WO2024077815A1 - Mutant de virus adéno-associé et son utilisation - Google Patents

Mutant de virus adéno-associé et son utilisation Download PDF

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WO2024077815A1
WO2024077815A1 PCT/CN2023/074186 CN2023074186W WO2024077815A1 WO 2024077815 A1 WO2024077815 A1 WO 2024077815A1 CN 2023074186 W CN2023074186 W CN 2023074186W WO 2024077815 A1 WO2024077815 A1 WO 2024077815A1
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cells
capsid protein
aav capsid
protein mutant
associated virus
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李华鹏
卜晔
钟育健
代志勇
张有为
陈君霖
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广州派真生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/015—Parvoviridae, e.g. feline panleukopenia virus, human parvovirus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biomedicine technology, and in particular to an adeno-associated virus mutant with inactivated T cell targeting and application thereof.
  • Adeno-associated virus is a small non-enveloped virus that encapsulates a linear single-stranded DNA genome. It belongs to the genus Dependovirus of the family Parvoviridae and requires a helper virus (usually adenovirus) to participate in replication.
  • the AAV genome is a single-stranded DNA fragment contained in a non-enveloped viral capsid and can be divided into three functional regions: two open reading frames (Rep gene, Cap gene) and inverted terminal repeats (ITR).
  • Recombinant adeno-associated virus vector rAAV is derived from non-pathogenic wild-type adeno-associated virus.
  • rAAV has been used in the study of gene therapy for a variety of diseases (including in vivo and in vitro experiments), such as gene function research, construction of disease models, preparation of gene knockout mice, etc.
  • Tumor immunotherapy is the use of the immune system's own specific ability to identify and kill tumor cells. It is one of the treatment methods with the most promising application prospects in the current field of tumor treatment. With the in-depth research on tumor immunotherapy in recent years, various types of therapies have shown good efficacy in refractory recurrent tumors, such as antibody-drug conjugates, dual-targeted antibodies, CAR-T therapy and TCR-T therapy. T cells play an important role in destroying diseased cells throughout the body. Related studies on immune checkpoint inhibitors and tumor-infiltrating lymphocytes have shown the potential of T cells in treating cancer. However, T cells need appropriate tumor specificity, sufficient numbers, and overcome any local immunosuppressive factors to be effective.
  • CAR-T therapy and TCR-T therapy are based on T cell modification methods. Although they have been clinically applied, there are still some problems.
  • the use of lentivirus for T cell modification involves the integration of the T cell genome, which requires a long period of cell in vitro culture and cumbersome quality control process. Long-term in vitro operation is not only costly, but also affects the activity of T cells.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide an adeno-associated virus mutant with inactivated T cell targeting and its application.
  • the adeno-associated virus mutant has better infection ability for inactivated T cells and has the advantages of low dosage, strong infectivity and good safety.
  • the present invention screens a heterologous peptide with targeting to unactivated T cells, wherein the amino acid sequence of the heterologous peptide is a sequence as shown in any one of SEQ ID No. 9 to 12.
  • the recombinant adeno-associated virus vector constructed by screening the AAV capsid protein mutant containing the heterologous peptide in the present invention has high efficiency in targeting unactivated T cells.
  • the nucleotide sequence of the heterologous peptide is a sequence shown in any one of SEQ ID No.13 to 16.
  • the present invention provides an AAV capsid protein mutant with non-activated T cell targeting, comprising the heterologous peptide.
  • the AAV capsid protein mutants screened by the present invention do not need to be integrated into the genome, and the recombinant adeno-associated virus constructed to infect unactivated T cells can achieve rapid infection and reinfusion, reducing unnecessary quality inspections and in vitro residence time, and has the advantages of low dosage, strong infectivity, and good safety.
  • the AAV capsid protein mutant described in the present invention is obtained by inserting or replacing 5 to 20 amino acids of the AAV capsid protein by the heterologous peptide.
  • the insertion site of the heterologous peptide is located between amino acids 588 and 589 of the AAV capsid protein.
  • its amino acid sequence is a sequence shown in any one of SEQ ID No.1 to SEQ ID No.4.
  • nucleotide sequence is a sequence shown in any one of SEQ ID No.5 to SEQ ID No.8.
  • the present invention provides a recombinant adeno-associated virus with inactivated T cell targeting, including the AAV capsid protein mutant.
  • the recombinant adeno-associated virus vector of the present invention has better infection ability for unactivated T cells, and has the advantages of low dosage, strong infectivity and good safety. Infection of unactivated T cells can achieve the advantages of collecting cells and infecting them on the same day, and reinfusing them on the same day, which greatly reduces the preparation cost and reduces the impact on T cell activity.
  • the recombinant adeno-associated virus of the present invention also includes a heterologous target gene.
  • the heterologous target gene encodes any gene product of interfering RNA, aptamer, endonuclease, and guide RNA.
  • the present invention uses the heterologous peptide, the AAV capsid protein mutant, and the recombinant adeno-associated virus in the preparation of a drug for delivering a gene product to cells of a subject.
  • the cells are immune cells.
  • the present invention uses the heterologous peptide, the AAV capsid protein mutant, and the recombinant adeno-associated virus to infect unactivated T cells.
  • the present invention uses the heterologous peptide, the AAV capsid protein mutant, and the recombinant adeno-associated virus in the preparation of tumor immunotherapy drugs.
  • the immunotherapy includes CAR-T therapy or TCR-T therapy.
  • the present invention has the following beneficial effects:
  • the recombinant adeno-associated virus vector constructed by the AAV capsid protein mutant obtained by the screening of the present invention has high efficiency in targeting unactivated T cells, and has the advantages of low dose, strong infectivity and good safety. Compared with the infection after stimulation by stimulating factors (48h) in the prior art, the infection of unactivated T cells can achieve the advantages of collecting cells and infecting on the same day, and reinfusing on the same day, which greatly reduces the preparation cost. And the unactivated T cells reduce the in vitro culture and processing time, which can reduce the impact on T cell activity.
  • the AAV capsid protein mutant obtained by the screening of the present invention does not need to integrate the genome, can achieve rapid infection and reinfuse, and reduce unnecessary quality inspection and in vitro residence time.
  • the present invention provides a method for delivering gene products to unactivated T cells. In view of the fact that the rapid expansion of activated T cells in vitro will lead to a decrease in anti-cancer activity, the effect is not as good as that of unactivated T cells.
  • the use of the AAV capsid protein mutant obtained by the screening of the present invention to infect unactivated T cells has great clinical value and commercial application scenarios.
  • Figure 1 shows the EGFP fluorescence intensity of T cells infected with different rAAV virions detected by fluorescence microscopy (72h);
  • a and B are control rAAV6 viruses, and
  • C to F are AAV6 capsid protein mutant viruses 1 to 4.
  • Figure 2 shows the EGFP fluorescence intensity of T cells infected with different rAAV virions detected by fluorescence microscopy (96h);
  • a and B are control rAAV6 viruses, and
  • C to F are AAV6 capsid protein mutant viruses 1 to 4.
  • FIG3 shows the relative intensity of EGFP mRNA of AAV6 capsid protein mutant virus 1 in T cells detected by RT-qPCR (72 h).
  • FIG. 4 is a flow cytometric analysis of the EGFP fluorescence intensity of AAV6 capsid protein mutant virus 1 in T cells (96 h).
  • FIG5 is a graph showing the relative intensity of EGFP mRNA in T cells detected by RT-qPCR for AAV6 capsid protein mutant virus 2 (72 h).
  • FIG6 is a flow cytometric analysis of the EGFP fluorescence intensity of AAV6 capsid protein mutant virus 2 in T cells (96 h).
  • FIG. 7 shows the relative intensity of EGFP mRNA of AAV6 capsid protein mutant virus 3 in T cells detected by RT-qPCR (72 h).
  • FIG8 is a flow cytometer-detected EGFP fluorescence intensity of AAV6 capsid protein mutant virus 3 in T cells (96 h).
  • FIG. 9 shows the relative intensity of EGFP mRNA in T cells detected by RT-qPCR of AAV6 capsid protein mutant virus 4 (72 h).
  • FIG. 10 is the flow cytometric detection of EGFP fluorescence intensity of AAV6 capsid protein mutant virus 4 in T cells (96 h).
  • GenBank accession number of AAV6 VP1 is AF028704.1.
  • Example 1 Screening for AAV6 mutants that effectively infect unactivated T cells
  • the AAV6 library backbone vector contains CAG promoter, Intron, mutant AAV6 CAP sequence [the sequence after T589 of the AAV6 CAP sequence is removed, and the T of N583 and the sequence in front of polyA constitute the site BsrGI (TGTACA) for subsequent restriction enzyme cleavage of the backbone] and poly A.
  • the above sequence is synthesized by gene synthesis and inserted between the ITRs of the rAAV vector to form the AAV6 library backbone vector.
  • the Rep-CAP vector By introducing a stop codon within the first 20 bp of the start codons of VP1, VP2, and VP3 of the CAP sequence in AAV6, the Rep-CAP vector expresses the Rep protein, but cannot express the VP1, VP2, and VP3 proteins of CAP, thereby avoiding contamination of the CAP sequence in the parent AAV6.
  • the above sequence is synthesized by gene synthesis and inserted to replace the CAP sequence of the Rep-CAP vector.
  • the upstream primer targeted the nucleic acid sequence after T589 of the template, and the downstream primer targeted the terminal sequence of CAP.
  • the 5' ends of the upstream and downstream primers both had homology arm sequences of more than 15 bp consistent with the backbone.
  • the upstream primer introduced a 21 bp nucleic acid sequence (7*NNK) between the homology arm sequence and the targeting sequence to introduce a random 7-peptide into the CAP sequence.
  • the base sequences of the two primers (5’->3’) are as follows:
  • the above primers were used to amplify the fragment containing the random sequence by PCR.
  • the fragment was subjected to gel electrophoresis and gel recovery to obtain the purified nucleic acid fragment of the random 7-peptide library;
  • the nucleic acid fragment was connected to the AAV6 library backbone vector (after BsrGI digestion and gel recovery purification) by Gibson homologous recombination connection;
  • the connected vector was purified by a PCR product purification kit and digested with Plasmid-Safe DNase enzyme to remove the unconnected fragments; finally, it was purified by a PCR product purification kit to obtain the constructed AAV6 vector library.
  • the mutant Rep-Cap plasmid, AAV6 vector library and pHelper plasmid were co-transfected into HEK-293T cells, and the adeno-associated virus was purified by iodixanol gradient ultracentrifugation.
  • the virus titer was measured to be 1 ⁇ 10 12 GC/mL to 1 ⁇ 10 13 GC/mL, and the AAV6 mutant virus library was obtained and stored at -80°C for use.
  • Preheat RPMI 1640 medium at 37°C take frozen CD3 + T cells and thaw quickly; aspirate the revived cells into a 50 mL centrifuge tube, add 12 mL of RPMI 1640 medium containing 1% FBS, and centrifuge at 300 g. Centrifuge for 10-15 minutes; resuspend the cells with 1 mL of RPMI 1640 medium containing 1% FBS, count the cells (trypan blue staining, count the total number of cells and the number of dead cells); add 1 ⁇ 10 6 cell suspension to each well of the cell culture plate; add AAV6 mutant virus library at a dose of 3 ⁇ 10 10 GC per well to infect unactivated T cells for 6 hours.
  • the cells were aspirated into a 1.5 mL centrifuge tube, centrifuged at 300 g for 15 min to collect the cells, and the liquid was aspirated.
  • the RNA extraction method was in accordance with the instructions of the TransZol Up Plus RNA Kit (Full Gold, ER501).
  • the extracted RNA sample was synthesized using PrimeScript TM IV 1st strand cDNA Synthesis Mix (Takara, 6215A) for the first strand cDNA.
  • NEB Q5 was used for 2 rounds of PCR amplification, the first round of amplification using outer primers, the second round of gel-recovered first round products as templates, amplified with NGS primers, and the gel-recovered PCR products of corresponding band sizes were sent to the company for NGS sequencing; after sequencing, sequencing data analysis was performed, and the sequences with the highest frequency of occurrence were selected for the construction of the AAV mutant sub-library, and the sub-library construction was subjected to another round of screening process, and the sub-vector library was constructed, packaged, screened, and the like according to the aforementioned construction process.
  • AAV capsid protein mutants 1 to 4 were screened out, and the amino acid sequences of their VP1 were shown as SEQ ID No.1 to SEQ ID No.4, and the nucleotide sequences were shown as SEQ ID No.5 to SEQ ID No.8, respectively; the amino acid sequences of the targeting peptides in VP1 were shown as SEQ ID No.9 to SEQ ID No.12, and the nucleotide sequences were shown as SEQ ID No.13 to SEQ ID No.16, respectively.
  • AAV capsid protein mutants 1 to 4 were constructed as follows:
  • the Rep-CAP plasmid was double-digested with Smi I and BshTI, and the fragment band of about 5000bp was cut out by gel electrophoresis and gel recovery to obtain the digested backbone fragment.
  • Primers were designed according to the Cap sequences of the target mutants 1 to 4 obtained by screening to construct the plasmids of the target mutant AAV.
  • the Rep-CAP plasmid of AAV6 was used as a template and the F1+R1 primers were used for PCR amplification to obtain the target product 1.
  • the Rep-CAP plasmid of AAV6 was used as a template and the F2+R2 primers were used for PCR amplification to obtain the target product 2.
  • the backbone and the fragments, and the fragments have homology arm sequences, and multiple fragments can be assembled into a complete vector by Gisbon.
  • the primers for PCR product 1 in the construction of AAV capsid protein mutant 1 vector are Cap-f+YJ191-R, and the primers for PCR product 2 are YJ191-F+cap-r.
  • the primer sequences involved (5'->3') are:
  • the primers for PCR product 1 in the construction of AAV capsid protein mutant 2 vector are Cap-f+YJ192-R, and the primers for PCR product 2 are YJ192-F+cap-r.
  • the primer sequences involved (5'->3') are:
  • the primers for PCR product 1 in the construction of AAV capsid protein mutant 3 vector are Cap-f+YJ193-R, and the primers for PCR product 2 are YJ193-F+cap-r.
  • the primer sequences involved (5'->3') are
  • the primers for PCR product 1 in the construction of AAV capsid protein mutant 4 vector are Cap-f+YJ194-R, and the primers for PCR product 2 are YJ194-F+cap-r.
  • the primer sequences involved (5'->3') are
  • primers Cap-f and Cap-r involved in the construction of the above-mentioned AAV capsid protein mutants 1 to 4 vectors are:
  • the bacterial solution was centrifuged at 12000rpm for 1 minute, and the supernatant medium was discarded; 250 ⁇ L of bufferP1/RNaseA mixture was added, and the bacteria were resuspended by high-speed vortexing; 250 ⁇ L of bufferP2 was added, and the solution was inverted 8-10 times; 350 ⁇ L of bufferP3 was added, and the solution was immediately inverted and mixed 8-10 times to completely neutralize the solution; centrifuged at 13000rpm for 10 minutes, and the supernatant was passed through the column; centrifuged at 12000 for 1 minute, the waste liquid was discarded, 500 ⁇ L of PW1 was added, and the waste liquid was discarded; 600 ⁇ L of PW2 was added, and the supernatant was discarded; 600 ⁇ L of PW2 was added, and the solution was centrifuged at 12000 for 1 minute, and the supernatant was discarded; the solution was idling at 12000rpm for 2 minutes; 30-50 ⁇ L of 55°C preheated
  • the obtained plasmid was tested for concentration, and 10 ⁇ L of the positive plasmid identified by enzyme digestion was sent for sequencing, and the positive plasmid was stored at -20°C. The sequencing results showed that the obtained plasmid could encode the variant capsid protein VP1. Finally, according to the amount of virus required for the later test, the relevant Helper plasmid, each group of Rep-Cap plasmid (AAV6, AAV6 mutant 1 to 4) plasmid and GOI plasmid (including scAAV, CAG, EGFP, WPREs, SV40pA) were extracted.
  • Rep-Cap plasmids, plasmids expressing green fluorescent protein (EGFP) and pHelper plasmids of each group were co-transfected into HEK-293T cells in appropriate amounts, and the AAV virus was purified by iodixanol gradient ultracentrifugation.
  • the virus titer was measured to be 1 ⁇ 10 12 GC/mL to 1 ⁇ 10 13 GC/mL, and the control AAV6 virus and AAV6 capsid protein mutant viruses 1 to 4 were obtained and placed at -80°C for use.
  • Preheat RPMI 1640 medium at 37°C Take the frozen CD3 + T cells and thaw them quickly; transfer the revived cells to 50 mL centrifuge tubes, add 12 mL of RPMI 1640 medium containing 1% FBS, and centrifuge at 300 g.
  • the cells were respectively aspirated into 1.5 mL centrifuge tubes, 1 mL of RPMI 1640 medium containing 1% FBS was added dropwise, centrifuged at 300 g for 8 min, the medium was aspirated, and this step was repeated once; the cells were resuspended with 100 uL of culture medium (10% FBS, 25 uL/mL CD3/CD28 T cell activator, 50 U/mL rhIL2) and cultured in a cell culture incubator for 72 h (37° C., 5% CO 2 ).
  • RNA extraction method was in accordance with the instructions of TransZol Up Plus RNA Kit (Full Gold, ER501). 200 ul of TranZol up was added to each tube of cells, and 40 ⁇ L of chloroform was added. The mixture was shaken vigorously for 30 s and incubated at room temperature for 3 min; centrifuged at 12,000 g and 4 ° C for 10 min.
  • the primer sequences (5'->3') are as follows:
  • EGFP-Tf GCTGGAGTACAACTACAAC
  • GAPDH101-F CTGGGCTACACTGAGCACC;
  • GAPDH101-R AAGTGGTCGTTGAGGGCAATG.
  • Collect the T cells of each group cultured for 96 hours collect the cells and supernatant of each well in a 1.5mL EP tube, add 1mL 2% FBS PBS dropwise, centrifuge at 300g for 8min, remove the supernatant, repeat washing once, remove the supernatant, add 100 ⁇ L 2% FBS to resuspend, and use a pipette tip to blow it into a single cell suspension, and place it on ice for upflow detection.
  • the relative mRNA expression showed ( Figure 3) that the AAV capsid protein mutant (MOI: 1E5) was about 45 times stronger than the rAAV6 (MOI: 1E5) control group, and was also much stronger than the rAAV6 (MOI: 1E6) control group.
  • the flow cytometer test results showed ( Figure 4) that the proportion of fluorescent cells in the AAV capsid protein mutant (MOI: 1E5) was over 90%, while that in the rAAV6 (MOI: 1E5) control group was less than 40%, and the fluorescence intensity of the main cell population (about 1E3) was about 100 times lower than that of the AAV capsid protein mutant (MOI: 1E5) (the fluorescence intensity of the main cell population was about 1E5).
  • the relative mRNA expression showed ( Figure 5) that the AAV capsid protein mutant (MOI: 1E5) was about 3.37 times stronger than the rAAV6 (MOI: 1E5) control group.
  • the flow cytometer test results showed ( Figure 6) that the proportion of fluorescent cells in the AAV capsid protein mutant (MOI: 1E5) at 96h was 85.5%, which was higher than 37.6% of rAAV6 (MOI: 1E5) and 51.1% of rAAV6 (MOI: 1E6), and the fluorescence intensity of the main cell population was also higher than that of rAAV6 (MOI: 1E5 and 1E6).
  • the relative mRNA expression showed ( Figure 7) that the AAV capsid protein mutant (MOI: 1E5) was about 4.41 times stronger than the rAAV6 (MOI: 1E5) control group.
  • the flow cytometer test results showed ( Figure 8) that the proportion of fluorescent cells in the AAV capsid protein mutant (MOI: 1E5) at 96h was 85.9%, which was higher than 37.6% of rAAV6 (MOI: 1E5) and 51.1% of rAAV6 (MOI: 1E6), and the fluorescence intensity of the main cell population was also higher than that of rAAV6 (MOI: 1E5 and 1E6).
  • AAV capsid protein mutant 4 the relative mRNA expression showed ( Figure 9) that AAV capsid protein mutant (MOI: 1E5) was about 5.62 times stronger than the rAAV6 (MOI: 1E5) control group.
  • the flow cytometer test results showed ( Figure 10) that the proportion of fluorescent cells in AAV capsid protein mutant (MOI: 1E5) at 96h was 59.8%, which was higher than 37.6% of rAAV6 (MOI: 1E5) and 51.1% of rAAV6 (MOI: 1E6), and the fluorescence intensity of the main cell population was also higher than that of rAAV6 (MOI: 1E5 and 1E6).
  • rAAV6 has a strong ability to infect immune cells, its ability to infect unactivated T cells is still very low. In the control group, even if the MOI of rAAV6 was increased to 1E6, there was no obvious improvement in the infection effect compared with MOI:1E5, indicating that rAAV6 cannot infect unactivated T cells very effectively.
  • the recombinant adeno-associated virus vector constructed by the AAV capsid protein mutant screened in the present invention has high efficiency in targeting unactivated T cells, and has the advantages of low dosage, strong infectivity and good safety. Compared with the prior art of infection after stimulation by stimulating factors (48h), infection of unactivated T cells can achieve the advantages of collecting cells and infecting them on the same day, and reinfusing them on the same day, which greatly reduces the preparation cost. The cells reduce the in vitro culture and processing time, which can reduce the impact on T cell activity.
  • the AAV capsid protein mutants screened by the present invention do not need to integrate the genome, can achieve rapid infection and reinfusion, and reduce unnecessary quality inspections and in vitro residence time.
  • the present invention provides a method for delivering gene products to unactivated T cells. Given that rapid expansion of activated T cells in vitro can lead to a decrease in anti-cancer activity, the effect is not as good as that of unactivated T cells. Using the AAV capsid protein mutants screened by the present invention to infect unactivated T cells has great clinical value and commercial application scenarios.

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Abstract

L'invention concerne un mutant de protéine capsidique de virus adéno-associé ayant une propriété de ciblage de lymphocyte T non activé et son utilisation. Le mutant comprend une séquence telle que représentée dans l'une quelconque des SEQ ID No 9 à 12. Le vecteur de virus adéno-associé recombinant construit à l'aide du mutant de protéine capsidique de virus adéno-associé a une efficacité élevée dans le ciblage de lymphocytes T non activés, et présente les avantages d'une faible dose, d'une forte infectivité et d'une bonne sécurité.
PCT/CN2023/074186 2022-10-09 2023-02-02 Mutant de virus adéno-associé et son utilisation WO2024077815A1 (fr)

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Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117820442B (zh) * 2023-06-09 2024-05-31 广州派真生物技术有限公司 腺相关病毒突变体及其应用
CN116836237B (zh) * 2023-06-16 2023-12-05 广州译码基因科技有限公司 提高视网膜靶向性的aav病毒衣壳蛋白突变体及其应用
CN116970041B (zh) * 2023-07-21 2024-02-09 广州派真生物技术有限公司 腺相关病毒突变体及其应用
CN118480097B (zh) * 2023-09-20 2024-11-05 广州派真生物技术有限公司 腺相关病毒突变体及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111349148A (zh) * 2020-03-13 2020-06-30 辉大(上海)生物科技有限公司 一种腺相关病毒载体及其用途
CN111836649A (zh) * 2017-09-20 2020-10-27 4D分子治疗有限公司 腺相关病毒变异衣壳和其使用方法
CN112029737A (zh) * 2005-04-07 2020-12-04 宾夕法尼亚大学托管会 增强腺相关病毒载体功能的方法
CN112813037A (zh) * 2021-01-08 2021-05-18 中国科学院动物研究所 一种高效感染原代小胶质细胞的重组突变腺相关病毒及其相关生物材料
CN113423434A (zh) * 2018-12-05 2021-09-21 阿贝奥纳治疗有限公司 用于基因递送的重组腺相关病毒载体
CN113717248A (zh) * 2020-09-30 2021-11-30 广州派真生物技术有限公司 腺相关病毒突变体及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7081767B2 (ja) * 2017-01-30 2022-06-07 学校法人日本医科大学 アデノ随伴ウイルス（ａａｖ）キャプシドタンパク質の変異体
CN109897831B (zh) * 2019-03-14 2021-05-28 苏州吉脉基因药物生物科技有限公司 具有突变衣壳的腺相关病毒病毒体及其应用
CN113748122A (zh) * 2019-04-24 2021-12-03 宝生物工程株式会社 具有脑靶向特性的aav突变体
CN111825772B (zh) * 2020-07-30 2023-10-20 中国科学院精密测量科学与技术创新研究院 具有变异衣壳蛋白的腺相关病毒及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112029737A (zh) * 2005-04-07 2020-12-04 宾夕法尼亚大学托管会 增强腺相关病毒载体功能的方法
CN111836649A (zh) * 2017-09-20 2020-10-27 4D分子治疗有限公司 腺相关病毒变异衣壳和其使用方法
CN113423434A (zh) * 2018-12-05 2021-09-21 阿贝奥纳治疗有限公司 用于基因递送的重组腺相关病毒载体
CN111349148A (zh) * 2020-03-13 2020-06-30 辉大(上海)生物科技有限公司 一种腺相关病毒载体及其用途
CN113717248A (zh) * 2020-09-30 2021-11-30 广州派真生物技术有限公司 腺相关病毒突变体及其应用
CN113754727A (zh) * 2020-09-30 2021-12-07 广州派真生物技术有限公司 腺相关病毒突变体及其应用
CN112813037A (zh) * 2021-01-08 2021-05-18 中国科学院动物研究所 一种高效感染原代小胶质细胞的重组突变腺相关病毒及其相关生物材料

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