Retina and muscle high-affinity adeno-associated virus (AAV) capsid protein and related application
A capsid protein, identity technology, applied in the field of gene therapy, can solve problems such as off-target and aggravating adverse reactions
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Embodiment 1
[0090] Example 1: Transformation and screening of AAV
[0091] Such as Figure 1A Shown: First, the engineered AAV library and helper plasmids were transfected into HEK293 cells. Next, AAV-containing HEK293 cell lysates were added to cultured C2C12 myoblasts together with Ad5 (adenovirus type 5). Cell lysates were enriched and repinfected C2C12 cells 4-5 times. Viral genomic bands of candidate AAV capsids were enriched by PCR on C2C12 cell lysates and sequenced. By screening, the serotype sequences with high enrichment were selected to obtain the serotype mutant AAVz2( Figure 1B ).
[0092] AAVz2 particles were purified by AAVX (Thermo Scientific) affinity chromatography plus iodixanol ultracentrifugation, and concentrated to 200-500 μl for the next experiment to determine the solidity rate and tissue targeting of the carrier.
Embodiment 2
[0093] Example 2: Yield and solid rate of AAVz2
[0094] Purify 5×10 by affinity chromatography plus iodixanol ultracentrifugation 7 AAV (AAVz2, AAV5, AAV8, and AAV9) virus particles produced in individual cells. Then, the virus particles were diluted 10,000 times, digested with DNase I at 37°C for 1 hour, and placed at 100°C for 10 minutes to inactivate DNase I. Virus yield was quantified by qPCR and silver staining experiments. Then, 6 titer gradients (1×10 10 , 1x10 9 , 1x10 8 , 1x10 7 , 1x10 6 , 1x10 5 vg / ml) as a standard, calculate the ratio of the qPCR quantitative results to the silver staining quantitative results to obtain the solid rate of AAV. Such as Figure 1C As shown, the number of solid AAVz2 viral genomes quantified by qPCR produced by HEK293 cells was comparable to that of wild-type AAV5, 8, and 9, indicating that the insertion of oligopeptides had no negative impact on virus production. also, Figure 1C showed that the solid rate of AAVz2 viru...
Embodiment 3
[0095] Example 3: C2C12 transduction specificity of mutant AAVz2
[0096] To study transduction specificity, cells were infected with AAV (AAVz2, AAV5, AAV8, and AAV9) carrying the GFP gene at a multiplicity of infection (MOI) of 1 × 10 5 vg / cell. The picture is acquired after 72h of AAV processing.
[0097] Such as Figure 2A As shown, wild-type AAV8 or AAV9 exhibited significant transduction in C2C12 myoblasts (myocyte progenitor cells), C28 / I2 (human chondrocyte lineage) cells, and Huh7 (human hepatoma cell lineage in the hepatocyte lineage) cells. guide, and wild-type AAV5 is almost non-infective to all cells. In contrast, AAVz2 showed stable and efficient GFP expression in C2C12 myoblasts, and only slightly transduced C28 / I2, Huh7 and HEK293 cells.
[0098] In addition, the infection specificity of mutant AAVz2 for C2C12 was further quantified by calculating the ratio of GFP-positive C2C12 cells to GFP-positive C28 / I2, Huh7, and HEK293 cells. The results showed tha...
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