Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes

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.

Figure 1

Human NPC1 Western blot of COS cell lysates removed 2 days following cell lipofection with Lipofectamine 2000 and 2 μg DNA per dish of either no plasmid (control), pPDGF-B-NPC1 plasmid DNA, or pCMV-NPC1 plasmid DNA. Duplicate lanes of each are shown. Lipofection with either NPC1 expression plasmid produces a 180–200 kDa immunoreactive protein, which corresponds with the size of the human NPC1 protein.

Figure 2

ELISA results of binding of either unconjugated recombinant TfRMAb or the DSPE-PEG²⁰⁰⁰-TfRMAb conjugate. The latter is formed following solubilization of TfRMAb-conjugated THLs with 0.2% Triton X-100. The thiolated TfRMAb forms a thio-ether linkage at the maleimide tip of the DSPE-PEG2000 phospholipid. The ED50 of binding was determined by non-linear regression analysis of the binding data (“Methods” section), and converted to nM based on the 150,000 Da molecular weight of the TfRMAb.

Figure 3

H&E stains of cerebral cortex (A–C), spleen (D–F), and liver (G–I) from NPC1^−/− mice or control mice at euthanasia. Panels (A, D, G) are NPC1^−/− mice treated with vehicle; panels (B, E, H) are NPC^−/− mice treated with TfRMAb/pPDGFB-NPC1 THLs. Panels (C, F, I) are control mice. Arrows in panels (A, D, G) point to cholesterol-laden cells in brain, spleen, and liver, respectively. Brain sections are through cortical layers I–IV. Magnification is the same for all panels and the panel A magnification bar = 100 microns.

Figure 4

Electron microscopy of cerebral cortex of vehicle (A, B) or THL (C, D) treated NPC1^−/− mouse at euthanasia. Multi-lamellated vacuoles of varying size are present in the cytoplasm of brain cells. Magnification in panels (A) and (C) is the same and magnification bar in panel (A) is 1,000 nm; magnification in panels (B) and D is the same and magnification bar in panel (B) is 1,000 nm.

Figure 5

GFAP immunocytochemistry of thalamus of NPC1^−/− mouse treated with vehicle (A) or the TfRMAb/pPDGFB-NPC1 THLs (B). The GFAP immunocytochemistry of thalamus from a control mouse is shown in (C). Sections are counter-stained with hematoxylin. Magnification of all 3 panels is the same and the panel (A) magnification bar = 100 microns.

Figure 6

NPC1 Western blot of the spleen of 3 vehicle treated NPC^−/− mice and 3 THL treated NPC^−/− mice. The spleens of the THL treated mice express the 200 kDa immunoreactive NPC1 protein.