Novel muscle and retina specific adeno-associated virus (AAV) capsid protein
A capsid protein, a new type of technology, applied in muscular system diseases, neuromuscular system diseases, viruses, etc., can solve problems such as off-target and aggravated adverse reactions
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Embodiment 1
[0089] Example 1: Transformation and screening of AAV
[0090] 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 AAVz1( Figure 1B ).
[0091] AAVz1 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 tissue targeting of the carrier.
Embodiment 2
[0092] Example 2: Validation of C2C12 transduction specificity of mutant AAVz1
[0093] To study transduction specificity, cells were infected with AAV (AAVz1, 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.
[0094] 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, AAVz1 showed stable and efficient GFP expression in C2C12 myoblasts, and only slightly transduced C28 / I2, Huh7 and HEK293 cells.
[0095] In addition, the infection specificity of mutant AAVz1 for C2C12 was further quantified by calculating the ratio of GFP-positive C2C12 cells to GFP-positive C28 / I2, Huh7, and HEK293 cells. The resu...
Embodiment 3
[0096] Example 3: Validation of Muscle Targeting of Mutant AAVz1
[0097] C57BL / 6 mice were injected with 1×10 13 AAV (AAVz1, AAV5, AAV8 and AAV9) virus particles carrying the GFP gene at vg / kg. After 4 weeks of intravenous injection of the virus, the liver, heart, lung, and spleen were isolated for immunostaining ( Figure 3A ). AAV (AAVz1, AAV5, AAV8 and AAV9) viruses carrying GFP gene (5×10 10 vg / mouse). Four weeks after virus injection, gastrocnemius (GA), tibialis anterior (TA) and soleus (SO) muscles were isolated for immunostaining ( Figure 3B ).
[0098] In addition, the specificity of mutant AAVz1 for muscle cells was further quantified by calculating the ratio of GFP-positive muscle cells (average of GA, TA, and SO) to GFP-positive hepatocytes, heart cells, lung cells, and splenocytes. The results showed that in the AAVz1 group, the transduction specificity of muscle relative to liver, lung, heart and spleen was significantly higher than that of the wild-typ...
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