Development of an Optimized rAAV2/6 Human Factor 8 cDNA Vector Cassette for Hemophilia a Gene Therapy
Bibliographic record
Abstract
Abstract Hemophilia A, which is caused by a mutation in the Factor 8 (F8) gene resulting in a deficiency or lack of the Factor VIII (FVIII) protein, is the most common inherited bleeding disorder in humans with an estimated worldwide incidence of half a million people. The disorder is X-linked and occurs in approximately 1 in 5,000 males; however there is also a growing appreciation of the impact on carrier females having a single mutant allele, with at least 10% of hemophilia A female carriers having less than normal clotting activity. Even modest increases in Factor V III activity (>1% of normal) can have a positive impact on patient lives, thus making the disease an ideal candidate for liver-directed gene therapy. Recombinant AAV (rAAV) has been used extensively for nearly 20 years as a gene therapy vector in preclinical and clinical studies where rAAV delivery to non-dividing tissues such as liver, brain and muscle affords stable, long-term transgene expression. However, there has been a lag in the clinical translation of a rAAV gene therapy approach for Hemophilia A/human F8 (hF8) compared to Hemophilia B/human Factor 9 due to poor yields of rAAV encoding a F8 transgene at clinical scale, and a requirement for large doses of rAAV F8 vector to achieve therapeutically relevant levels of circulating human FVIII (hFVIII), with the attendant risk of inducing an AAV-directed immune response requiring transient immunosuppression. To address these issues we optimized a rAAV F8 cDNA vector cassette to improve both virus yields and liver-specific hFVIII expression. The rAAV F8 cDNA vector cassette optimization required multi-factorial modifications to the liver-specific promoter module, hF8 transgene, synthetic polyadenylation signal and vector backbone sequence. This iterative process resulted in improved vector yields at research scale and greater than five-fold improvement in vector yields at clinical scale using our proven manufacturing process. Administration of the optimized rAAV hF8 cDNA packaged in serotype AAV2/6 at a dose of ~7.2E+12 vg/kg to both wild type and Hemophilia A mice resulted in robust circulating hFVIII levels and activity (levels in wild type mice were 241.6% of normal, and activity in Hemophilia A mice were 330.9% of normal). An analysis of hF8 mRNA levels in different tissues following dosage with our optimized vector demonstrated that hF8 expression from the modified promoter module was restricted to the liver. Notably there was a striking impact on hemostasis in the Hemophilia A mice treated with the optimized rAAV hF8 cDNA, with a reduction in bleeding time from 38.3 minutes to 2.5 minutes in treated mice (n = 12, p-value < 0.0001), which is in line with bleeding times in wild type mice. Initial studies in non-human primates (NHPs) resulted in supraphysiological levels of circulating hFVIII with mean peak values of 400-600% of normal levels. A follow up dose-ranging study was performed in NHPs with a rAAV2/6 F8 cDNA vector manufactured using our GMP clinical manufacturing process. Administration of vector doses ranging from 6E+11 vg/kg to 6E+12 vg/kg resulted in therapeutic circulating levels of hFVIII that were 5% - 229% of normal levels. The peak circulating hFVIII levels achieved in this dose-ranging study using GMP clinical-scale vector exceeds the levels previously reported in NHPs by several fold on an AAV vector dose basis. The high potency of this enhanced rAAV F8 cDNA cassette could significantly reduce the dose required to achieve therapeutically relevant levels in human subjects and reduce the potential of developing immune responses to AAV capsid requiring immunosuppression. Disclosures Riley: Sangamo BioSciences Inc: Employment. Boonsripisal:Sangamo BioSciences Inc: Employment. Goodwin:Sangamo BioSciences Inc: Employment. Garces:Sangamo BioSciences Inc: Employment. Ballaron:Sangamo BioSciences Inc: Employment. Tran:Sangamo BioSciences Inc: Employment. Kang:Sangamo BioSciences Inc: Employment. Zhang:Sangamo BioSciences Inc: Employment. Meyer:Sangamo BioSciences Inc: Employment. Greengard:Sangamo BioSciences Inc: Employment. Surosky:Sangamo BioSciences Inc: Employment. Ando:Sangamo BioSciences Inc: Employment. Lillicrap:bayer: Research Funding; biogen: Research Funding; CSL: Research Funding; Octapharma: Research Funding; Sangamo Biosciences Inc: Research Funding. Nichol:Sangamo BioSciences Inc: Employment. Holmes:Sangamo BioSciences Inc: Employment.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.003 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".