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BACKGROUND: A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein has been evaluated. The microcapsules are implanted intraperitoneally. In order to prevent cell immune rejection, cells are enclosed in non-antigenic biocompatible alginate microcapsules prior to their implantation into mice. It has been shown that encapsulated myoblasts can deliver therapeutic levels of Factor IX (FIX) in mice. The delivery of human Factor VIII (hFVIII) in mice using microcapsules was evaluated in this study. METHODS: Mouse C2C12 myoblasts and canine MDCK epithelial kidney cells were transduced with MFG-FVIII (B-domain deleted) vector. Selected recombinant clones were enclosed in alginate microcapsules. Encapsulated recombinant clones were subsequently implanted intraperitoneally into C57BL/6 and immunodeficient SCID mice. RESULTS: Plasma of mice receiving C2C12 and encapsulated MDCK cells had transient therapeutic levels of FVIII in immunocompetent C57BL/6 mice (up to 20% and 7% of physiological levels, respectively). In addition, FVIII delivery in SCID mice was also transient, suggesting that a non-immune mechanism must have contributed to the decline of hFVIII in plasma. Quantitative RT-PCR analysis confirmed directly that the decline of hFVIII is due to a reduction in steady-state hFVIII mRNA, consistent with transcriptional repression. Furthermore, encapsulated cells retrieved from implanted mice were viable, but secreted FVIII ex vivo at three-fold lower levels than the pre-implantation levels. In addition, antibodies to hFVIII were detected in immunocompetent C57BL/6 mice. CONCLUSIONS: Implantable microcapsules can deliver therapeutic levels of FVIII in mice, suggesting the potential of this gene therapy approach for haemophilia A. The findings suggest vector down-regulation in vivo.

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L-HES is a rare disease characterized by symptoms linked with the persistence and proliferation of a CD4+ T-cell clone producing cytokines that induce polyclonal hypereosinophilia. This disease shares several characteristics with allergic diseases (allergic rhinitis, asthma, etc…), that exhibit hypereosinophilia driven by allergen specific CD4+ Th2 cells. The CD3-CD4+ T-cell clones detected in L-HES patients however are not directed to a specific antigen, thereby excluding the possibility of anti-idiotype or tolerization therapy. The work presented in this thesis addresses the question of what mechanisms are deregulated in the CD3-CD4+ T cells that allow them to persist and expand in L-HES patients, putting them at significant risk for the development of a T-cell lymphoma. The first aim of this study was to characterize the phenotypic and immunological traits of the CD3-CD4+ T cell-clone found in the majority of L-HES patients. Furthering our understanding of the nature of the underlying abnormal T-cell population provides a basis for improved diagnosis and ultimately tailoring treatment for L-HES patients. This objective was achieved by producing a complete profile of the CD3-CD4+ T-cells, including their immunophenotype, cytokine and chemokine secretion pattern, infectious status and T cell receptor rearrangement. The second aim of this work was to identify the genetic alterations linked with the sustained survival and expansion of the CD3-CD4+ T-cell clone. This goal was accomplished at the chromosomal, molecular and cellular levels by assessing the karyotype, the evolving molecular profiles as well as designing functional experiments to characterize mechanisms underlying the persistent expansion of the L-HES T-cell clones. Among the genetic aberrations detected, our ultimate aim was to identify potential suppressor genes and decipher the role they play in the normal apoptotic and proliferative mechanisms responsible for controlling CD4+ T cell growth. Understanding these events should provide new and improved diagnostic tools and therapeutic targets for L-HES associated T-lymphomas. (SBIM 3) -- UCL, 2010