Significance: Niemann Pick Type C1 (NPC1) is a devastating degenerative orphan disease of the brain caused by mutations in the NPC1 gene, which encodes for an intracellular membrane cholesterol transporter. There is currently no approved treatment for NPC1. A potentially curative treatment is gene therapy aimed at delivery of a gene encoding the NPC1 transporter. However, the NPC1 gene is too large for the vector backbone of current viral vectors for gene therapy. An alternative approach is non-viral gene therapy of NPC1, 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 20 kb, can be encapsulated in the interior of a 100-150 nm liposome. The surface of the liposome is conjugated with several thousand strands of polyethyleneglycol (PEG), a process called pegylation. The tips of 1-2% 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 R&D stage over the last 15 years, and has been reduced to practice in multiple animal models of neural disease, including a lysosomal storage disorder, experimental Parkinson'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 R&D stage to a commercial stage that can support human clinical trials of NPC1. 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-5 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 120 nm.
Specific Aims : First, the HIRMAb and a plasmid DNA encoding the human NPC1 cDNA will be produced to support THL manufacturing at a 100X scale-up over past R&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, NPC1 gene delivery in brain and peripheral organs, anti-drug antibody response, and tissue histology, at 3 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 C1, an autosomal recessive, progressive, lethal neurodegenerative disease, for which there is no current therapy.
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 C1 disease.