Hyperactivity of factor IX Padua (R338L) depends on factor VIIIa cofactor activity
Open accessAbstract
Adeno-associated virus (AAV) vector liver-directed gene therapy (GT) for hemophilia B (HB) is limited by a vector dose–dependent hepatotoxicity. Recently, this obstacle has been partially circumvented by the use of a hyperactive factor IX (FIX) variant, R338L (Padua), which has an 8-fold increased specific activity compared with FIX-WT. FIX-R338L has emerged as the standard for HB GT. However, the underlying mechanism of its hyperactivity is undefined; as such, safety concerns of unregulated coagulation and the potential for thrombotic complications have not been fully addressed. To this end, we evaluated the enzymatic and clotting activity as well as the activation, inactivation, and cofactor dependence of FIX-R338L relative to FIX-WT. We observed that the high specific activity of FIX-R338L requires factor VIIIa (FVIIIa) cofactor. In a potentially novel system using emicizumab, an FVIII-mimicking bispecific antibody, the hyperactivity of both recombinant FIX-R338L and AAV-mediated transgene-expressed FIX-R338L from HB GT subjects was ablated without FVIIIa activity. We conclude that the molecular regulation of activation, inactivation, and cofactor dependence of FIX-R338L is similar to FIX-WT but that the FVIIIa-dependent hyperactivity of FIX-R338L is the result of a faster rate of factor X activation. This mechanism helps mitigate safety concerns of unregulated coagulation and supports the expanded use of FIX-R338L in HB therapy.
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All HA clinical trials use a B-domain-deleted FVIII transgene, and HB trials have universally adapted the FIX-Padua (FIX-R338L) transgene, which affords approximately 8-fold greater specific activity relative to wild-type FIX.
“All HA clinical trials use a B-domain-deleted FVIII (BDD-FVIII) transgene, which retains full procoagulant function while meeting AAV packaging constraints (∼4.7 kb) and enhancing expression.187, 188, 189”
From Current clinical applications of in vivo gene therapy with AAVs (2021) · cites this paper as [193]
Biochemical data suggest FIX-R338L is activated and inactivated analogous to wild-type FIX and is not inherently prothrombotic.
“available biochemical data suggest FIX-R338L is activated and inactivated analogous to wild-type FIX and not inherently prothrombotic.193”
From Current clinical applications of in vivo gene therapy with AAVs (2021) · cites this paper as [193]
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Current clinical applications of in vivo gene therapy with AAVs
Mendell, J. R., Al-Zaidy, S. A., Rodino-Klapac, L. R., Goodspeed, K., Gray, S. J., Kay, C. N., Boye, S. L., Boye, S. E., George, L. A., Salabarria, S., et al. (2021) · Molecular Therapy · doi:10.1016/j.ymthe.2020.12.007 · Cited by 743 · see citing works
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- Authors
- Samelson-Jones, B. J., Finn, J. D., George, L. A., Camire, R. M., & Arruda, V. R.
- Affiliations
- The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
- Children's Hospital of Philadelphia (US)
- Corresponding author
- Arruda, Valder R.
- ORCID
- Venue
- JCI Insight
- Published
- 2019-06-20
- Volume
- 4
- Issue
- 14
- ISSN
- 2379-3708 (ISSN-L)
- PMC
- PMC6675543
- OpenAlex
- W2949083906
- Cited by
- 44
- References
- 7
- Citations by year
- 2026: 2 · 2025: 8 · 2024: 5 · 2023: 6 · 2022: 8 · 2021: 12 · 2020: 3
- FWCI
- 4.06
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- JCI Insight (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- NHLBI (P01HL64190, P01HL139420, 1K08HL140078)
- Pfizer (Aspire)
- HTRS (Mentored Research Award)
- Bayer (Bayer Hemophilia Program Fellowship Award)
- Topics
- Health Sciences › Medicine › Hematology › Hemophilia Treatment and Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Health Sciences › Medicine › Hematology › Coagulation, Bradykinin, Polyphosphates, and Angioedema
- Keywords
- hemophilia B, factor X activation, emicizumab, thrombotic complications, recombinant factor IX
- MeSH terms
- Dependovirus, Blood Coagulation, Hemophilia B, Factor IX, Genetic Vectors, Humans, Kinetics, Genetic Therapy, Factor VIIIa, HEK293 Cells
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