Jeannine M. Scott

Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin

Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult mdx (dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin expression cassettes are able to efficiently transduce muscles of 1-yr-old mdx mice. Single i.m. injections of viral vector restored dystrophin production to 25–30% of mouse limb muscle 1 mo after injection. Furthermore, functional tests of virally transduced muscles revealed almost 40% correction of their high susceptibility to contraction-induced injury. Our results show that functional abnormalities of dystrophic muscle can be corrected by delivery of full-length dystrophin to adult, immunocompetent mdx mice, raising the prospects for gene therapy of muscular dystrophies.

Viral vectors for gene transfer of micro-, mini-, or full-length dystrophin

Gene therapy for Duchenne muscular dystrophy will require methods to deliver gene constructs encoding functional versions of dystrophin to the vast majority of a patients musculature. Obstacles to achieving these goals include identifying which forms of dystrophin would be effective in a clinical setting and developing gene delivery shuttles capable of carrying and expressing dystrophin cassettes without toxic or adverse immunologic consequences. We review here recent work from our laboratory to identify sequences within dystrophin that are required to prevent development of dystrophic changes in muscle or which might be able to correct pre-existing damage. We also describe work aimed at developing viral shuttle vectors able to carry and express these dystrophin cassettes at high levels and in a muscle-specific fashion. While great challenges remain in developing methods for systemic gene delivery, we show that a variety of viral vectors are able to carry and express therapeutic levels of dystrophin when delivered directly to mouse skeletal muscle.

Role of the ras-MAPK signaling pathway in the DNA methyltransferase response to DNA hypomethylation

Our group reported that inhibiting DNA methylation in human T cells increases DNA methyltransferase expression and activity, and suggested that this may represent a response to DNA hypomethylation. The increase correlates with increases in Ha-ras and c-jun, suggesting that increased signaling through the ras-MAPK pathway, due to overexpression of some elements, may be responsible. However, whether human DNA MTase is regulated by the ras-MAPK pathway, and whether overexpression of elements in this pathway will increase DNA MTase, is unknown. We report that treating cells with a DNA methylation inhibitor increases transcription regulated by a putative DNA MTase promoter, and that this increase requires AP-1 sites. Additional studies demonstrate that overexpression of an unmutated Ha-ras causes an increase in DNA MTase, and that human T cell DNA MTase can be decreased by inhibiting signaling through the ras-MAPK pathway. Together, these studies suggest that human T cell DNA MTase is regulated through the ras-MAPK pathway, and that overexpression of Ha-ras is sufficient to increase DNA MTase expression. These results thus provide a mechanism for the increase in DNA MTase observed after inducing DNA hypomethylation, a response which may have relevance to some disease states.

Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses.

Helper‐dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high‐titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo.

Impaired DNA Methylation in Lupus T Cells

Although lupus is commonly thought of as a B-cell disease, evidence from murine models of lupus-like syndromes and our understanding of how T cells coordinate the overall immune response suggest that T cells play a fundamental role in this disorder. Our group has developed a model that directly implicates T cells in the initiation of drug-induced and idiopathic lupus, and suggests molecular mechanisms relevant to this process. These studies derive from experiments aimed at determining the importance of DNA methylation in the regulation of T-cell function and gene expression. One particularly interesting outcome of these experiments is that hypomethylated CD4+ cells are no longer dependent on antigen for activation, and respond to antigen-presenting cells (APCs) lacking specific antigen, thus demonstrating characteristics of autoreactivity. We have used this observation to develop a working model for the study of both idiopathic and drug-induced lupus, in which methylation changes in the promoter sequences of critical genes lead to autoreactivity, and the autoreactive T cells then induce an autoimmune disease. This chapter reviews the current concepts regarding DNA methylation and its role in gene regulation, and relates this information to our model of T-cell—induced lupus-like disease. Finally, this chapter describes our current work for consideration as an area of research to be pursued to understand fully the molecular events initiating lupus.