阅读说明：本技术 腺相关病毒突变体及其应用 (Adeno-associated virus mutant and application thereof ) 是由 裴晓磊 张磊 于 2020-05-22 设计创作，主要内容包括：本发明提供了腺相关病毒突变体及其应用,所述腺相关病毒突变体的外壳蛋白包括SEQ ID NO:1～5所示的氨基酸序列,或与SEQ ID NO:1～5具有98％以上同一性、且具有相同或相似生物学功能的氨基酸序列。本发明通过对野生型腺相关病毒的外壳蛋白编码基因进行拼接重组,构建腺相关病毒库,采用抗AAV外壳蛋白的中和抗体筛选得到腺相关病毒突变体,对人源肝脏细胞的感染能力强,并能够躲避中和抗体的中和作用,有针对性地解决了血友病治疗领域存在的技术障碍。(The invention provides an adeno-associated virus mutant and application thereof, wherein the coat protein of the adeno-associated virus mutant comprises an amino acid sequence shown in SEQ ID NO 1-5, or an amino acid sequence which has more than 98% of identity with SEQ ID NO 1-5 and has the same or similar biological functions. According to the invention, the coat protein coding genes of wild type adeno-associated virus are spliced and recombined to construct adeno-associated virus library, and the adeno-associated virus mutant is obtained by screening neutralizing antibodies against AAV coat protein, so that the mutant has strong infection capability on human liver cells and can avoid the neutralizing effect of the neutralizing antibodies, and the technical obstacle existing in the field of hemophilia treatment is specifically solved.)

1. An adeno-associated virus mutant is characterized in that the coat protein of the adeno-associated virus mutant comprises an amino acid sequence shown in SEQ ID NO 1-5, or an amino acid sequence which has more than 98% of identity with SEQ ID NO 1-5 and has the same or similar biological functions.

2. A nucleic acid molecule encoding the adeno-associated virus mutant according to claim 1, or encoding a protein having the same or similar biological function as the adeno-associated virus mutant according to claim 1;

preferably, the nucleic acid molecule comprises a nucleic acid sequence shown in SEQ ID NO. 6-10 and/or a complementary sequence of SEQ ID NO. 6-10.

3. An expression vector comprising a wild-type adeno-associated viral vector having the nucleic acid molecule of claim 2 inserted therein;

preferably, the wild-type adeno-associated viral vector comprises any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV 2.

4. An adeno-associated virus library, wherein the adeno-associated virus library comprises the adeno-associated virus mutant of claim 1.

5. A method of constructing the adeno-associated virus library according to claim 4, wherein the method comprises:

(1) amplifying a coat protein coding gene of the wild type adeno-associated virus by PCR;

(2) digesting the amplification product by using DNA enzyme, and selecting DNA fragments with the length of 100-300 bp for splicing again;

(3) inserting the spliced product into a wild type adeno-associated virus vector to obtain an adeno-associated virus vector library;

(4) and co-transfecting the adeno-associated virus vector library and helper plasmids to mammalian cells, and preparing to obtain the adeno-associated virus library.

6. The method according to claim 5, wherein the PCR primer of step (1) comprises a nucleic acid sequence shown as SEQ ID NO. 11-12;

preferably, the splicing method in the step (2) is to mix the DNA fragment with the length of 100-300 bp with DNA polymerase, perform gradient cooling within the range of 96-41 ℃, and obtain a spliced product through Shuffling PCR;

preferably, the wild-type adeno-associated viral vector of step (3) comprises any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV 2.

7. A method for screening the adeno-associated virus mutant according to claim 1, wherein the method comprises:

incubating the adeno-associated virus library of claim 4 in admixture with human immunoglobulin for intravenous injection prior to introduction into a liver cell humanized mouse;

introducing adenovirus, feeding for a period of time, separating human cells in mouse liver, and extracting DNA;

performing PCR amplification by using a primer pair shown in SEQ ID NO. 13-14, inserting an amplification product into a wild type adeno-associated virus vector, and co-transfecting a mammalian cell with an auxiliary plasmid; and (3) cracking the mammalian cells after culture to obtain the adeno-associated virus mutant.

8. A method for preparing an adeno-associated virus mutant, which comprises co-transfecting a mammalian cell with the expression vector of claim 3 and a helper plasmid; and (3) cracking the mammalian cells after culture to obtain the adeno-associated virus mutant.

9. A pharmaceutical composition comprising any one of the adeno-associated virus mutant of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3, or the adeno-associated virus library of claim 4, or a combination of at least two thereof;

preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

10. Use of the adeno-associated virus mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3, the adeno-associated virus library according to claim 4 or the pharmaceutical composition according to claim 9 for the preparation of a medicament for the treatment of a gene deficiency disease;

preferably, the gene deficiency disease comprises any one of hemophilia, congenital black , duchenne muscular dystrophy or spinal muscular atrophy, or a combination of at least two of them.

Technical Field

The invention belongs to the technical field of genetic engineering and biological engineering, and relates to an adeno-associated virus mutant and application thereof.

Background

In recent years, adeno-associated virus (AAV) has been used as a vector for gene therapy in clinical treatment of various gene defects, such as hemophilia B, progressive DMD atrophy, SMA dyskinesia, etc., with exciting results. In particular, the successful use of AAV in hemophilia therapy is expected to save thousands of patients who are ineffective for conventional therapies. It has been reported in the literature that after injection of AAV carrying the nine-factor gene, hemophilia patients do not have severe immune responses, and the damage of AAV to liver is transient; after AAV is injected, the nine-factor level in the peripheral blood of the patient is recovered to about 10% of the normal level, and the expression is stable for a long time, and the dependence on the nine-factor recombinant protein is basically separated. Preliminary clinical trials are underway for hemophilia a caused by eight-factor deficiency. However, the gene encoding factor VIII is long, about 4.5kbp, and the length of the whole gene exceeds the packaging range of AAV due to the addition of promoter and termination sequences, which is a major obstacle of AAV gene therapy in the treatment of hemophilia A and is a research hotspot at present.

However, AAV gene therapy also has several limitations in the treatment of hemophilia. One of them is that a certain proportion of neutralizing antibodies against AAV coat protein is present in peripheral blood of normal persons and hemophiliacs, and therefore, it is necessary to first detect the presence of neutralizing antibodies in peripheral blood before evaluating whether patients can be treated with AAV gene therapy, and if neutralizing antibodies against AAV are present in patients, they are not suitable for AAV gene therapy. Given the wide prospect of AAV gene therapy as a treatment for hemophilia, it is particularly important to help AAV evade clearance by neutralizing antibodies in vivo.

In response to this problem, researchers have proposed a variety of methods including neutralization of the vacant coat protein, pretreatment of immunosuppressants, neutralization of small fragments of DNA, mutation of AAV coat protein, and the like. The neutralizing method of the capsid protein is to mix a large amount of the AAV capsid protein when the AAV is injected, so as to offset the pressure of the clearance of the AAV by the neutralizing antibody in vivo, thereby increasing the infection efficiency. However, the research finds that the AAV capsid protein is more easily presented by antigen presenting cells, strong T cell immune response is stimulated, the clearing effect of the T cells on the AAV is enhanced, and finally the transduction efficiency of the AAV is reduced. Immunosuppressive pretreatment is a method in which a patient is injected with an appropriate amount of an immunosuppressive agent such as dexamethasone prior to injection of AAV, and although this method can improve the transduction efficiency of AAV to some extent, the improvement is limited because dexamethasone is widely used for immunosuppression and cannot specifically clear neutralizing antibodies against AAV and recognize T/B cells of AAV. The small-fragment DNA neutralization method is characterized in that small-fragment DNA capable of specifically blocking AAV neutralizing antibodies is used, the application prospect is good, but no relevant clinical application report is found. The AAV coat protein mutation method refers to that some sites in the AAV coat protein amino acid sequence recognized by a neutralizing antibody are mutated to reduce the recognition capability of the neutralizing antibody on AAV, so that the clearance effect of the neutralizing antibody is avoided.

Therefore, the AAV is modified so as to avoid the clearing effect of in vivo neutralizing antibodies, the application range of the AAV is improved, and the AAV has important significance and wide application prospect in the field of gene defect disease treatment.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides the adeno-associated virus mutant and the application thereof, wherein the adeno-associated virus mutant has strong infection capacity on human liver cells, can avoid the neutralization effect of a neutralizing antibody, and purposefully solves the problem that a hemophilia patient with an anti-AAV neutralizing antibody encounters when the patient receives gene therapy.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an adeno-associated virus mutant, wherein the coat protein of the adeno-associated virus mutant comprises an amino acid sequence shown in SEQ ID NO. 1-5.

In the invention, splicing recombination is carried out on coat protein coding genes of wild type adeno-associated virus to construct an adeno-associated virus library, five adeno-associated virus mutants are obtained by screening neutralizing antibodies against AAV (adeno-associated virus), the coat proteins of the adeno-associated virus mutants comprise amino acid sequences shown in SEQ ID NO: 1-5, and the obtained adeno-associated virus mutants have the capability of escaping from the neutralizing antibodies against AAV, have high-efficiency specific infection capability on human liver cells and have wide application prospect in the field of hemophilia gene therapy.

The five adeno-associated virus mutants obtained by screening have high sequence homology, and are found by sequence comparison with wild type AAV9 that the amino acids from the 100 th to the 200 th at the N end of the AAV coat protein amino acid sequence can be a high-frequency recognition region of an anti-AAV neutralizing antibody.

Preferably, the coat protein of the adeno-associated virus mutant also comprises an amino acid sequence which has more than 98% of identity with SEQ ID NO. 1-5 and has the same or similar biological function, for example, the coat protein of the adeno-associated virus mutant can reach 98% of identity with SEQ ID NO. 1-5 and has the capability of escaping anti-AAV neutralizing antibodies.

In a second aspect, the present invention provides a nucleic acid molecule encoding the adeno-associated virus mutant according to the first aspect, or encoding a protein having the same or similar biological function as the adeno-associated virus mutant according to the first aspect.

Preferably, the nucleic acid molecule comprises a nucleic acid sequence shown in SEQ ID NO. 6-10 and/or a complementary sequence of SEQ ID NO. 6-10.

In the invention, the nucleic acid sequence shown by SEQ ID NO. 6 is the coding sequence of the amino acid sequence shown by SEQ ID NO. 1, the nucleic acid sequence shown by SEQ ID NO. 7 is the coding sequence of the amino acid sequence shown by SEQ ID NO. 2, the nucleic acid sequence shown by SEQ ID NO. 8 is the coding sequence of the amino acid sequence shown by SEQ ID NO. 3, the nucleic acid sequence shown by SEQ ID NO. 9 is the coding sequence of the amino acid sequence shown by SEQ ID NO. 4, and the nucleic acid sequence shown by SEQ ID NO. 10 is the coding sequence of the amino acid sequence shown by SEQ ID NO. 5.

In a third aspect, the present invention provides an expression vector comprising a wild-type adeno-associated viral vector into which has been inserted a nucleic acid molecule according to the second aspect.

Preferably, the wild-type adeno-associated viral vector comprises any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV 2.

In a fourth aspect, the present invention provides an adeno-associated virus library comprising the adeno-associated virus mutant according to the first aspect.

Preferably, the adeno-associated virus library further comprises wild-type and/or mutant adeno-associated viruses that do not have the ability to evade neutralizing antibodies against adeno-associated virus coat proteins.

In a fifth aspect, the present invention provides a method for constructing an adeno-associated virus library according to the fourth aspect, wherein the method comprises:

(1) amplifying a coat protein coding gene of the wild type adeno-associated virus by PCR;

(2) digesting the amplification product by using DNA enzyme, and selecting DNA fragments with the length of 100-300 bp for splicing again;

(3) inserting the spliced product into a wild type adeno-associated virus vector to obtain an adeno-associated virus vector library;

(4) and co-transfecting the adeno-associated virus vector library and helper plasmids to mammalian cells, and preparing to obtain the adeno-associated virus library.

Preferably, the PCR primer in the step (1) comprises a nucleic acid sequence shown as SEQ ID NO. 11-12;

SEQ ID NO:11：5’-ATAAAGCGAGTAGTC-3’；

SEQ ID NO:12：5’-GAGGGTATGCGACAT-3’。

preferably, the splicing method in the step (2) is to mix the DNA fragment with the length of 100-300 bp with DNA polymerase, perform gradient cooling within the range of 96-41 ℃, and obtain a spliced product through Shuffling PCR.

In the invention, the DNA fragment with the length of 100-300 bp is selected as the splicing fragment, so that not only is the sequence diversity of the splicing product enriched, the virus amount in an adeno-associated virus library improved, but also the splicing efficiency of Shuffling PCR is ensured, the splicing success rate of the splicing product is improved, and the method is beneficial to efficiently screening and obtaining the adeno-associated virus mutant capable of avoiding the neutralizing effect of a neutralizing antibody.

Preferably, the wild-type adeno-associated viral vector of step (3) comprises any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, preferably AAV 2.

In a sixth aspect, the present invention provides a method for screening the adeno-associated virus mutant according to the first aspect, wherein the method comprises:

incubating the adeno-associated virus library of the fourth aspect in a mixture with human immunoglobulin for intravenous injection, and then introducing the incubated mixture into a liver cell humanized mouse;

introducing adenovirus, feeding for a period of time, separating human cells in mouse liver, and extracting DNA;

performing PCR amplification by using a primer pair shown in SEQ ID NO. 13-14, inserting an amplification product into a wild type adeno-associated virus vector, and co-transfecting a mammalian cell with an auxiliary plasmid; cracking the mammalian cells after culturing to obtain the adeno-associated virus mutant;

SEQ ID NO:13：CAACTCCATCACTAGGGGTTC；

SEQ ID NO:14：CATGGGAAAGGTGCCAGA。

in the present invention, the upstream primer SEQ ID NO. 13 is designed based on AAV2-rep gene, and the downstream primer SEQ ID NO. 14 is designed based on the downstream consensus sequence of AAV2-ITR plasmid.

In a seventh aspect, the present invention provides a method for preparing an adeno-associated virus mutant, the method comprising co-transfecting a mammalian cell with the expression vector of the third aspect and a helper plasmid; and (3) cracking the mammalian cells after culture to obtain the adeno-associated virus mutant.

In an eighth aspect, the present invention provides a pharmaceutical composition comprising any one of the adeno-associated virus mutant according to the first aspect, the nucleic acid molecule according to the second aspect, the expression vector according to the third aspect, or the adeno-associated virus library according to the fourth aspect, or a combination of at least two of them;

preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In a ninth aspect, the present invention provides an application of the adeno-associated virus mutant according to the first aspect, the nucleic acid molecule according to the second aspect, the expression vector according to the third aspect, the adeno-associated virus library according to the fourth aspect, or the pharmaceutical composition according to the eighth aspect in preparing a medicament for treating gene deficiency diseases.

Preferably, the gene deficiency disease comprises hemophilia.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention constructs an adeno-associated virus library by splicing and recombining coat protein coding genes of wild adeno-associated viruses, obtains five adeno-associated virus mutants by screening neutralizing antibodies against AAV coat proteins, has the capability of escaping the neutralizing antibodies against AAV, and has high-efficiency specific infection capability on human liver cells;

(2) the five adeno-associated virus mutants obtained by screening have high sequence homology, and are found by sequence comparison with wild type AAV9 that the amino acids from the 100 th position to the 200 th position at the N end of the AAV coat protein amino acid sequence can be a high-frequency recognition region of an anti-AAV neutralizing antibody;

(3) the adeno-associated virus mutant has wide application prospect and great market value in the field of hemophilia gene therapy.

Drawings

FIG. 1 shows the source of the coat protein gene sequence in AAV mutants and the point mutations present in the sequence;

FIG. 2 is an evolutionary relationship between AAV mutants and wild type;

FIG. 3 is a graph of the packaging efficiency of AAV wild type and mutant in HEK293 cell lines;

FIG. 4 is a graph of AAV wild type and mutant in vitro infection efficiency of Huh7 cells;

FIG. 5 is a graph of the evasion capacity of AAV wild-type and mutant extrasomal neutralizing antibodies to IVIG;

FIG. 6(A) is the ability of AAV wild type and mutant type to evade in vitro neutralizing antibodies in normal human peripheral blood serum, and FIG. 6(B) is the ability of AAV wild type and mutant type to evade in vitro neutralizing antibodies in peripheral blood serum of hemophilia patients;

FIG. 7 is the efficiency of infection of human liver cells in vivo with mutant 2-10;

FIG. 8(A) is a schematic view showing the gene therapy process, and FIG. 8(B) is the therapeutic effect.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.