The Leading Publisher in Biotechnology

ABSTRACT

Therapeutic gene delivery in severe genetic skin disease may require production of a uniformly corrected population of cells capable of regeneration of normal skin elements when returned to the host. To achieve this, we have used lamellar ichthyosis (LI), a disorder of epidermal differentiation recently associated with defects in keratinocyte transglutaminase (TGase1), as a prototype. We have used a high-efficiency retroviral delivery approach to uniformly restore normal levels of TGase1 expression to primary keratinocytes from severely affected LI patients previously lacking TGase1. Delivered TGase1 was correctly targeted to membrane association and restored patient cell transglutaminase activity levels to normal. Corrected primary LI patient keratinocytes also demonstrated restoration of previously defective involucrin cross-linking and in vitro measures of cornification to levels found in normal cells. These results indicate that efficient TGase1 delivery to early passage keratinocytes can produce a population of corrected LI patient cells. The capability to produce such cells may provide a basis for future efforts at gene therapy for genetic skin disease.

Overview summary

Production and characterization of uniformly corrected primary cells in vitro suitable for therapeutic return to patients with monogenic diseases can pose a significant challenge. The recently characterized transglutaminase 1 (TGase1)-deficient disorder, lamellar ichthyosis (LI), offers a prototype for development of this capability using keratinocytes from patients with this severe genetic skin disease. We show here that primary cells from LI patient skin can be rapidly engineered at high efficiency and without subsequent selection via a retroviral expression vector for TGase1. It is shown that this gene delivery restores measures of cellular functions to normal. This rapid and high-efficiency approach for corrective gene delivery to primary patient cells in vitro may serve as a model for efforts in the skin and other tissues.