Sayda M. Elbashir

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called ‘non-druggable’ targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV).

Small interfering RNAs (siRNAs) work through RNA interference (RNAi), the natural RNA inhibitory pathway, to down-regulate protein production by inhibiting targeted mRNA in a sequence-specific manner. ALN-RSV01 is an siRNA directed against the mRNA encoding the N-protein of respiratory syncytial virus (RSV) that exhibits specific in vitro and in vivo anti-RSV activity. The results of two safety and tolerability studies with ALN-RSV01 involving 101 healthy adults (65 active, 36 placebo, single- and multiple dose, observer-blind, randomized dose-escalation) are described. Intranasal administration of ALN-RSV01 was well tolerated over a dose range up through 150mg as a single dose and for five daily doses. Adverse events were similar in frequency and severity to placebo (normal saline) and were transient, mild to moderate, with no dose-dependent trend. The frequency or severity of adverse events did not increase with increasing ALN-RSV01 exposure. All subjects completed all treatments and assessments with no early withdrawals or serious adverse events. Physical examinations, vital signs, ECGs and laboratory tests were normal. Systemic bioavailability of ALN-RSV01 was minimal. ALN-RSV01 appears safe and well tolerated when delivered intranasally and is a promising therapeutic candidate for further clinical development.

RNA Interference-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Antiviral Strategy

ABSTRACT We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.

Activation of LDL Receptor Expression by Small RNAs Complementary to a Noncoding Transcript that Overlaps the LDLR Promoter

Low-density lipoprotein receptor (LDLR) is a cell-surface receptor that plays a central role in regulating cholesterol levels. Increased levels of LDLR would lead to reduced cholesterol levels and contribute to strategies designed to treat hypercholesterolemia. We have previously shown that duplex RNAs complementary to transcription start sites can associate with noncoding transcripts and activate gene expression. Here we show that duplex RNAs complementary to the promoter of LDLR activate expression of LDLR and increase the display of LDLR on the surface of liver cells. Activation requires complementarity to the LDLR promoter and can be achieved by chemically modified duplex RNAs. Promoter-targeted duplex RNAs can overcome repression of LDLR expression by 25-hydroxycholesterol and do not interfere with activation of LDLR expression by lovastatin. These data demonstrate that small RNAs can activate LDLR expression and affect LDLR function.