Implantable Microcapsules for Gene Therapy for Hemophilia
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abstract
Hemophilia A is an X-linked recessive bleeding disorder caused by the deficiency of coagulation factor VIII [1]. Current treatment for hemophilia A consists of prophylactic or on demand replacement therapy of either plasma-derived or recombinant FVIII concentrates [2]. Albeit effective, there are several limitations associated with factor concentrates, including high cost that limits its availability for close to 80% of hemophilia patients in developing countries [3-5]. An alternative treatment would thus be desirable. Gene therapy for hemophilia has seen many successes in animal models and represents a more cost-effective alternative to the current treatment modalities [6]. In the current work, I present a gene therapy system for hemophilia that uses mouse fetal myoblasts engineered to secrete FVIII, enclosed in immuno-protective alginate-poly-L-lysine-alginate (APA) microcapsules, as a sustained source of FVIII. In this study, a thorough examination of the encapsulated myoblasts using a novel flow cytometry assay was performed. This method yielded an accurate and precise method for encapsulated cell viability calculation, and also allowed for analysis of several other parameters such as health (cell morphology), cell size and distribution. Flow cytometry was also used to monitor the time-course proliferation profile of encapsulated myoblasts secreting cFVIII, using the division tracking dye CFSE. We found that encapsulated cells display a decreased proliferation rate as well as lower viability than non-encapsulated cells. Implantation of encapsulated G8 myoblasts secreting cFVIII into hemophilia A mice resulted in maximum plasma levels of protein on day 1 ( ~18% of normal canine FVIII levels). Delivery of cFVIII in hemophilic mice also offered protection against blood loss after the mice were subjected to injury (as measured by hematocrit levels); indicating that biologically functional cFVIII continued for at least 7 days post-capsule implantation. Low levels of FVIII antigen returned on day 28 after a transient disappearance on day 14. However, the presence of antigen must be reconciled with appearance of anti-cFVIII antibodies that were detected in the plasma of treated mice at the end of five weeks. The neutralizing nature of these antibodies still needs to be characterized by Bethesda assay Overall, our study demonstrates the feasibility of delivering therapeutic levels of FVIII using encapsulated G8 fetal myoblasts. The presence of functional FVIII protein on day 7, suggests that this treatment was not met by transcriptional repression in vivo, thereby overcoming one of the major obstacles faced by using the transformed C2C12 cell line secreting hFVIII [7] If such levels of FVIII were achieved in humans, it would be sufficient to convert a severe hemophiliac into a mild phenotype. Thus, this gene therapy strategy may be a suitable therapeutic alternative for hemophilia patients. Further work ought to focus on the long-term persistence of FVIII in hemophilia A mice, and also determining the protection following trauma over time to determine if the FVIII remains functional. Other cell lines should be explored for higher expression, reduced immunogenicity and improved viability Still, there is a need to develop human cells expressing high levels of biologically active hFVIII with similar properties to the fetal cells described in this study [8, 9]
