Gene delivery to differentiated neurotypic cells with RGD and HIV Tat peptide functionalized polymeric nanoparticles
Introduction
Gene therapy is a potential strategy to combat neurodegenerative disorders, such as Parkinson's Disease (PD) [1]. For example, successful delivery of genes encoding glial cell line-derived neurotrophic factor (GDNF) [2] and/or tyrosine hydroxylase (TH) (involved in dopamine synthesis) [3] may prevent disease progression and maintain proper dopamine levels despite cell loss caused by PD. GDNF and TH gene delivery using viral vectors, including adenovirus [3], [4] and adeno-associated virus [5], provided efficient normalization of function and an increase in survival rate of dopamine-producing neurons in rodents. As an ex vivo gene therapy approach, efficient genetic modification of cells was also achieved using recombinant retroviruses [6].
Despite current progress with virus-mediated gene delivery in the CNS, continued development of nonviral vectors is attractive due to the potential for improved safety, reduced immunogenicity, ease of manufacturing and scale up, and the ability to accommodate larger DNA plasmids compared to viral vectors [7], [8]. Unfortunately, synthetic systems such as polymer-, lipid- and peptide-based gene carriers are typically much less efficient in delivering genes to primary neurons compared to viral vectors [9]. Thus, identifying bottleneck(s) to efficient nonviral gene delivery in transfection-resistant neurons and testing of new vectors are priorities.
Polyethylenimine (PEI) is currently the most popular polymer used to deliver genes into various cell types [10], [11], [12], [13], including neurons [10], [11]. PEI is able to condense genes into small nanoparticles [14] and protect the DNA from degradation by nucleases [15]. In addition, the cationic nature of PEI facilitates entry of these gene vectors into cells by binding to negatively charged heparan sulfate proteoglycans on the cell surface [16]. Following internalization, PEI/DNA nanocomplexes are known to efficiently transport toward the perinuclear region of cells [17], [18], including primary neurons [19]. PEI is hypothesized to escape endosomes using a proton-sponge effect [20], [21], although the mechanism is controversial [22].
In this study, dopaminergic SH-SY5Y neuroblastoma cells were used as an in vitro cell model of neurons since they are widely used in PD pathogenesis studies [23], [24] and they can be differentiated into neurotypic cells by treatment with retinoic acid (RA) [25]. The mechanism of gene transfer with PEI/DNA nanocomplexes was compared between transfection-permissive undifferentiated [26] and transfection-restrictive differentiated [26], [27] SH-SY5Y cells. To improve gene delivery into differentiated neurotypic cells, PEI/DNA nanocomplexes were coated with poly(ethylene glycol) (PEG) after complex formation and bioactive ligands, specifically RGD and HIV-1 Tat peptides, were attached to exposed PEG ends to overcome identified cellular barriers.
Section snippets
Cell culture
The SH-SY5Y human neuroblastoma cell line was maintained in a 1:1 mixture of Eagle's minimum essential medium and Ham's F12 nutrient mixture supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml) and 2 mm glutamine. Cells were incubated at 37 °C in a humidified environment with 5% CO2 atmosphere and passaged every third day with a subcultivation ratio of 1:5.
Cell differentiation
Cells were treated with all-trans RA (Sigma, St. Louis, MO) to induce differentiation [25]. RA was
Effect of N/P ratio on gene transfection
We first sought to determine the nitrogen to phosphate ratio (N/P) of PEI/DNA nanocomplexes that led to enhanced transfection of undifferentiated SH-SY5Y cells by measuring YFP expression. PEI/DNA nanocomplexes formulated at N/P=20 demonstrate approximately 20-fold higher percentage of transfected cells compared to DNA alone (Fig. 1, , ANOVA). Complexes produced at N/P=10 and 30 also increase the percentage of transfected cells compared to DNA alone, by 3.4- and 10-fold, respectively (
Discussion
Differentiated cells are often relatively resistant to high level of gene transfection with nonviral vectors [32], [33], [34], [35], a property observed here with differentiated neurotypic cells. To investigate the mechanistic reason(s) for this result, and in the process begin to uncover the bottlenecks to gene delivery into differentiated neurons, we compared the gene delivery process between differentiated and undifferentiated SH-SY5Y cells. Remarkably, differentiated neurotypic cells
Conclusion
We show that modification of nonviral gene vectors with RGD or Tat peptides via covalently attached PEG chains can enhance gene delivery into differentiated neurons by up to 14-fold in vitro. An increase in cellular uptake was observed with RGD- and Tat-modified vectors, and an increase in endosome escape was observed with RGD-modified gene vectors. Further modification of gene vectors to improve their endosome escape and nuclear import may improve gene transfer into differentiated neurons.
Acknowledgements
The authors thank Drs. Suk Jin Hong and Ted Dawson (Johns Hopkins University, Department of Neuroscience) for helpful discussions. Funding was provided by the NSF (BES 9978160 and 0346716), NIH (T32-GM07057) and an ARCS fellowship to J. Suh.
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