Platform Delivery Technology for Plasmid DNA Therapeutics

Abstract SignificanceThis research will develop the Trojan horse liposomeTHLplatform technology for delivery to organs in vivo of non viral plasmid DNA therapeuticsTHLsin combination with tissue specific gene promotersenable the expression of the therapeutic gene in distant sites of the bodyincluding the brain following a non invasive intravenous administrationThe technology is particularly suited to therapeutic DNA delivery to brain across the blood brain barrierBBBThe THL platform can deliver an unlimited number of therapeutic genes for treatment of monogenic diseasessuch as severe inborn errors of metabolism and orphan diseasesor inherited blindnessas well as polygenic disease such as neurodegenerationTHLs can deliver plasmid DNA encoding short hairpin RNA to enable RNA interferenceHypothesisTHLs have been investigated previouslybut only at the microscale Randamp D levelThe present research plan is based on the hypothesis that THL manufacturing can be scaled by orders of magnitudefrom the Randamp D stageand that manufactured THLs will have an acceptable shelf life to enable commercializationThis is achievable with the combined use of a pressurized stainless steel extruder and re formulation of THLs as a lyophilized freeze dried preparationTHLs made with this new manufacturing model will be reduced to practice with a `use case andaposwhich will treat the mouse model of Krabbe diseasea lysosomal storage disease caused by a missense mutation in the gene encoding the galactosylceramidaseGALClysosomal enzymePreliminary DataPrior work has shown that THLs can be targeted to brain with monoclonal antibodiesMAbagainst either the insulin receptor or the transferrin receptorTfRfor delivery of plasmid DNA to either the monkey brain or the mouse brainincluding mouse models of lysosomal storage diseaseSpecific AimsPhase IA plasmid DNA encoding the human GALC enzyme will be genetically engineered under the influence of akb gene promoterin parallel with production of the monoclonal antibodyMAbTrojan horseTHLs targeted with the monoclonal antibody and encapsulated with the GALC plasmid DNA will be produced with a large scale extruder device suitable for production of clinical trial quantities of THLsThe biologic activity of the monoclonal antibodytargeted THLsencapsulating the GALC plasmid DNAwill be confirmed in human Krabbe disease fibroblastswith both immune detection of the GALC enzymeas well as measurements of intracellular GALC enzyme activitySpecific AimsPhase IIThe Manufacturing Module is comprised ofGenetic engineering of a humanized form of the targeting MAb andmg gigapreps of low endotoxin GALC plasmid DNAMAb production from either myeloma cells or stably transfected CHO cellsLarge scale THL production with the pressurized extruder and a programmed freeze dry processThe Preclinical Module is comprised ofA collaboration with The Jackson Lab for treatment of a twitcher missense mouse model of Krabbe diseaseA collaboration with a CRO for execution of aweek dose ranging study of THLs in the non human primate Project Narrative Gene therapy is possible with non viral plasmid DNA based therapeuticsonly if a functional platform delivery technology is developedTrojan horse liposomesTHLsprovide the needed platform technology for delivery of plasmid DNA therapeuticsTHLsin combination with tissuespecific gene promoterscan enable the expression of a therapeutic gene in distant targets sites of the bodyincluding the brainfollowing a non invasive intravenous administration