Gene therapy in skin disease

Journal of Drugs in Dermatology, Oct, 2007 by Sarwar Zahid, Isaac Brownell

Gene therapy holds great promise for the treatment of many dermatologic diseases. (1-4) However, practical application of gene delivery has proven to be challenging. Numerous hurdles must be overcome if gene therapy is to become a viable treatment. Vector efficiency, mode of delivery, and side effects are among the leading concerns. Some highly publicized adverse events have prompted increased caution when starting human trials. These include the 1999 death of a patient after injection of an adenoviral vector designed to correct his ornithine transcarbamoylase deficiency, and 2 patients treated for X-SCID developing leukemia in 2003 due to oncogene activation by retroviral insertion. Nonetheless, numerous trials have reported encouraging results for gene therapy. This review discusses some of the approaches, vectors, and constructs available for gene therapy in skin disease, including recent successes and future prospects.

Approaches

In vivo vs. ex vivo

The 2 primary approaches to gene therapy are in vivo and ex vivo. In vivo therapy involves introduction of genetic material to a patient's skin. (5) Although this approach has its benefits, it suffers from low efficiencies of gene transfer. This will be a limiting factor in treating inherited skin disorders involving the entire cutaneous surface.

In contrast, the ex vivo approach has been shown to exhibit higher levels of gene transfer. Ex vivo therapy involves in vitro gene transfer to cultured cells from the patient, expansion of the genetically modified cells, and grafting back onto the patient. (5) Cultured keratinocytes can be expanded up to 10,000-fold, and the grafting of unmodified epithelial sheets generated in culture is presently used to treat large burns. (6) Efficiency can be further enhanced by including a selectable marker in the transgene construct and enriching for the genetically-modified cells. (7) Disadvantages of the ex vivo approach include the need to ablate the diseased skin prior to grafting, and that multiple grafts are required to cover the entire cutaneous surface.

Targeting Stem Cells

Permanent correction of heritable skin disease will require treating cutaneous stem cells. In the skin, certain cells in the basal keratinocyte layer and the hair follicles have been classified as stem cells in that they divide infrequently and are the source of differentiated epidermal keratinocytes. (8) Both in vivo and ex vivo approaches seek to target these cells. in vivo gene therapy, however, is presently impractical for stem cell targeting due to low gene transfer frequencies. In contrast, ex vivo protocols can employ culture techniques to select for stem cells prior to gene transfer. The lack of a definitive marker to prospectively identify stem cells necessitates the use of progeny analysis to identify stem cells. Cells whose daughter clones contain minimal numbers of terminally differentiated cells are considered to be keratinocyte stem cells. (9) Importantly, since stem cells divide infrequently, a vector capable of infecting nondividing cells with a high efficiency is preferable when targeting stem cells. Furthermore, in order for the therapy to persist, the vector must integrate genetic material into the host cell genome.

Vectors and Delivery Systems

The various techniques used to deliver genetic material into cells each have their own optimal conditions. The efficiency in vivo and ex vivo, and selectivity toward specific cell populations are important considerations in choosing a modality. Likewise, potential side effects and the ability to affect permanent change are also crucial. The choice of vector depends on treatment goals and the nature of the pathology under treatment. The ideal vector to correct inherited skin diseases would efficiently integrate DNA into the genome of cutaneous stem cells. Most strategies, however, do not integrate into the genome and are more suited for tasks that only require temporary gene expression. These include DNA vaccines, expression of targeted DNA repair constructs, and providing cytokines to assist with immune responses or wound healing.

Mechanical

Introduction of naked DNA to the skin for gene therapy can be accomplished via jet injection, microseeding, direct injection, or particle-mediated gene transfer (gene guns). Direct needle injection of DNA in the dermis has been shown to lead to localized expression in the epidermis in both pig and human skin. (10) Although cheap and flexible, it is impractical for the treatment of large surfaces.

Microseeding uses microneedles that are repeatedly driven into the skin to deliver genetic material. In pigs, gene transfer experiments have established the superiority of microseeding to direct injection in the transfection of partial thickness wounds. (11) Gene guns are another option--genetic material is coated onto metal particles and accelerated by an electric field. The effectiveness of using plasmid-coated gold particles in transfecting mouse skin has been demonstrated in vivo. (12)