Gene therapy in skin disease

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

Constructs

The vectors used in gene therapy deliver genetic material into cells. Most often, this DNA encodes for a protein whose function will help ameliorate the condition that is being treated. Thus, the nature of the condition dictates the design of the genetic construct that is delivered.

Gene Replacement

In the skin, numerous diseases are caused by mutations resulting in loss of gene function--gene replacement is required in these cases. In junctional epidermolysis bulosa (JEB), for example, a single mutation in the [beta]3 subunit of laminin-5 (LAMB3) can disrupt the dermal-epidermal basement membrane (BM) resulting in blisters. Gene therapy providing basal keratinocytes with a functional copy of this gene would cure the observed clinical defect. Use of endogenous promoters in gene replacement can theoretically restrict transgene expression to the appropriate cells and also reduce the risk of gene silencing. Unfortunately, both retroviruses and AAVs have limited amounts of DNA that can be packaged into them, making it difficult to integrate both regulatory and coding regions for replacement genes.

Gene Targeting

Gene targeting involves correcting mutations within the host genome. In contrast to gene replacement, this approach lacks transgene integration and restores the wild type genomic state. Potential strategies include triple-helix-forming oligonucleotides, (23) zinc-finger nucleases, (4,24) and RNA/DNA chimeric oligonucleotides to modify genomic DNA. (25) Use of topical and injected RNA/DNA oligonucleotides caused local correction of a tyrosinase mutation in albino mice in vivo. (25) The proposed mechanism for these interesting phenomena involves site-specific recruitment of DNA repair proteins to the mutated genomic locus.

Gene Silencing

Gene silencing is particularly important for mutant proteins that act through dominant negative mechanisms. While many approaches exist for gene silencing, RNAi is the most pervasive method for silencing mutant alleles. (26) In addition to using small interfering RNAs (siRNAs) to knockdown gene expression, it may also be possible to use promoter-targeting siRNAs to elicit epigenetic silencing of transcription through changes in chromatin and histone methylation. An alternative approach is to use ribozyme-mediated cleavage of the aberrant mRNA. (27)

Recent Research and Clinical Trials

Junctional Epidermolysis Bulosa (JEB)

Recently, ex vivo gene therapy using an oncovirus successfully treated a patient with JEB. (28) This patient carried a null allele for LAMB3 and a mutant allele with a single point mutation (E210K). The therapy consisted of isolating the patient's keratinocytes from a skin biopsy, selecting stem cell rich holoclones in culture, and infecting these with oncovirus containing the full-length LAMB3 gene. Transduced cells were grown into epithelial sheets that were grafted onto wound beds surgically created on the patient's thighs. Within 8 days, the grafted skin had regenerated and one year later the area produced histologically normal epidermis containing the vector DNA and normal levels of laminin-5.