A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus

Abstract

RNA interference (RNAi) is a natural mechanism regulating protein expression that is mediated by small interfering RNAs (siRNA). Harnessing RNAi has potential to treat human disease; however, clinical evidence for the effectiveness of this therapeutic approach is lacking. ALN-RSV01 is an siRNA directed against the mRNA of the respiratory syncytial virus (RSV) nucleocapsid (N) protein and has substantial antiviral activity in a murine model of RSV infection. We tested the antiviral activity of ALN-RSV01 in adults experimentally infected with wild-type RSV. Eighty-eight healthy subjects were enrolled into a randomized, double-blind, placebo-controlled trial. A nasal spray of ALN-RSV01 or saline placebo was administered daily for 2 days before and for 3 days after RSV inoculation. RSV was measured serially in nasal washes using several different viral assays. Intranasal ALN-RSV01 was well tolerated, exhibiting a safety profile similar to saline placebo. The proportion of culture-defined RSV infections was 71.4 and 44.2% in placebo and ALN-RSV01 recipients, respectively (P = 0.009), representing a 38% decrease in the number of infected and a 95% increase in the number of uninfected subjects. The acquisition of infection over time was significantly lower in ALN-RSV01 recipients (P = 0.007 and P = 0.03, viral culture and PCR, respectively). Multiple logistic regression analysis showed that the ALN-RSV01 antiviral effect was independent of other factors, including preexisting RSV antibody and intranasal proinflammatory cytokine concentrations. ALN-RSV01 has significant antiviral activity against human RSV infection, thus establishing a unique proof-of-concept for an RNAi therapeutic in humans and providing the basis for further evaluation in naturally infected children and adults.

Fig. 1.

Study design for randomized phase II trial of intranasal ALN-RSV01 versus placebo in subjects experimentally infected with RSV. d, study day; h, hours; i.n., intranasal; RSV, inoculation with RSV, with quantity of inoculum administered to subjects in each cohort indicated in the boxes labeled “Cohort 1” and “Cohorts 2–6”; Rx, dosing with ALN-RSV01 or placebo. Cohorts 1–6 consisted of 8, 8, 18, 16, 24, and 14 subjects, respectively. Three subjects (1 active, 2 placebo) were withdrawn from cohort 6 due to food-related gastroenteritis having received one study drug dose and without receiving RSV inoculation. Thus, 88 were evaluated for safety and 85 for antiviral effect and clinical efficacy. Subjects were quarantined from day −2 through day 11. Nasal washes were obtained daily during quarantine except on days −1, 0, and 1 so as not to affect study drug or RSV inoculation.

Fig. 2.

Reverse Kaplan–Meier estimates of RSV infection, according to treatment assignment. Reverse Kaplan–Meier curves of the percentage of subjects infected with RSV are shown for ALN-RSV01 (dashed line) and placebo (solid line), with infection determined either by (A) culture (plaque assay) (P = 0.0069) or (B) qRT-PCR (P = 0.0321). Inoculation with RSV occurred at day 0, whereas daily treatment with intranasal ALN-RSV01 or placebo continued from day −1 through day 3. This thus represents a cumulative plot of infection. When an individual subject first meets the definition of RSV infected, the line rises 1 unit vertically.

Fig. 3.

Quantitative viral and disease measures in subjects treated with ALN-RSV01 or placebo. (A–D) Results shown are for both infected and uninfected subjects combined in each treatment arm, with infection measured either by quantitative culture or qRT-PCR. (A and B) The bars show the mean and standard error for viral AUC (area under the curve is calculated as the sum of the areas of each trapezoid, formed by the borders of the previous daily viral load and the subsequent day’s viral load, from day 2 through day 11) and peak viral load over days 2–11 following viral inoculation on day 0. (C and D) The curves show the mean and standard error for viral load on each day from day 2 to day 11. (E–G) Results are for all subjects in each treatment arm, with individual results (dots) and mean for the group (horizontal dashed line) shown for (E) mean total symptom score, (F) mean total directed physical examination (DPE) score, and (G) mean mucus weight. The components of the symptom scoring and the DPE scoring systems are found in Tables S2 and S3. (H and I) Mean daily symptom score and standard error for ALN-RSV01 or placebo by study day, with results shown for (H) all subjects or (I) infected subjects, with infection confirmed by quantitative culture or qRT-PCR. Inoculation with RSV occurred on day 0, and the period of treatment with either ALN-RSV01 or placebo is shown by the solid horizontal bar.