Promising New Frontiers for RNAi Therapy

In 2001 Elbashir and Tuschl (1) published they had managed to silence gene expression in mammalian cells using small interfering RNA (siRNA). This was the catalyst for an explosion of research using siRNA to demonstrate the effect of knocking out targets without the need to identify compounds capable of doing this job. Potentially highly specific, the opportunity to use these as therapeutics was rapidly explored. Ten years later the first clinical trials are coming through; impressively fast.

Probably the largest obstacle to therapeutic siRNA has been delivery, which often is either toxic, or not very effective – as any of us who have tried to transfect siRNA into primary cells will be able to appreciate. Advances in materials science are seemingly solving this problem encapsulating siRNA in nanoparticles, resulting in safe and effective delivery. CALAA-01 (Calando Pharmaceuticals) lead the way  being the first to deliver siRNA therapeutically demonstrating phase I efficacy and safety, as well as localisation to melanoma metastases.

One particular advantage of siRNA is that once delivery has been optimised, it is possible for several different siRNAs for different targets to be contained within on package. This could enable simultaneous delivery of different targets simultaneously effecting different aspects of the same disease, i.e. metastasis and well as tumour growth. By also hitting a known resistance pathway this duel delivery could enable the chemotherapy to be more effective.

A recently published article (2) has done just this, delivering two siRNAs in lipid nanoparticles (known as ALN-VSP) for both VEGF-A (vascular endothelial growth factor-A) and kinesin spindle protein (KSP) for the treatment of advanced solid tumours with liver metastases.  KSP is involved in cancer proliferation and VEGF-A in the growth of new blood vessels.

The study was phase I, dose escalation, on patients who had already been heavily pre-treated with chemotherapy and/or anti VEGF/VEGFR agents with the ALN-VSP administered as 15 minute IV every 2 weeks for 1 month.

The safety profile was very encouraging with ALN-VSP being generally well-tolerated with mainly low-grade fatigue, nausea and fever noted in 15-24% of patients. The lipid nanoparticle of ALN-VSP distributes primarily to the liver and spleen and the delivery was also excellent. Liver biopsies were performed on 12 patients before the first dose and then at 2 and 7 days post dose. qPCR identified VEGF siRNA present in all 12 patients and KSP siRNA present in 11 of the 12.

Of the patients treated, 7 had no disease progression (measured by computerized tomography [CT] scan) after the treatment cycles and continued onto an extension study. One patient in particular with endometrial cancer achieved a complete response after 20 months of treatment

This is clearly fantastic progress for ALN-VSP, and specifically for the handful of patients who were positively affected from participation in the trial. The results from this study also demonstrate the ability for safe delivery of multiple siRNA to specific sites tumour and this extends the promising start for these methods of siRNA delivery which may open up previously un-druggable targets.


1.           Elbashir SM, Harborth J, Lendeckel W, Yalcin a, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–8, 2001.

2.           Tabernero J, Shapiro GI, Lorusso PM, Cervantes A, Schwartz GK, Weiss GJ, Paz-Ares L, Cho DC, Infante JR, Alsina M, Gounder MM, Falzone R, Harrop J, Seila White AC, Toudjarska I, Bumcrot D, Meyers RE, Hinkle G, Svrzikapa N, Hutabarat RM, Clausen V a, Cehelsky J, Nochur S V, Gamba-Vitalo C, Vaishnaw AK, Sah DWY, Gollob J a, Burris H a. First-in-Man Trial of an RNA Interference Therapeutic Targeting VEGF and KSP in Cancer Patients with Liver Involvement. Cancer discovery (January 28, 2013). doi: 10.1158/2159-8290.CD-12-0429.




Identification of a small molecule inhibitor of BLM helicase

BLM helicase is a member of the RecQ helicase family of ATP-dependent proteins that separate the two strands of duplex DNA, allowing DNA replication and repair processes to proceed. Mutations in this protein result in Bloom’s syndrome (BS), characterised by genomic instability, particularly an increased frequency of sister chromatid exchanges (SCEs), and a predisposition to cancer development, amongst other clinical features. Patients often develop leukaemia’s and lymphoma’s although most types of cancer have been observed in this group. BLM has also been demonstrated to be involved in a process known as Alternative Lengthening of the Telomeres (ALT). This is a homologous recombination-mediated mechanism by which new telomeric DNA is synthesised from an existing DNA template. The finer details of this pathway are unknown but it is known that depletion of BLM by siRNA in ALT+ cells results in telomere shortening.

Telomeres are ‘caps’ at the ends of the chromosomes and consist of repetitive DNA sequence and proteins. They function to protect the chromosome end from degradation or association with other chromosomes. The length of the telomere shortens with each cell division, resulting in replicative  senescence when a critical telomere length is reached. Telomeres are maintained in cancer cells either by up regulation of Telomerase, an enzyme that adds telomeric repeats, or via the use of the ALT pathway, enabling cells to proliferate aberrantly. Approximately 10% of all tumours rely on the ALT pathway for their continued growth and 66% of Osteosarcoma tumours are ALT+. Repression of ALT in ALT+ immortal cell lines results in senescence and cell death, making inhibition of this pathway an attractive therapeutic target.

Nguyen et al., recently described the identification of a novel small molecule BLM inhibitor. The group developed a high-throughput screening assay using a labelled partly duplex DNA substrate. Quenching of the substrate was lost upon separation of the two strands, resulting in increased fluorescence with increasing BLM helicase activity. Hits from this primary screen were then confirmed in a helicase gel assay. Medicinal chemistry optimisation of a hit compound resulted in a lead molecule termed ML216 which inhibited BLM with an IC50 value of 3µM. They went on to show that ML216 affects the DNA binding activity of BLM . Further, the group demonstrated that ML216 inhibited the proliferation of cells transfected with BLM, but not the isogenic BLM negative control cells. Treatment with ML216 also sensitised the BLM+ cells to the replication inhibitor, aphidicolin but with no sensitising effect on the BLM negative controls.  A further important observation was that treatment of cells with ML216 increased the frequency of SCE’s observed, but only in the BLM expressing cells. The group also observed that ML216 could inhibit WRN, a related DNA helicase, in in vitro gel assays. However, they found no inhibitory effects on WRN in the cell-based assays conducted, indicating that the primary target of this compound in human cells is BLM. There is currently no available crystal structure of BLM for further analysis of ML216 binding.sw3sw4


The identification of this novel small molecule could now enable studies of the effects of BLM helicase inhibition in ALT+ tumour cells and potentially uncover a new treatment strategy for ALT+ tumours.