Delivery of a Secretable Adenosine Deaminase Through Microcapsules–A Novel Approach to Somatic Gene Therapy

Abstract

Many current gene therapy protocols require genetic modification of autologous cells. An alternate approach is to use universal recombinant cell lines engineered to secrete in vivo the desired gene products. Enclosing these cells within immunoprotective devices before implantation would prevent rejection of the nonautologous donor cells. To overcome the limitation that not all therapeutic gene products are secreted, we now propose to fuse a signal sequence to the amino terminus of a nonsecreted protein such as human adenosine deaminase (ADA), thus directing the product into a secretory pathway for release from the cells. A fusion gene constructed between the cDNA of the β-lactamase signal sequence and human ADA expressed a product after in vitro transcription and translation that was immunologically similar to the human protein. Mouse fibroblasts transfected with the fusion gene demonstrated secreted ADA activity that resembled the human cytosolic enzyme in its heat stability, pH optimum, K_M, electrophoretic mobility, and immunologic reactivity. Hence, the secreted enzyme expressed from the fusion gene is antigenically and enzymatically similar to the authentic human form. When transfected mouse fibroblasts or myoblasts were enclosed in permselective alginate-poly-l-lysine alginate microcapsules, ADA activity was secreted from the microcapsules and the cells remained viable for over 5 months. Hence, a secretable and functional human ADA has been constructed that can be delivered from recombinant cells within immunoprotective capsules. The success of this strategy provides the prototype for engineering nonsecreted gene products for therapy via this novel method of somatic gene therapy. We have demonstrated recently the feasibility of a novel approach to somatic gene therapy described as nonautologous somatic gene therapeutics. Universal cell lines engineered to secrete therapeutic products are enclosed in permselective devices. Upon implantation into nonautologous hosts, these cells are immunologically protected and continue to secrete the gene product of interest. However, this approach is not applicable to gene products such as adenosine deaminase (ADA) that are not naturally secreted. To overcome this limitation, we now demonstrate that by fusing a signal sequence to its amino terminus, this cytosolic enzyme can be redirected into a secretory pathway. The secreted enzyme is immunologically and catalytically similar to the intracellular form. This work illustrates a strategy that permits a broader range of gene products to be delivered through this novel method of somatic gene therapeutics. It is conceivable that other desirable properties can be conferred similarly through molecular engineering to deliver a truly “designer molecule.”