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Carbosilane Dendrimers to Transfect Human Astrocytes with Small Interfering RNA Targeting Human Immunodeficiency Virus

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Abstract

Background

HIV infection of the CNS is the principle cause of HIV-associated dementia in adults and encephalopathy in children. Gene therapy techniques such as small interfering RNA (siRNA) possess great potential in drug development, but first they must overcome the key obstacle of reaching the interior of the affected cells. A successful delivery vector for anti-HIV drugs that is capable of crossing the blood-brain barrier (BBB) could provide a way of addressing this issue. Non-viral vectors such as dendrimers offer a means for effectively delivering and transfecting siRNA to the target cells.

Objective

To evaluate the application of gene therapy for reducing HIV replication in human astrocytes.

Methods

We used the 2G-NN16 amino-terminated carbosilane dendrimer as a method for delivering siRNA to HIV-infected human astrocytes. We tested the cytotoxicity in human astrocytoma cells caused by 2G-NN16 and dendriplexes formed with siRNA (siRNA/2G-NN16) by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium-bromide (MTT) and lactate dehydrogenase assays. The ability to transfect human astrocytes with siRNA/2G-NN16 dendriplexes was tested by flow cytometry and immunofluorescence microscopy. To assess the potential capability of siRNA/2G-NN16 dendriplexes for crossing the BBB, we used an in vitro transcytosis assay with bovine brain microvascular endothelial cells. HIV-1 inhibition assays using 2G-NN16 and siRNA/2G-NN16 dendriplexes were determined by quantification of the viral load from culture supernatants of the astrocytes.

Results

A gradual time-controlled degradation of the 2G-NN16 dendrimer and liberation of its siRNA cargo between 12 and 24 hours was observed via gel electrophoresis. There was no cytotoxicity in HIV-infected or non-infected human astrocytoma cells when treated with up to 24 μg/mL of 2G-NN16 dendrimer or siRNA/2G-NN16 dendriplexes, and siRNA/2G-NN16 dendriplexes were seen to successfully transfect human astrocytes even after crossing an in vitro BBB model. More interestingly, transfected siRNA was observed to exert a biologic effect, as dendriplexes were shown to down-regulate the housekeeping gene GAPDH and to reduce replication of HIV-1 strains X4-HIV NL4-3 and R5-HIV BaL in human astrocytes.

Conclusions

The 2G-NN16 dendrimer successfully delivers and transfects siRNA to HIV-infected human astrocytes and achieves gene silencing without causing cytotoxicity.Both authors have contributed equally to this paper.

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Acknowledgments

This work was supported by grants from Fundación para la Investigación y Prevención del SIDA en España, FIPSE (240800/09), Red Temática de Investigación Cooperativa Sanitaria ISCIII (RETIC RD06/0006/0035), MNT-ERA NET 2007 (NAN2007-31198-E), Fondos de Investigación Sanitaria (FIS PS09/02029; PS09/02669), Fundación Caja Navarra, Co-munidad Autónoma de Madrid (S-SAL-0159-2006), and COST Action (TD0802) to Dr Muñoz-Fernóndez. Authors from the Universidad de Alcaláwere supported by MNT-ERA NET 2007 (NAN2007-31135-E) and Fondo de Investigación Sanitaria (PI040993). Dr José; Luis Jiménez was supported by FIS PI081495 and Programa de Investigación de la Consejería Sanidad de la Comunidad de Madrid, and Dr López-Fernández was supported by FIS CP06/0267 Miguel Servert Program. Maria Isabel Clemente is the holder of a fellowship from Fondo de Investigaciones Sanitarias (FI0501093). We would like to thank Laura Diaz for her assistance with flow cytometry analysis.

The authors have no conflicts of interest that are directly relevant to the content of this study.

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Authors and Affiliations

  1. Plataforma de Laboratorio, Hospital General Universitario Gregorio Marañón, C/Doctor Esquerdo 46, Madrid, Spain

    Jose Luis Jiménez & Javier Sanchez

  2. CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Spain

    Jose Luis Jiménez, María Isabel Clemente, Nick D. Weber, Paula Ortega, F. Javier de la Mata, Rafael Gómez, Dolores García, Luis A. López-Fernández &  María Ángeles Muñoz-Fernández

  3. Laboratorio Inmunobiología Molecular, Hospital General Universitario Gregorio Marañón, C/Dr. Esquerdo 46, 28007, Madrid, Spain

    María Isabel Clemente, Nick D. Weber, Dolores García &  María Ángeles Muñoz-Fernández

  4. Departamento de Química Inorgánica, Universidad de Alcalá, Campus Universitario, Edificio de Farmacia, Alcalá de Henares, Spain

    Paula Ortega, F. Javier de la Mata & Rafael Gómez

  5. Laboratorio de Farmacogenética y Farmacogenómica, Hospital General Universitario Gregorio Marañón, C/Doctor Esquerdo 46, Madrid, Spain

    Luis A. López-Fernández

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  1. Jose Luis Jiménez
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  2. María Isabel Clemente
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  3. Nick D. Weber
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  9. Luis A. López-Fernández
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  10. María Ángeles Muñoz-Fernández
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Correspondence to María Ángeles Muñoz-Fernández.

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Jiménez, J.L., Clemente, M.I., Weber, N.D. et al. Carbosilane Dendrimers to Transfect Human Astrocytes with Small Interfering RNA Targeting Human Immunodeficiency Virus. BioDrugs 24, 331–343 (2010). https://doi.org/10.2165/11538400-000000000-00000

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