Background: Immunogenicity is a key safety parameter of protein drugs. Clear conceptual differences exist regarding immunogenicity in the setting of gene therapy, where a continuous supply of the transgene product (i.e., immunogen) is presented to the immune system from endogenous sources in contrast to conventional intravenous needle delivery of most protein drugs.

Aims: In the current study, we examined the immunogenicity of coagulation factor VIII (fVIII) as a model gene therapy transgene product in the setting of a common gene therapy approach, liver-directed adeno-associated viral (AAV) vector delivery, using a fVIII-deficient hemophilia A mouse model.

Methods: Several independent variables were tested including promoter design (varying strength), transgene nucleic acid and encoded amino acid sequence (varying biosynthetic efficiency), and vector dose (>3 orders of magnitude). A longitudinal, pre-randomized dose-response study was performed in which fVIII activity, anti-fVIII IgG and Bethesda (inhibitor) titer incidence, magnitude and kinetics were followed (Figure 1).

Longitudinal dose-response study of four AAV-FVIII vectors in fVIII-deficient hemophilia A mice.

Results: Independent of the other variables, initial fVIII production rate (ko) was identified as the strongest predictor of inhibitor development after AAV-FVIII delivery (Figure 2. 50% inhibitor development threshold [ID50] = 4.49 IU/day, 95%CI [1.860, 8.818]; logistic regression threshold p<0.0001). Furthermore, the majority of mice that did not have inhibitors and possessed steady-state fVIII levels around 1.5 – 4.5 IU/mL were protected from developing inhibitors upon subsequent challenge with intravenous delivery of recombinant fVIII protein. The majority of mice making less than 1.5 IU/mL developed inhibitors upon challenge.

Pharmacokinetic model defines a fVIII immunogenicity threshold based on initial fVIII production rate.

Conclusions: Therefore, the initial fVIII expression kinetics and subsequent steady-state fVIII levels reveal an ideal therapeutic window wherein immunogenicity risk is lower and the probability of achieving and maintaining tolerance to the transgene product is higher. Similarly designed studies for other gene therapies are warranted to explore the translatability of these findings and validate the critical immunogenicity determinants identified for this broad class of rapidly advancing genetic medicines.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/pharmacokinetic-analysis-of-fviii-exposure-identifies-an-immunogenicity-threshold-after-aav-fviii-gene-therapy-in-a-murine-hemophilia-a-model/