Influence of Vector Dose on Factor IX-Specific T and B Cell Responses in Muscle-Directed Gene Therapy

Abstract

Intramuscular injection of an adeno-associated virus (AAV) vector has resulted in vector dose-dependent, stable expression of canine factor IX (cF.IX) in hemophilia B dogs with an F.IX missense mutation (Herzog et al., Nat. Med. 1999;5:56-63). The use of a species-specific transgene allowed us to study risks and characteristics of antibody formation against the therapeutic transgene product. We analyzed seven dogs that had been injected at a single time point at multiple intramuscular sites with varying vector doses (dose per kilogram, dose per animal, dose per site). Comparison of individual animals suggests an increased likelihood of inhibitory anti-cF.IX (inhibitor) development with increased vector doses, with dose per site showing the strongest correlation with the risk of inhibitor formation. In six of seven animals, such immune responses were either absent or transient, and therefore did not prevent sustained systemic expression of cF.IX. Transient inhibitory/neutralizing anti-cF.IX responses occurred at vector doses of 2 x 10(12)/site, whereas a 6-fold higher dose resulted in a longer lasting, higher titer inhibitor. Anti-cF.IX was efficiently blocked in an eighth animal that was injected with a high vector dose per site, but in addition received transient immune suppression. Inhibitor formation was characterized by synthesis of two IgG subclasses and in vitro proliferation of lymphocytes to cF.IX antigen, indicating a helper T cell-dependent mechanism. Anti-cF.IX formation is likely influenced by the extent of local antigen presentation and may be avoided by limited vector doses or by transient immune modulation.

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 AAV vector expressing canine F.IX. 11 Consequently, the dose per site of injection was limited in a Phase I clinical trial on muscle-directed AAV-F.IX gene transfer in subjects with severe hemophilia B. 5 …”

“…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.)…”

“…administration, which did not require immune suppression unless very high vector doses were injected. 4,11 After comparing vector doses per site of injection between different treated hemophilia B dogs and experiments with a more efficient AAV serotype-1 vector, we suspected local F.IX antigen doses in transduced muscle to be a critical parameter for the risk of inhibitor formation. 7,11 Based on these observations, only subjects with F.IX missense mutations were enrolled in the trial based on muscle delivery, and the vector dose per site of i.m.…”

Immunity to adeno-associated virus vectors in animals and humans: a continued challenge

“…38,41,42 Since both B-and T-cell responses to AAV-encoded transgenes rely on uptake by APCs, whether or not an immune response is mounted depends in a large part on the abundance of the transgene, the kinetics of its expression, as well as its immunogenicity rather than on the vector serotype. Therefore, responses are generally dose dependent 43 and can be avoided by a more restricted expression, for example by using tissuespecific promoters as opposed to systemic general promoters. [44][45][46] Independent from these findings, evidence is accumulating that targeting AAV vectors specifically to the liver seems to induce tolerance to the transgene product.…”