Trojan horse liposomes (THLs) are a form of ligandtargeted nanomedicine, where a plasmid DNA is encapsulated in the interior of a 100−150 nm pegylated liposome, and the tips of a fraction of the surface pegylated strands are covalently linked to a receptorspecific monoclonal antibody (MAb) via a thio-ether linkage. The goal of this work was to develop a lyophilization methodology that enables retention of the structure and function of the THLs following the freezedrying/hydration process. THL fusion and leakage of plasmid DNA were observed with several lyoprotectants, including trehalose, hyaluronic acid, γ-cyclodextrin, or sulfobutylether-β-cyclodextrin. However, the use of hydroxypropyl-γ-cyclodextrin, at a 40:1 wt/wt ratio relative to the THL phospholipid, eliminated liposome fusion and produced high retention of encapsulated plasmid DNA and THL-mediated gene expression after lyophilization followed by hydration. The freeze-dried THL cake was amorphous without cavitation, and the diameters and functional properties of the THLs were preserved following hydration of cakes stored for at least six months. Intravenous administration of the hydrated freeze-dried THLs in the Rhesus monkey demonstrated the safety of the formulation. Blood plasmid DNA was measured with a quantitative polymerase chain reaction method, which enabled a pharmacokinetics analysis of the blood clearance of the THL-encapsulated plasmid DNA in the primate. The work shows that optimization of the lyoprotectant enables long-term storage of the MAb-targeted DNA encapsulated liposomes in the freeze-dried state.
Niemann-Pick C1 (NPC1) is a lysosomal cholesterol storage disorder, that severely affects the brain, and is caused by mutations in the NPC1 gene, which encodes an intracellular membrane transporter of non-esterified cholesterol. Therapeutic options for NPC1 are few, and classical enzyme replacement therapy with the recombinant protein is not possible as the NPC1 gene product is an insoluble membrane protein, which increases the need for development of gene therapy for NPC1. While viral based gene therapy is under development, it is important to investigate alternative approaches to brain gene therapy without viral vectors. The present work develops a plasmid DNA approach to gene therapy of NPC1 using Trojan horse liposomes (THLs), wherein the plasmid DNA is encapsulated in 100 nm pegylated liposomes, which are targeted to organs with a monoclonal antibody against the mouse transferrin receptor. THLs were encapsulated with a 8.0 kb plasmid DNA encoding the 3.9 kb human NPC1 open reading frame, under the influence of a 1.5 kb platelet derived growth factor B (PDGFB) promoter. THLs were administered weekly beginning at 6-7 weeks in the NPC1 −/− null mouse, and delivery of the plasmid DNA, and NPC1 mRNA expression in brain, spleen, and liver were confirmed by quantitative PCR. THL treatment reduced tissue inclusion bodies in brain, and peripheral organs, but did not prolong lifespan in these mice. The work suggests that early treatment after birth may be required to reverse this disease model with NPC1 gene replacement therapy. Niemann-Pick C1 (NPC1) disease is an inherited lysosomal storage disorder caused by mutations in the gene encoding the NPC1 protein, which is a 200 kDa intracellular membrane transporter of non-esterified cholesterol. Cell cholesterol accumulation leads to intracellular inclusion bodies, which cause neurodegeneration, and hepatosplenomegaly 1. The diseases causes early mortality in most cases, and is associated with dementia, seizures, ataxia, and decreased audition 1. There is no approved therapy for NPC1. New treatments may be tested in the NPC1 null mouse (NPC1 −/−) such as the NPC1 m1N mouse 2,3 , and the systemic administration of large doses of hydroxypropyl beta cyclodextrin (HPβCD) to NPC1 −/− mice prolongs lifespan 4. HPβCD does not cross the blood-brain barrier (BBB) 5 , and HPβCD administration to NPC1 patients uses intrathecal administration via injections into the lumbar cerebrospinal fluid (CSF) 6. However, intrathecal drug delivery to brain only allows for drug exposure at the CSF surface of the brain 7 , and intrathecal HPβCD has not been approved. NPC1 gene therapy with adeno-associated virus (AAV) serotypes, e.g. AAV9, is possible, particularly with selfcomplementary AAV (scAAV), as these serotypes cross the BBB 8. However, the maximal size of the expression cassette that can be inserted in the scAAV is < 2.3 kb 9 , and the size of the NPC1 open reading frame alone is 3.9 kb. Single stranded AAV (ssAAV) traverses the BBB less efficiently 9,10 , but this viral genome will acce...
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