Muscle-Directed Gene Transfer and Transient Immune Suppression Result in Sustained Partial Correction of Canine Hemophilia B Caused by a Null Mutation

Abstract

The X-linked bleeding disorder hemophilia B is caused by absence of functional blood coagulation factor IX (F9) and can be treated by adeno-associated viral (AAV) mediated gene transfer to skeletal muscle. The safety of this approach is currently being evaluated in a phase I clinical trial. Efficacy of this and several other gene therapy strategies has been addressed in hemophilia B dogs, an important preclinical model of the disease. While previously published data demonstrated sustained expression of canine F9 in dogs with a missense mutation in the gene F9, we show here that AAV-mediated canine F9 gene transfer to skeletal muscle of hemophilia B dogs carrying a null mutation of F9 (causing an early stop codon and an unstable mRNA) results in induction of inhibitory anti-canine F9 at comparable vector doses (1 x 10(12) vector genomes/kg). Thus, the risk of inhibitor formation following AAV-mediated F9 gene therapy may be influenced by the nature of the underlying mutation in F9. Transient immune suppression with cyclophosphamide at the time of vector administration blocked formation of anti-canine F9 antibodies in the one animal treated with this approach. Treatment with this combination of gene transfer and transient immune modulation has resulted in sustained expression (>8 months) of canine F9 at levels sufficient for partial correction of coagulation parameters.

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

“…9,14 Transient immune suppression using cyclophosphamide successfully prevented inhibitor formation. 8,10,11 In contrast, sustained expression of canine F.IX in hemophilia B dogs with a F.IX missense mutation was obtained after i.m. administration, which did not require immune suppression unless very high vector doses were injected.…”

“…injection of vector in animals that lacked tolerance to the F.IX transgene product such as mice with F.IX gene deletion, dogs with F.IX nonsense mutation, or animals that received transfer of a F.IX transgene that was not species-specific. 8,10,12,14 Use of a muscle-specific promoter did not prevent antibody formation to F.IX. 15 Anti-F.IX formation upon muscle gene delivery is due to an adaptive, CD4 + T-celldependent immune response which is absent in CD4-or Rag1-deficient mice.…”

“…8,10 Additional studies are required to characterize antigen presentation and activation signals, which ultimately lead to T-cell activation in draining LNs of skeletal muscle.…”

“…6 However, several studies demonstrated formation of antibodies to the F.IX transgene product after AAV-mediated gene transfer. [7][8][9][10][11][12] The risk for such immune responses depended on the underlying F.IX mutation, route of vector administration, vector dose and serotype. 13 Inhibitor formation to F.IX was most frequently observed after intramuscular (i.m.)…”

Intravenous administration of an AAV-2 vector for the expression of factor IX in mice and a dog model of hemophilia B

“…administration in animal models has yielded low circulating FIX levels and induced a robust anti-FIX antibody response in some immunocompetent hosts. [10][11][12] Preclinical studies in which gene delivery was targeted to the liver, the natural site of FIX production, resulted in higher levels of FIX expression with few immunological complications. Currently, direct rAAV delivery to the liver via a catheter to the hepatic artery is being investigated in human trials.…”