Adeno-associated virus vector for treating mucopolysaccharide storage disease type II, medicine and application of adeno-associated virus vector
A technology for mucopolysaccharidosis and viral vectors, which is applied in the field of adeno-associated virus vectors for the treatment of mucopolysaccharidosis type II, which can solve the problems of high surgical mortality, long-term injection of ERT, and inability to pass through the central nervous system
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Embodiment 1
[0062] Embodiment 1: Construction of plasmid vector
[0063] In this example, the pAAV2.1 single-chain recombinant AAV vector (its sequence is shown in SEQ ID NO: 12) owned by our laboratory was used as the vector backbone to construct. By replacing the eukaryotic expression elements on the original backbone with CB promoter (its sequence is shown in SEQ ID NO: 3), SV40 intron (its sequence is shown in SEQ ID NO: 2), Kozak sequence (5' -GCCACC-3'), original human IDS (hIDS) (whose sequence is shown in SEQ ID NO: 7) or optimized human IDS (ohIDS), and bGH poly A (whose sequence is shown in SEQ ID NO: 6 ). Regarding the ohIDS sequence, the inventor independently designed the optimized coding sequence of hIDS, that is, the ohIDS sequence (its sequence is shown in SEQ ID NO: 1), and analyzed the optimized sequence by RNAfold.
[0064] The synthetic hIDS and the ohIDS sequence shown in SEQ ID NO: 1 were digested with FseI and BstBI restriction endonucleases (New England Biolabs) ...
Embodiment 2
[0065] Example 2: Expression and functional analysis of pCB-hIDS and pCB-ohIDS vectors in cells
[0066] Well-grown HEK293 cells were evenly spread in six-well plates, 1×10 per well 6 Cells were inoculated in a medium volume of about 2 mL. When the growth confluence of the cells reached 80-90%, transfection was performed with polyethyleneimine (PEI, HEDE Biotech), and the ratio of DNA to PEI reagent was 1:2. Three wells were set up: 5 μg pCB-hIDS plasmid + 10 μL PEI; 5 μg pCB-ohIDS plasmid + 10 μL PEI; non-transfected blank control. After 48 hours of transfection, the cells and supernatant were collected to determine the specific expression of the hIDS gene in the pCB-hIDS vector and pCB-ohIDS vector at the cellular level.
[0067] (1) Real-time quantitative PCR analysis of the expression of hIDS mRNA:
[0068] Real-time quantitative PCR was used to determine the specific expression of hIDS gene at the mRNA level in the cells. The results showed that after pCB-ohIDS transfe...
Embodiment 3
[0076] Example 3: Preparation and in vivo functional analysis of AAV9-ohIDS and AAV9-hIDS
[0077] This example delivered 1E13 vg / kg AAV9-CB-ohIDS and AAV9-CB-hIDS to 2-month-old wild-type (WT) male mice by tail vein injection (n=4). Blood was collected twice at 1 week and 1 month after the injection, and enzyme activities in the brain and liver tissues of the mice were analyzed at 1 month. The results showed that hIDS activity could be detected 7 days after virus injection, and the AAV9-CB-ohIDS group was significantly higher than the AAV9-hIDS group ( Figure 2A ). In the blood of mice 1 month after injection, the activity of hIDS in the injection group was significantly higher than that 7 days after injection, and the activity of AAV9-CB-ohIDS group was significantly higher than that of AAV9-CB-hIDS group ( Figure 2B ). Analysis of the enzyme activity in the brain tissue of the mice one month after injection showed that the enzyme activity of AAV9-CB-hIDS was increased ...
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