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. 2011 Jan 19;3(66):66ra6.
doi: 10.1126/scitranslmed.3001581.

An aptamer-siRNA chimera suppresses HIV-1 viral loads and protects from helper CD4(+) T cell decline in humanized mice

Charles Preston Neff  1 , Jiehua Zhou, Leila Remling, Jes Kuruvilla, Jane Zhang, Haitang Li, David D Smith, Piotr Swiderski, John J Rossi, Ramesh Akkina
Affiliations

An aptamer-siRNA chimera suppresses HIV-1 viral loads and protects from helper CD4(+) T cell decline in humanized mice

Charles Preston Neff et al. Sci Transl Med. .

Abstract

Therapeutic strategies designed to treat HIV infection with combinations of antiviral drugs have proven to be the best approach for slowing the progression to AIDS. Despite this progress, there are problems with viral drug resistance and toxicity, necessitating new approaches to combating HIV-1 infection. We have therefore developed a different combination approach for the treatment of HIV infection in which an RNA aptamer, with high binding affinity to the HIV-1 envelope (gp120) protein and virus neutralization properties, is attached to and delivers a small interfering RNA (siRNA) that triggers sequence-specific degradation of HIV RNAs. We have tested the antiviral activities of these chimeric RNAs in a humanized Rag2(-/-)γc(-/-) (RAG-hu) mouse model with multilineage human hematopoiesis. In this animal model, HIV-1 replication and CD4(+) T cell depletion mimic the situation seen in human HIV-infected patients. Our results show that treatment with either the anti-gp120 aptamer or the aptamer-siRNA chimera suppressed HIV-1 replication by several orders of magnitude and prevented the viral-induced helper CD4(+) T cell decline. In comparison to the aptamer alone, the aptamer-siRNA combination provided more extensive inhibition, resulting in a significantly longer antiviral effect that extended several weeks beyond the last injected dose. The aptamer thus acts as a broad-spectrum HIV-neutralizing agent and an siRNA delivery vehicle. The combined aptamer-siRNA agent provides an attractive, nontoxic therapeutic approach for treatment of HIV infection.

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Figures

Fig. 1
Fig. 1
Schematic of anti-HIV-1 gp120 aptamer-siRNA chimera (Ch A-1). The aptamer and sense strand segment of the siRNAs contain nuclease-resistant 2′-fluoro uridine triphosphate and 2′-fluoro cytidine triphosphate. The aptamer portion of the chimera binds to gp120 protein, whereas the 27-mer Dicer substrate siRNA duplex is targeted to the HIV-1 tat/rev common exon. A linker (UU) between the aptamer and siRNA is indicated in green.
Fig. 2
Fig. 2
Aptamer A-1 and chimera Ch A-1, but not a mutant chimera, suppress viral loads in HIV-1–infected RAG-hu mice. Mice were infected with HIV-1 at week 0, and 5 weeks later, weekly treatments were started. The treatment period is indicated by the yellow region. (A) Viral loads in uninfected mice (n = 2), untreated mice (n = 5), tat/rev siRNA only–treated mice (n = 6), and ChA-1–treated mice (n = 6). (B) In a separate experiment, viral loads in uninfected mice (n = 3), untreated mice (n = 3), A-1 aptamer–treated mice (n = 3), Ch A-1 chimera–treated mice (n = 3), andmutantA-5 chimera–treated mice (n = 3). The viral RNA was detected through qRT-PCR as described in Materials and Methods. Thus, 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. P values for the effects of A-1 and Ch A-1 are indicated and were calculated as described in Materials and Methods. The data for individual mice are presented in fig. S3 and table S1.
Fig. 3
Fig. 3
The detection and function of tat/rev siRNA in PBMCs from treated RAG-hu mice. (A) Detection of the siRNA sequences in PBMCs during (weeks 5 and 7 after infection) and after (week 12) treatment with Ch A-1 and siRNA only, and in untreated mice and uninfected mice (animals from Fig. 2A). One minus RT control, J274 (−RT), also was set up. The synthetic 27- and 21-mer tat/rev siRNAs were used as positive controls. The background copy number of siRNA is <106 (gray area). Error bars indicate SD (n = 4 measurements per sample). Some of the error bars are not discernible in the graph because their relative percentage was less than 5%. (B) Expression of tat/rev gene transcripts at the first and third weeks of treatment (weeks 5 and 7 after infection) with Ch A-1 and siRNA only and in untreated and uninfected animals (animals from Fig. 2A). Gene expression was normalized to that of a representative HIV-1–infected untreated mouse.
Fig. 4
Fig. 4
5′-RACE PCR analysis of in vivo tat/rev siRNA delivered by Ch A-1. (A) Nested PCR products were resolved in an agarose gel; specific siRNA-mediated RACE PCR cleavage mRNA products are marked by a red arrow. (B) DNA sequence analyses of cloned RACE PCR products. The positions of the two different siRNA-directed cleavage sites in the tat/rev target RNA are indicated. nt, nucleotides.
Fig. 5
Fig. 5
Protection of RAG-hu mice from CD4+ T cell loss by the Ch A-1 chimera. CD4+ T cell levels were assessed by FACS at each indicated week before and after siRNA treatment. BL, baseline measurement. Treatment duration is indicated by the yellow region. (A) Mice from the first experiment (shown in Fig. 2A) group. Uninfected mice (n = 2), untreated mice (n = 5), tat/rev siRNA–treated mice (n = 6), and chimera Ch A-1–treated mice (n = 6). (B) Mice from second experiment (shown in Fig. 2B). CD4-CD3 T cell ratios were assessed by FACS at each indicated week before and after treatment. P values for both experiments were determined as described in Materials and Methods. For baseline measurements, each individual mouse was bled twice before HIV-1 infection and the CD4-CD3 values were averaged within treatment groups to establish a baseline CD4-CD3 level. The data for individual mice are presented in fig. S4 and table S2.
Fig. 6
Fig. 6
Effect of in vivo administration of Ch A-1 on type I IFN. (A) The effect of aptamer A-1 and chimera A-1 on expression of type I IFN response genes (p56 and OAS1) at week 7 after infection. IFN-α-treated, infected human PBMCs were used as a positive control. Gene expression was normalized to the gapdh mRNA. Error bars indicate SD (n =4). (B) Expression of IFN-α 2 or 24 hours after RNA injection as measured by an ELISA. Poly I:C-treated, HIV-infected RAG-hu mice were used as a positive control. Error bars indicate SD (n = 3).
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