Carbosilane dendrimers to transfect human astrocytes with small interfering RNA targeting human immunodeficiency virus.
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 microg/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.
Jiménez
Jose Luis
JL
Plataforma de Laboratorio, Hospital General Universitario Gregorio Marañón, C/Doctor Esquerdo 46, Madrid, Spain.
Clemente
María Isabel
MI
Weber
Nick D
ND
Sanchez
Javier
J
Ortega
Paula
P
de la Mata
F Javier
FJ
Gómez
Rafael
R
García
Dolores
D
López-Fernández
Luis A
LA
Muñoz-Fernández
María Angeles
MA
D015526:AIDS Dementia Complex / Q000235:genetics / Q000473:pathology / Q000628:therapy; D000328:Adult; D000818:Animals; D019380:Anti-HIV Agents / Q000627:therapeutic use; D001253:Astrocytes / Q000502:physiology / Q000821:virology*; D001812:Blood-Brain Barrier / Q000166:cytology / Q000502:physiology / Q000821:virology; D002417:Cattle; D002478:Cells, Cultured; D002648:Child; D050091:Dendrimers / Q000633:toxicity; D055808:Drug Discovery; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000633:toxicity; D006678:HIV / Q000235:genetics*; D006801:Humans; D034741:RNA, Small Interfering; D012821:Silanes / Q000633:toxicity; D052416:Targeted Gene Repair; D014779:Virus Replication / Q000235:genetics*
D019380:Anti-HIV Agents; D050091:Dendrimers; D034741:RNA, Small Interfering; D012821:Silanes; C504072:carbosilane