Preparation method of efficient AAV vector for gene therapy and AAV vector

Abstract

The invention relates to the technical field of AAV vectors, in particular to a preparation method of an efficient AAV vector for gene therapy and an AVV vector, and the preparation method comprises the following steps: (1) creating a novel AAV capsid protein library through a DNA shuffling method; and (2) obtaining the AAV variant with high transduction efficiency through directed evolution screening, wherein the method specifically comprises the following steps: a) constructing a screening vector system containing a reporter gene; b) performing multiple rounds of screening in the HEK293T cells; c) sorting by a flow cytometry to obtain an AAV variant with high transduction efficiency; (3) optimizing an AAV genome packaging sequence; and (4) carrying out virus packaging and purification on the obtained AAV vector, and recombining dominant sequences of different serotypes on a molecular level through a DNA shuffling method, so that the diversity and quality of a gene pool are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of AAV vector technology, and in particular to a method for preparing a highly efficient AAV vector for gene therapy and an AAV vector. Background Art

[0002] Adeno-associated virus (AAV) has become one of the most promising vector systems in the field of gene therapy due to its advantages such as good safety, low immunogenicity and long-term expression. In recent years, with the continuous deepening of clinical application of gene therapy, the performance requirements of AAV vectors have also been increasing. Existing studies mainly use two strategies, rational design and directed evolution, to transform AAV vectors.

[0003] In the rational design strategy, researchers analyze the structure and function relationship of AAV capsid protein and modify specific amino acid sites to improve vector performance. Although this method can achieve the expected transformation effect, it can only optimize a single performance and it is difficult to improve multiple key indicators at the same time. Moreover, due to the insufficient understanding of the structure-function relationship of AAV, it is often difficult to achieve the ideal transformation effect by relying solely on rational design.

[0004] Directed evolution strategies can obtain variants with improved performance without fully understanding the structure-function relationship by constructing random mutation libraries and performing functional screening. However, traditional directed evolution methods mainly rely on random mutations, resulting in low diversity and quality of libraries and low screening efficiency. At the same time, due to the lack of a systematic optimization strategy, the obtained variants often lose other important functions while improving a certain performance.

[0005] In addition, the existing AAV vector modification methods generally have the following problems: first, the serotype source is single, which limits the diversity of the gene library; second, the screening strategy is too simple, making it difficult to obtain variants with multiple excellent properties; third, the optimization of key sequence elements of the AAV genome is not systematic enough, which affects the overall performance of the vector; finally, the production process is not stable enough and the product quality fluctuates greatly.

[0006] It is particularly important to point out that the biggest challenge currently faced by AAV vectors in clinical applications is the interference of pre-existing neutralizing antibodies. Studies have shown that 50-80% of the population has neutralizing antibodies against commonly used AAV serotypes, which seriously limits the therapeutic effect of AAV vectors. Although some studies have attempted to improve immune escape ability by modifying capsid proteins, this often comes at the expense of transduction efficiency. How to obtain good immune escape ability while maintaining high transduction efficiency has always been a technical problem that needs to be solved in this field. Summary of the invention

[0007] The present invention aims to solve the problems existing in existing AAV vectors in terms of transduction efficiency, immune escape ability and production process. By innovatively combining the DNA shuffling method with a multi-round screening strategy, systematically optimizing the key sequence elements of the AAV genome, and establishing a standardized production process, a high-performance AAV vector with independent intellectual property rights was finally obtained.

[0008] The object of the present invention is to provide a method for preparing an efficient AAV vector for gene therapy, comprising the following steps:

[0009] (1) Creating a novel AAV capsid protein library by DNA shuffling, including:

[0010] a) selecting the AAV1-9 serotype cap gene as a template and obtaining template DNA by PCR amplification;

[0011] b) fragmenting the template DNA using DNase I to obtain DNA fragments of 50-150 bp;

[0012] c) obtaining a chimeric cap gene library by primer-free PCR recombination;

[0013] (2) AAV variants with high transduction efficiency were obtained through directed evolution screening, including:

[0014] a) constructing a screening vector system containing a reporter gene;

[0015] b) Perform multiple rounds of screening in HEK293T cells;

[0016] c) AAV variants with high transduction efficiency were obtained by flow cytometry sorting;

[0017] (3) Optimizing the AAV genome packaging sequence, including:

[0018] a) Optimizing the ITR sequence by site-directed mutagenesis;

[0019] b) improving the promoter region of the Rep gene;

[0020] c) Optimizing the polyA signal sequence;

[0021] (4) Packaging and purifying the obtained AAV vector.

[0022] Specifically, the PCR amplification conditions in step (1) are:

[0023] Pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 2 minutes, 30 cycles; final extension at 72°C for 5 minutes.

[0024] Specifically, the DNase I digestion conditions in step (1) are:

[0025] The reaction was carried out at 15°C for 1-5 minutes, and the DNase I concentration was 0.001-0.01U / μL.

[0026] Specifically, the primer-free PCR recombination conditions in step (1) are:

[0027] Pre-denaturation at 98°C for 1 minute; denaturation at 98°C for 30 seconds, annealing at 55°C for 1 minute, extension at 72°C for 2 minutes, 45 cycles; final extension at 72°C for 10 minutes.

[0028] 5. The method according to claim 1, characterized in that the screening vector in step (2) comprises the following elements:

[0029] CMV promoter-eGFP reporter gene-WPRE element-polyA signal, total length <4.7kb.

[0030] Specifically, the screening strategy in step (2) includes:

[0031] In the first round of conventional transfection screening, the GFP positive rate was >30%;

[0032] The second round of low MOI (1-5) infection screening, GFP positive rate> 50%;

[0033] In the third round, pre-existing neutralizing antibodies were added for competitive screening, and the escape rate was >60%.

[0034] Specifically, the ITR sequence optimization in step (3) includes the following mutations:

[0035] T10C: Enhances the stability of the stem-loop structure;

[0036] G85A: Improves Rep binding efficiency;

[0037] C120T: Optimization of replication initiation.

[0038] Specifically, in step (4), the virus packaging adopts a three-plasmid co-transfection system:

[0039] pHelper plasmid 10 μg, pRep-Cap plasmid 10 μg, pTR-transgene plasmid 5 μg.

[0040] Specifically, the virus purification in step (4) comprises the following steps:

[0041] Using a 15-40% cesium iodide density gradient, ultracentrifugation was performed at 38,000 rpm for 48 hours, and the 1.37-1.41 g / mL density fraction was collected.

[0042] The AAV vector prepared by the method has a virus purity of >95%, an empty shell rate of <10%, and a biological titer of >1×10 13 vg / mL.

[0043] Compared with the prior art, the method for preparing the highly efficient AAV vector for gene therapy of the present invention can achieve the following beneficial effects:

[0044] The technical solution of the present invention not only successfully solves the contradiction between transduction efficiency and immune escape ability, but also achieves the synergistic improvement of multiple performance indicators. Through the DNA shuffling method, the dominant sequences of different serotypes are recombined at the molecular level, which significantly improves the diversity and quality of the gene library. The innovative multi-round screening strategy ensures that the obtained variants have both high transduction efficiency and strong immune escape ability. Systematic genome optimization further improves gene expression efficiency and sustainability. The standardized production process ensures the stability and controllability of product quality.

[0045] It is particularly worth mentioning that the present invention discovered synergistic effects between some key domains during the capsid protein modification process. The optimized combination of these domains not only improved the stability of the virus particles, but also unexpectedly reduced the overall immunogenicity. At the same time, the improved ITR sequence and the optimized promoter element produced a significant superposition effect, greatly improving the gene expression level and sustainability. These findings provide new ideas for the in-depth development of AAV vectors.

[0046] The AAV vector system of the present invention has the following significant advantages: first, the transduction efficiency is 2.5-3.5 times higher than that of the prior art, and it performs well in a variety of cell types; second, it still maintains 65-75% transduction activity in the presence of high concentrations of pre-existing antibodies, which is significantly better than the existing vectors; third, the duration of gene expression is extended by 50-80%, meeting the needs of long-term treatment; fourth, the production process is stable and controllable, the product purity is >99%, and the empty shell rate is <5%, which lays the foundation for clinical application. The realization of these excellent properties provides important technical support for the clinical transformation of gene therapy. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0048] Example 1 Preparation of highly efficient AAV vector based on DNA shuffling method

[0049] This example provides a method for creating a diverse AAV capsid protein library by DNA shuffling and combining multiple rounds of screening to obtain a highly efficient AAV vector. The specific implementation steps are as follows:

[0050] First, in the gene library construction stage, the cap genes of two serotypes, AAV1 and AAV2, were selected as starting templates. Among them, the cap gene sequence of AAV1 is derived from the nt2203-4407 region of GenBank sequence number AF063497.1, and the cap gene sequence of AAV2 corresponds to the nt2201-4410 region of GenBank sequence number AF043303.1. Based on these two sequences, the following primers were designed and synthesized:

[0051] AAV1-F:5'-ATGGCTGCCGATGGTTATCTTC-3'

[0052] AAV1-R:5'-TTACAGATTACGAGTCAGGTAT-3'

[0053] AAV2-F:5'-ATGGCTGCCGATGGTTATCTTC-3'

[0054] AAV2-R:5'-TTACAGATTACGGGTGAGGTA-3'

[0055] Furthermore, the target gene was amplified by PCR. In a 50 μL reaction system, 5 μL of 10× High Fidelity Buffer, 1 μL of 10 mM dNTP Mix, 1 μL of the above forward primer and reverse primer (10 μM), 50 ng of template DNA, and 0.5 μL of Q5 High-Fidelity DNA polymerase were added in sequence, and finally the mixture was filled to 50 μL with nuclease-free water. The PCR reaction program was optimized as follows: pre-denaturation at 98°C for 30 seconds; followed by 30 cycles of amplification (denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 2 minutes); and finally, final extension at 72°C for 5 minutes.

[0056] After obtaining the PCR product, DNA fragmentation was performed. Specifically, 2 μg of purified PCR product, 10 μL of 10×DNase I Buffer, and 1 μL of DNase I (0.1 U / μL) were added to a 100 μL reaction system, and the mixture was supplemented with nuclease-free water. The reaction was carried out at 15°C for 2 minutes, and then 10 μL of 0.5 M EDTA was added to terminate the reaction. It is worth noting that in order to ensure the appropriate size distribution of DNA fragments, 2% agarose gel electrophoresis was used for monitoring, and the electrophoresis conditions were 80 V and 45 minutes.

[0057] After electrophoresis, 50-150 bp DNA fragments were selected for recovery. Preferably, Qiagen Gel Extraction Kit (Cat. No. 28704) was used for gel recovery, and finally 30 μL TE buffer (pH 8.0) was used to elute the DNA fragments. The concentration of the recovered DNA fragments was about 50 ng / μL as measured by a spectrophotometer, and the A260 / A280 ratio was between 1.8 and 2.0, indicating good purity.

[0058] Subsequently, the obtained DNA fragments were recombined by primer-free PCR. Specifically, 5 μL of 10× High Fidelity Buffer, 1 μL of 10 mM dNTP Mix, 200 ng of the above purified DNA fragment mixture, and 1 U of Phusion DNA polymerase were added to the 50 μL reaction system in sequence, and supplemented with nuclease-free water. The PCR program was set as follows: 98°C pre-denaturation for 1 minute; 45 cycles of recombination reaction (98°C denaturation for 30 seconds, 55°C annealing for 1 minute, and 72°C extension for 2 minutes); and finally 72°C final extension for 10 minutes.

[0059] After completing DNA shuffling to obtain a chimeric cap gene library, this example further constructed a reporter gene vector system for screening. The vector contains the following functional elements: a modified CMV promoter, an eGFP reporter gene, a WPRE element, and a polyA signal, and the total length is controlled within 4.5 kb. Among them, the CMV promoter sequence has been optimized, and some CpG sites have been removed to reduce immunogenicity; the WPRE element uses a modified papaya virus post-transcriptional regulatory element sequence, which can significantly increase the gene expression level.

[0060] In terms of establishing a cell screening platform, this example uses the HEK293T cell line for multiple rounds of screening. The cell culture adopts the following optimized formula: 10% FBS, 1× non-essential amino acids, 1× penicillin-streptomycin, 2mM L-glutamine, and 10mM HEPES (pH 7.4) are added to the DMEM basal medium (containing 4.5g / L glucose). During cell passage, 0.25% trypsin-EDTA was used for digestion for 3 minutes, followed by washing twice with pre-warmed PBS, 10mL each time. The trypan blue exclusion method was used to count cells, and the planting density was strictly controlled at 2×10 5 cells / cm 2 .

[0061] The screening strategy adopted a three-round progressive scheme. In the first round, conventional transfection method was used to adjust the cell density to 1×10 6 cells / well (6-well plate), the amount of transfected DNA was 4μg / well, and the mass ratio of PEI to DNA was 3:1. The specific operation was as follows: DNA was dissolved in 500μL serum-free DMEM, and PEI was dissolved in an equal volume of serum-free DMEM, incubated at room temperature for 5 minutes, mixed the two, and added to the cells after incubation at room temperature for 15 minutes. Flow cytometry was performed 48 hours after transfection, and no less than 10,000 cells were collected on the BD FACSCalibur instrument. The data were analyzed using FlowJo v10.0 software, and the results showed that the GFP positive rate reached 35%.

[0062] In the second round of screening, the virus MOI was adjusted to 2 for infection. The culture medium was replaced every 24 hours during the infection process. After 72 hours, the GFP positivity rate was found to increase to 55%, indicating that the AAV variants obtained by screening have a high transduction efficiency. To further evaluate the immune escape ability, a 1:20 diluted pre-existing neutralizing antibody was added in the third round of screening. The specific operation is: after incubating the virus with the diluted neutralizing antibody at 37°C for 1 hour, add it to the cells for infection. After testing, 65% of the cells were still successfully transduced in the presence of neutralizing antibodies, indicating that the obtained AAV variants have good immune escape ability.

[0063] After completing the cell screening, this example systematically optimized the AAV genome. First, three key mutations were introduced into the ITR sequence: T10C was used to enhance the stability of the stem-loop structure, G85A was used to improve the Rep binding efficiency, and C120T was used to optimize the replication start site. The effects of these mutations were predicted by the Mfold software for secondary structure, and their thermal stability was verified by 60-95°C gradient PCR. Among them, the T10C mutation reduced the △G value of the stem-loop structure by 2.3 kcal / mol, indicating that the structural stability was significantly improved.

[0064] Furthermore, the p5 promoter region was modified. The optimized SP1 binding site sequence was changed from GGGGCGGAGCTTGGGG to GGGGTGGAGCTTGGGG, the YY1 binding site was changed from CGCCAT TTTGACCGTG to CGCCATTTTGACAGTG, and the TATA box sequence was changed from TATAT A to TATAAA. The activity of the modified promoter was verified by the Dual-Luciferase reporter gene system, which was 45% higher than that of the original sequence. In addition, Real-time PCR and Western blot analysis confirmed that both transcription and expression levels were significantly improved.

[0065] In terms of polyA signal optimization, the GC content upstream of the core sequence was first reduced to 35%, and potential splicing sites were removed. Specific GT-rich sequences (GTGTGTTGGTT GTGTGTGTTT) and T-rich sequences (TTTTTCTTTTTTTTTTTGTTTT) were introduced into the downstream region. RNA stability analysis after Actinomycin D treatment showed that the optimized polyA signal could extend the mRNA half-life by about 1.8 times.

[0066] Finally, this example uses a three-plasmid co-transfection system for virus packaging. Specifically, 3×106 HEK293T cells were inoculated in a T175 culture flask, and transfection was performed when the cell confluence reached 80%. Three plasmids, pHelper, pRep-Cap, and pTR-transgene, were used, with a total DNA amount of 25 μg and a plasmid ratio of 10:10:5. After transfection, the cells were cultured at 37°C and 5% CO2 for 72 hours.

[0067] The virus harvesting and purification process uses a modified protocol. First, cells are treated with an optimized lysis buffer (pH 7.5), which consists of 50mM Tris, 150mM NaCl, 2mM MgCl2, 1% Triton X-100, 1× protease inhibitor cocktail, 50U / mL Benzonase nuclease, and 2mM DTT. Subsequently, purification is performed by 15-40% cesium iodide density gradient centrifugation at 38,000rpm, 18°C, and 48 hours. Virus particles in the density range of 1.37-1.41g / mL are strictly collected during aliquoting.

[0068] The final viral vector was subjected to comprehensive quality testing. 10% SDS-PAGE and silver staining analysis showed a purity of 98%, and electron microscopy observation (60,000× magnification) showed that the empty shell rate was only 8%. The biological titer was determined using the qPCR method. The specific reaction system (20μL) contained: 2×TaqMan Universal PCR Master Mix 10μL, ITR-specific primers (10μM) 0.4μL each, TaqMan probe (5μM) 0.4μL, template DNA 2μL, and nuclease-free water was added. The PCR program was: 95℃ for 10 minutes; 95℃ for 15 seconds, 60℃ for 1 minute, and 45 cycles. The final viral titer was 2.5×10 13 vg / mL.

[0069] The transduction efficiency test results showed that the AAV vector was 2.8 times higher than the original type. The transduction experiments conducted in HEK293T and HeLa cells (5×10 4 cells / well, 96-well plate) showed that the transduction activity was still high even in the presence of pre-existing neutralizing antibodies. Flow cytometry analysis (BD FACSCalibur, FL1 channel) confirmed that the expression level was stable at different time points (24h, 48h, 72h).

[0070] Example 2 Optimization of preparation of highly efficient AAV vectors based on multi-serotype cap genes

[0071] Based on Example 1, this example further expanded the diversity of the starting templates, and selected the cap genes of three serotypes, AAV2, AAV8 and AAV9, for DNA shuffling. Based on the sequence information in the GenBank database (AAV2: AF043303.1, AAV8: AF513852.1, AAV9: AY530579.1), specific primers were designed and optimized respectively.

[0072] In the PCR amplification stage, this embodiment uses a larger volume reaction system. Specifically, 10 μL of 10× High Fidelity Buffer, 2 μL of 10 mM dNTP Mix, 2 μL of each serotype-specific primer (10 μM), 30 ng of template DNA, and 1 U of Q5 High-Fidelity DNA polymerase are added to the 100 μL reaction system in sequence. Preferably, the PCR program is adjusted to: 98°C pre-denaturation for 1 minute; followed by 28 cycles of amplification (98°C denaturation for 15 seconds, 60°C annealing for 30 seconds, 72°C extension for 2.5 minutes); and finally 72°C final extension for 8 minutes.

[0073] It is worth noting that this embodiment uses a modified DNase I digestion system in the DNA fragmentation process. Specifically, 3 μg of purified PCR product, 15 μL of 10×DNase I Buffer, and 1.5 μL of DNase I (0.1 U / μL) are added to a 150 μL reaction system. The reaction is carried out at 15°C for 3 minutes in a precisely temperature-controlled water bath, and then 15 μL of 0.5M EDTA is immediately added to terminate the reaction. The DNA fragments of 80-120 bp are preferably recovered by separation through 2% agarose gel electrophoresis.

[0074] The purified DNA fragments were then recombined by primer-free PCR. In a 75 μL reaction system, 10× High Fidelity Buffer 7.5 μL, 10 mM dNTP Mix 1.5 μL, DNA fragment mixture 250 ng and Phusion DNA polymerase 1.5 U were included. The PCR program was optimized as follows: 98°C pre-denaturation for 90 seconds; 40 cycles of recombination reaction (98°C denaturation for 30 seconds, 56°C annealing for 90 seconds, 72°C extension for 2 minutes); and finally 72°C final extension for 12 minutes.

[0075] In terms of screening vector construction, this example selected an optimized CMV promoter-eGFP-WPRE-BGH polyA signal combination, with a total length of 4.2kb. Among them, 85% of the CpG sites were removed from the CMV promoter sequence, and the WPRE element used a codon-optimized version. In order to improve the screening efficiency, a modified HEK293T cell line was selected, and the FBS concentration in the culture medium was increased to 12%, and 1×GlutaMAX and 1mM sodium pyruvate were supplemented.

[0076] The optimization scheme was adopted in the screening strategy. In the first round, liposome transfection was used and the cell density was adjusted to 1.2×10 6 cells / well, DNA dosage 3.5μg / well, liposome to DNA ratio 2.5:1. After 48 hours of transfection, the GFP positive rate reached 40% by flow cytometry. In the second round of infection screening, the MOI was increased to 3, the culture time was extended to 72 hours, and the GFP positive rate was successfully increased to 60%. In the third round of neutralizing antibody screening, the pre-stored antibody dilution was adjusted to 1:25, and infection was performed after neutralization at 37℃ for 90 minutes, and finally a 70% escape rate was obtained.

[0077] In terms of genome optimization, this example further improves the sequence modification strategy. In addition to the introduction of three basic mutations, T10C, G85A and C120T, a new G140A mutation site was added. The new mutation combination increased the binding activity of the Rep protein by about 2.5 times, as verified by the EMSA method. At the same time, the p5 promoter region was fully modified, and the optimized promoter activity was increased by 65% ​​compared with the original sequence, and this result was verified by the luciferase reporter gene system.

[0078] A modified three-plasmid co-transfection system was used for virus production. 3.5×106 HEK293T cells were inoculated in a T175 culture flask, and transfection was performed 22 hours later when the cell confluence reached 85%. The plasmid dosage was adjusted to pHelper 12μg, pRep-Cap 12μg, and pTR-transgene 6μg. After transfection, the culture temperature was lowered to 33℃ and the culture time was extended to 72 hours.

[0079] Virus purification uses an improved lysis buffer (pH 7.4): 40mM Tris, 130mM NaCl, 2.5mM MgCl2, 0.5% Triton X-100. Ultracentrifugation is performed through an 18-45% cesium iodide gradient, the speed is increased to 40,000rpm, the temperature is reduced to 16°C, and the centrifugation time is 44 hours. Virus particles in the 1.36-1.42g / mL density range are collected and processed through an optimized dialysis system.

[0080] The final product reached ultra-high purity (99%), the empty shell rate was reduced to 7%, and the biological titer reached 3.2×10 13 vg / mL. The transduction efficiency test conducted by HEK293T and HeLa cell lines showed that it was 3.2 times higher than the original type and still maintained more than 65% activity in the presence of high concentrations of neutralizing antibodies.

[0081] Example 3 In-depth optimization preparation of high transduction efficiency AAV vector

[0082] Based on the research foundation of the previous two embodiments, this embodiment deeply optimizes the DNA shuffling method and virus purification process to obtain an AAV vector with better performance.

[0083] First, in the selection of the starting template, this embodiment simultaneously uses the cap genes of four serotypes, AAV1, AAV2, AAV8 and AAV9. Preferably, an 80 μL amplification system is used: 8 μL of 10× High Fidelity Buffer, 1.6 μL of 10 mM dNTPMix, 1.6 μL of each forward and reverse primer optimized by sequence alignment (10 μM), 25 ng of template DNA of each serotype, and 0.8 U of Q5 High-Fidelity DNA polymerase. The PCR program is precisely optimized: 98°C pre-denaturation for 45 seconds; 32 cycles of amplification (98°C denaturation for 12 seconds, 62°C annealing for 25 seconds, 72°C extension for 2.2 minutes); and finally 72°C final extension for 7 minutes.

[0084] It is worth noting that this embodiment uses a modified DNase I digestion scheme in the DNA fragmentation process. In a 120 μL reaction system, 2.5 μg of purified PCR product, 12 μL of 10×DNase I Buffer, and 1.2 μL of DNase I (0.1 U / μL) were used, and the reaction was carried out in a constant temperature water bath at 15°C for 4 minutes. The size distribution of DNA fragments was monitored by Coomassie Brilliant Blue G250 staining to ensure that DNA fragments in the target range (70-130 bp) were obtained.

[0085] Furthermore, optimized reaction conditions were used in the primer-free PCR recombination stage. The 60μL reaction system contained 10×High Fidelity Buffer 6μL, 10mM dNTP Mix 1.2μL, DNA fragment mixture 220ng, and PhusionDNA polymerase 1.2U. The PCR program was adjusted to: 98℃ pre-denaturation for 75 seconds; 42 cycles (98℃ denaturation for 25 seconds, 57℃ annealing for 75 seconds, 72℃ extension for 1.8 minutes); and finally 72℃ final extension for 15 minutes. It is worth mentioning that a slow cooling strategy (0.1℃ / second) was used in the annealing stage, which significantly improved the recombination efficiency of DNA fragments.

[0086] In terms of screening vector construction, this example developed a new screening vector, which includes an enhanced CMV promoter-eGFP-optimized WPRE-SV40 polyA signal, with a total length of 4.3kb. Among them, the enhanced CMV promoter has added multiple transcription enhancement elements through bioinformatics analysis; the WPRE element has been codon-optimized and potential splicing sites have been removed; and the SV40 polyA signal has increased the processing efficiency by increasing the GT / T enrichment region.

[0087] The cell screening platform uses a modified HEK293T cell line. The FBS concentration in the optimized culture medium formula is increased to 15%, and 1mM sodium pyruvate, 1×GlutaMAX and 20mM HEPES are added. To improve cell viability, the culture medium is replaced every 48 hours, and the trypsin digestion time is strictly controlled to no more than 3 minutes during the passage process.

[0088] The screening strategy was further optimized as follows: the first round of calcium phosphate transfection was used, and the cell density was adjusted to 1.5×10 6 cells / well, DNA dosage 4.2μg / well. Preferably, the transfection mixture is slowly dripped at room temperature, and fresh culture medium is replaced after 24 hours. After 48 hours, the GFP positive rate reaches 45% by flow cytometry detection. In the second round of infection screening, the MOI is increased to 4, and the culture time is extended to 96 hours. During this period, fresh culture medium with 2% FBS is supplemented every 24 hours, and the final GFP positive rate reaches 65%. In the third round of neutralizing antibody screening, the concentration of the pre-stored antibody is increased to a dilution of 1:15, and the neutralization time is extended to 2 hours, and finally a 75% escape rate is obtained.

[0089] In terms of AAV genome optimization, this example introduces more innovative designs. Five mutation sites, T10C, G85A, C120T, G140A, and C160T, were added to the ITR sequence. Atomic force microscopy analysis confirmed that the new mutation combination made the secondary structure of ITR more stable and the thermal denaturation temperature increased by 3.2°C. At the same time, a new transcription factor binding site was introduced into the p5 promoter region, and its binding activity was verified by ChIP-seq. The transformation of the polyA signal region was more comprehensive, including the precise control of the GC content of the upstream sequence at 28%, and the design of a new downstream regulatory sequence.

[0090] The virus packaging used an improved four-plasmid system, and 4×106 modified HEK293T cells were inoculated in a T225 culture flask. When the cell confluence reached 88-90%, transfection was performed, and the plasmid dosage was optimized to pHelper:pRep:pCap:pTR-transgene=10μg:8μg:8μg:4μg. After transfection, the culture temperature was reduced to 32℃, which can significantly improve the assembly efficiency of viral particles. The culture time was extended to 96 hours, and fresh culture medium containing 2% FBS was supplemented every 24 hours.

[0091] In terms of virus purification, this embodiment developed an innovative dual gradient centrifugation scheme. The first round used a 20-40% cesium iodide gradient, 38,000rpm, 18°C ​​centrifugation for 36 hours; the second round used a 35-55% cesium iodide gradient, 42,000rpm, 16°C centrifugation for 24 hours. The 1.37-1.40g / mL density segment was collected and then buffer exchanged through a modified dialysis system. It is worth noting that the temperature was strictly controlled between 4-8°C throughout the purification process, which significantly improved the stability of the virus particles.

[0092] The final viral vector obtained reached ultra-high purity (>99.5%), the empty shell rate dropped to less than 5%, and the biological titer was as high as 4.5×10 13 vg / mL. The transduction efficiency is 3.5 times higher than that of the original type, and it still maintains more than 70% activity in the presence of high concentrations of pre-stored antibodies. More importantly, a three-month stability study showed that the activity loss under -80°C storage conditions does not exceed 10%, which lays the foundation for the clinical application of the product.

[0093] The above three examples fully demonstrate the implementation effects of the present invention under different process parameter combinations. The results show that through the systematic optimization of DNA shuffling method, screening strategy, genome optimization and production process, AAV vectors with high transduction efficiency, strong immune escape ability and good stability were successfully obtained. In particular, significant progress has been made in key indicators such as transduction efficiency, immune escape ability and product quality.

[0094] The examples of the present invention show that a high-performance AAV vector with independent intellectual property rights has been successfully obtained by combining the DNA shuffling method with directed evolution screening. The vector not only has excellent transduction efficiency and immune escape ability, but also has a stable and controllable production process, providing important technical support for the field of gene therapy.

[0095] In order to comprehensively evaluate the performance characteristics of the AAV vector of the present invention, we designed a series of systematic test experiments. Through these experiments, we not only verified the various technical indicators of the present invention, but also discovered some unexpected synergistic effects.

[0096] 1. DNA shuffling efficiency assessment

[0097] To verify the effectiveness of the DNA shuffling strategy of the present invention, we performed deep sequencing analysis on the gene libraries obtained in different embodiments. The results showed that the optimized DNA shuffling scheme can significantly improve the efficiency of gene recombination. The specific data are shown in Table 1:

[0098] Table 1 Comparison of DNA shuffling efficiency in different embodiments

[0099] Example Number of templates Recombinant fragment size Recombination efficiency Chimeric sequence diversity index* 1 2 50-150bp 45％ 0.68 2 3 80-120bp 62％ 0.82 3 4 70-130bp 78％ 0.91

[0100] *The diversity index ranges from 0 to 1, with larger values ​​indicating higher diversity

[0101] Further analysis revealed that the slow cooling strategy (0.1°C / second) used in Example 3 played a key role in improving the recombination efficiency. High-throughput sequencing revealed that this strategy not only increased the recombination frequency, but more importantly, increased the uniformity of the recombinant fragments, which laid a good foundation for subsequent screening.

[0102] 2. Evaluation of transduction efficiency and immune escape ability

[0103] We evaluated the transduction efficiency of the obtained AAV variants in various cell lines and investigated their immune escape ability in the presence of pre-existing antibodies. The results are shown in Table 2:

[0104] Table 2 Test results of AAV variant transduction efficiency and immune escape ability

[0105]

[0106] *Fold relative to wild-type AAV

[0107] Test conditions: MOI = 10, detection 72 hours after transduction

[0108] It is worth noting that the AAV variant obtained in Example 3 not only showed the highest transduction efficiency, but also showed excellent immune escape ability. Western blot and immunofluorescence analysis showed that this enhanced immune escape ability was closely related to amino acid mutations in specific regions of the capsid protein surface, which changed the conformation of key antigenic epitopes but did not affect the cellular uptake efficiency of the virus.

[0109] 3. Evaluation of gene expression persistence

[0110] In order to evaluate the long-term effect of gene therapy, we conducted a 6-month in vitro sustained expression study on the AAV vectors obtained in different embodiments. The results are shown in Table 3:

[0111] Table 3 Comparison of persistence of AAV-mediated gene expression

[0112]

[0113] * The expression level at 1 month is 100%

[0114] Test conditions: MOI = 5, collect samples for testing every month

[0115] Surprisingly, the optimized AAV vector of the present invention not only exhibited higher initial transduction efficiency, but also showed significantly enhanced expression persistence. Through RT-qPCR and epigenetic analysis, it was found that this improvement in persistence was mainly attributed to two aspects: first, the optimized ITR sequence improved the stability of the genome; second, the improved promoter design reduced the occurrence of epigenetic silencing.

[0116] 4. Virus Particle Stability Study

[0117] To evaluate the storage stability of the product, we conducted accelerated stability tests and long-term stability studies. The results are shown in Table 4:

[0118] Table 4 AAV vector stability test results

[0119]

[0120] * Taking initial activity as 100%

[0121] Through dynamic light scattering (DLS) and transmission electron microscopy analysis, it was found that the improved stability of the virus particles in Example 3 was mainly due to the optimized capsid protein structure, which significantly improved the thermal stability and pH tolerance of the particles while maintaining the virus's ability to infect.

[0122] Unexpected technical effects:

[0123] 1. Synergistic effect: The most important discovery of this invention is that there is a significant synergistic effect between multiple optimization strategies. For example, the optimization of ITR sequence not only improves the replication efficiency of the genome, but also produces a synergistic effect with the improved capsid protein, which jointly improves the packaging efficiency of the virus, reducing the empty shell rate of the final product to an unprecedented level below 5%. This synergistic effect has not been reported in previous studies.

[0124] 2. Improved broad-spectrum: Surprisingly, the AAV variants obtained by the present invention not only performed well in known target cells, but also showed significant improvement in transduction ability in a variety of difficult-to-transduce cells. This improvement in broad-spectrum may be related to the special conformation of the new chimeric capsid protein, which provides new possibilities for expanding the application range of AAV vectors.

[0125] 3. Reduced immunogenicity: Through in vitro and in vivo immunogenicity assessments, it was found that the AAV vector optimized by the present invention unexpectedly showed significantly reduced immunogenicity while maintaining high transduction efficiency. In-depth research found that this may be related to the change in the surface charge distribution of the optimized capsid protein, making it less likely to be recognized by the immune system.

[0126] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a highly efficient AAV vector for gene therapy, characterized in that , including the following steps: (1) Creating a novel AAV capsid protein library by DNA shuffling, including: a) selecting the AAV1-9 serotype cap gene as a template and obtaining template DNA by PCR amplification; b) fragmenting the template DNA using DNase I to obtain DNA fragments of 50-150 bp; c) obtaining a chimeric cap gene library by primer-free PCR recombination; (2) AAV variants with high transduction efficiency were obtained through directed evolution screening, including: a) constructing a screening vector system containing a reporter gene; b) Perform multiple rounds of screening in HEK293T cells; c) AAV variants with high transduction efficiency were obtained by flow cytometry sorting; (3) Optimizing the AAV genome packaging sequence, including: a) Optimizing the ITR sequence by site-directed mutagenesis; b) improving the promoter region of the Rep gene; c) Optimize the polyA signal sequence; (4) Packaging and purifying the obtained AAV vector.

2. The method according to claim 1, characterized in that , the PCR amplification conditions in step (1) are: Pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 2 minutes, 30 cycles; final extension at 72°C for 5 minutes.

3. The method according to claim 1, characterized in that , the DNase I digestion conditions in step (1) are: The reaction was carried out at 15°C for 1-5 minutes, and the DNase I concentration was 0.001-0.01U / μL.

4. The method according to claim 1, characterized in that , the primer-free PCR recombination conditions in step (1) are: Pre-denaturation at 98°C for 1 minute; denaturation at 98°C for 30 seconds, annealing at 55°C for 1 minute, extension at 72°C for 2 minutes, 45 cycles; final extension at 72°C for 10 minutes.

5. The method according to claim 1, characterized in that , the screening vector in step (2) comprises the following elements: CMV promoter-eGFP reporter gene-WPRE element-polyA signal, total length <4.7kb.

6. The method according to claim 1, characterized in that , the screening strategy in step (2) includes: In the first round of conventional transfection screening, the GFP positive rate was >30%; The second round of low MOI (1-5) infection screening, GFP positive rate> 50%; In the third round, pre-existing neutralizing antibodies were added for competitive screening, and the escape rate was >60%.

7. The method according to claim 1, characterized in that , the ITR sequence optimization in step (3) includes the following mutations: T10C: Enhances the stability of the stem-loop structure; G85A: Improves Rep binding efficiency; C120T: Optimized replication initiation.

8. The method according to claim 1, characterized in that In step (4), the virus packaging adopts a three-plasmid co-transfection system: pHelper plasmid 10 μg, pRep-Cap plasmid 10 μg, pTR-transgene plasmid 5 μg.

9. The method according to claim 1, characterized in that , the virus purification in step (4) comprises the following steps: Using a 15-40% cesium iodide density gradient, ultracentrifugation was performed at 38,000 rpm for 48 hours, and the 1.37-1.41 g / mL density fraction was collected.

10. The AAV vector prepared by the method according to any one of claims 1 to 9, characterized in that : Virus purity>95%, empty shell rate<10%, biological titer>1×1013vg / mL.

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