Functional in vivo delivery of multiplexed anti-HIV-1 siRNAs via a chemically synthesized aptamer with a sticky bridge

Abstract

One of the most formidable impediments to clinical translation of RNA interference (RNAi) is safe and effective delivery of the siRNAs to the desired target tissue at therapeutic doses. We previously described in vivo cell type-specific delivery of anti-HIV small-interfering RNAs (siRNAs) through covalent conjugation to an anti-gp120 aptamer. In order to improve the utility of aptamers as siRNA delivery vehicles, we chemically synthesized the gp120 aptamer with a 3' 7-carbon linker (7C3), which in turn is attached to a 16-nucleotide 2' OMe/2' Fl GC-rich bridge sequence. This bridge facilitates the noncovalent binding and interchange of various siRNAs with the same aptamer. We show here that this aptamer-bridge-construct complexed with three different Dicer substrate siRNAs (DsiRNAs) results in effective delivery of the cocktail of DsiRNAs in vivo, resulting in knockdown of target mRNAs and potent inhibition of HIV-1 replication. Following cessation of the aptamer-siRNA cocktail treatment, HIV levels rebounded facilitating a follow-up treatment with the aptamer cocktail of DsiRNAs. This follow-up injection resulted in complete suppression of HIV-1 viral loads that extended several weeks beyond the final injection. Collectively, these data demonstrate a facile, targeted approach for combinatorial delivery of antiviral and host DsiRNAs for HIV-1 therapy in vivo.

Figure 1

Schematic of anti-HIV-1 gp120 aptamer-DsiRNA conjugates (N-1 design). The aptamer portion of the conjugate binds to gp120. A GC-rich “sticky bridge” facilitates the interchange of three different DsiRNAs (one against the HIV-1 tat/rev transcripts, and two siRNAs targeting the HIV-1 host-dependency factors (CD4 and TNPO3)) with the same aptamer. The 3′-end of the aptamer and one of the two siRNA strands were chemically conjugated with complementary sticky sequences, thereby, allowing the aptamer and siRNA portions to be noncovalently conjugated via Watson-Crick base-pairing by simple mixing. A 7 unit three-carbon linker (C3) between the aptamer/siRNA and stick is indicated in green. For clarity, the N-1 design is shown as an example, in which the 3′-end of the antisense strand of the DsiRNA was appended to a bridge complementary sequence. DsiRNA, Dicer substrate small-interfering RNA.

Figure 2

Aptamer-cocktailed DsiRNA conjugates suppress viral loads in HIV-1–infected RAG-hu mice. HIV-1 viral loads at different weeks post-infection and treatment are indicated. The treatment period is indicated by the yellow framed in region. Weeks post-injection and the time point of treatment start and end are indicated. (a) The first treatment includes five weekly injections: The viral loads of uninfected mice (n = 2), non-treated mice (n = 8), naked cocktail DsiRNA-treated mice (n = 5), and aptamer-cocktailed DsiRNA conjugates treated mice (n = 8) are indicated. (b) The re-treatment including twice weekly injections with aptamer-cocktailed DsiRNA conjugates: The viral loads of uninfected mice (n = 2), non-treated mice (n = 5), and aptamer-cocktailed DsiRNA conjugates treated mice (n = 4) are indicated. P values for both experiments were determined as described in Materials and Methods and Supplementary Table S1c,d. The viral RNA was detected through qRT-PCR as described in Materials and Methods. 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. DsiRNA, Dicer substrate small-interfering RNA; qRT-PCR, quantitative reverse transcription-PCR.

Figure 3

The detection and function of tat/rev siRNA in PBMCs from treated RAG-hu mice. (a,b) Detection of the tat/rev siRNA sequences at weeks (a) 9 and (b) 13 post-infection using naked cocktailed DsiRNAs versus aptamer-cocktailed DsiRNA conjugates treated animals from Figure 2a. The background copy number of siRNA is <10⁷ or 10⁵ (gray). Error bars indicate SD (n = 4 measurement per sampe). (c,d) Expression levels of targeted tat/rev, CD4, and TNPO3 gene transcripts at weeks 8 and 9 post-infection are shown relative to (c) HIV-1–infected, non-treated animal samples and (d) naked siRNA-treated animal samples, respectively. DsiRNA, Dicer substrate small-interfering RNA; PBMC, peripheral blood mononuclear cell; RT, reverse transcriptase.

Figure 4

Aptamer-cocktailed DsiRNA complexes protect against CD4+ T-cell loss in RAG-hu mice. CD4+ T-cell levels were assessed by FACS at each indicated week pre- and post-siRNA treatment. Start and end of treatments are indicated by the yellow framed in region. Mice from the first treatment are depicted in Figure 2a. Uninfected mice (n = 2), non-treated mice (n = 7), naked cocktailed DsiRNA-treated mice (n = 5), and aptamer-cocktailed DsiRNA conjugates treated mice (n = 8) are indicated. P values for the experiment are indicated and were determined as described in Materials and Methods and Supplementary Table S2b. DsiRNA, Dicer substrate small-interfering RNA; FACS, fluorescence-activated cell sorting.

Figure 5

In vivo administration of aptamer-cocktailed DsiRNA conjugates does not induce type I interferon (IFN). (a,b) The expression of type I IFN response genes (P56 and OAS1) at week (a) 8 and (b) 9 post-infection after treatment with naked cocktailed siRNAs and aptamer-cocktailed DsiRNA conjugates. IFN-α–treated, HIV-1–infected human 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 or 24 hours after aptamer-cocktailed DsiRNA conjugates injections as measured by an ELISA are shown. Poly (I:C)-treated infected Rag-hu mice were used as a positive control. Error bars indicate SD (n = 3). DsiRNA, Dicer substrate small-interfering RNA; PBMC, peripheral blood mononuclear cell; RT, reverse transcription.