My laboratory is interested in utilizing recombinant adeno-associated virus mediated (rAAV) gene transfer to develop meaningful treatments for genetic and infectious diseases.  Current research focuses on three principle areas: (i) use of antibody gene transfer as a means to augment host immunity; (ii) use of RNA based anti-sense therapeutics to achieve exon-skipping of the dystrophin gene for treatment of DMD; and (iii) development of scalable rAAV production platforms using novel AAV serotypes to support clinical trials.  Antibody gene transfer:  The focus of this project is to develop AAV delivery vectors that efficiently express antibodies of predetermined specificity within host tissue.  Because of the inherent flexibility of this system, light and heavy chain antibody genes can be incorporated into a single rAAV vector, and the antibody-expressing vector can then be used to transduce target tissue in vivo (muscle, liver, and pleural cavity).  This in turn, leads to sustained expression of biologically active antibody molecules in the circulation.  We have shown that human anti-HIV-1 monoclonal antibodies can be expressed in mice and non-human primates – endowing the host with anti-HIV-1 in vitro neutralization activity.  Thus, this approach allows for predetermination of antibody affinity and specificity prior to "immunization", and avoids the need for an active humoral immune response.  Beyond the current application for HIV-1 infection, this strategy might be useful for other situations where antibodies with predetermined specificities need to be delivered in vivo.  Currently, we are evaluating methods to further augment in vivo levels using various AAV serotypes and routes of delivery.  Exon-Skipping:  The goal of this project is to develop a meaningful therapy to treat muscular dystrophy caused by mutations within the dystrophin gene that ablate the reading frame.  We have constructed several rAAV gene transfer vectors that express anti-sense splice sequences linked to a modified U7 small nuclear RNA (snRNA).  Expression of these RNAs block inclusion of the mutated dystrophin exon into the mRNA transcript.  This results in the generation of a functional (albeit slightly smaller) dystrophin mRNA and protein product.  Currently, we are evaluating exonic splice enhancers (ESEs) as potential targets for efficient skipping and methods of vascular delivery to the muscles of the lower limbs.  Lastly, we are developing scalable production and purification methods using stable cell lines and transient transfection technologies for the production of multiple AAV serotypes (1-9).  Current research has focused on developing a cGMP “friendly” approach to large-scale production that can be transitioned into our production facility upon its completion.

Clark KR, Voulgaropoulou F, and Johnson PR (1996).  A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors.  Gene Therapy 3, 1124-1132.