CN115991743A - Adeno-associated virus mutant capable of efficiently infecting HT-22 cells - Google Patents

Abstract

The invention relates to packaging and screening of virus vectors, in particular to packaging and screening of AAV mutants, and specifically relates to an adeno-associated virus mutant capable of efficiently infecting HT-22 cells; through constructing a peptide mutation library of AAV9, screening and verifying to obtain a novel AAV9 mutant inserted with 7 amino acids, the mutant can effectively infect HT-22 cells under the condition of MOI=1E+5, achieves a higher infection effect than that of natural AAV9 serotypes MOI=1E+5, and the AAV9-HT01 obtained through screening has the best effect, obviously improves the infection positive rate relative to a control AAV9 serotypes under the same MOI condition, and statistics of multiple of the infection positive rate is 4.4 times through luciferase (RLU) value detection, thereby effectively reducing the use amount of AAV infected cells, saving experimental cost and simultaneously enabling the research on related mechanisms of central nervous disease gene therapy to be feasible in HT-22 cells.

Description

Adeno-associated virus mutant capable of efficiently infecting HT-22 cells

Technical Field

The invention relates to packaging and screening of virus vectors, in particular to packaging and screening of AAV mutants, and in particular relates to an adeno-associated virus mutant capable of efficiently infecting HT-22 cells.

Background

Central nervous system diseases (Central nervous system, CNS) seriously affect physical and mental health and quality of life of patients, and due to the complexity of the central nervous system itself, the Blood-brain barrier (BBB) is a barrier to drug delivery, it is still difficult to treat the nervous system diseases with common surgical operations or traditional drugs, so scientists in various fields are currently researching the feasibility of treating nervous system diseases based on gene therapy methods.

Adeno-associated virus (AAV) is a tiny, envelope-free virus with an icosahedral structure, and is the simplest single-stranded DNA-defective virus presently discovered. Because of the characteristics of good safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, long time for expressing exogenous genes in vivo and the like, the gene expression system has become an important platform for in vivo gene therapy delivery gradually. However, the efficiency of delivery of gene vectors is an important obstacle to the development of AAV-mediated central nervous system gene therapy. AAV is different from other viral vectors in that its different capsid proteins are capable of recognizing different host cell surface receptors, enabling rAAV viral vectors to specifically infect different tissues. There have been many studies on the selection of AAV capsid proteins, i.e., AAV serotypes, capable of efficient infection in a variety of models using cell lines, primary cells, mice or non-human primates, respectively.

HT-22 cells, which are a good model for in vitro studies of glutamate toxicity, are well-used in many neurodegenerative diseases, such as Alzheimer's Disease and Parkinson's Disease, and many researchers have used this cell line for related in vitro mechanism studies. We have found earlier that infection of HT-22 by AAV9 wild-type serotypes is inefficient, which makes in vitro studies with larger amounts of virus and has a certain impact on cell status after increasing amounts of virus.

Disclosure of Invention

Aiming at the defects in the prior art, the invention designs and constructs a peptide fragment mutation library of AAV9, and AAV mutants capable of efficiently infecting HT-22 cell lines are obtained through library screening so as to meet the requirement of HT-22 cell lines as in-vitro research application.

The invention discloses an AAV9 serotype capsid protein mutant inserted with heterologous peptide;

the heterologous peptide is as follows: the amino acid sequence is a polypeptide consisting of SVADRMY (SEQ ID NO: 12);

the AAV9 serotype capsid protein amino acids 588-589 are inserted by the heterologous peptide.

The invention discloses nucleic acid molecules encoding the AAV9 serotype capsid protein mutants described above.

Nucleic acid vectors operably linked to the above nucleic acid molecules are disclosed.

The invention discloses a host cell containing the nucleic acid vector.

The invention discloses a composition or a kit, which contains the AAV9 serotype capsid protein mutant, a nucleic acid molecule or a nucleic acid vector.

The invention discloses the use of the AAV9 serotype capsid protein mutant, nucleic acid molecule or nucleic acid vector described above for infecting HT-22 cells, said use being of non-diagnostic and therapeutic interest.

The invention discloses application of the AAV9 serotype capsid protein mutant, nucleic acid molecule or nucleic acid vector in preparing a medicament for treating central nervous system diseases.

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

according to the technical scheme, a peptide fragment mutation library of AAV9 is constructed, screening verification shows that a novel AAV9 mutant inserted with 7 amino acids is obtained, the mutant can effectively infect HT-22 cells under the condition of MOI=1E+5, a higher infection effect than that of natural AAV9 serotypes MOI=1E+5 is achieved, the effect of AAV9-HT01 obtained through screening is best, under the same MOI condition, the infection positive rate is obviously improved relative to that of a control AAV9 serotypes, the factor of the infection positive rate is 4.4 times through luciferase (RLU) value detection, the use amount of AAV infected cells is effectively reduced, the experimental cost is saved, and meanwhile, the research on gene therapy related mechanisms of central nervous diseases in HT-22 cells becomes feasible.

Drawings

FIG. 1 is a block diagram showing pAAV-shortUBC-mScarlet-polyA-P40-AAV9-Cap-FLEX-SV40polyA expression in example 1;

FIG. 2 is a pAAV-shortUBC-mScarlet-polyA-P40-AAV9-Cap-FLEX-SV40polyA-WPRE map of example 1;

FIG. 3 is a flowchart of AAV9 library construction and screening;

FIG. 4 is a fluorescence of HT-22 infected cells of the different serotypes of example 2;

FIG. 5 is a graph of firefly luciferin values (RLU) for HT-22 cells infected with different serotypes in example 2.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1, AAV97-mer random peptide insertion library construction:

the library consisted of the following vectors:

library shuttle vector pAAV-short UBC-mScarlet-polyA-P40-AAV9-Cap-FLEX-SV40polyA, as shown in FIGS. 1-2.

Example 2, AAV9 library construction and screening procedure:

by constructing a library of random mutations of peptide fragments of AAV9, novel AAV9 mutants having 7 amino acids inserted therein were screened, as shown in FIG. 3.

1. Mutant library preparation

1.1 chemical Synthesis of AAV9-7mer-NNS two fragments:

5'CCACCAGAGTGCCCAANNSNNSNNSNNSNNSNNSNNSGCACAGGCGCAG 3'(SEQ ID NO:1)；

5'CGGTCTGCGCCTGTGCSNNSNNSNNSNNSNNSNNSNNTTGGGCACTCTG 3'(SEQ ID NO:2)；

wherein NNS represents a random coding sequence.

1.2 annealing of the synthesized AAV9-7mer-NNS plus 10. Mu.L of each of the forward and reverse primers (final primer concentration 10 mM) to obtain AAV9-7mer-NNS template. The annealing procedure is as follows: 95 ℃ for 5min;95 ℃ for 1min;92min,1min;4 ℃ for 60min. Wherein, in the second step and the third step, each cycle is reduced by 3 ℃ for 25 cycles.

1.3 plasmid pAAV-short UBC-mScarlet-polyA-P40-AAV9-Cap-FLEX-SV40polyA (structure and insertion site are shown in FIG. 1) was subjected to single cleavage with BsmBI, and cleavage system (50 uL) is shown in Table 1.

TABLE 1

After digestion for 4h at 55℃1% agarose gel electrophoresis was performed, and the large fragment was excised with a knife under an ultraviolet lamp and recovered for purification.

1.4 the purified cleavage product obtained in step 1.3 and the AAV9-7mer-NNS nucleotide sequence obtained in step 1.2 were ligated using T4 DNA library. Ligation T4 DNA ligase using Takara, 10. Mu.L of the reaction system is shown in Table 2, and the ligation is performed overnight at 4 ℃.

TABLE 2

1.5 adding 10. Mu.L of the enzyme-linked product into 50. Mu.L of library-specific electrotransformation competent cells (purchased from Lucigen corporation), mixing, placing, transferring into a precooled electrode cup, performing electrotransformation by using an electrotransformation instrument of Berle corporation, adding 1mL of SOC liquid culture medium preheated at 37 ℃ to the inside after electrotransformation, recovering at 37 ℃ for 1 hour, and performing centrifugal coating.

1.6 repeating steps 1.4-1.5 untilThe number of clones reached 5X 10 ¹¹ 。

2. Library virus packaging and screening

2.1AAV mutant library viral packaging: 1.5X10 per dish ⁷ The 293AAV packaging cells are inoculated into a 15cm cell culture dish and cultured for 18-24 hours, and transfection can be started after the cells are attached. pAAV-short UBC-mScarlet-polyA-P40-AAV9-Cap-FLEX-SV40polyA-insertion expression vector library containing AAV9-7mer-NNS inserts was transfected with PEI transfection reagent, plasmids were packaged, helper plasmids were transferred into 293AAV cells, and after 72h transfection, the proportion of vector library cells in AAV-293 cells was counted under a fluorescence microscope to determine virus packaging efficiency. After the virus is packaged, repeatedly blowing the cells by using a gun head, so that all the cells are completely separated from the culture dish, and collecting all the cell samples.

2.2 purification of virus: repeatedly freezing and thawing the collected cell sample at-80 ℃ and 37 ℃, centrifuging, collecting cell supernatant, removing cell fragments by using a PVDF filter with the thickness of 0.45 mu m, and purifying the collected recombinant AAV by using an AAV purification kit to obtain the recombinant AAV.

2.3 determination of recombinant AAV viral titers: taking 20 mu L of concentrated virus liquid, adding 1 mu L of RNase-free DNase, mixing uniformly, incubating for 30min at 37 ℃, centrifuging at 10000rpm for 10min, taking 20 mu L of supernatant, adding 80 mu L of dilution Buffer into another sterile tube, mixing uniformly, and reacting for 10min in a metal bath at 100 ℃. Naturally cooling to room temperature, adding 3 mu L of proteinase K, incubating at 37 ℃ for 60min, reacting in a metal bath at 100 ℃ for 10min, and cooling to room temperature. The sample is diluted and used as a template, and the recombinant AAV titer is determined by adopting a real-time quantitative PCR detection method. The qPCR reaction system and the reaction conditions are as follows: 95 ℃ for 10min;95 ℃ for 30s;60 ℃,30s,35 cycles.

2.4AAV infection of HT-22 (mouse hippocampal neurons) cells:

2.4.1 cell plating: HT-22 cells were seeded at 40% confluence into 10cm cell dishes at 5X 10 per dish ⁶ Cells were plated and multiple cell culture dishes were plated.

2.4.2 viral infection: HT-22 cells were infected with pAAV-short UBC-mScarlet-polyA-P40-AAV9-Cap-FLEX-SV40polyA-insertion expression vector library virus.

2.5 collecting the infected cells, performing fluorescence sorting by using a flow cytometry, and selecting the cells with the red fluorescence brightness of 5% at the front as the screened target cells for collection. The cells after sorting and collection are paved into a cell dish, and the cells are collected after being amplified.

2.6 extracting the genome from the collected cells, performing PCR amplification, and sequencing the PCR products in high throughput.

Amplification primers:

AAV9-F: AACTACTAACCCGGTAGCAACGG (SEQ ID NO: 3) (forward primer on vector)

AAV9-R: CGTCCGTGTGAGGAATTTTGG (SEQ ID NO: 4) (reverse primer on vector) high throughput sequencing with addition of adaptor and index sequences primers were used as follows:

NGS-AAV9-F：

TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGG CTAGAAACTACTAACCCGGTAGCAACGG(SEQ ID NO:5)

NGS-R1：

GTTCGTCTTCTGCCGTATGCTCTACACTGACCTCAAGTCTGCACACGAGAAGG CTAGCGAGTAATCGTCCGTGTGAGGAATTTTGG(SEQ ID NO:6)

NGS-R2：

GTTCGTCTTCTGCCGTATGCTCTACACTGACCTCAAGTCTGCACACGAGAAGG CTAGTCTCCGGACGTCCGTGTGAGGAATTTTGG(SEQ ID NO:7)

NGS-R3：

GTTCGTCTTCTGCCGTATGCTCTACACTGACCTCAAGTCTGCACACGAGAAGG CTAGAATGAGCGCGTCCGTGTGAGGAATTTTGG(SEQ ID NO:8)

NGS-R4：

GTTCGTCTTCTGCCGTATGCTCTACACTGACCTCAAGTCTGCACACGAGAAGG CTAGGGAATCTCCGTCCGTGTGAGGAATTTTGG(SEQ ID NO:9)

NGS-R5：

GTTCGTCTTCTGCCGTATGCTCTACACTGACCTCAAGTCTGCACACGAGAAGG CTAGTTCTGAATTTCCGTCCGTGTGAGGAATTTTGG(SEQ ID NO:10)

the expected sequencing sequences obtained are:

AACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAANNSNNSNNSNNSNNSNNSNNSGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACG(SEQ ID NO:11)

analyzing the high-throughput sequencing result, and respectively naming mutants with high occurrence frequency as AAV9-HTxx; if the peptide fragment 01 is: AAV9-HT01.

3. Screening and validation of AAV9 mutants

Construction of 3.1AAV9 mutant

The AAV2/9 of the natural serotype is taken as a vector, fragments such as AAV9-HTxx of candidate mutants are inserted at the positions of 588-589 serving as amino acids, so that new serotype vectors such as AAV9-HTxx and the like are obtained, and more than 30 mutants are constructed according to the result of high-throughput sequencing.

3.2 AAV viruses of various mutant serotypes were obtained by packaging with AAV9-HTxx et al as serotype vectors using the shuttle vector pAAV-CBh-mScarlet-P2A-Luc 2.

3.3 viral titer assays were performed on the above viruses using WPRE primers, and expression of viruses VP1, VP2, VP3 was confirmed using cowling.

3.4 AAV9-HTxx et al viruses of pAAV-CBh-mScarlet-P2A-Luc2 were expressed as MOI=1X10 ⁵ The infection results of 5 different mutant serotypes of peptide fragments are selected, the fluorescence diagram of 72h of the infection results and the control AAV9 are shown in figure 4, the infection efficiency of AAV9-HT01 mutant serotypes of virus is obviously higher than that of the control AAV9, meanwhile, the luciferase (RLU) value of the detection cells using firefly luciferase is shown in figure 5, the infection efficiency of AAV9-HT01 is higher than that of the control AAV9, and the RLU value is improved by 4.4 times compared with that of the control AAV 9. The AAV9-HT01 obtained by screening has the best effect, and under the same MOI condition, the infection positive rate is obviously improved relative to the control AAV9 serotype, and the detection statistics of luciferase (RLU) value shows that the infection positive rate is 4.4 times.

Claims (7)

1. An AAV9 serotype capsid protein mutant inserted with a heterologous peptide, characterized in that,

the heterologous peptide is as follows: the amino acid sequence is a polypeptide composed of SVADRMY;

the AAV9 serotype capsid protein amino acids 588-589 are inserted by the heterologous peptide.

2. A nucleic acid molecule encoding the AAV9 serotype capsid protein mutant of claim 1.

3. A nucleic acid vector operably linked to the nucleic acid molecule of claim 2.

4. A host cell comprising the nucleic acid vector of claim 3.

5. A composition or kit comprising the AAV9 serotype capsid protein mutant of claim 1, the nucleic acid molecule of claim 2 or the nucleic acid vector of claim 3.

6. Use of an AAV9 serotype capsid protein mutant according to claim 1, a nucleic acid molecule according to claim 2 or a nucleic acid vector according to claim 3 for infecting HT-22 cells, said use being of non-diagnostic and therapeutic interest.

7. Use of an AAV9 serotype capsid protein mutant according to claim 1, a nucleic acid molecule according to claim 2 or a nucleic acid vector according to claim 3 in the manufacture of a medicament for the treatment of a central nervous system disorder.

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