Edmund J. Niedzinski

Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation

Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, nonviral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable the development of enhanced nonviral gene transfer in salivary glands and other tissues. One potential barrier is the degradation of plasmid DNA by endonucleases. To test this hypothesis, we coadministered two endonuclease inhibitors ((zinc and aurintricarboxylic acid (ATA)) with plasmid DNA, containing the secreted alkaline phosphatase gene (SEAP), to the submandibular glands of rats. The effect of zinc and ATA on SEAP expression, tissue accumulation of plasmid DNA, and plasmid DNA stability was then characterized. We observed that mixtures containing zinc/DNA, ATA/DNA, and zinc/ATA/DNA significantly enhanced both systemic transgene expression and the amount of plasmid DNA associated with treated tissues. The relative endonuclease inhibitory activity of zinc, ATA, and zinc/ATA correlated with the observed effects on transfection efficacy. The use of zinc/ATA enhanced the efficacy of salivary gland transfection by at least 1000-fold versus DNA alone. Importantly, this improved performance resulted in robust systemic secretion of an exogenous protein (SEAP), thus demonstrating the potential this nonviral gene transfer technology has as a method to treat systemic protein deficiencies.

Zinc enhancement of nonviral salivary gland transfection

Gene transfer to exocrine glands, including the major salivary glands, presents an attractive method to deliver proteins for therapeutic applications. Previous efforts using nonviral gene delivery to these glands have resulted in limited success. In this report, zinc and other divalent transitions were coadministered with plasmid DNA in an effort to improve nonviral salivary gland transfection efficiency. The inclusion of zinc into plasmid DNA solutions resulted in a 20-fold enhancement in transgene expression without noticeable inflammation compared to a solution of plasmid DNA only. This observed enhancement in transgene expression was dependent upon the DNA dose and correlated with the accumulation of plasmid DNA by salivary gland tissues.

Gastroprotection of DNA with a synthetic cholic acid analog

The oral delivery of functional DNA to the gastrointestinal system would constitute a desirable, noninvasive method for potentially treating a variety of diseases. The digestive process, however, remains a formidable barrier. This dilemma may be addressed by using targeted liposomes both to protect the polynucleotide and to deliver the therapeutic DNA with high tissue specificity. The present study represents the initial steps toward developing a novel gene delivery system designed to interact with the enterohepatic receptors of the small intestine. Two cholic acid esters were synthetically modified at position C(3) to incorporate a DNA-binding domain. These novel compounds were evaluated for their ability to protect DNA from the nucleases found in gastrointestinal segments. Additionally, the compounds were screened as a component of a gene delivery vector. Formulations containing the new bile salt derivatives protected DNA from degradation for more than 2 h and were capable of transfecting cultured NIH 3T3 cells.

147. Chemical enhancers of non-viral salivary gland gene transfer

Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, non-viral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable enhanced non-viral gene transfer in salivary glands and other tissues. Our efforts to identify and transcend these barriers have resulted in the discovery of a group of compounds that enhance non-viral, salivary gland gene transfer by four orders of magnitude relative to unformulated DNA. These data as well as data directed towards understanding the mechanism-of-action responsible for the observed enhancement in non-viral gene transfer will be presented.