| First-generation adenovirus (Ad) vectors are unsuitable for gene replacement therapy, due to immune responses and low cloning capacity. These problems may be exacerbated in the inflammatory milieu of dystrophic muscle. It is hoped that gutted Ad vectors, which do not carry viral genes, may provide effective gene transfer without eliciting acute toxicity or a longer-term, antigen-specific anti-adenovirus response. Unfortunately, gutted Ad vectors share some drawbacks with conventional Ad vectors and present new challenges. First, gutted vectors share the tropism of conventional adenovirus and could elicit an anti-transgene response in patients with null alleles. Second, clinical grade production of gutted Ad vectors has not been achieved.; The goal of this thesis is to determine whether these two problems with gutted adenovirus, low virus yield and immune response to transgene products, can be overcome. Two hypotheses were tested: (1) poor growth of gutted adenovirus vectors in tissue culture is caused primarily by failure of sub-optimal replication origins to compete for replication factors; and (2) in healthy muscle tissue, but not necessarily in dystrophic tissue, the immune response to transgene products delivered by a vector with promiscuous tropism can be eliminated by strict tissue-specific gene expression.; We explored whether gutted virus production would be facilitated by transfection into cells expressing replication factors or by use of synthetic terminal protein-DNA complex. Both methods were effective, together yielding a 1000-fold increase in gutted virus titer.; Muscle tissue from the mdx mouse, a model for Duchenne muscular dystrophy (DMD), is characterized by infiltrating antigen-presenting cells (APCs). We found that mdx mouse muscles mounted a stronger immune response to antigens that can be directly presented by those APCs. However, we did not detect an immune response to β-galactosidase expressed specifically in muscle by an Ad vector, even at high expression levels. This suggests that cross-presentation is not dramatically more effective in mdx mouse muscle, and that targeted vectors and tissue-specific promoters may be useful tools for evasion of the immune response in dystrophic muscle.; The results of our studies indicate that gutted vectors carrying muscle-specific transgenes have great potential for gene therapy of DMD. |
