Long-term Transgene Expression in the Central Nervous System Using DNA Nanoparticles

Abstract

This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1-3 weeks after injection.

GDNF Gene Delivery via a 2-(Dimethylamino)ethyl Methacrylate Based Cyclized Knot Polymer for Neuronal Cell Applications

“…14,15 Trojan horse liposomes with surface coatings of poly(ethylene glycol) (PEG) and compacted DNA nanoparticles based on PEG substituted lysine peptides have been used to successfully produce GDNF transgene expression in the mammalian central nervous system (CNS). 16,17 PEG modified poly(amido amine) (PAMAM) dendrimers have also been used to deliver the GDNF encoding gene, 18 whereby conjugating targeting peptides (lactoferrin and transferrin) allowed brain uptake of 0.1% and 0.14% (respectively) post systemic delivery. 19 Two different structures of the polymer vector, polyethyleneimine (PEI), have been used to deliver marker genes to the brain by direct injection.…”

intranasal GDNF gene therapy approach for treating Parkinson's disease

“…This suggests that plasmid DNA can transfect cells without the aid of vectors, especially in areas of presumably high tissue concentration, such as observed in frontal brain areas closest to nasal cavity in our study. Others have reported successful transfection after injection of naked pDNA directly into tissue 239,317,318 , although the use of nanoparticle vectors which unimolecularly compact the DNA typically increase transfection efficiency, as we have observed here.…”

“…Although both constructs generated similar levels of GDNF one week after intranasal administration (Figure 25), GDNF expression may have been more sustained over the one month after dosing with the NPs than with the naked plasmid, thereby generating more neuroprotection during the course of lesion development. In support of this conclusion, DNA NPs were previously shown to yield longer--lasting expression than the naked plasmid after injection into the rat brain 239,242 . While an acute, neurotoxin--induced lesion of SN dopamine neurons is not mechanistically or temporally comparable to the gradual, progressive course of human PD, these results confirm the tremendous potential of intranasal pGDNF_1b, and the nanoparticle vector, as means of protecting and rescuing dopamine neurons from a severe neurotoxic insult.…”

“…Another possibility is that microglia may be migrating to these sites by phagocytic attraction to the NPs themselves. This seems less 239 . Deciphering the means of recruitment and the meaning of these clusters of transfected cells will require double--label IHC using a microglial specific antibody, such as ionized calcium binding adaptor molecule 1 (Iba1), to establish whether they are indeed microglia.…”

“…Copernicus specializes in producing 10 kDa polyethylene glycol--substituted lysine 30--mer plasmid DNA nanoparticles (NPs), whose cationic properties allow them to compact a single plasmid of negatively--charged DNA into a very small diameter particle, ranging from just 8--10 nm 239 . Furthermore, these NPs are non--immunogenic and non--inflammatory 284,285 and can enter the nucleus of cells via the nuclear membrane pore complexes 236 .…”