Polynucleotides
A polynucleotide and nucleotide technology, applied in the direction of enzymes, biochemical equipment and methods, single-stranded DNA viruses, etc., can solve problems such as high treatment burden
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Embodiment 1
[0393] Example 1 - Method
[0394] In the Examples described below, the following general methodology was followed unless otherwise stated.
[0395] Generation of rAAV
[0396] AAV2 / 8 particles were generated by transient transfection of HEK293T cells with plasmids encoding AAV Rep and Cap, adenoviral helper functions, and a recombinant genome containing the GBA construct. AAV2 / 8 particles were purified by aPOROS CaptureSelect affinity columns, titrated by qPCR, and characterized by alkaline gel analysis.
[0397] mouse study design
[0398] AAV viral particles carrying the GBA transgene under the transcriptional control of a hepatocyte-specific promoter were administered into the tail vein of 6-8 week old wild-type (C57BL / 6) male mice. AAV doses ranged from 6 × 10 11 vg / kg to 6×10 12 vg / kg. For each experiment, another group of animals received no treatment, serving as a control for the effect of the treatment. To assess the kinetics and persistence of transgene expres...
Embodiment 2
[0410] Example 2 - GBA constructs
[0411] To assess whether a liver-directed gene therapy approach could be used to treat Gaucher disease (GD), the human full-length GBA coding sequence (found at GenBank accession number NM_000157.3; SEQ ID NO:9) was cloned into a liver-specific promoter-driven gland-associated viral (AAV) vector. In the FLF-PL01 AAV construct ( figure 1 In A), the GBA wild-type sequence (GBAwt, not codon-optimized) is driven by a liver-specific promoter referred to herein as "LSP-S" (SEQ ID NO: 10). To determine the most suitable sequence for expression, sequences were designed using a number of different codon optimization strategies. In one example AAV construct (FLF-PL28), the GBA codon sequence was optimized and driven by the same liver-specific promoter LSP-S ( figure 1 B). The FLF-PL64 construct contains the same GBA codon-optimized sequence as FLF-PL28, but differs by containing a longer transcriptional regulatory element referred to herein as "LS...
Embodiment 3
[0412] Example 3——Wild type GBA transgene expression analysis
[0413] To assess whether the (wild type) GBA construct FLF-PLOl would lead to hepatic expression and subsequent secretion of β-glucocerebrosidase (GCase) into the bloodstream, FLF-PL01 was pseudotyped to AAV2 / 8. rAAV particles were prepared and titrated as described above and characterized by alkaline gel analysis prior to use in mice. Take 6×10 11 vg / kg to 6×10 12 Eight-week-old wild-type (C57BL / 6) mice were treated with a single injection of AAV2 / 8-FLF-P01 at a dose of vg / kg. Control (naive) mice were untreated. Serum samples were collected at 4, 8, and 12 weeks after AAV injection for assessment of circulating active GCase levels. GCase activity was assayed and immunohistochemical staining was performed as described above. Sections were counterstained with hematoxylin.
[0414] Injection of wild-type mice with AAV2 / 8-FLF-PL01 resulted in increased expression of human GCase in the liver of treated animals ...
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