167. Partial correction of the hemophilic phenotype in mice implanted with encapsulated human primary myoblasts secreting FIX

Top of pageAbstract Hemophilia B is a bleeding disease caused by a defective factor IX (FIX). Current treatment includes infusion of concentrated FIX derived from plasma or recombinant FIX. Alternative treatments would be highly desirable. We propose a gene therapy strategy based on the use of recombinant cell enclosed in the implantable alginate microcapsules. Previously, we have reported a transient delivery of hFIX with implantation of encapsulated C2C12 myoblasts and sustained delivery of hFIX with implantation of encapsulated mouse primary myoblasts in immune competent mice. As the purpose of the study of the gene therapy is for treatment of human patients, in this study we report on the feasibility of encapsulated genetically engineered human primary myoblasts to correct the bleeding disorder of FIX deficient mice. Human primary myoblasts were transfected with plasmid pKL4M-hFIX containing hFIX cDNA driven by the ubiquitous β-actin promoter. Transfected cells were selected with G418 and clones secreting high levels of hFIX selected by ELISA. Clone pKL4M-hFIX-C12, secreting 900 ng hFIX/106/24hr in vitro, was used in this study. The recombinant human primary myoblasts were enclosed in 1.7% alginate-PLL microcapsules with a diameter of 500–600 μm. Encapsulated cells were subsequently implanted intraperitoneally into immune competent C57BL/6 (6–8 weeks of age) hemophilic mice. Each mouse received 20 × 106 cells. Circulating level of hFIX in treated mice as detected by ELISA was on average 68 ng/ml, with a peak of ~120 ng/ml on day one after implantation. The level of circulating hFIX was reduced by day 7 and could not be detected after day 14. A plausible cause for the transient delivery of hFIX might be the anti-hFIX antibodies that were detected in the treated mice following the xenotransplant. At the same time, APTT values in the treated mice were gradually reduced on average from 88.9 sec before treatment to 74.6 sec after treatment. Mice were also subjected to a tail transection test to assess the protection of treated mice against severe trauma. Importantly, tail-cut bleeding time decreased from more than 15 min before treatment to 5 min post-treatment. Control non-hemophilic mice stop bleeding in approximately 3 min. Interestingly, in selected treated hemophilic mice, bleeding was completely corrected (i.e. tail-cut bleeding time went back to normal). Taken together, these results indicate that the hFIX secreted from the encapsulated cells in mouse plasma and tissue has strong biological activity. Therefore, our findings would support the feasibility of human encapsulated myoblasts as a gene therapy strategy for hemophilia B.