Journal: Blood Advances

Article Title: Identification of coagulation factor IX variants with enhanced activity through ancestral sequence reconstruction

doi: 10.1182/bloodadvances.2021004742

Figure Lengend Snippet: AAV-AnFIX gene therapy. (A) AAV vectors incorporating AAV2 inverted terminal repeats, either the HCB (146 nt) or HHS4 (294 nt) promoter, a minute virus of mice (MVM) intron (92 nt), a FIX transgene, and a synthetic β-globin polyadenylation signal are depicted. The predicted HCB- and HHS4-containing ssDNA AAV genome sizes are 2003 and 2151 nt, respectively. (B) Hemophilia B mice were injected IV with 5 × 1012 vg/kg AAV2/8 encoding An96 Padua-LCO (closed circles) or hFIX-Padua (open triangles) driven by the highly compact, liver-directed HCB promoter (n = 6 per group). Plasma was collected biweekly for 12 weeks, and FIX activity was determined by 1-stage clotting assay. (C) Hemophilia B mice were injected IV with a 37-fold lower dose (1.4 × 1011 vp/kg) of AAV2/8-HHS4-An96-Padua (closed circles), AAV2/8-HHS4-An96 (closed squares), or AAV2/8-HHS4-hFIX-Padua (open triangles; n = 4-6 per group). Plasma was collected biweekly for 12 weeks, and FIX activity was determined by 1-stage clotting assay (C) or chromogenic assay (Rox Factor IX; Diapharma, West Chester, OH) (D). (E) AAV vector copy number was determined from liver tissue genomic DNA using qPCR. No significant differences were observed among the groups (1-way analysis of variance [ANOVA]; P = .2). (F) Saphenous vein bleeding challenge was performed on mice from each group (from left to right, open diamonds represent wild-type C57Bl/6; closed diamonds, untreated hemophilia B; open triangles, hFIX-Padua transgene; closed circles, An96-Padua transgene; closed squares, An96 transgene). The average time to clot for each mouse was measured over 30 minutes. Comparisons among the groups were made by 1-way ANOVA and post hoc Holm-Sidak testing. All groups were significantly different to the untreated hemophilia B (negative control) group (P < .05). (G) Dose-response curves were generated by administration of log10 doses of AAV2/8-HHS4-An96-Padua at 1.4 × 109 (circles), 1.4 × 1010 (squares), or 1.4 × 1011 vp/kg (triangles) to hemophilia B mice (n = 5-7 per group). Plasma FIX activity was measured by 1-stage clotting assay at biweekly intervals.

Article Snippet: On the basis of the amino acid sequences inferred for An102, An97, An96, An88, An84, An70, An65, and An63, complementary DNA (cDNA) sequences were generated using a liver codon optimization (LCO) algorithm described previously and de novo synthesized by GenScript (Piscataway, NJ) to contain flanking 5' XhoI and 3' NotI restriction sites for subcloning into the mammalian expression vectors ReNeo and pcDNA 3.4. as well as recombinant adeno-associated and lentiviral vector expression plasmids.4 Human FIX-T148 as selected as the wild-type FIX control based on its higher allele frequency and prior functional characterization by our laboratory.5.

Techniques: Virus, Injection, Clinical Proteomics, Activity Assay, Coagulation, Chromogenic Assay, Plasmid Preparation, Negative Control, Generated