Use of Engineered Autologous BOEC for Gene Therapy of Canine Hemophilia A.

Abstract Hemophilia A is an attractive candidate disease for corrective gene therapy because relatively small amounts of the missing protein, FVIIII, have a significant biological effect. We previously described a novel approach: intravenous administration of ex vivo expanded BOEC (blood outgrowth endothelial cells) engineered to express FVIII. Robust therapeutic results were obtained using NOD/SCID mice and human BOEC stably transfected to express human FVIII (Blood99:457, 2002). We are now conducting scale-up studies in the canine hemophilia A model. Based on the previous mouse data, we estimated that 400 million cells would be required to achieve a therapeutic effect in the dog. First, two normal dogs were infused with autologous unmanipulated BOEC, which revealed removal from the circulation via single-pass kinetics. Then, BOEC cultures were obtained from three hemophilia A dogs, and an onco-retroviral vector (MIRG/cFVIII) was used to obtain transduced BOEC producing B-domainless canine FVIII. Then, for each treatment, autologous engineered BOEC were given IV to the original donor dogs, with dose divided over 3 daily infusions, and whole blood clotting time (WBCT, in minutes) was followed. Dog Date # cells infused pre Rx WBCT effect on WBCT D28 12/04 274x106 >60 nadir 36 at day 14, Still 39 at day 185 D28 6/05 386x106 39 nadir 24.5 at day 23, now at 30 at day 44, being followed E64 7/04 220x106 >60 nadir 34.5 at day 3, >60 min by day 38 E64 12/04 274x106 >60 still 37.5 at day 189 E64 6/05 185x106 37.5 nadir 34.5 at day 2, at 36 on day 32, being followed H17 6/05 272x106 >60 nadir 29 at day 44, being followed WBCT (nl = 8-12 min) needs to be <20 min to reflect >0.1% FVIII activity; but the observed WBCT are clearly improved compared to baseline. One dog (E64) was treated for a mouth bleeds on d37 in cycle 2, and d36 in cycle 3. One animal (E64) developed hypotension and tachypnea during one of the infusions (probably because cells were not kept sufficiently in suspension) but recovered. Four of the infusions were associated with mild transient thrombocytopenia. Based on these initial scale-up experiments, it appears that use of engineered BOEC for gene therapy is an approach worthy of continued study, as cell doses can be increased and other improvements in the method are readily envisioned. It has a number of advantages, including use of autologous carrier cells with which any expression vector can be paired, ex vivo exposure to vector rather than in vivo, use of BOEC expansion in culture to simultaneously achieve vector expansion, and the ability to chose a clone of engineered cells having a single, known (and studied) insertion site.

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