In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver.

Mutant proteins have the potential to exert dominant-negative effects that might limit the therapeutic efficacy of their wild-type counterparts after gene transfer. For ornithine transcarbamylase (OTC) deficiency, in vitro studies have suggested the presence of dominant-negative effects, however, supporting in vivo studies have not been conducted. In this study, we exploited the capacity of recombinant adeno-associated virus (rAAV) 2/8 vectors to deliver transgenes to the mouse liver with high efficiency to determine whether expression of selected OTC mutant proteins exert inhibitory effects on endogenous wild-type OTC enzymatic activity. Using site-directed mutagenesis we constructed three OTC mutants with a theoretical or reported in vitro capacity to exert dominant-negative effects, and delivered these to the liver using rAAV2/8. Each mutation had been earlier identified in patients with OTC deficiency. Treated mice showed no increase in urinary orotic acid levels or reduction in OTC activity despite supra-physiological expression of the mutant proteins, consistent with an absence of dominant-negative effects. These data have important implications for the development of gene therapy strategies for OTC deficiency and validate a model system in which potential dominant-negative effects of specific mutations in prospective patients can be examined empirically before gene therapy.

Antigen-specific humoral tolerance or immune augmentation induced by intramuscular delivery of adeno-associated viruses encoding CTLA4-Ig-antigen fusion molecules.

This study initially sought to investigate the immunostimulatory properties of recombinant adeno-associated virus (rAAV) with a view to developing a genetic vaccine for malaria using muscle as a target tissue. To augment humoral immunity, the AAV-encoded antigen was genetically fused with CTLA4-Ig, a recombinant molecule that binds B7 costimulatory molecules. At 10(9) vg, CTLA4-Ig fusion promoted the humoral immune response 100-fold and was dependent on CTLA4-Ig binding with B7 costimulatory molecules, confirming plasmid DNA models using this strategy. In distinct contrast, 10(12)-10(13) vg of rAAV1 specifically induced long-lived humoral tolerance through a mechanism that is independent of CTLA4-Ig binding with B7. This finding was unexpected, as rAAV delivery to muscle, unlike liver, has shown that this tissue provides a highly immunogenic environment for induction of humoral immunity against rAAV transgene products. An additional unpredicted consequence of antigen fusion with CTLA4-Ig was the enhancement of antigen expression by approximately one log, an effect mapped to the hinge and Fc domain of IgG(1,) but not involving antigen dimerization or the neonatal Fc receptor. Collectively, these findings significantly advance the potential of rAAV both as a vaccine or immunotherapeutic platform for the induction of antigen-specific humoral immunity or tolerance and as a gene therapeutic delivery system.

Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct.

Gene transfer vectors encoding two or more genes are potentially powerful research tools and are poised to play an increasingly important role in gene therapy applications. Common strategies employed to express more than one transgene per vector include the use of multiple promoters, internal ribosome entry site (IRES) elements, splicing signals and fusion proteins. Of these, the IRES elements and multiple promoters have been most widely used. The use of multiple promoters, however, may be compromised by interference between promoters, promoter silencing and vector rearrangements or deletions. In this study, we demonstrate promoter interference between two internal heterologous promoters in the context of a late-generation lentiviral vector. The interference, involving the human cytomegalovirus-immediate-early promoter and human elongation-factor-1alpha promoter, occurred bidirectionally with both promoters markedly impairing expression of the adjacent transcription unit. The data presented not only highlight the potential for interference between these widely-used promoters, but also the value of a sequential approach to vector construction that allows such effects to be recognized.

Fibroblasts modulate cardiomyocyte excitability: implications for cardiac gene therapy.

In an earlier study exploring the potential of gene transfer to repair myocardial conduction defects, we observed that myotubes, generated by forced expression of MyoD, exhibit reduced excitability when also modified to express connexin43 (Cx43). We hypothesized that this effect was caused by gap junction-mediated coupling between myotubes and the underlying fibroblast feeder layer. This intriguing possibility has important implications for ongoing efforts to develop strategies for repairing myocardial conduction defects by gene transfer, and also provides novel insights into the electrophysiological function of naturally occurring heterologous cell coupling within the heart. Although a conductive function for fibroblasts through heterologous coupling has previously been reported, the current study provides novel evidence that fibroblasts can modulate cardiomyocyte excitability in a Cx43-dependent manner. In a co-culture study system, neonatal rat cardiomyocytes were grown on monolayers of mouse fibroblasts with genetically altered Cx43 expression and the effect on intrinsic beat frequency examined. Cardiomyocytes grown on wild-type (WT) fibroblasts expressing native levels of Cx43 beat significantly slower than cells grown on fibroblasts devoid of this molecule (germline knockout) or with dominant-negative functional suppression. Expression of Cx43 in fibroblasts from Cx43 knockout mice restored cardiomyocyte beat frequency, to rates comparable with those observed in co-culture with WT fibroblasts.

Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

The TetA(K) tetracycline/H(+) antiporter from Staphylococcus aureus: mutagenesis and functional analysis of motif C.

Conserved motif C, identified within members of the major facilitator superfamily (MFS) of transport proteins that mediate drug export, was examined in the tetracycline resistance efflux protein TetA(K) from Staphylococcus aureus; motif C is contained within transmembrane segment 5. Using site-directed mutagenesis, the importance of the conserved glycine (G151, G155, G159, and G160) and proline (P156) residues within this motif was investigated. Over 40 individual amino acid replacements were introduced; however, only alanine and serine substitutions for glycine at G151, G155, and G160 were found to retain significant levels of tetracycline resistance and transport activity in cells expressing mutant proteins. Notably, P156 and G159 appear to be crucial, as amino acid replacements at these positions either significantly reduced or abolished tetracycline/H(+) activity. The highly conserved nature of motif C and its distribution throughout drug exporters imply that the residues of motif C play a similar role in all MFS proteins that function as antiporters.

Membrane topology of the metal-tetracycline/H+ antiporter TetA(K) from Staphylococcus aureus.

A series of fusions to the reporter proteins alkaline phosphatase and beta-galactosidase have been constructed in the predicted periplasmic and cytoplasmic loops of TetA(K), a protein responsible for efflux-mediated tetracycline resistance in Staphylococcus aureus. The results support a topological model of 14 transmembrane segments for TetA(K).