WO2024044892A1 - 一种用于基因靶向与表达的aav-8型血清型的改造型载体及其构建方法及应用 - Google Patents

一种用于基因靶向与表达的aav-8型血清型的改造型载体及其构建方法及应用 Download PDF

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WO2024044892A1
WO2024044892A1 PCT/CN2022/115529 CN2022115529W WO2024044892A1 WO 2024044892 A1 WO2024044892 A1 WO 2024044892A1 CN 2022115529 W CN2022115529 W CN 2022115529W WO 2024044892 A1 WO2024044892 A1 WO 2024044892A1
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aav
cells
virus
serotype
amino acid
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French (fr)
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张仕卿
吴小江
顾莉萍
余虹
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上海目镜生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

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  • the invention belongs to the fields of genetic engineering and biotechnology, and specifically relates to the screening, construction and application of a modified AAV-8 serotype for gene targeting and expression.
  • AAV is a non-pathogenic parvovirus with DNA defects.
  • Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus. It has weak immunogenicity, good safety, wide host range and infection With high efficiency and strong tissue specificity, it is an ideal gene expression vector and has been approved by the FDA for clinical experiments.
  • AAV viral coat proteins are used in AAV virus packaging systems.
  • Traditional wild-type AAV type 1, 2, 5, 8 and 9 coat proteins are mostly used, and have different tissue or cell specificities.
  • This type of natural wild-type AAV virus shell can effectively target AAV to target tissues for the expression of foreign genes.
  • AAV1 can efficiently infect skeletal muscle cells
  • AAV2 can infect retinal cells
  • AAV8 can efficiently deliver foreign genes.
  • AAV9 has the ability to cross the blood-brain barrier and express foreign genes in the central nervous system and brain, etc.
  • the natural affinity of viruses determines the basis for targeted delivery therapy.
  • it also provides a platform for us to modify these viral shells to have higher penetration capabilities, longer lasting expression times, and lower immunogenicity. .
  • AAV has been widely used in preclinical research on various diseases
  • the top priority is the targeted modification and optimization of the natural wild-type AAV coat protein.
  • AAV capsid proteins including DNA shuffling technology, capsid protein amino acid site-directed mutation and artificial insertion, deletion of amino acid sequence modifications, capsid protein modification, etc.
  • the phage display system technology developed in recent years is an efficient and feasible screening method that selects specifically targeted short peptides from a random peptide library and inserts them into specific sites on the wild-type AAV coat protein.
  • AAV-mediated treatment of ophthalmic diseases has broad clinical application prospects. Its advantages are that the eyeball has a clear tissue structure, a transparent refractive stroma, and is easy to observe, position and operate. In addition, eyeball tissue also has immune immunity, that is, it is difficult to reject foreign substances, such as adenovirus (AdV), adeno-associated virus (AAV), etc., making the virus highly feasible for treating eye diseases whether it is a single gene or multiple genes. Has potential. Epidemiological data show that most people around the world have been infected with wild-type AAV, and AAV2 antibodies are already present in newborn babies.
  • AdV adenovirus
  • AAV2 antibodies are already present in newborn babies.
  • AAV2 is more likely to cause autoimmune reactions and acquired immune reactions in the body
  • AAV8 was originally derived from the Ganges River. Isolated from monkeys, it is serologically unique, has minimal cross-reactivity with other serotypes, and has much lower immunogenicity than AAV.
  • the patient's retina is usually relatively fragile, so in clinical practice, infection is often caused by intravitreal injection of drugs rather than injection into the subretinal space.
  • Relevant experiments have shown that the transmission efficiency of wild-type AAV8 from the vitreous body to the retina and other posterior segments of the eye is relatively weak, and it has also been found in clinical applications of actual ophthalmic diseases.
  • One of the technical problems to be solved by the present invention is to screen a modified vector of AAV-8 serotype for gene targeting and expression.
  • the second technical problem to be solved by the present invention is to provide a method for constructing the modified vector.
  • the present invention selects a new AAV-8 modified shell AAV8-590RGD that can infect the retina and retinal pigment epithelial cells through vitreous injection of the eyeball. That is, by using the in vitro evolutionary screening method, 10 amino acids were inserted between the 590th amino acid and the 591st outer amino acid of the AAV-8 wild-type shell, including 7 random amino acids, 2 protected amino acids LA and 3 at the 5' end. 'End 1 protected amino acid A. After two steps of virus packaging, each AAV virus shell has only a unique 10 amino acid sequence insertion, corresponding to its AAV genome sequence. This virus is injected into the vitreous body of mice, and the small cells are removed after about 1 week. The mouse retina and choroid were extracted and analyzed to see whether AAV virus penetrated from the vitreous into the retina and retinal pigment epithelium.
  • the third technical problem to be solved by the present invention is to provide the application of the modified vector AAV8-590RGD.
  • the present invention adopts the following technical solutions:
  • a modified vector of AAV-8 serotype for gene targeting and expression in which the AAV-8 serotype coat protein shown in SEQ ID NO. Insert the 10 amino acid sequence shown in SEQ ID NO.3 between the 590th amino acid and the 591st amino acid: LARGDSTKSA, in which the 1st, 2nd and 10th amino acids are protective amino acids, and the 3rd to 9th amino acids Amino acid sequences obtained for screening.
  • the nucleotide sequence of the modified vector of the AAV-8 serotype used for gene targeting and expression is shown in SEQ ID NO. 1.
  • the amino acid sequence LARGDSTKSA inserted into the modified vector is used as a shell for AAV virus packaging, or for the connection and targeting of biological macromolecules, antibody drugs, peptides and small chemical molecules.
  • the present invention constructs an AAV vector containing a 30-base sequence corresponding to the 10 amino acid sequences (7 random amino acid sequences and 3 protected amino acids) shown in SEQ ID NO. 3, including the CAP gene and the REP gene, selectively Mark the Ampicillin resistance gene; insert the corresponding 30 bases as shown in SEQ ID NO. 4 between the bases corresponding to the 590th amino acid and the 591st amino acid of the CAP protein.
  • Ampicillin resistance gene is an antibiotic resistance gene. Its purpose is to make the bacteria successfully introduced into the vector resistant to antibiotics, so that AAV vectors can be screened and amplified.
  • a method for constructing the above-mentioned vector includes the following steps:
  • the first step is to synthesize 21 random bases and add protective bases TTGGCT and GCC at the 5' and 3' ends, and insert them into the base sequence corresponding to the 590th and 591st amino acids of the AAV-8 Cap gene.
  • Form AAV shell vector
  • the above-mentioned AAV shell vector is electroporated into several competent states.
  • Each competent state is cultured in Luria-Bertani (LB) medium overnight.
  • LB Luria-Bertani
  • the bacterial solution from each culture medium is mixed and inoculated into the LB medium.
  • the bacteria were shaken overnight, and the remaining bacterial liquid was stored in glycerol (random fragment library); the plasmid was extracted to obtain a mixed vector library, named pAAV8-590-7aa.
  • pAAV8-590-7aa, AAV-8 Cap plasmid and helper plasmid are used for AAV virus packaging and purification.
  • This virus is named AAV library transfer shuttles and co-infects Hek293T cells with adenovirus for the second round of AAV. packaging and purification of viruses;
  • the fourth step is to administer the second round of AAV virus into the eyes of C57BL/6J strain mice, remove the retinal layer and choroidal layer, extract the genomic DNA of the retinal layer and choroidal layer, and detect the 590th amino acid of the AAV-8 Cap gene
  • the 21 amino acids following the corresponding base sequence are sequenced;
  • the fifth step is to analyze the sequencing results and PCR amplify the random sequence and repeat the first step for the second round of screening. Analyze the sequencing results to determine the 10 amino acid sequences and insert them into positions 590 and 591 of the AAV-8 Cap gene. , AAV virus packaging, intravitreal injection to infect the retina, brain stereotaxic injection to infect the hippocampus, and in vitro infection experiments of each cell to compare the infection differences between AAV-8 and AAV8-590RGD.
  • the AAV library transfer shuttles virus and the adenovirus co-infect Hek293T cells.
  • the AAV library transfer shuttles virus and the adenovirus are co-infected with the adenovirus at a ratio of infection multiplicity value of 1. Hek293T cells were co-infected together.
  • the application of the modified carrier is also provided.
  • the application of the modified vector in the preparation of products for infected retina wherein the infected retina can be administered through eye drops or intravitreal injection, but is not limited to these two administration methods.
  • the application of the modified vector in the preparation of products that infect the cerebellum, hippocampus, motor cortex, and striatum can be injected through stereotaxic brain injection.
  • the modified vector is used to infect retinal ganglion isolated cells, Neuro2A cells, U251 cells, ARPE-19 cells, SH-SY-5Y cells, BV2 cells, HBMEC primary isolated cells, JURKAT cells, K562 cells and THP1 cells in vitro. (but not limited to the above cells).
  • AAV capsid vector capable of expressing adeno-associated virus protein capsid, also known as capsid.
  • Capsids are oligomers formed by viral capsid protein subunits. The function of the capsid is to encapsulate the genetic material of the virus.
  • AAV-8 A type of AAV that was originally isolated from rhesus monkeys. It is serologically unique, has minimal cross-reactivity with other serotypes, and is less immunogenic than AAV2.
  • AAV-8 serotype coat protein The protein coat of the AAV-8 virus, which encapsulates the genetic material of AAV-8.
  • Wild-type AAV-8 serotype The antigen possessed by the natural AAV-8 virus that has not been modified or transformed.
  • Gene targeting and expression Targeted gene delivery to target cells or tissues and gene expression.
  • Protective amino acids connecting amino acids that connect the inserted amino acids to the original shell amino acids and are used to stabilize protein conformation and function.
  • Random amino acid sequence a random base sequence synthesized from a random base sequence, which corresponds to the corresponding random amino acid sequence.
  • the vector of the present invention includes a serotype coat amino acid sequence, an insertion site and an insertion amino acid sequence.
  • the AAV-8 wild-type serotype 7 Random amino acid fragments and 3 protected amino acids, a total of 10 amino acids, are inserted between amino acids 590-591 of the AAV-8 wild-type shell, inserted into a random short peptide display library, and expressed in the target tissue through in vitro expression
  • the random short peptide library was screened out quickly and effectively to identify new AAV modified shells with good penetration, strong infectivity and low immunogenicity that can be infected into the retina through intravitreal injection.
  • the modified vector of the present invention has a stronger fluorescence effect and can be observed clearly and intuitively.
  • the foreign gene can efficiently express the target tissue in the body. When using the same amount of virus, it has better results than the wild-type serotype. expression effect and had less hepatic leakage than the wild-type serotype upon intravitreal injection of the eye.
  • AAV-8 engineered serotypes were better able to infect mouse retinas via intravitreal injection (Fig. 3) (Fig. 4), and had less leaky expression in the liver (Fig. 5).
  • AAV-8 engineered serotypes are better able to infect in vitro including but not limited to retinal ganglion isolated cells (Figure 6), Neuro2A cells (Figure 7), U251 cells (Figure 8), SH-SY-5Y cells (Figure 10), HBMEC primary isolated cells (Figure 12), JURKAT cells (Figure 13).
  • AAV-8 engineered serotypes are better able to be injected stereotactically through the brain, including but not limited to the mouse cerebellum (Figure 16), hippocampus (Figure 17) and striatum (Figure 19).
  • Figure 1 is a schematic structural diagram of the AAV8-590RGD vector in Example 1 of the present invention.
  • Figure 2 is a schematic diagram of the base sequencing results of the AAV8-590RGD serotype screened in Example 3 of the present invention.
  • Figure 3 is a schematic diagram of the situation in which the AAV8-590RGD serotype packaged virus screened in Example 5 of the present invention and the wild AAV8 serotype packaged virus are respectively injected into the vitreous cavity and infected the retina (tiled); wherein, Figure 3 (A) represents the wild AAV8 serotype packaged virus; Figure 3(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 4 is a schematic diagram of the situation in which the AAV8-590RGD serotype packaged virus screened in Example 5 of the present invention and the wild AAV8 serotype packaged virus are respectively injected into the vitreous cavity and infect the retina (slice) area; wherein, Figure 4 (A) represents the wild AAV8 serotype packaged virus; Figure 4(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 5 is a schematic diagram of in vivo imaging of the AAV8-590RGD serotype packaged virus screened in Example 5 of the present invention, and the wild AAV8 serotype packaged virus, respectively injected into the vitreous cavity, infecting the eyeball, brain and various organs;
  • Figure 5 (A) represents the wild AAV8 serotype packaged virus
  • Figure 5 (B) represents the AAV8-590RGD serotype packaged virus selected in the present invention.
  • Figure 6 is a schematic diagram of the virus packaged by the AAV8-590RGD serotype screened in Example 4 of the present invention, and the virus packaged by the wild AAV8 serotype, infecting isolated retinal ganglion cells;
  • Figure 6(A) represents the wild type AAV8 serotype packaged virus;
  • Figure 6(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 7 is a schematic diagram of the infection of Neuro2A cells by the AAV8-590RGD serotype packaged virus screened in Example 4 of the present invention and the wild AAV8 serotype packaged virus.
  • Figure 7(A) represents the wild AAV8 serum. type packaged virus;
  • Figure 7(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 8 is a schematic diagram of the infection of U251 cells by the AAV8-590RGD serotype packaged virus screened in Example 4 of the present invention and the wild AAV8 serotype packaged virus.
  • Figure 8(A) represents the wild AAV8 serum. type packaged virus;
  • Figure 8(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 9 is a schematic diagram of the infection of ARPE-19 cells by the AAV8-590RGD serotype packaged virus screened in Example 4 of the present invention and the wild AAV8 serotype packaged virus; wherein Figure 9(A) represents the wild AAV8 serotype packaged virus. AAV8 serotype packaged virus; Figure 9(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 10 is a schematic diagram of the infection of SH-SY-5Y cells by the AAV8-590RGD serotype packaged virus screened in Example 4 of the present invention and the wild AAV8 serotype packaged virus; wherein, Figure 10(A) represents Wild AAV8 serotype packaged virus; Figure 10(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 11 is a schematic diagram of the infection of BV2 cells by the AAV8-590RGD serotype packaged virus screened in Example 4 of the present invention and the wild AAV8 serotype packaged virus.
  • Figure 11(A) represents the wild AAV8 serum. type packaged virus;
  • Figure 11(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 12 is a schematic diagram of the infection of the AAV8-590RGD serotype packaged virus screened in Example 4 of the present invention and the wild AAV8 serotype packaged virus infecting HBMEC primary isolated cells; wherein, Figure 12(A) represents the wild type AAV8-590RGD serotype packaged virus. AAV8 serotype packaged virus; Figure 12(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 13 is a schematic diagram of the infection of JURKAT cells by the AAV8-590RGD serotype packaged virus screened in Example 4 of the present invention and the wild AAV8 serotype packaged virus.
  • Figure 13(A) represents the wild AAV8 serum. type packaged virus;
  • Figure 13(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 14 is a schematic diagram of the infection of K562 cells by the AAV8-590RGD serotype packaged virus screened in Example 4 of the present invention and the wild AAV8 serotype packaged virus.
  • Figure 14(A) represents the wild AAV8 serum. type packaged virus;
  • Figure 14(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 15 is a schematic diagram of the infection of THP1 cells by the AAV8-590RGD serotype packaged virus screened in Example 4 of the present invention and the wild AAV8 serotype packaged virus.
  • Figure 15(A) represents the wild AAV8 serum. type packaged virus;
  • Figure 15(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 16 is a schematic diagram of the virus packaged by the AAV8-590RGD serotype screened in Example 5 of the present invention, and the virus packaged by the wild AAV8 serotype, infecting the mouse cerebellum; wherein, Figure 16(A) represents the wild AAV8 Serotype packaged virus; Figure 16(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 17 is a schematic diagram of how the AAV8-590RGD serotype packaged virus screened in Example 5 of the present invention and the wild AAV8 serotype packaged virus infect the hippocampus of mice;
  • Figure 17(A) represents the wild AAV8 Serotype packaged virus;
  • Figure 17(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 18 is a schematic diagram of the situation in which the AAV8-590RGD serotype packaged virus screened in Example 5 of the present invention and the wild AAV8 serotype packaged virus infect the motor cortex of mice; wherein, Figure 18(A) represents the wild type AAV8 serotype packaged virus; Figure 18(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • Figure 19 is a schematic diagram of the virus packaged by the AAV8-590RGD serotype screened in Example 5 of the present invention and the virus packaged by the wild AAV8 serotype, infecting the striatum of mice; wherein, Figure 19(A) represents the wild AAV8 serotype packaged virus; Figure 19(B) represents the AAV8-590RGD serotype packaged virus screened in the present invention.
  • the PCR system is as follows: 32.5 ⁇ L H2O, 10 ⁇ L 5 ⁇ Buffer (containing Mg2+), 4 ⁇ L dNTPs (2.5mM each), 1 ⁇ L forward primer Primer1(+), 1 ⁇ L reverse primer Primer2( -) (10 ⁇ M), 1 ⁇ L target gene template DNA, and 0.5 ⁇ L PrimeSTAR enzyme to form a reaction system;
  • the PCR program was as follows: denaturation at 98°C for 3 minutes; annealing at 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 1 minute, repeated for 30 cycles; and extension at 72°C for 10 minutes.
  • step (2) Recombine the PCR product of step (2) and the vector skeleton of step 1), transform into E. coli, screen positive bacteria and extract their plasmids to obtain the recombinant vector.
  • step 1) the enzyme digestion system is: BsmBI: 1 ⁇ L, buffer: 3 ⁇ L, Rep-AAV8-Cap plasmid: 1 ⁇ g, supplement water to 30 ⁇ L; enzyme digestion at 37°C for 4 hours.
  • step 2) the recombination system is: recombinase: 15 ⁇ L, recovered PCR product DNA: 40 ng, recovered plasmid: 20 ng; after 30 min in a 42°C water bath, transform into E. coli.
  • pAAV8-590-7aa-F AGGACCCTGTTACCGCCAAC, as shown in SEQ ID NO:5
  • the completed pAAV8-590RGD vector structure contains the ampicillin resistance gene, the AAV replication gene, and the AAV8 coat protein gene. It contains a random base sequence corresponding to 10 amino acid sequences: LARGDSTKSA , as shown in SEQ ID NO:3.
  • AAV-293 cells will experience decreased growth status, mutations, etc.
  • cell cryopreservation solution (70% complete culture medium + 20% FBS + 10% DMSO) to resuspend the cells at a density of 3 x 106 cells/mL.
  • the cells grow to a confluence rate of 80% to 90%, the cells need to be passaged to expand the number of cells and maintain good growth status of the cells.
  • the constructed AAV vector, packaging plasmid and helper plasmid need to be extracted in large quantities, and the concentration is greater than 1 ⁇ g/ ⁇ L.
  • A260/280 can only be used to encapsulate the virus when the A260/280 is between 1.7-1.8. It is recommended to use the Qiagen large-scale extraction kit for large-scale endotoxin-free extraction of plasmids.
  • transfection is performed on the second day, 9-10 million cells/T75 should be plated; if transfection is performed on the third day, 3.5-4 million cells/T75 should be plated.
  • LipofiterTM transfection reagent is a product of Hanheng Biotech.
  • LipofiterTM manual please refer to the LipofiterTM manual.
  • Viral particles are present in both packaging cells and culture supernatant. Both cells and culture supernatant can be collected for best yield.
  • AAV virus packaging titer determination (using Q-PCR method)
  • Adeno-associated virus can be used for experiments within a short period of time after receiving the virus solution.
  • the virus can be temporarily stored at 4°C; if long-term storage is required, please place it at -80°C (the virus is placed in a cryopreservation tube and a sealing film is used) seal).
  • the virus can be stored at -80°C for more than 6 months; however, if the virus is stored for more than 6 months, it is recommended that the virus titer needs to be re-measured before use.
  • centrifugation is required, use a well-sealed centrifuge tube, or seal with a sealing film and centrifuge, and try to use a centrifuge in the tissue culture room.
  • Example 3 AAV capsid-packaged virus containing random sequences, intravitreal injection and retinal extraction in mice
  • Mydriatic solution dilates pupils, and methylcellulose keeps the eye surface moist
  • Each AAV shell contains a specific amino acid sequence of the AAV shell protein library virus, and slowly inject it into the vitreous cavity of the mouse. After injection, leave the needle for 0.5-1 minutes and quickly withdraw it. Needle;
  • mice were anesthetized and killed, the target tissue retina and retinal pigment epithelium were taken, their genomes were extracted, and the AAV shell sequence that penetrated into the target tissue was sequenced and analyzed;
  • the box represents the inserted 30 bases containing 21 random base sequences obtained by sequencing. Among them, the main peak of the 21 random base sequence sequencing results is more obvious. , read and analyze to obtain the ttggctagaggtgatagcacaaagtctgcc base sequence.
  • Example 4 Viruses packaged in AAV-8 and AAV8-590RGD shells respectively, cell infection and fluorescence comparison
  • the previous step can also be replaced by observing the cells become round under the microscope;
  • Figure 6- Figure 6- Figure 15 represents viruses packaged in AAV-8 (A) and AAV8-590RGD (B) capsids with the same viral amounts, respectively, infecting retinal ganglion isolated cells ( Figure 6), Neuro2A cells (Figure 7), and U251 cells ( Figure 8), ARPE-19 cells ( Figure 9), SH-SY-5Y cells (Figure 10), BV2 cells ( Figure 11), HBMEC primary isolated cells (Figure 12), JURKAT cells ( Figure 13), K562 cells ( Figure 14), THP1 ( Figure 15) fluorescence of cells.
  • AAV8-590RGD coated virus infected retinal ganglion isolated cells Figure 6
  • Neuro2A cells Figure 7
  • U251 cells Figure 8
  • SH-SY-5Y cells Figure 10
  • HBMEC primary isolated cells Figure 12
  • JURKAT cells Figure 13
  • Example 5 AAV-8 and AAV8-590 RGD shell-packed viruses, intravitreal injection and brain positioning injection in mice, comparison of infected areas
  • anesthetics such as sodium pentobarbital, chloral hydrate or isoflurane/oxygen gas mixture to anesthetize the mouse to a moderate degree
  • mice After the experiment, place the mice in a place with a suitable temperature (around 25°C) (such as a constant-temperature heating plate) to recover. When the mice wake up, they can be put back into the cage and raised;
  • a suitable temperature around 25°C
  • mice injected with the virus were raised for 3-4 weeks, they were killed by cervical dislocation, the brains were removed, fixed with 4% paraformaldehyde for about 1 day, and dehydrated with 20% and 30% sucrose solutions;
  • Figure 3 Figure 4, and Figure 5 represent viruses packaged in AAV-8 (A) and AAV8-590RGD (B) shells with the same viral amount, injected through the vitreous body, and retinal bottles ( Figure 3) and retinal frozen sections ( Figure 3) were taken. 4) and in vivo imaging ( Figure 5) to observe the expression effect and found that the virus packaged in the AAV8-590RGD shell has better infection efficiency and lower organ leakage.
  • the virus packaged by AAV8-590RGD shell has better infection efficiency than the virus packaged by AAV-8 shell in infecting mouse cerebellum ( Figure 16), mouse hippocampus ( Figure 17) and mouse brain striatum ( Figure 19).

Abstract

本发明公开了一种用于基因靶向与表达的腺相关病毒（AAV）8型血清型（AAV-8）的改造型载体，包括血清型外壳氨基酸序列，插入位点及插入氨基酸序列；在如SEQ ID NO.2所示的AAV-8型血清型外壳蛋白的第590位氨基酸与第591位氨基酸之间插入如SEQ ID NO.3所示的10个氨基酸序列:LARGDSTKSA,其中第1、第2和第10位的氨基酸为保护氨基酸，第3至第9位氨基酸为筛选获得的氨基酸序列。此外，本发明还公开了该改造型载体的构建方法和应用。本发明改造型载体的荧光效果更强，能够明显且直观的观察，外源基因能够在体内对靶向组织进行高效表达，在使用相同病毒量的情况下，比野生型血清型有更好的表达效果，并且在通过眼球玻璃体注射比野生型血清型有更少的肝脏渗漏表达。

Description

一种用于基因靶向与表达的AAV-8型血清型的改造型载体及其构建方法及应用 技术领域
本发明属于基因工程和生物技术领域，具体涉及一种用于基因靶向与表达的AAV-8型血清型的改造型的筛选、构建及应用。
背景技术
AAV是一种有DNA缺陷的非致病性细小病毒，重组腺相关病毒载体(rAAV)源于非致病的野生型腺相关病毒，具有免疫原性弱、安全性好、宿主范围广、感染效率高且组织特异性强，是理想的基因表达载体，已经被FDA批准用于临床实验。
目前AAV病毒包装系统中使用AAV病毒外壳蛋白，较多使用的是传统的野生型的AAV1、2、5、8和9型外壳蛋白，并且具有不同的组织或细胞的特异性。这类天然野生型AAV病毒外壳，虽然能够将AAV有效地靶向目的组织进行外源基因的表达，如AAV1可以高效感染骨骼肌细胞，AAV2能够感染视网膜细胞，AAV8型可将外源基因高效传递至肝细胞，AAV9具有跨血脑屏障的能力并将外源基因在中枢神经系统以及脑部表达等等。病毒的天然亲嗜性决定了靶向传递治疗的基础，另一方面也为我们改造这些病毒外壳，使其具有更高的渗透能力，更持久的表达时间以及更低的免疫原性提供了平台。
鉴于AAV已被广泛运用于各类疾病的临床前研究，当务之急是针对天然野生型AAV外壳蛋白的靶向性改造修饰与优化。目前，用于AAV衣壳蛋白进行改造优化的方法有很多，包括DNA shuffling技术、衣壳蛋白氨基酸定点突变和人工插入、删除氨基酸序列改造衣壳蛋白修饰等等。其中，近年来发展而来的基于噬菌体展示系统技术，从随机肽段库中筛选特异靶向的短肽段插入野生型AAV外壳蛋白的特定位点，是一种高效，可行的筛选方法。
AAV介导的眼科疾病的治疗，在临床上具有很广阔的运用前景，其优势在于眼球组织结构清晰、具有透明的屈光间质，易于观察、定位和操作。此外眼球组织还具有免疫赦免性即不易排斥外来物质的特质，例如腺病毒(AdV)、腺相关病毒(AAV)病毒等，使得病毒不论是单一基因还是多基因治疗眼部疾病的可行性都极具潜力。流行病学资料表明，世界各地大多数人感染过野生型AAV，刚出生的婴儿体内就已存在AAV2抗体，因此AAV2较易引起人体自身免疫反应及获得性免疫反应，而AAV8最初是从恒河猴中分离出来的，在血清学上比较独特，与其他血清型的交叉反应最小，免疫原性要比AAV低得多。在许多眼科疾病中，病人的视网膜通常是比较脆弱的，因此临床实践中往往通过玻璃体注射药物的方式而不是视网膜下腔的注射方式进行感染。相关实验表明，野生型AAV8在眼内玻璃体向视网膜等眼后段的传递效率比较弱，并且在实际眼科疾病临床运用中也发现。因此我们拟用定向进化的方法，将7个随机氨基酸片段及3个保护氨基酸，总共10个氨基酸，一同插入AAV-8型野生型外壳的第590-591位氨基酸之间，插入随机短肽展示库，并通过体外表达的方法在目的组织中筛选出随机短肽库中迅速有效筛选出能够通过玻璃体注射感染到视网膜的穿透性好，感染性强，免疫原性低的新型AAV改造型外壳。
发明内容
本发明要解决的技术问题之一是筛选一种用于基因靶向与表达的AAV-8型血清型的改造型载体。
本发明要解决的技术问题之二是提供该改造型载体的构建方法。本发明通过眼球玻璃体注射，筛选到一个能感染到视网膜及视网膜色素上皮细胞的新型AAV-8改造型外壳AAV8-590RGD。即通过使用体外进化筛选的方法，在AAV-8野生型外壳的第590位氨基酸和第591外氨基酸之间插入10个氨基酸，其中包括7个随机氨基酸、5’端2个保护氨基酸LA和3’端1个保护氨基酸A。经过两步病毒包装，使得每个AAV病毒外壳只带有独特唯一的10个氨基酸序列插入，并对应其AAV的基因组序列，将这一病毒注射在小鼠的玻璃体中，约1周后取小鼠的视网膜及脉络膜，提取并分析其基因组，观察是否有AAV病毒从玻璃体渗透到视网膜及视网膜色素上皮层。
本发明要解决的技术问题之三是提供该改造型载体AAV8-590RGD的应用。
为解决上述技术问题，本发明采用如下技术方案：
在本发明的第一方面，提供一种用于基因靶向与表达的AAV-8型血清型的改造型载体，在如SEQ ID NO.2所示的AAV-8型血清型外壳蛋白的第590位氨基酸与第591位氨基酸之间插入如SEQ ID NO.3所示的10个氨基酸序列:LARGDSTKSA,其中第1、第2和第10位的氨基酸为保护氨基酸，第3至第9位氨基酸为筛选获得的氨基酸序列。
所述如SEQ ID NO.3所示的10个氨基酸序列，其对应的碱基序列如SEQ ID NO.4所示。
所述用于基因靶向与表达的AAV-8型血清型的改造型载体的核苷酸序列如SEQ ID NO.1所示。
所述改造型载体插入的氨基酸序列LARGDSTKSA用于AAV病毒包装用的外壳，或者用于生物大分子、抗体药物、肽段和化学小分子的连接及靶向。
本发明构建含有对应如SEQ ID NO.3所示的10个氨基酸序列(7个随机氨基酸序列及3个保护氨基酸)的30个碱基序列的AAV载体，包括CAP基因、REP基因，选择性的标记Ampicillin抗性基因；在所述CAP蛋白的第590位氨基酸与第591位氨基酸对应的碱基之间插入如SEQ ID NO.4所示的相对应的30个碱基。Ampicillin抗性基因即抗生素抗性基因，其目的是使成功导入载体的细菌对抗生素产生抗性，从而能够筛选并扩增AAV载体。
在本发明的第二方面，还提供上述载体的构建方法，该方法包括如下步骤：
第一步，合成随机的21个碱基以及在5’端和3’端添加保护碱基TTGGCT和GCC，插入AAV-8 Cap基因的第590位和591位氨基酸对应的碱基序列之中，形成AAV外壳载体；
第二步，将上述AAV外壳载体，电击转化若干个感受态，每个感受态在Luria-Bertani(LB)培养基培养过夜，次日于每个培养基中取菌液混合接种于LB培养基摇菌过夜，剩余菌液甘油保存(随机片段库)；质粒抽提，得到混合载体库，命名为pAAV8-590-7aa。
第三步，pAAV8-590-7aa与AAV-8 Cap质粒与辅助质粒一起进行AAV病毒包装、纯化，此病毒命名为AAV library transfer shuttles，与腺病毒一起共感染Hek293T细胞，进行第二轮的AAV病毒的包装和纯化；
第四步，将第二轮的AAV病毒进行C57BL/6J品系小鼠的滴眼给药，取视网膜层和脉络膜层，提取视网膜层和脉络膜层的基因组DNA，检测AAV-8 Cap基因590位氨基酸对应的碱基序列后的21个氨基酸并测序；
第五步，分析测序结果并PCR扩增随机序列并重复第一步，进行第二轮筛选，分析测序结果，确定10个氨基酸序列，将其插入AAV-8 Cap基因的第590位和591位，AAV病毒包装，玻璃体腔注射感染视网膜，脑立体定位注射感染海马，各细胞体外感染实验，比较AAV-8与AAV8-590RGD的感染差异。
作为本发明优选的技术方案，第三步中，所述AAV library transfer shuttles病毒与腺病毒一起共感染Hek293T细胞具体为，所述AAV library transfer shuttles病毒以感染复数值为1的比例，与腺病毒一起共感染Hek293T细胞。
在本发明的第三方面，还提供该改造型载体的应用。所述的改造型载体在制备感染视网膜的产品中的应用，其中感染视网膜可以通过滴眼给药或者玻璃体注射，但不限于这两种给药方式。所述的改造型载体在制备感染小脑、海马、运动皮层、纹状体的产品中的应用，所述感染小脑、海马、运动皮层、纹状体可以通过脑立体定位注射。所述的改造型载体通过体外感染视网膜神经节分离细胞、Neuro2A细胞、U251细胞、ARPE-19细胞、SH-SY-5Y细胞、BV2细胞、HBMEC原代分离细胞、JURKAT细胞、K562细胞和THP1细胞(但不仅限于上述细胞)中的应用。血清型与野生型AAV-8行血清型，在感染不同组织及细胞中的应用与比较。
以上术语说明如下：
AAV外壳载体：能够表达腺相关病毒蛋白质外壳,又称为壳体。衣壳是由病毒衣壳蛋白亚基所形成的寡聚体。衣壳的作用是用于包裹病毒的遗传物质。
AAV-8型：AAV的一种，最初是从恒河猴中分离出来，在血清学上比较独特，与其他血清型的交叉反应最小，免疫原性要比AAV2低。
AAV-8型血清型外壳蛋白:AAV-8病毒的蛋白质外壳，包裹了AAV-8的遗传物质。
野生型AAV-8血清型:未经修饰或改造的天然AAV-8病毒所具有的抗原。基因靶向与表达:将目的基因定向递送至目标细胞或者组织并进行基因表达。
保护氨基酸:连接插入的氨基酸与原外壳氨基酸的连接氨基酸，用于稳定蛋白构象及功能。
随机氨基酸序列:由随机碱基序列合成的随机碱基序列，其对应相应的随机氨基酸序列。
与现有技术相比，本发明具有如下有益效果：本发明载体包括血清型外壳氨基酸序列，插入位点及插入氨基酸序列，在所述的AAV-8野生型血清型的基础上，将7个随机氨基酸片段及3个保护氨基酸，总共10个氨基酸，一同插入AAV-8型野生型外壳的第590-591位氨基酸之间，插入随机短肽展示库，并通过体外表达的方法在目的组织中筛选出随机短肽库中迅速有效筛选出能够通过玻璃体注射感染到视网膜的穿透性好，感染性强，免疫原性低的新型AAV改造型外壳。本发明改造型载体的荧光效果更强，能够明显且直观的观察，外源基因能够在体内对靶向组织进行高效表达，在使用相同病毒量的情况下，比野生型血清型有更好的表达效果，并且在通过眼球玻璃体注射比野生型血清型有更少的肝脏渗漏表达。
与野生型AAV-8血清型相比，本发明AAV8-590RGD血清型改造型载体在感染不同组织及细胞中的应用与比较实验显示，本发明具有如下有益效果：
AAV-8改造型血清型能够更好地通过玻璃体注射，感染小鼠视网膜(图3)(图4)，并且在肝脏中有更少的渗漏表达(图5)。
AAV-8改造型血清型能够更好地通过体外感染包括但不仅限于视网膜神经节分离细胞(图6)、Neuro2A细胞(图7)、U251细胞(图8)、SH-SY-5Y细胞(图10)、HBMEC原代分离细胞(图12)、JURKAT细胞(图13)。
AAV-8改造型血清型能够更好地通过脑立体定位注射，感染包括但不仅限于小鼠小脑(图16)、海马(图17)和纹状体(图19)。
附图说明
图1为本发明实施例1中AAV8-590RGD载体结构示意图。
图2为本发明实施例3中筛选到的AAV8-590RGD血清型碱基测序结果示意图。
图3为本发明实施例5中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，分别注射玻璃体腔，感染视网膜(平铺)的情况示意图；其中，图3(A)代表野生的AAV8血清型包装的病毒；图3(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图4为本发明实施例5中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，分别注射玻璃体腔，感染视网膜(切片)区域的情况示意图；其中，图4(A)代表野生的AAV8血清型包装的病毒；图4(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图5为本发明实施例5中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，分别注射玻璃体腔，感染眼球，大脑及各个脏器的活体成像情况示意图；其中，图5 (A)代表野生的AAV8血清型包装的病毒；图5(B)代表代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图6为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染视网膜神经节分离细胞的情况示意图；其中，图6(A)代表野生的AAV8血清型包装的病毒；图6(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图7为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染Neuro2A细胞的情况示意图；其中，图7(A)代表野生的AAV8血清型包装的病毒；图7(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图8为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染U251细胞的情况示意图；其中，图8(A)代表野生的AAV8血清型包装的病毒；图8(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图9为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染ARPE-19细胞的情况示意图；其中，图9(A)代表野生的AAV8血清型包装的病毒；图9(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图10为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染SH-SY-5Y细胞的情况示意图；其中，图10(A)代表野生的AAV8血清型包装的病毒；图10(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图11为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染BV2细胞的情况示意图；其中，图11(A)代表野生的AAV8血清型包装的病毒；图11(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图12为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染HBMEC原代分离细胞的情况示意图；其中，图12(A)代表野生的AAV8血清型包装的病毒；图12(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图13为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染JURKAT细胞的情况示意图；其中，图13(A)代表野生的AAV8血清型包装的病毒；图13(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图14为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染K562细胞的情况示意图；其中，图14(A)代表野生的AAV8血清型包装的病毒；图14(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图15为本发明实施例4中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型 包装的病毒，感染THP1细胞的情况示意图；其中，图15(A)代表野生的AAV8血清型包装的病毒；图15(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图16为本发明实施例5中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染小鼠小脑的情况示意图；其中，图16(A)代表野生的AAV8血清型包装的病毒；图16(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图17为本发明实施例5中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染小鼠海马的情况示意图；其中，图17(A)代表野生的AAV8血清型包装的病毒；图17(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图18为本发明实施例5中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染小鼠运动皮层的情况示意图；其中，图18(A)代表野生的AAV8血清型包装的病毒；图18(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
图19为本发明实施例5中筛选到的AAV8-590RGD血清型包装的病毒，与野生的AAV8血清型包装的病毒，感染小鼠纹状体的情况示意图；其中，图19(A)代表野生的AAV8血清型包装的病毒；图19(B)代表本发明筛选到的AAV8-590RGD血清型包装的病毒。
具体实施方式
下面结合具体实施例进一步阐述此发明。应理解的是，在此描述的特定实施方式通过举例的方式来表示，并不作为对本发明的限制。在不偏离本发明范围的情况下，本发明的主要特征可以用于各种实施方式。
实施例1载体构建
序列设计和合成的具体方法和步骤如下：
(1)根据GeneBank中AAV8型包装质粒的Cap的基因信息，设计BsmBI-AAV8 Cap(482-655aa)-BsmBI基因DNA片段，合成双链DNA分子；
(2)分别用合成的引物pAAV8-590-7aa-F(如SEQ ID NO.5所示，即正向引物Primer1)和pAAV8-590-7aa-R(如SEQ ID NO.6所示，即反向引物Primer2)来PCR步骤(1)合成的双链DNA分子，得到PCR产物。
步骤(2)中，所述PCR的体系如下：32.5μLH2O，10μL 5×Buffer缓冲液(含有Mg2+)，4μL dNTP(各2.5mM)，1μL正向引物Primer1(+)，1μL反向引物Primer2(－)(10μM)，1μL目的基因模板DNA，以及0.5μL的PrimeSTAR酶，组成反应体系；
所述PCR程序如下：98℃，变性3分钟；退火98℃10秒，55℃15秒，72℃1分钟，重复30个循环；延伸72℃10分钟。
将序列插入位点的具体方法和步骤如下：
1)用限制性内切酶BsmBI酶切病毒载体Rep-AAV8-Cap，回收载体骨架；
2)将步骤(2)的PCR产物和步骤1)的载体骨架重组，转化进大肠杆菌，筛选阳性菌并提取其质粒，得到重组载体。
步骤1)中，所述酶切体系为：BsmBI:1μL，缓冲液:3μL，Rep-AAV8-Cap质粒:1μg，补充水至30μL；37℃酶切4小时。
步骤2)中，所述重组体系为：重组酶:15μL，回收的PCR产物DNA:40ng，回收的质粒:20ng；42℃水浴30min后，转化进大肠杆菌。
pAAV8-590-7aa-F：AGGACCCTGTTACCGCCAAC，如SEQ ID NO:5所示
pAAV8-590-7aa-R：GATGTTTCAGGCCAAAGCCG，如SEQ ID NO:6所示
如图1所示，构建完成的pAAV8-590RGD载体结构，其中包含有氨苄的抗性基因、AAV的复制基因、AAV8的外壳蛋白基因，其中包含有随机碱基序列，对应10个氨基酸序列：LARGDSTKSA，，如SEQ ID NO:3所示。
实施例2AAV病毒包装
(一)AAV-293细胞的冻存
随着传代的次数增加，AAV-293细胞会出现生长状态下降、突变等。为了防止此类现象的出现，我们需要在开始就对细胞进行大量冻存，以保证实验的稳定性和持续性。在细胞对数生长期进行冻存，增加细胞复苏成活率。
1、去掉
细胞培养上清液，加入PBS洗去残留的培养基；
2、加入0.25％的胰酶，消化1-2min后，镜下观察细胞变圆，细胞间间隙加大时，去除胰酶，加入新鲜培养基吹打混匀，移入离心管中。
3、细胞计数，将细胞全部晃下，加入3mL 37℃预热的10％DMEM，用10mL移液管进行吹打，较大力吹打6-8次即可，不留死角，之后，将所有细胞吸出，置于15mL离心管中，取50μL混匀后的细胞于1.5mLeppendorf管中，加入450μL 10％DMEM，即为10倍稀释，混匀，取10μL细胞于计数板中计数。计数板上共4大格，每大格16小格。计数时，4大格均计数，总数除以4(得每大格细胞数)，再乘以10(10倍稀释)，即为实际n万/mL细胞浓度。
4、细胞离心，1000rpm/min，5min。去掉上清。
5、根据细胞计数,结果加入细胞冻存液(70％完全培养基+20％FBS+10％DMSO)重悬细胞，密度为3 x  106个/mL。
6、分装进细胞冻存管，放入冻存盒中，放入-80℃超低温冰箱。
7、第二天将细胞放于液氮罐中长久保存，并作记录。保存过程中，要不时复苏细胞检测细胞存活率，观察细胞状态等。
(二)AAV-293细胞的传代
当细胞生长到汇合率达到80％～90％时需要对细胞进行传代操作，以扩大细胞数量，维持细胞良好的生长状态。
1、消化细胞，方法同细胞冻存。
2、细胞离心结束后，加入完全培养基重悬。
3、根据具体情况，将细胞分到10cm培养皿中，每个培养皿补足到10mL培养基。
(三)AAV-293细胞的复苏
当细胞传代次数过多，细胞状态变差时，或者细胞出现污染事故时，需要丢弃并对最初冻存的细胞进行复苏。
1、设置温度为37～42℃的水浴。
2、查看细胞库记录，根据记录从液氮罐中取出冻存的细胞(需戴上棉手套，防止被冻伤)，迅速丢入水浴锅中并快速晃动，尽量在1～2min内使细胞溶液完全
溶解。
3、将细胞溶液转移到15mL离心管中，并在其中加上1mL新鲜的完全培养基，混匀后离心，1000rpm/min，5min。
4、去掉上清，加入5mL新鲜的完全培养基，混匀沉淀后，转入6cm培养皿。
5、将培养皿平稳放入37℃、5％CO2和95％相对湿度的培养箱中培养。
6、第二天观察细胞存活率。给细胞换一下培养基。以后每天观察细胞生长情况。
(四)AAV包装和浓缩
1.质粒扩增
构建好的AAV载体、包装质粒和辅助质粒需经过大量抽提，浓度大于1μg/μL，A260/280在1.7-1.8间方可用以包毒。推荐使用Qiagen大抽试剂盒进行质粒的大量去内毒素抽提。
2.传AAV-293细胞
将培养AAV-293细胞T75瓶中的培养基吸净，加入2mL 4度冰箱取出的0.25％胰酶，使其均匀覆盖瓶底，置于37度培养箱中3-5min，取出，摇晃可发现细胞于底部脱离，将其全部晃下，加入3mL 37度水浴中预热的10％DMEM，移液枪用10mL移液管进行吹打，较大力吹打6-8次即可， 不留死角，瓶口处较难吹打可将移液管对准培口，小力将培养基打出即可覆盖到接近瓶口的细胞。之后，将所有细胞吸出，置于15mL离心管中，取50μL混匀后的细胞于1.5mL eppendorf管中，加入450μL 10％DMEM，即为10倍稀释，混匀，取10μL细胞于计数板中计数。计数板上共4大格，每大格16小格。计数时，4大格均计数，总数除以4(得每大格细胞数)，再乘以10(10倍稀释)，即为实际n万/mL细胞浓度。传代当天记为第一天，若第二天进行转染，铺900-1000万/T75；若第三天转染，铺350-400万/T75。每瓶T75加10mL 10％DMEM培养基。转染当天观察细胞密度，80-90％满即可进行转染。转染前无需换培养基。
3.做脂转complex
Figure PCTCN2022115529-appb-000001
注：LipofiterTM转染试剂为汉恒生物产品，使用说明参考LipofiterTM说明书。
4.AAV病毒收毒：
病毒颗粒同时存在于包装细胞和培养上清中。可以将细胞和培养上清都收集下来以获得最好的收率。
1)准备一个干冰乙醇浴(将乙醇倾入装有干冰的泡沫盒即可，也可用液氮替代干冰乙醇浴)和37℃水浴；
2)将产毒的细胞连同培养基一同收集到一个15mL的离心管中。收集细胞时，将培养盘倾斜一定角度将细胞刮到培养基中；
3)1000rpm/min，离心3分钟，分离细胞和上清，将上清另外存放，细胞用1mL PBS重悬；
4)将细胞悬浮液在干冰乙醇浴和37℃水浴中反复转移，冻融四次。每次融解后稍加震荡。注意：每次凝固和解冻大概需要十分钟的时间。
5.AAV病毒浓缩：
1)10,000g离心去除细胞碎片，将离心上清转移到一个新离心管中。
2)将两次收集的上清混合在一起，用0.45μm滤器过滤除杂质
3)加入1/2体积的1M NaCl，10％PEG8000溶液，混合均匀，4度过夜
4)12,000rpm离心2h，弃上清，病毒沉淀用适量的PBS溶液溶解，待完全溶解后用0.22μm滤器过滤除菌。
5)加入Benzonase核酸酶消化去除残留的质粒DNA(终浓度为50U/mL)。合上管盖，颠倒几次以 充分混合。在37℃孵育30分钟；
6)用0.45μm过滤头过滤，取滤出液，即为浓缩的AAV病毒。
6.AAV的纯化
1)向病毒浓缩液中添加固体CsCl直到密度为1.41g/mL(折射率为1.372)；
2)将样品加入到超速离心管中，用预先配好的1.41g/mL CsCl溶液将离心管剩余空间填满；3)在175,000g下离心24小时，以形成密度梯度。按顺序分步收集不同密度的样品，取样进行滴度测定。收集富集有AAV颗粒的组分；
4)重复上述过程一次。
5)将病毒装入100kDa的透析袋，4度透析脱盐过夜。此即为纯化的AAV病毒
AAV病毒包装滴度测定(采用Q-PCR法)
1)取20μL浓缩病毒液，加入1μL RNAse-free DNAse，混匀，37℃水浴反应30min。
2)4℃，12000rpm/min，离心10min，取10μL上清到另一个无菌的1.5mL EP管中。
3)加入90μL Dilution Buffer(1mM Tris-HCl,pH 8.0，0.1mM EDTA，150mM NaCl)，混匀，37℃金属浴反应30min。
4)自然冷却至室温，加入1μL蛋白酶K，65℃水浴反应1h。
5)100℃金属浴反应10min，自然冷却至室温。
6)进行Q-PCR检测滴度。
AAV病毒的储存、稀释
1.病毒的储存：
收到病毒液后在很短时间内即使用腺相关病毒进行实验，可以将病毒暂时放置于4℃保存；如需长期保存请放置于-80℃(病毒置于冻存管，并使用封口膜封口)。
1)病毒可以存放于-80℃6个月以上；但如果病毒储存时间超过6个月，建议在使用前需要重新测定病毒滴度。
2)反复冻融会降低病毒滴度：每次冻融会降低病毒滴度10％；因此在病毒使用过程中应尽量避免反复冻融，为避免反复冻融，建议收到病毒后按照每次的使用量进行分装。
2.病毒的稀释：
如果需要稀释病毒，请将病毒取出置于冰浴融解后，使用PBS缓冲液或培养目的细胞无血清培养基(含血清或含双抗不影响病毒感染)。混匀分装后4℃保存(请尽量在三天内用完)分装后使用。AAV使用安全注意事项
1.病毒操作时最好使用生物安全柜。如果使用普通超净工作台操作病毒，请不要打开排风机。
2.病毒操作时请穿实验服，带口罩和手套。
3.操作病毒时特别小心不要产生气雾或飞溅。如果操作时超净工作台有病毒污染，请立即用70％乙醇加1％SDS溶液擦拭干净。接触过病毒的枪头、离心管、培养板、培养液等请用84消毒液或1％SDS中浸泡过夜后弃去。
4.用显微镜观察细胞感染情况时应遵从以下步骤：拧紧培养瓶或盖紧培养板。用70％乙醇清理培养瓶外壁后到显微镜处观察拍照。离开显微镜实验台之前，用70％乙醇清理显微镜实验台。
5.如需要离心，应使用密封性好的离心管，或者用封口膜封口后离心，而且尽量使用组织培养室内的离心机。
6.脱掉手套后，用肥皂和水清洗双手。
实施例3含有随机序列的AAV外壳包装的病毒，小鼠玻璃体腔注射及视网膜提取
1.麻醉：4.3％水合氯醛0.01mL/g；
2.散瞳液散瞳，甲基纤维素保持眼表湿润；
3.调整小鼠头位，注射之位置：角膜缘后约1mm；
4.用33G注射器做切口，针尖垂直进入，随后倾斜，将每个AAV外壳都含有特定氨基酸序列的AAV外壳蛋白库病毒，缓慢推注入小鼠玻璃体腔，注射后，留针0.5-1min，迅速出针；
5.约一周后麻醉处死小鼠，取目的组织视网膜及视网膜色素上皮，提取其基因组，测序分析目的组织中渗透进入的AAV外壳序列；
由测序结果分析基因组中所含AAV序列(见图2)，方框表示测序获得的插入的含有21个随机碱基序列的总共30个碱基，其中21个随机碱基序列测序结果主峰比较明显，阅读分析得到ttggctagaggtgatagcacaaagtctgcc碱基序列。
实施例4 AAV-8及AAV8-590RGD外壳分别包装的病毒，细胞感染及荧光比较
1.在细胞库表格里查询好所需细胞的位置；
2.将细胞从液氮罐中取出，迅速置于37度水浴锅，不停轻轻摇晃；
3.将内部液体完全融化的冻存管，置于离心机中以800RPM离心5min；
4.离心结束后，将离心管中上清倒去；
5.向冻存管中加入1mL相应的培养基，轻轻吹打均匀，使成为单细胞悬液；
6.取合适大小的皿(通常为10cm或6cm皿)，加入培养基；
7.将冻存管中已经吹打均匀的细胞悬液加入皿中；
8.以米子形手法摇晃细胞培养皿，使其均匀混合；
9.将摇晃好的细胞培养皿置于37度培养箱；
10.第二天，将皿取出置于显微镜下观察，进行换液等后续操作；
11.将密度为80％的细胞从培养箱取出；
12.吸掉皿中原有培养基；
13.加入3mLPBS缓冲液，均匀摇晃皿，使PBS可清洗到皿每个角落；
14.洗掉清洗用的PBS缓冲液；
15.加入1mL胰酶，均匀摇晃皿，使胰酶可均匀接触到皿每个角落；
16.将加完胰酶已摇晃均匀的皿放回37度培养箱；
17.消化一定时间(一般为1min到2min之间)，取出细胞皿；
18.将皿拿于左手，用右手沿着皿壁轻轻拍打，看到细胞有滑落即为消化好；
19.上一步操作也可换为在显微镜下见细胞变圆即可；
20.加入2mL相应培养基与10cm皿中终止消化；
21.均匀摇晃已经加入终止液的细胞皿；
22.使用1mL移液器，对细胞进行吹打，使其成为单细胞悬液；
23.吹打结束后，吸取全部液体到5mL离心管；
24.将离心管标记好之后，置于离心机中以800RPM离心5min；
25.离心结束后，将离心管中上清倒去；
26.加入2mL相应培养基重悬细胞使其成为单细胞悬液；
27.以一定数量将细胞分到孔板中；
28.将孔板中细胞和培养基混匀；
29.将孔板置于37度培养箱培养；
30.侵染前一天，细胞铺孔板(见上述步骤)；
31.胞铺板后12小时即可以侵染；
32.细胞铺板12小时后，准备好相应病毒/丁酸钠；
33.按照细胞对应的MOI值，取适量病毒于2％血清的培养基混匀；
34.按照1:1000的比例，加入丁酸钠；；
35.将孔板换液为2％血清培养基
36.将病毒-丁酸钠混合液加入到孔板中，摇晃均匀；
37.将孔板置于37摄氏度培养箱培养；
38.培养6小时后，将孔板取出，吸掉里面的所有液体；
39.加入一定量新鲜的培养基，继续放置于37摄氏度培养箱培养；
40.一定时间后进行(荧光)拍照，送样；
图6-图15代表相同病毒量的AAV-8(A)和AAV8-590RGD(B)外壳分别包装的病毒，感染视网膜神经节分离细胞(图6)、Neuro2A细胞(图7)、U251细胞(图8)、ARPE-19细胞(图9)、SH-SY-5Y细胞(图10)、BV2细胞(图11)、HBMEC原代分离细胞(图12)、JURKAT细胞(图13)、K562细胞(图14)、THP1(图15)细胞的荧光情况。其中AAV8-590RGD外壳包装的病毒感染视网膜神经节分离细胞(图6)、Neuro2A细胞(图7)、U251细胞(图8)、SH-SY-5Y细胞(图10)、HBMEC原代分离细胞(图12)、JURKAT细胞(图13)比AAV-8外壳包装的病毒具有更好的感染效率。
实施例5 AAV-8及AAV8-590RGD外壳包装的病毒，小鼠玻璃体腔注射、脑定位注射，感染区域比较
(一)玻璃体腔注射(见实施例3)
(二)小鼠脑立体定位注射步骤如下：
1.麻醉
1)用麻药如戊巴比妥钠、水合氯醛或异氟烷/氧气混合气体将小鼠麻醉，麻醉程度适中；
2.固定
1)打开冷光源提供照明，将已麻醉的小鼠固定在脑立体定位注射仪上；
2)固定头颅：先将一侧耳棒轻轻插入外耳道，碰到骨性外耳道底后固定耳棒，继之同样插入固定另一耳棒，检查小鼠头部固定是否稳定，松斜，两侧耳棒刻度是否对称，轻移耳棒使两侧刻度一致头位完全居中，再次固定耳棒；
3)固定上颌：将小鼠的上门牙塞进上齿固定板的槽内，旋紧螺丝。从各方向推压动物头部，均不应出现移动。通过定位针的测量调节前后囟在同一矢状线上，并使前囟(Bregma)和后囟(Lambda)尽量处于同一水平面上；
3.钻孔
1)将小鼠头部的毛用宠物剃毛刀剃掉，然后用医用酒精和碘酒对头部消毒，防止感染；
2)将眼药膏抹在动物眼睛上，保持眼睛湿润，防止动物眼睛由于长时间处于干涩状态而失明；
3)用医用剪刀剪开从两眼间到两耳根间的头皮；
4)用止血钳将开口撑大，用蘸有双氧水的棉棒擦拭并去除颅骨表面硬脑膜(Dura)；
5)利用定位仪，保证前囟(Bregma，X＝0,Y＝0,Z＝0)和后囟(Lambda)处于同一水平面上(X、Z值相差皆小于0.1)；
6)根据脑图谱，确定待注射脑区的位置参数；
7)利用定位仪找到要注射病毒的位置，用记号笔在颅骨上做好标记；
8)用颅骨转在注射位点处轻磨颅骨，将颅骨慢慢打薄，当颅骨出现裂缝的时候，用医用注射器的针头小心挑破，防止损伤；
4.病毒注射
1)用PBS冲洗微量注射器(5μL规格)3-5次；
2)先吸取约1μL空气，再吸取约1μL稀释好的病毒，在空气中测试注射器是否通畅；
3)将微量注射泵，微量注射器组装好，置于钻好的孔上方，针尖与颅骨平行(Z＝0)，微调注射器位置使之与之前钻孔时位置相同；
4)根据定好的深度将注射针缓慢下降；
5)以0.05μL/min的速度注射病毒，待还剩0.5μL时停止注射；
6)注射完毕后，将注射针停留在注射位置10min待病毒扩散，然后缓慢提针；
7)用PBS清洗微量注射器5次备用；
8)注意注射过程中若有出血立刻用棉签吸走，以免带出病毒；
5.缝合
1)注射针完全拔出来后将头皮缝合；
2)实验结束后，将小鼠放在温度合适(25℃左右)的地方(如恒温加热板)恢复，等小鼠清醒即可放回笼子中饲养；
6.检测
1)注射过病毒的小鼠饲养3-4周后，脱颈处死，取脑，4％多聚甲醛固定约1天，20％和30％蔗糖溶液脱水；
2)冰冻切片，厚度为10μm。荧光显微镜下观察荧光；
图3、图4、图5代表相同病毒量的AAV-8(A)和AAV8-590RGD(B)外壳分别包装的病毒，通过玻璃体注射，取视网膜瓶体(图3)和视网膜冰冻切片(图4)和活体成像(图5)观察表达效果，发现AAV8-590RGD外壳包装的病毒具有更好的感染效率和更低的脏器渗漏。脑立体定位注射，感染小鼠小脑(图16)、小鼠海马(图17)、小鼠脑运动皮层(图18)和小鼠脑纹状体(图19)的荧光情况。其中AAV8-590RGD外壳包装的病毒感染鼠小脑(图16)、小鼠海马(图17)和小鼠脑纹状体(图19)比AAV-8外壳包装的病毒具有更好的感染效率。

Claims (9)

  1. 一种用于基因靶向与表达的AAV-8型血清型的改造型载体，特征在于：在如SEQ ID NO.2所示的AAV-8型血清型外壳蛋白的第590位氨基酸与第591位氨基酸之间插入如SEQ ID NO.3所示的10个氨基酸序列:LARGDSTKSA,其中第1、第2和第10位的氨基酸为保护氨基酸，第3至第9位氨基酸为筛选获得的氨基酸序列。
  2. 如权利要求1所述的载体，其特征在于，所述如SEQ ID NO.3所示的10个氨基酸序列，其对应的碱基序列如SEQ ID NO.4所示。
  3. 如权利要求1所述的载体，其特征在于，所述用于基因靶向与表达的AAV-8型血清型的改造型载体的核苷酸序列如SEQ ID NO.1所示。
  4. 如权利要求1所述的载体，其特征在于，所述改造型载体插入的氨基酸序列LARGDSTKSA用于AAV病毒包装用的外壳，或者用于生物大分子、抗体药物、肽段和化学小分子的连接及靶向。
  5. 如权利要求1-4任一项所述的改造型载体的构建方法，包括如下步骤：
    第一步：合成随机的21个碱基，并在5’端和3’端添加保护碱基TTGGCT和GCC，并插入AAV-8Cap基因的第590位和591位氨基酸对应的碱基序列之中，形成AAV外壳载体；
    第二步，将上述AAV外壳载体，电击转化若干个感受态，每个感受态在LB培养基培养过夜，次日于每个培养基中取菌液混合接种于LB培养基摇菌过夜，剩余菌液甘油保存；质粒抽提，得到混合载体库，命名为pAAV8-590-7aa；
    第三步，pAAV8-590-7aa与AAV-8 Cap质粒与辅助质粒一起进行AAV病毒包装、纯化，此病毒命名为AAV library transfer shuttles，与腺病毒一起共感染Hek293T细胞，进行第二轮的AAV病毒的包装和纯化；
    第四步，将第二轮的AAV病毒进行C57BL/6J品系小鼠的滴眼给药，取视网膜层和脉络膜层，提取视网膜层和脉络膜层的基因组DNA，检测AAV-8 Cap基因590位氨基酸对应的碱基序列后的21个氨基酸并测序；
    第五步，分析测序结果并PCR扩增随机序列并重复第一步，进行第二轮筛选，分析测序结果，确定10个氨基酸序列，将其插入AAV-8 Cap基因的第590位和591位，AAV病毒包装，玻璃体腔注射感染视网膜，脑立体定位注射感染海马，各细胞体外感染实验，比较AAV-8与AAV8-590RGD的感染差异。
  6. 如权利要求5所述的方法，其特征在于，第三步中，所述AAV library transfer shuttles病毒与腺病毒一起共感染Hek293T细胞具体为，所述AAV library transfer shuttles病毒以感染复数值为1的比例，与腺病毒一起共感染Hek293T细胞。
  7. 一种如权利要求1-4任一项所述的载体在制备感染视网膜的产品中的应用。
  8. 一种如权利要求1-4任一项所述的载体在制备通过脑立体定位注射，感染小脑、海马、运动皮层、纹状体的产品中的应用。
  9. 一种如权利要求1-4任一项所述的载体通过体外感染视网膜神经节分离细胞、Neuro2A细胞、U251细胞、ARPE-19细胞、SH-SY-5Y细胞、BV2细胞、HBMEC原代分离细胞、JURKAT细胞、K562细胞和THP1细胞中的应用。
PCT/CN2022/115529 2022-08-29 2022-08-29 一种用于基因靶向与表达的aav-8型血清型的改造型载体及其构建方法及应用 WO2024044892A1 (zh)

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