Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model

Abstract

In earlier work, we showed that adenoassociated virus-mediated delivery of a Factor IX gene to skeletal muscle by direct intramuscular injection resulted in therapeutic levels of circulating Factor IX in mice. However, achievement of target doses in humans proved impractical because of the large number of injections required. We used a novel intravascular delivery technique to achieve successful transduction of extensive areas of skeletal muscle in a large animal with hemophilia. We provide here the first report of long-term (> 3 years, with observation ongoing), robust Factor IX expression (circulating levels of 4%-14%) by muscledirected gene transfer in a large animal, resulting in essentially complete correction of the bleeding disorder in hemophilic dogs. IntroductionIn previous work, we showed that intramuscular injection of a recombinant adeno-associated virus 2 (AAV-2) vector expressing blood coagulation Factor IX (F.IX) resulted in long-term expression of F.IX, as judged by circulating F.IX levels and by immunohistochemistry of biopsied injected muscle, in mice and hemophilic dogs. 1,2 We also showed that safety and efficacy considerations imposed an upper limit on the amount of vector that could be injected into a single site. Skeletal muscle has only a limited capacity to execute essential posttranslational modifications, 3 the likelihood of forming neutralizing antibodies to F.IX, an undesirable consequence, rises as the AAV dose per site is increased, 4 and vector uptake into target cells is receptor-mediated and saturable. 5 This work formed the basis for a clinical trial in which an AAV-F.IX vector was administered by direct intramuscular injection into skeletal muscle of adults with severe hemophilia B. 6,7 The study had a dose escalation design; based on preclinical studies, the dose per site in the clinical trial was held to less than 2 ϫ 10 12 vector genomes (vg) per injection site. The first subjects enrolled received only about 10 injections, but the limitation on dose per site meant that subjects in the third and highest dose cohort required close to 100 intramuscular injections in order to receive a dose of 2 ϫ 10 12 vg/kg. Based on preclinical studies in mice and dogs, the dose required for efficacy was approximately 10 13 vg/kg, but the number of injections required for this (ϳ500 sites) seemed prohibitive from a feasibility standpoint, and the target dose was not reached. Instead, the study was stopped after finding subtherapeutic levels of F.IX (an outcome consistent with dose-finding studies in the dog model) in 2 human subjects injected at a dose of 2 ϫ 10 12 vg/kg. Three important conclusions from the human study were that (1) intramuscular injection of AAV-F.IX at doses up to 2 ϫ 10 12 vg/kg in humans was safe, with no evidence of toxicity; (2) the characteristics of skeletal muscle transduction by AAV-2 were similar in mice, dogs, and humans; and (3) transgene expression appeared stable over time, as judged by immunofluorescent staining of muscle biopsies examined up to 10 months ...

Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

“…The kinetics of IgG and IgM generation and IgG subclass switching further supports that AAV types mount a T cell-dependent B cell response (46). It is tempting to speculate that blocking T-B cell interactions might represent an effective approach to overcome AAV NAb activity and allow repeat administration (1,19,29,42).…”

“…AAV2 vectors induce long-term transgene expression in mice, dogs, and primates (1,17,59,67). Persistent stable transgene expression has been observed in rhesus monkeys over 6 years and in dogs over 3 years (1,59). Such animal studies suggest that a single application of AAV vector may induce stable lifelong therapeutic gene expression that might exclude the need for repeat administration.…”

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer

“…10 Preclinical experiments with AAV in animal models for different diseases have proven the long-term stable transgene expression in liver, muscle, and central nervous system. [11][12][13][14][15][16][17] Several early-phase clinical trials with AAV vectors have been initiated and shown to be safe. [18][19][20][21] Among the at least eight different AAV natural serotypes discovered so far, AAV vector derived from serotype 2 (AAV2) has been the most extensive studied and widely used AAV vector in the past 20 years.…”

Progress and Prospects: Gene Therapy Clinical Trials (Part 1)

“…Once again, there is careful focus on the potential that immune responses to AAV capsid proteins could limit transgene expression. 13 Future directions for clinical AAT gene therapy are likely to rest with either a greater distribution of vector to a larger muscle mass through limb perfusion technology 14 or with some form of immune modulation, should immune responses prove to be a limiting factor. There is also considerable promise from a large number of new AAV serotypes that might be used to transduce either muscle or a range of other tissues with higher efficiency.…”