Physiologically-Regulated Expression Vectors for Gene Therapy

Abstract

Gene-replacement gene therapy has been under development for a number of years. In spite of the large amount of research invested into developing gene therapy for the treatment of recessive genetic disorders only a limited number of patients world-wide have received the benefits. In addition, several high profile adverse events in gene therapy trials have lead to an increasing awareness of the challenges facing gene therapy treatments before they become established in the clinic. This has necessitated the development of novel advances in gene therapy vector design and delivery. This chapter will focus on the development of gene expression vectors incorporating native genomic regulatory elements that ensure transgene expression is physiologically relevant. Three main advances will be discussed here in detail; the use of whole genomic DNA loci to ensure physiologically-regulated transgene expression; development of viral vectors based on the herpes simplex virus type 1 for delivery of whole genomic DNA loci; and the development of genomic mini-gene vectors that contain native regulatory regions for the physiologically-regulated expression of cDNA mini-genes. The principal aim of gene-replacement gene therapy is to complement the loss of function of an endogenous gene by supplying an exogenous ‘working’ copy in trans. The conventional approach to this is to supply a wild-type cDNA copy of the gene in a small vector in which transgene expression is controlled by a strong heterologous promoter, such as the immediate early promoter of cytomegalovirus (pCMV). The advantage of this approach is that the vectors are easy to use, have high levels of transgene expression, and fit easily into most viral delivery systems such as lentivirus and adenovirus. However, expression from these vectors is characteristically short-term and wide-spread with no tissue specificity or temporal regulation. One alternative to heterologous expression vectors for gene therapy is to utilise native genomic DNA regulatory elements to ensure gene expression that is both spatially and temporally regulated. A highly effective means of achieving gene expression that is physiologically-regulated is through the use of whole genomic loci which contain all introns, exons and regulatory regions in the correct genomic context. Expression from whole genomic loci has been proven to recapitulate endogenous expression. In the context of gene therapy, delivery of whole genomic loci using bacterial artificial chromosomes (BAC) has been shown to be an effective means of complementing gene deficiencies. Delivery of BAC vectors carrying complete loci encoding, for example, the genes for the human low density lipoprotein receptor (LDLR), the Friedreich's ataxia (FRDA) frataxin protein (FXN),