Hemophilia B Gene Therapy Modelled in Null and Missense Mutant Mice: Kinetics of Transgenic Factor IX Expression Influences Tolerance to a Greater Extent Than Dose or Serotype of Adeno-Associated Virus (AAV) Vector.

Abstract A major concern regarding the safety of gene therapy for protein deficiencies, including hemophilia, is the possibility of immune responses against the therapeutic transgenic protein. We have previously reported the use of hemophilic mice expressing defective missense human factor IX (hFIX) protein (Cross-reactive material positive, CRM +) or having a complete deletion of FIX product (CRM-) to examine the influence of the underlying mutation upon the risk of inhibitor development following intramuscular FIX gene therapy. When inhibitor antibodies have developed following adeno-associated virus (AAV) virus FIX gene therapy, the vector dose per injection site has been implicated as an important influence. To examine this, we treated CRM + missense R333Q-hFIX strain hemophilic mice with a single IM injection of AAV serotype2.hFIX. Doses of 1 x 1011 and 8 x 1011 vector genomes/animal resulted in increases of 50 ng/ml and 100 ng/ml hFIX, respectively, without inhibitor development. Identical vector doses given to the FIXKO CRM- strain resulted in inhibitors in all animals, predominantly IgG1 subclass, and zero circulating FIX. Using identical gene sequences and injection parameters, 1 x 1011 vg/animal of an AAV1 serotype hFIX vector was given to R333Q-hFIX mice and FIXKO mice. This resulted in physiologic hFIX levels in all animals at two weeks (1st timepoint examined), peaking at 3.9 μg/ml in FIXKO mice and 21.8 μg/ml in R333Q-hFIX mice. No mice developed inhibitors, despite the development of non-inhibitory IgG1 and IgG2 anti-factor IX. Despite subsequent challenge with repeated IV hFIX protein injections and with 1 x 1011 AAV2.hFIX, inhibitor antibodies could not be elicited in these animals. To examine the role of kinetics of trangene expression upon inhibitor development, stepwise decreases in the AAV1 vector dose were used, to try to reproduce the level of expression achieved in AAV2-treated animals that developed inhibitors [see Table]. At doses of 1 x 109 and 1 x 1010 AAV1hFIX, onset of expression was slow in R333Q-hFIX, averaging only10 ng/ml and 60 ng/ml above background at 2 weeks, and remaining below 300 ng/ml over months. Although lowering the vector dose of AAV1 reproduced the pattern of slow, low level transgene expression seen with the higher doses of AAV2, no inhibitors ever developed in the mice with CRM+ background. Nevertheless, FIXKO CRM- mice failed to achieve tolerance after the lower AAV1 doses. Neither the AAV1 serotype nor the vector particle number independently determined the inhibitor trigger. The results suggest the influence of the kinetics of onset and level of transgenic protein achieved are of primary importance in establishing tolerance in this application. Factor IX Expression and Inhibitor Formation Relative to Underlying FIX Mutation Mouse strain/Dose vector (vg/animal) FIX incr: 2 weeks (μg/ml) FIX incr: Peak (μg/ml) Bethesda Inhibitor (BIU range) R333Q, AAV2 1x10e11 0.01 0.05 0/5 mice (0 BIU) FIXKO, AAV2 1x10e11 0 0 5/5 mice (3–34 BIU) R333Q, AAV1 1x10e11 15.8 21.8 0/5 mice (0 BIU) R333Q, AAV1 1x10e10 0.06 0.25 0/4 mice (0 BIU) R333Q, AAV1 1x10e9 0.02 0.12 0/4 mice (0 BIU) FIXKO, AAV1 1x10e11 2.5 3.9 0/5 mice (0 BIU) FIXKO, AAV1 1x10e10 0.04 0.4 4/5 mice (2–16 BIU) FIXKO, AAV1 1x10e9 0 0 3/4 mice (2–6 BIU)