Keratinocyte Gene Therapy for Adenosine Deaminase Deficiency: A Model Approach for Inherited Metabolic Disorders

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Abstract
Disorders in which there is toxic buildup of circulating substrate may be treated by furnishing an enzyme reservoir capable of metabolically processing the excess substrate. The epidermal keratinocyte is a potential site for such a reservoir. In this study, we explore the capacity of genetically modified keratinocytes to metabolize extracellular substrate in a culture model that resembles in vivo epidermal architecture. Keratinocytes from adenosine deaminase (ADA)-deficient patients were transduced with a retroviral vector encoding the human ADA gene and the capacity of this tissue to deaminate deoxyadenosine (dAdo) in vitro was measured. The results show that at a substrate concentration of 10 μM, ADA-corrected keratinocytes deaminate dAdo at a rate of 0.38 nmol/min · 106 cells. These results indicate that keratinocytes process extracellular substrate at rates that suggest complete substrate conversion in a single pass. This study provides a strong indication that the epidermis, the largest and most accessible tissue of the body, is a valuable site for designing clinically relevant gene therapies. To develop epidermal gene therapy for metabolic disorders, keratinocytes must be capable of metabolizing a significant amount of circulating substrate. To study this potential, keratinocytes derived from two adenosine deaminase-deficient patients were transduced using a retroviral vector encoding the human adenosine deaminase gene. The resulting keratinocytes were grown using a culture system that resembles intact epidermis. Transduced cells were observed to metabolize deoxyadenosine substrate at rates that suggest that genetically modified keratinocytes could serve as a reservoir of enzymatic activity capable of producing therapeutic benefit.