Brain DNA Therapeutics with Trojan Horse Liposomes

Abstract Significance Niemann Pick Type C NPC is a devastating degenerative orphan disease of the brain caused by mutations in the NPC gene which encodes for an intracellular membrane cholesterol transporter There is currently no approved treatment for NPC A potentially curative treatment is gene therapy aimed at delivery of a gene encoding the NPC transporter However the NPC gene is too large for the vector backbone of current viral vectors for gene therapy An alternative approach is non viral gene therapy of NPC which is the goal of this project However the limiting factor in the drug development of plasmid DNA for brain is the blood brain barrier BBB delivery technology Brain delivery of plasmid DNA therapeutics is possible with the use of the Trojan horse liposome THL technology A plasmid DNA as large as kb can be encapsulated in the interior of a nm liposome The surface of the liposome is conjugated with several thousand strands of polyethyleneglycol PEG a process called pegylation The tips of of the PEG strands is conjugated with a receptor specific monoclonal antibody MAb that targets a receptor mediated transport system on the BBB such as the human insulin receptor HIR The HIRMAb binds the endogenous insulin receptor on the BBB to trigger receptor mediated transport into brain binds the endogenous insulin receptor on brain cells to trigger receptor mediated endocytosis into cells of the brain and the HIRMAb causes triage of the plasmid DNA to the nuclear compartment of brain cells The THL technology has been developed at the Randamp D stage over the last years and has been reduced to practice in multiple animal models of neural disease including a lysosomal storage disorder experimental Parkinsonandapos s disease brain cancer and gene delivery to the retina Hypothesis The hypothesis tested in the present work is that the manufacturing of THLs can be advanced from the Randamp D stage to a commercial stage that can support human clinical trials of NPC This is enabled by the proposed modifications of THL production a use of a large pressurized extruder that can produce liposomes in large volumes and b formulation of the THLs as a freeze dried power to be reconstituted in saline on the day of infusion Preliminary Data Feasibility studies using the C mechanical extruder show that THLs can be successfully manufactured with this device with high levels of DNA encapsulation and final diameters of the THLs of approximately nm Specific Aims First the HIRMAb and a plasmid DNA encoding the human NPC cDNA will be produced to support THL manufacturing at a X scale up over past Randamp D production Second HIRMAb THLs encapsulating the DNA will be produced to support an initial primate study Each lot of HIRMAb plasmid DNA and THL will be assayed with multiple test methods with defined acceptance criteria Third a dose ranging study in adult Rhesus monkeys will be performed to determine the plasma pharmacokinetics NPC gene delivery in brain and peripheral organs anti drug antibody response and tissue histology at doses of THLs infused weekly If successful this work will provide the basis for the first human therapeutic using the THL technology which will seek to deliver to brain the gene that is mutated in Niemann Pick type C an autosomal recessive progressive lethal neurodegenerative disease for which there is no current therapy Project Narrative The development of plasmid DNA as new therapeutics for brain diseases is limited by the technology that enables delivery of the DNA across the blood brain barrier BBB The present work focus on the Trojan horse liposome THL technology for BBB delivery of plasmid DNA and develop a manufacturing methodology that can be later transferred to GMP production of BBB penetrating THL based DNA therapeutics for brain The technology can be used to treat inherited diseases of the brain and the first clinical application will be the treatment of Niemann Pick Type C disease