Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants

Abstract

Human Immunodeficiency Virus-type 1 (HIV-1) binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA) transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV-1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA) sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α), a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

Keywords: HIV-1; heterosexual transmission; nanoparticles.; virus infection; women.

Figure 1

Timeline of the experimental protocols using human female reproductive tract explants. Samples of female reproductive tract tissues were received, dissected, and placed into culture on day 0, the same day they were transfected with nanoparticles containing siRNA. On day 2, the explants were thoroughly washed to remove residual siRNA nanoparticles, and some sections were exposed to cell-free HIV-1BaL. On day 3, the HIV-exposed explants were again thoroughly washed to remove any unincorporated HIV-1. CCR5 and CD4 mRNA levels were measured on uninfected ectocervical explants on days 1, 2, 3, 4, 6, and 8, and only on days 3 and 6 in explants from endometrium and endocervix. HIV-1 DNA was measured on the HIV-1 exposed explant cultures on day 8.

Figure 2

siRNA suppresses both CD4 and CCR5 expression in human female reproductive tract explants. Female reproductive tract tissue explants from the endometrium (EM), endocervix (CX) and ectocervix (ECX) were treated with nanoparticles containing either an irrelevant siRNA (20 nM) or a combination of CCR5- and CD4-specific siRNA (10 nM each, black bars). Shown are CD4 (Panel A) and CCR5 (Panel B) expression on days 3 and 6 for EM and CX, and days 3, 4, 6 and 8 for ECX. The data represent the average of the mean relative expression of 6 subjects for CD4 and 4 subjects for CCR5. The expression from each subject was assayed in triplicate, normalized to GAPDH expression, and expressed relative to values for explants treated with irrelevant siRNA from that patient (irrelevant siRNA control = 100%). The bars represent the average of the relative mean expression levels from 4 or 6 subjects and the error bars are the standard deviation of the means. A single asterisk (*) denotes statistically significant differences at P<0.05 and double asterisks (**) denotes statistically significant differences at P<0.005 between explants treated with irrelevant and receptor-specific siRNAs. Bars with no asterisk did not reach statistical significance at the 95% confidence level.

Figure 3

Timeline of siRNA effects on CD4 and CCR5 expression in human female reproductive tract ectocervical explants. Ectocervical explants were treated with siRNA nanoparticles containing irrelevant siRNA (20 nM, thin line), the combination of both CD4- and CCR5-specific siRNAs (10 nM each, heavy line), or left untreated (dashed line). On days 1, 2, 3, 4, 6 and 8 of culture, tissues were harvested and assessed for levels of CD4 transcripts (panel A, top) or CCR5 transcripts (panel B, bottom). The data is from a single representative subject and shows transcript levels in arbitrary units normalized to expression of GAPDH. Error bars show standard deviation of triplicate assays.

Figure 4

Nanoparticles containing CCR5 and CD4 specific siRNAs inhibit HIV-1 reverse transcription in female reproductive tract explants. Female reproductive tract tissue explants from the endometrium (EM), endocervix (CX) and ectocervix (ECX) were either left untreated (grey bars), or treated with nanoparticles containing irrelevant siRNA (20 nM, white bars), or the combination of both CCR5 and CD4-specific siRNA (10 nM each, black bars) for 48 hrs prior to exposure to HIV-1BaL. Explants were assessed for HIV-1 reverse transcripts 5 days after virus exposure. Data are displayed by setting the levels of HIV-1 DNA from the irrelevant siRNA treated explants to 100%. Data are a compilation from four subjects, each of which was assayed in triplicate. Error bars show the standard deviation of the means relative to the irrelevant siRNA control. The single asterisk (*) denotes statistically significant differences among bracketed bars at the P<0.05 level, and the double asterisks (**) denote significance at the P<0.005 level. Bars with no asterisks did not reach statistical significance at the 95% confidence level.

Figure 5

Nanoparticles containing siRNA to murine Cd4 reduce Cd4 expression but not Cd45 expression in murine uterine tissues. Nanoparticles containing siRNA to an irrelevant sequence or to murine Cd4 (10 µg/mouse) were instilled directly into each uterine horn. On days 1, 2, 3 and 5, the mice were euthanized and uterine tissues assessed by real-time PCR for Cd4 expression (Panel A) or for Cd45 expression (Panel B) in triplicate and normalized to murine β-actin expression. Average relative expression of Cd4 or Cd45 was compared between groups of mice treated with the irrelevant siRNA control (white bars) or Cd4-specific siRNA (grey bars). Two mice are represented in each group for days one and two and four mice are represented in each group for days 3 and 5. Error bars show the standard deviation of the means among the mice in each group. A statistically significant difference (as denoted by a single asterisk) between the irrelevant and Cd4-specific siRNA treated groups was observed on day 3 (P<0.05). Bars with no asterisk did not reach statistical significance at the 95% confidence level.

Figure 6

siRNA transfection upregulates IFN-α expression. A) IFN-α secreted by PBMC left untreated (grey bars) or treated with nanoparticles containing irrelevant siRNA (20 nM, white bars) or the combination of CCR5 & CD4-specific siRNA (10 nM each, black bars) were quantified at time 0, day 1, day 2, and day 4 post-transfection. Error bars show the standard deviation of the replicates. Asterisks (***) denote statistically significant differences (P<0.001) in IFN-α production between untreated and siRNA-treated PBMCs. B) siRNA stimulates IFN-α transcription in ectocervical explants. Total RNA was extracted from ECX explants on days 1, 2 and 4 either left untreated (grey bars), or after transfection with nanoparticles containing irrelevant siRNA (20 nM, white bars), or CCR5 plus CD4 specific siRNA (10 nM each, black bars). Levels are expressed as mean IFN-α transcripts relative to that observed at culture initiation (day 0). Error bars the show standard deviation of the means. Asterisks (*) denote statistically significant differences (P<0.05) between explants treated with siRNA as compared to untreated explants. Bars with no asterisk did not reach statistical significance at the 95% confidence level.