Daniel G. Miller, MD, PhD
Keratin Gene Targeting for the Treatment of Epidermolysis Bullosa Simplex
Epidermolysis Bullosa Simplex (EBS) is a debilitating, dominantly inherited skin blistering condition without an effective treatment. We propose to correct the phenotype of EBS-patient keratinocytes and prepare them for autologous transplantation by using a gene targeting approach. This method allows gene mutations that result in proteins with dominant negative effects to be repaired, and allows appropriate transcriptional regulation of corrected sequences at the endogenous locus. Alternatively transcription from genes producing toxic proteins can be disrupted by targeted insertion of vector sequences into the mutant allele. Until recently, efficient methods for the delivery of DNA recombination templates to the nuclei of primary cells have been lacking. Adeno-Associated Virus (AAV) vectors efficiently transduce primary cells in culture and tissues in vivo, and their single stranded DNA genomes have been shown to recombine with homologous chromosomal sequences. Furthermore, keratinocytes expressing abnormal keratins may be at a growth disadvantage making gene targeting strategies a plausible approach because of the growth advantage conferred to repaired cells. The concept of growth-rate differences of cells expressing normal and abnormal keratins is further supported by reports of revertant somatic mosaicism in patients with epidermolysis bullosa suggesting that even infrequent back mutations, or second site “suppressor” mutations can be therapeutic.
Dux4 Gene Expression Differences in Facioscapulohumeral Muscular Dystrophy
Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating genetic condition manifest by weakness of facial and upper extremity musculature that presents in the 2nd decade of life. The causative genetic event is a contraction of a subtelomeric array of repeated 3.3 kb sequence units residing on one of two common alleles of chromosome 4. How this array contraction translates into cellular differences that result in weakness of select muscle groups is a fascinating question that is not presently understood. Each D4Z4 repeat unit contains a large open reading frame that encodes a putative double homeodomain containing protein named Dux4 making aberrant expression, or expression of aberrant Dux4 isoforms an attractive mechanism for FSHD pathology. Our long term objectives are to understand how muscle strength is compromised as a result of molecular events initiated by these contractions. We have begun transgenic mouse experiments that will define the normal expression pattern of the Dux4 gene in vivo, and characterize RNA transcripts that arise from Dux4 and related loci in human cell- culture. We hope these studies will help identify the genetic and molecular changes associated with this debilitating muscular dystrophy and suggest areas of research focus for treatment.