of interest is located in a region selected from the group consisting of: loop between beta B and beta C, HI loop, variable region I, variable region II, variable region IV, variable region V, variable region VI, variable region VII, variable region VIII, and variable region IX of the VP1 protein, the VP2 protein, and / or the VP3 protein.

29. The nucleic acid combination of any one of claims 26-28, wherein the insertion site of the protein of interest is located in a region selected from the group consisting of: variable region I, variable region II, variable region IV, variable region V, variable region VI, variable region VII, variable region VIII, and variable region IX.

30. The nucleic acid combination of claim 29, wherein the insertion site of the protein of interest is selected from one or more of the group consisting of: between G453 and S454, between A589 and Q590, between N498 and S499, between G530 and E531.

31. The nucleic acid combination of any one of claims 26-30, wherein the protein of interest comprises a reporter protein, a therapeutic protein, and / or a prophylactic protein.

32. The nucleic acid combination of any one of claims 26-31, wherein the protein of interest comprises an antigen binding protein.

33. The nucleic acid combination of claim 32, wherein the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

34. The nucleic acid combination of claim 33, wherein the antibody comprises a nanobody.

35. The nucleic acid combination of any one of claims 1-34, further comprising a third nucleic acid molecule comprising a nucleotide sequence capable of expressing one or two proteins selected from the group consisting of VP1, VP2, and VP3, and the third nucleic acid molecule and the first nucleic acid molecule and the second nucleic acid molecule express different VP1, VP2, and VP3 protein species.

36. The nucleic acid combination of claim 35, wherein the first nucleic acid molecule comprises a genetic mutation resulting in an amino acid mutation selected from the group consisting of T138A and M203L, the second nucleic acid molecule comprises a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L and M203L, and the third nucleic acid molecule comprises a genetic mutation resulting in an amino acid mutation selected from the group consisting of M1L and T138A.

37. The nucleic acid combination of any one of claims 1-36, wherein the first nucleic acid molecule and / or the second nucleic acid molecule comprises a promoter, preferably a strong promoter, more preferably a CMV promoter.

38. The nucleic acid combination of any one of claims 1-37, further comprising a nucleic acid molecule encoding an assembly-activated protein (AAP) protein.

39. The nucleic acid combination of any one of claims 1-38, wherein the first nucleic acid molecule and / or the second nucleic acid molecule further comprises a further genetic mutation, preferably a genetic mutation that alters tissue and / or organ targeting.

40. The nucleic acid combination of any one of claims 1-39, wherein the first nucleic acid molecule and / or the second nucleic acid molecule comprises a nucleic acid molecule encoding an endosomal escape peptide.

41. The nucleic acid combination of claims 1-40, wherein the first nucleic acid molecule and the second nucleic acid molecule are located on different expression cassettes.

42. The nucleic acid combination of any one of claims 1-41, wherein the first nucleic acid molecule and the second nucleic acid molecule are located on the same molecule in a tandem format.

43. The nucleic acid combination of any one of claims 1-42, wherein the first nucleic acid molecule and the second nucleic acid molecule are located on different molecules.

44. A vector comprising the first nucleic acid molecule of the nucleic acid combination of any one of claims 1-43 and / or the second nucleic acid molecule of the nucleic acid combination of any one of claims 1-43.

45. The vector of claim 44, which is an expression vector.

46. The vector of any one of claims 44-45, which is a plasmid.

47. The vector of any one of claims 44-46, which is an adeno-associated virus (AAV).

48. A vector combination comprising a first vector comprising the first nucleic acid molecule of the nucleic acid combination of any one of claims 1-43 and a second vector comprising the second nucleic acid molecule of the nucleic acid combination of any one of claims 1-43.

49. The vector combination of claim 48, wherein the first vector or the second vector is an expression vector.

50. The vector combination of any one of claims 48-49, wherein the first vector or the second vector is a plasmid.

51. The vector combination of any one of claims 48-50, wherein the first vector or the second vector is an adeno-associated virus (AAV).

52. The vector combination of any one of claims 48-51, further comprising a third vector comprising the third nucleic acid molecule of the nucleic acid combination of claim 35 or 36.

53. A recombinant AAV virion packaged from expression of the first nucleic acid molecule of the nucleic acid combination of any one of claims 1-43 and the second nucleic acid molecule of the nucleic acid combination of any one of claims 1-43.

54. The recombinant AAV virion of claim 53, further packaged from expression of the third nucleic acid molecule of the nucleic acid combination of claim 35 or 36.

55. A cell comprising the nucleic acid combination of any one of claims 1-43, the vector of any one of claims 44-47, and / or the vector combination of any one of claims 48-52.

56. A pharmaceutical composition comprising the nucleic acid combination of any one of claims 1-43, the vector of any one of claims 44-47, the vector combination of any one of claims 48-52, the recombinant AAV virion of any one of claims 53-54, and / or the cell of claim 55, and optionally a pharmaceutically acceptable carrier.

57. A method of preventing, alleviating, and / or treating a disease comprising administering to a subject in need thereof an effective amount of the nucleic acid combination of any one of claims 1-43, the vector of any one of claims 44-47, the vector combination of any one of claims 48-52, the recombinant AAV virion of any one of claims 53-54, the cell of claim 55, and / or the pharmaceutical composition of claim 56.

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