Analysis of testes and semen from rabbits treated by intravenous injectionwith a retroviral vector encoding the human factor VIII gene: No evidence of germ line transduction

In a phase 1 clinical trial, we are evaluating a murine leukemia virus (MuL V)-based retroviral vector encoding the human factor VIII gene [hFVIII(V)], administered intravenously, as a therapy for hemophilia A. Preclinical bio localization studies in adult rabbits revealed vector-specific PCR signals in testis tissue at low levels. In follow-up animal studies we used PCR to (1) estimate the frequency with which a given cell in testis tissue is tran sduced, and (2) determine whether a positive PCR signal could be detected i n semen samples from animals treated with hFVIII(V). Using the 99% confiden ce bound, results indicate that the probability that a given cell within th e testis was transduced is less than 1/709,000 (97 days after treatment). T his probability decreased with time after hFVIII(V) administration. Moreove r, the rate of provector sequence detection in semen samples collected week ly throughout two cycles of spermatogenesis was 3/4281 reactions (0.07%), w hich is lower than the rate of false positives (1/800, 0.125%) observed for control animals. Using PCR assays with single-copy sensitivity, we have sh own that the small number of transduced cells present in testis tissue does not give rise to detectable transduced cells in semen.