Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Dopamine-deficient (DD) mice cannot synthesize dopamine (DA) in dopaminergic neurons due to selective inactivation of the tyrosine hydroxylase gene in those neurons. These mice become hypoactive and hypophagic and die of starvation by 4 weeks of age. We used gene therapy to ascertain where DA replacement in the brain restores feeding and other behaviors in DD mice. Restoration of DA production within the caudate putamen restores feeding on regular chow and nest-building behavior, whereas restoration of DA production in the nucleus accumbens restores exploratory behavior. Replacement of DA to either region restores preference for sucrose or a palatable diet without fully rescuing coordination or initiation of movement. These data suggest that a fundamental difference exists between feeding for sustenance and the ability to prefer rewarding substances.

A current model for transcription-coupled DNA repair is that RNA polymerase, arrested at a DNA lesion, directs the repair machinery to the transcribed strand of an active gene. To help elucidate this role of RNA polymerase, we constructed DNA templates containing the major late promoter of adenovirus and a cyclobutane pyrimidine dimer (CPD) at a specific site. CPDs, the predominant DNA lesions formed by ultraviolet radiation, are good substrates for transcriptioncoupled repair. A CPD located on the transcribed strand of the template was a strong block to polymerase movement, whereas a CPD located on the nontranscribed strand had no effect on transcription. Furthermore, the arrested polymerase shielded the CPD from recognition by photolyase, a bacterial DNA repair protein. Transcription elongation factor SIU (also called TFIIS) facilitates read-through of a variety of transcriptional pause sites by a process in which RNA polymerase II cleaves the nascent transcript before elongation resumes. We show that SU1 induces nascent transcript cleavage by RNA polymerase H stalled at a CPD. However, this cleavage does not remove the arrested polymerase from the site of the DNA lesion, nor does it facilitate translesional bypass by the polymerase. The arrested ternary complex is stable and competent to resume elongation, demonstrating that neither the polymerase nor the RNA product dissociates from the DNA template.Helix-distorting lesions are produced in cellular DNA by various endogenous and exogenous agents. Among such lesions is the cyclobutane pyrimidine dimer (CPD), the most prevalent lesion formed by short-wavelength UV radiation. CPDs can block DNA replication and transcription, leading to cell death. If unrepaired, the DNA damage can lead to mutagenesis, activation of protooncogenes, and ultimately carcinogenesis. One mechanism to remove these lesions is nucleotide excision repair (NER), common to a wide range of species from Escherichia coli to humans. The basic mechanism of NER is well understood in E. coli (1). Recognition of the damage is followed by incision of the damaged DNA strand on both sides of the lesion. The damage-containing oligonucleotide is removed, the resultant gap is filled in by DNA polymerase, and DNA ligase completes the process by joining the repair patch to the contiguous DNA strand. Although the detailed mechanism of NER in eukaryotes is not established as firmly, it appears to have the same essential features. A striking property of NER is the intragenomic heterogeneity of repair efficiency (2). Expressed genes are repaired more rapidly than the overall genome in rodent (3) and human (4) cells in culture. Furthermore, this preferential repair is largely due to efficient repair of the transcribed strand of an active gene compared to the nontranscribed strand or unexpressed DNA sequences (5). In addition to mammalian cells, preferential repair of transcribed DNA strands has been demonstrated in E. coli (6) and Saccharomyces cerevisiae (7-9), so it is likely to be a universal phenome...

Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAVhFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.

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