No abstract

Most evidence indicates that nitric oxide plays a role in normal wound repair; however, involvement of inducible nitric oxide synthase (iNOS) has not been established. Experiments were carried out to determine the requirement for iNOS in closing excisional wounds. Wound closure was delayed by 31% in iNOS knockout mice compared with wild-type animals. An identical delay in wound closure was observed in wild-type mice given a continuous infusion of the partially selective iNOS inhibitor N6-(iminoethyl)-L-lysine. Delayed wound healing in iNOS-deficient mice was completely reversed by a single application of an adenoviral vector containing human iNOS cDNA (AdiNOS) at the time of wounding. Reverse transcription PCR identified iNOS mRNA expression in wild-type mice peaking 4-6 d after wounding, and confirmed expression of human iNOS in the adenoviral vector containing human iNOS cDNA-treated animals. These results establish the key role of iNOS in wound closure, and suggest a gene therapy strategy to improve wound healing in iNOS-deficient states such as diabetes, and during steroid treatment.

The human adenovirus serotype 5 (Ad5) is used widely for applications in human gene therapy. Cellular attachment of Ad5 is mediated by binding of the carboxyl-terminal knob of its fiber coat protein to the Coxsackie adenovirus receptor (CAR) protein. However, Ad5 binding to CAR hampers the development of adenovirus vectors capable of specifically targeting (diseased) tissues or organs. Through sequence analysis and mutagenesis, a conserved receptor-binding region was identified on the side of three divergent CAR-binding knobs. The feasibility of simultaneous CAR ablation and redirection of an adenovirus to a new receptor is demonstrated.

Adenovirus (Ad) is used as a vector for gene delivery in therapies involving genetic disease, vascular disease, and cancer. The first step for efficient gene transfer is effective virus binding to the target cells. We have found that Ad-mediated gene delivery to multiple cell types is much less efficient compared to epithelial-derived cells. Low gene delivery to nonepithelial cell types was directly correlated to a deficiency of the cellular receptor which mediates Ad binding. To overcome this inefficiency we constructed a new virus, AdPK, that contains a heparin-binding domain that targets the virus to broadly expressed, heparan-containing cellular receptors. AdPK delivers genes to multiple cell types at markedly higher efficiencies than unmodified Ad. Viruses with enhanced attachment characteristics significantly improve gene transfer efficiency and may expand the tissues amenable to efficient Ad-mediated gene therapy.