Defining the therapeutic efficacy, tolerogenic potential, and genotoxicity of liver-targeted AAV gene therapy for hemophilia A

PROJECT SUMMARY: Gene therapy (GT) for the hemophilias is poised to fulfill the promise of a safe, affordable,
lifelong correction following a single treatment. Yet clinical trials employing AAV vectors to treat adult hemophilia
A (HA) patients have underscored the risk of hepatoxicity and genotoxicity in adult patients receiving GT. In
addition, the use of AAV-based GT is prevented by pre-existing AAV immunity in a high percentage of patients.
While FVIII inhibitors have not yet been seen in GT trials, inhibitors usually appear within the first 50 exposure
days to FVIII, and since all enrolled patients were selected based upon an inhibitor-free clinical history, it is likely
that the patients were not “inhibitor-prone”, leaving unanswered the critical question of whether GT will avoid
inhibitor induction. GT to treat HA at birth provides the advantage of correcting bleeding events sooner, but
whether neonatal GT would avoid FVIII inhibitor induction and hepatic inflammation is, at present, unknown.
Recent studies have shown a 59% incidence of neutralizing antibodies to AAV at birth. Thus, treatment during
the neonatal period will likely be hampered by anti-AAV immunity. One approach that could circumvent both
AAV- and FVIII-related immune hurdles while enabling correction prior to disease onset is in utero GT (IUGT).
Using the same preclinical animal model (sheep) used to delineate conditions to cure SCID patients prenatally,
we showed that the highly proliferative state and relative immune-immaturity of the fetus can be exploited to
achieve clinically relevant levels of gene transfer to multiple tissues and induce durable tolerance to vector-
encoded proteins. Besides its inherent advantages as a GT recipient, its rapid cellular turnover makes the fetus
a highly sensitive system in which to define the potential genotoxicity and risk of tumorigenesis following AAV
GT. This rapid cellular proliferation also provides a rigorous testbed to define the incidence and locations of AAV
genomic integration, determine the duration of AAV persistence, and identify the optimal target cell(s) within the
liver to achieve long-term expression of a fVIII transgene and resultant therapeutic benefit. The overall goal of
this proposal is to use fetal sheep to address critical unanswered biological and safety questions that plague
liver-targeted AAV-based GT for HA and test the hypothesis that performing AAV-based IUGT will overcome
the immune hurdles inherent to postnatal GT, transduce sufficient numbers of the appropriate hepatic cells to
mediate long-term therapeutic plasma FVIII levels, and induce durable tolerance to FVIII. We propose 3 Aims: i)
Define the in vivo tissue tropism of two AAV serotypes in use in clinical trials for HA (AAV5 and AAV3B) following
administration into fetal and neonatal recipients; ii) Define the impact of fVIII-encoding AAV transduction on fetal
vs. neonatal liver function; and iii) Determine the duration of fVIII-AAV persistence and fVIII transgene
expression, and whether durable immune tolerance is induced to FVIII and/or AAV following in utero vs. neonatal
GT. We hope that these studies will elucidate whether IUGT or neonatal GT for HA will be safe, and whether or
not the therapy will be long-lasting, prevent breakthrough bleeds, and avoid FVIII immune responses.

Grant Number: 5R01HL161290-04
NIH Institute/Center: NIH
Principal Investigator: Graca Almeida-Porada