577. DNA Ministrings: Linear-Covalently Closed Minivectors for Use in Non-Viral Gene Therapy with Applications Toward Ovarian Cancer

Abstract

Gene therapy depends on the successful transfer of genetic material into the nuclei of targeted human cells. Non-viral strategies address the safety concerns of viral gene delivery, but generally suffer from low transfection efficiencies. We previously constructed an enhanced linear covalently closed (LCC) minivector, a DNA ministring, which confers improved efficiency and safety over its plasmid, and even its isogenic circular counterparts. DNA minivectors are comprised solely of the eukaryotic expression cassette without the bulk of an immunogenic bacterial backbone, thus ensuring greater bioavailability, higher transfection efficiency, and prolonged duration of gene expression. The linear nature of DNA ministrings minimizes the potential for insertional mutagenesis from random genomic integration.DNA ministrings are produced through a bacteriophage (phage) -derived recombination system in recombinant Escherichia coli (Fig. 1A). The phage-derived protelomerase, Tel, regulated via a temperature-inducible expression system, acts on a parent plasmid to produce DNA ministrings in vivo, later purified for gene transfer applications. We report further development in DNA ministring production. By encoding a homing endonuclease under control of a separate promoter, and its respective target site onto the backbone of the parent plasmid, we eliminate residual parent plasmid and LCC backbone, simplifying ministring purification.We also began construction of a novel platform utilizing filamentous phage M13 to produce phage-encapsulated DNA ministrings as a one-step therapeutic production system, termed BEAM (Bacteriophage Encapsulated & Assisted Ministring) (Fig. 1B). Phages are exploited for drug delivery because they are safe, easy to produce, and genetically versatile. Since M13 packages single-stranded DNA (ssDNA), the parent plasmid must produce an ssDNA molecule that encodes both the ministring base sequence and its complementary sequence, which can anneal to form the double-stranded DNA (dsDNA) ministring prior to packaging. We report our initial developments for this plasmid.Last, we examine the applicability of DNA ministrings towards ovarian cancer therapy. We encoded negative dominant topoisomerase alleles, hypothesized to produce DNA-damaging derivatives mimicking the effect of common chemotherapeutics such as topotecan. We present the results of ministring delivery of such alleles in the treatment of ovarian cancer cell lines. View Large Image | Download PowerPoint Slide