Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Dec;19(12):2228-38.
doi: 10.1038/mt.2011.207. Epub 2011 Sep 27.

Systemic administration of combinatorial dsiRNAs via nanoparticles efficiently suppresses HIV-1 infection in humanized mice

Jiehua Zhou  1 , C Preston Neff, Xiaoxuan Liu, Jane Zhang, Haitang Li, David D Smith, Piotr Swiderski, Tawfik Aboellail, Yuanyu Huang, Quan Du, Zicai Liang, Ling Peng, Ramesh Akkina, John J Rossi
Affiliations

Systemic administration of combinatorial dsiRNAs via nanoparticles efficiently suppresses HIV-1 infection in humanized mice

Jiehua Zhou et al. Mol Ther. 2011 Dec.

Abstract

We evaluated the in vivo efficacy of structurally flexible, cationic PAMAM dendrimers as a small interfering RNA (siRNA) delivery system in a Rag2(-)/-γc-/- (RAG-hu) humanized mouse model for HIV-1 infection. HIV-infected humanized Rag2-/-γc-/- mice (RAG-hu) were injected intravenously (i.v.) with dendrimer-siRNA nanoparticles consisting of a cocktail of dicer substrate siRNAs (dsiRNAs) targeting both viral and cellular transcripts. We report in this study that the dendrimer-dsiRNA treatment suppressed HIV-1 infection by several orders of magnitude and protected against viral induced CD4(+) T-cell depletion. We also demonstrated that follow-up injections of the dendrimer-cocktailed dsiRNAs following viral rebound resulted in complete inhibition of HIV-1 titers. Biodistribution studies demonstrate that the dendrimer-dsiRNAs preferentially accumulate in peripheral blood mononuclear cells (PBMCs) and liver and do not exhibit any discernable toxicity. These data demonstrate for the first time efficacious combinatorial delivery of anti-host and -viral siRNAs for HIV-1 treatment in vivo. The dendrimer delivery approach therefore represents a promising method for systemic delivery of combinations of siRNAs for treatment of HIV-1 infection.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Structure of a flexible poly(amidoamine) (PAMAM) dendrimer with a triethanolamine core. At pH ≤7.4, the terminal amino groups (NH2) possess positive charges via protonation. For clarity, the generation 3 (G3) dendrimer is shown as an example.
Figure 2
Figure 2
Dendrimer-dicer substrate siRNAs (dsiRNA) nanoparticles mediate specific gene silencing in vitro. Inhibition of CD4 expression by dendrimer G5-mediated delivery of 21-mer or 27 mer anti-CD4 siRNAs (at an N/P ratio of 5) was assayed using quantitative real-time (qRT)-PCR 2 days post-treatment in culture (a) CEM T-cells and (b) human peripheral blood mononuclear cells (PBMCs). Dendrimer–small interfering RNA (siRNA) complexes inhibit HIV-1 infection in (c) CEM T-cells and (d) human PBMCs previously infected with HIV-1. HIV-1 infected cells were incubated at 37 °C with the G5-27-mer anti-tat/rev dsiRNA complexes or G5-cocktail siRNAs (combination of 27-mer anti-tat/rev dsiRNA and anti-CD4 dsiRNA) complexes. The culture supernatants were collected at 3 days following addition of the complexes for HIV p24 antigen analyses. Data represent the average of triplicate measurements of p24 and the average of three replicates.
Figure 3
Figure 3
Dendrimer–dicer substrate siRNAs (dsiRNA) complexes suppress viral loads in HIV-1 infected RAG-hu mice. HIV-1 viral loads at different weeks postinfection and treatment are indicated. The treatment period is indicated by the yellow framed in region. Viral loads at each indicated week pre- and post-treatment. Weeks postinjection and the time point of treatment start and end are indicated. (a) The first-treatment included five weekly injections: The viral loads of uninfected mice (n = 2), nontreated mice (n = 7), naked cocktail dsiRNA-treated mice (n = 5), G5-mutant-tat/rev dsiRNA complex-treated mice (n = 3), G5-tat/rev dsiRNA complexes treated mice (n = 6) and G5-cocktail small interfering RNA (siRNA) complex treated mice (n = 6) are indicated. (b) The retreatment including twice weekly injections with G5-cocktail siRNA complexes: The viral loads of uninfected mice (n = 2), nontreated mice (n = 4), G5-tat/rev dsiRNA complex treated mice (n = 5) and G5-cocktail dsiRNA complex treated mice (n = 1) are indicated. P values for both experiments were determined as described in Materials and Methods section. The viral RNA was detected through quantitative real-time (qRT)-PCR as described in Materials and Methods section. If there was no detectable viral RNA we established this as a value of 1 (100) to allow for the use of logarithmic values on the y-axis.
Figure 4
Figure 4
The detection and function of small interfering RNAs (siRNAs) in blood cells of HIV-1 infected RAG-hu mice and in vivo biodistribution. (a,b) Detection of the tat/rev small interfering RNA (siRNA) sequences at weeks 9 and 13 postinfection usin naked siRNAs versus G5–dicer substrate siRNAs (dsiRNA) complex treated animals from Figure 3a. The background copy number of siRNA is <107 (gray). Error bars indicate SD (n = 4). (c,d) Expression levels of targeted tat/rev, CD4, and TNPO3 gene transcripts at weeks 8 and 9 postinfection are shown relative to HIV-1 infected, (c) nontreated animal samples and (d) naked siRNA-treated animal samples, respectively. (e) In vivo biodistribution analyses. The delivery of the anti-tat/rev siRNA was monitored using Taqman quantitative real-time (qRT)-PCR on RNAs isolated from various tissues and peripheral blood mononuclear cells (PBMCs) following systemic administration of the G5 dendrimer-dsiRNA nanoparticles in RAG-hu mice. The background copy number of siRNA is <5 × 106 (gray). Error bars indicate SD (n = 3).
Figure 5
Figure 5
Dendrimer–dicer substrate siRNAs (dsiRNA) complexes protect RAG-hu mice from CD4+ T-cell loss. CD4+ T-cell levels were assessed by fluorescence-activated cell sorting (FACS) at each indicated week pre- and post-small interfering RNA (siRNA) treatment. Start and end of treatments are indicated by the yellow framed in region. Mice from the first treatment depicted in Figure 3a. Uninfected mice (n = 2), nontreated mice (n = 7), G5-mutant-tat/rev dsiRNA complex treated mice (n = 3), G5-tat/rev dsiRNA complex-treated mice (n = 6) and G5-cocktail dsiRNA complex-treated mice (n = 6) are indicated. P values for the experiment are indicated and were determined as described in Materials and Methods section. BL (base line): each individual mouse was bled two times before HIV-1 infection and the CD4:CD3 levels were averaged within treatment groups to establish a baseline CD4:CD3 level.
Figure 6
Figure 6
In vivo administration of dendrimer–dicer substrate siRNAs (dsiRNA) complexes do not induce interferon or toxicities. (a,b) The expression of type I interferon response genes (P56 and OAS1) at (a) week 8 and (b) 9 postinfection after treatment with siRNAs and G5–dsiRNA complexes. Interferon (IFN)-α treated, HIV-1-infected human peripheral blood mononuclear cells (PBMCs) were used as a positive control. Gene expression was normalized to the gapdh mRNA. Error bars indicate SD (n = 4). (c) The expression levels of IFN-α at 2 hours or 24 hours after G5-dsiRNA nanoparticle injections as measured by an enzyme-linked immunosorbent assay (ELISA) are shown. Poly (I:C) treated infected Rag-hu mice were used as a positive control. Error bars indicate SD (n = 3). (d) The levels of LDH (lactate dehydragenase) after G5-dsiRNA nanoparticles injections are shown. Cellular lysate-treated human PBMCs were used as a positive control. The data are cumulative from three mice per experimental group.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE., and, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806–811. - PubMed
    1. Zamore PD, Tuschl T, Sharp PA., and, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000;101:25–33. - PubMed
    1. Kim DH., and, Rossi JJ. Strategies for silencing human disease using RNA interference. Nat Rev Genet. 2007;8:173–184. - PubMed
    1. de Fougerolles A, Vornlocher HP, Maraganore J., and, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov. 2007;6:443–453. - PMC - PubMed
    1. Rossi JJ. RNAi as a treatment for HIV-1 infection. BioTechniques. 2006;Suppl:25–29. - PubMed

Publication types

MeSH terms

Substances