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

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.