UCSF

Non-viral gene therapy vectors have not yet achieved the gene transfer efficiency of viral vectors but continue to be an attractive alternative to viruses due to reduced toxicity and ease of preparation. Successful improvements to non-viral vectors will likely require rationally designed strategies that consider the many barriers that nucleic acid drugs must overcome, including distribution to target organs, internalization into cells, endosomal escape, transport in the cytoplasm, and nuclear uptake. Studies that increase our understanding of these barriers will therefore also represent a significant contribution to the development of new vectors.

This dissertation presents a series of studies related to the intracellular delivery of plasmid DNA to the nucleus. First, a new strategy was introduced to increase the cytoplasmic mobility of plasmid DNA: Biomolecular Adaptor for Retrograde Transport (BART) peptides designed to hijack endogenous molecular motors. Proof-of-concept experiments showed that a BART peptide can bind to a dynein light chain and that BART-bound cargo can be actively transported along microtubules by dynein in a reconstituted system. Second, an assay was developed to quantify the amount of plasmid DNA that is delivered to cell nuclei using various delivery conditions. Application of this assay revealed that cells transfected with Lipofectamine^TM lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus and that lipoplex-delivered plasmids were more efficiently expressed, based on protein expression per plasmids in the nucleus, than polyplex-delivered plasmids. Third, optimized conditions were established for a robust high throughput assay to screen for small molecule enhancers of transfection. Enhancer molecules discovered with this screen could help to elucidate rate-limiting steps in the transfection process and could also be used as adjuvants in gene therapy formulations. These studies summarize my efforts to design new tools and gauges to improve both the design of and our understanding of gene vectors.

In addition, the final report of a field study in the Dominican Republic to assess the feasibility of needle-free injection technology for that country's immunization program is included at the end of this dissertation. This report presents recommendations on how current devices could be improved and how the technology could be implemented.