Abstract
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.
Highlights
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
Lipofection of COS cells with the pPDGFB-NPC1 plasmid DNA resulted in a level of expression of the 180–200 kDa NPC1 protein comparable to the expression produced with the pCMV-NPC1 plasmid DNA (Fig. 1)
The optical density (OD)/μL was 0.16 ± 0.08, in the vehicle treated group with no sex differences, whereas the OD/μL was 1.57 ± 0.02 in the Trojan horse liposomes (THLs) treated group with no sex differences. These anti-drug antibody (ADA) titers are low compared to a significant ADA response, which can produce ADA titers ≫ 100 OD/μL19. This investigation attempts to treat the NPC1m1N null mouse with intravenous plasmid DNA therapy using THLs that target the mouse TfR1, and an expression plasmid DNA, designated pPDGFB-NPC1, encoding the 3.9 kb human NPC1 open reading frame driven by a 1.5 kb promoter taken from the human platelet derived growth factor-B (PDGFB) 5′-flanking sequence
Summary
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. The TfRMAb is conjugated on the surface of the THL and acts as a molecular Trojan horse to ferry the liposome-encapsulated plasmid DNA across both the BBB and the brain cell plasma membrane, followed by delivery of the plasmid DNA to the nuclear compartment[12,13]. Plasmid DNAs as large as 22 kb can be encapsulated in THLs, and genes encoded in such large plasmid DNAs are expressed. A large therapeutic gene such as NPC1 can be placed under the influence of a large promoter that is specific for neurons. 6 week old NPC1m1N mice were treated with weekly IV administration of either vehicle or TfRMAb targeted THLs encapsulating a 8 kb expression plasmid DNA encoding the 1.5 kb PDGFB promoter and the 3.9 kb human NPC1 open reading frame. The IV injection dose was 6 μg plasmid DNA per mouse, which was shown by quantitative PCR to deliver multiple copies of the plasmid DNA per brain cell
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