For a new technology, siRNAs have moved into the clinic at an unprecedented pace. Some examples of the diseases and siRNA-targeting strategies that are currently under investigation are described below.
The first siRNA protocol granted investigational new drug (IND) status and tested in a human clinical trial is the vascular endothelial growth factor (VEGF
)-targeted siRNA Bevasiranib (Acuity Pharmaceuticals, Philadelphia, Pennsylvania) for the treatment of wet age-related macular degeneration (see for a summary of ongoing siRNA clinical trials). This involves the overgrowth of blood vessels behind the retina, and causes severe and irreversible loss of vision; it affects 1.6 million people in the United States alone, and it is predicted that 11 million individuals worldwide will have the disease by 2013. Preclinical studies of Bevasiranib in mice showed reduced neovascularization resulting from downregulation of Vegf
expression after direct ocular injection of the siRNA69
. This siRNA, which is now in a phase III trial, is also in a phase II clinical trial for the treatment of diabetic macular oedema. By the conclusion of these trials, several hundred patients will have received the siRNA treatments.
Current clinical trials of RNAi-based therapeutics
Two other companies are also focusing on siRNA-based treatments against macular degeneration: Merck’s Sirna Therapeutics (San Francisco, California) with an siRNA (Sirna-027) that targets the VEGF receptor VEGFR1, and Quark Pharmaceuticals (Fremont, California) in collaboration with Silence Therapeutics (London and Berlin; previously SR Pharma), with one targeted against a hypoxia-inducible gene, RTP801 (also known as DDIT4), that is known to be involved in disease progression. This siRNA, RTP801i-14, has been licensed to Pfizer, UK, which is now running a phase I/IIA clinical trial. Quark Pharmaceuticals has also received IND status for another preclinical trial, in which it is currently enrolling patients. This trial is for an siRNA targeting TP53 mRNA (which encodes the protein p53), inhibition of which delays the induction of cell-death pathways and thereby reduces acute kidney injury after surgery.
Calando Pharmaceuticals (Pasadena, California), meanwhile, has initiated a phase I clinical trial for solid tumours using an siRNA that targets a subunit of ribonucleotide reductase (RRM2), an enzyme required for the synthesis of DNA building blocks. Importantly, this trial is the first to utilize receptor-mediated delivery of siRNAs, which are encapsulated in cyclodextrin particles decorated with transferrin. This results in uptake by cells expressing the transferrin receptor, which is highly expressed on cancer cell surfaces.
The clinical trials performed by Acuity Pharmaceuticals and Merck’s Sirna Therapeutics successfully stabilized patients’ conditions against further degeneration and improved their vision without adverse effects. These results engendered great optimism for intravitreal injection of siRNAs, but in a stunning turn of events a report by Kleinman et al
. demonstrated that the observed decrease in vascularization could be a consequence not of an siRNA-specific effect on angiogenesis, but rather a nonspecific activation of Toll-like receptor 3 (TLR3) and subsequent activation of interferon-γ and interleukin 12, which, in turn, downregulate VEGF70
. In other words, both the targeted and the control siRNAs mediated nonspecific inhibition of angiogenesis through a direct interaction of the siRNAs with TLR3. Cellular uptake is not necessary for this effect, and because TLR3 is involved in several other cellular pathways the finding has highlighted another level of concern for safe clinical use of siRNAs.
Alnylam Pharmaceuticals (Cambridge, Massachusetts) is a well-established siRNA-therapeutics company whose leading candidate siRNA, ALN-RSV01, is now in a phase II clinical trial. This siRNA targets respiratory syncytial virus — which affects almost 300,000 people every year in the United States alone — by silencing the virus’s nucleocapsid ‘N’ gene, a gene essential to viral replication. ALN-RSV01 was the first antiviral siRNA to enter clinical trials, and trials will soon be expanded to paediatric patients. Thus far it has been shown to be effective and well tolerated. Recently, Alnylam Pharmaceuticals formed an exclusive alliance with Kyowa Hakko Kogyo to develop and commercialize ALN-RSV01 in Japan and other Asian countries.
Also in development at Alnylam Pharmaceuticals are siRNAs directed against genes implicated in hypercholesterolaemia, Huntington’s disease (in a joint venture with Medtronic of Minneapolis, Minnesota), hepatitis C (in a joint venture with Isis Pharmaceuticals in Carlsbad, California), progressive multifocal leukoencephalopathy (in a joint venture with Biogen Idec of Cambridge, Massachusetts) and pandemic flu (in a joint venture with the Swiss company Novartis).
The International Pachyonychia Congenita Consortium (IPCC), in collaboration with TransDerm (Santa Cruz, California), has developed an siRNA to allow the correct production of keratin as a treatment for a rare skin disorder called pachyonychia congenita.
The City of Hope National Medical Center in Duarte, California, in collaboration with Benitec (Melbourne, Australia), has started a phase I trial for the treatment of AIDS lymphoma. This trial uses a Pol III promoter-expressed shRNA targeting the HIV tat
shared exons. The shRNA has been incorporated into an HIV-based lentiviral vector, which in turn has been used to insert the shRNA gene (along with two other RNA-based anti-HIV genes) into blood stem cells71
. The gene-modified stem cells have been infused into HIV-positive patients in a trial that uses autologous bone marrow transplantation to treat AIDS-related lymphomas. Four patients have now been treated in this trial.
As indicated above, partnerships have become quite accepted in the field of siRNA biotechnology. These consortia are considerably increasing the capital available for these efforts and are shortening the time involved in commercializing siRNA-based drugs.
Some companies, such as Regulus Therapeutics (Carlsbad, California), have chosen to focus on miRNAs as therapeutic targets. Santaris Pharma in Hørsholm, Denmark, has recently started the first phase I trial to target a human miRNA (miR-122). In this trial, miR-122 is being targeted for downregulation with a locked nucleic acid (LNA) anti-miRNA (SPC3649). LNA is a backbone modification that enhances the hybridization of the oligonucleotide with its target and protects it from nuclease degradation. The approach is intended to treat hepatitis C virus infection because miR-122 facilitates replication of this virus in the liver72,73
. Downregulation of miR-122 is also potentially useful in the treatment of hypercholesterolaemia. Targeting miRNAs expressed in the heart, such as miR-208, which regulates cardiac hypertrophy and fibrosis74
, may have an advantage, because in the medical field there is a considerable experience in delivering drugs directly into this organ.
Gain or loss of miRNA function has been linked to the onset and progression of various diseases75-77
. Protein function can be regulated either directly or indirectly by miRNAs, and alterations in miRNA expression can have profound effects on gene regulation. In instances in which disease results from altered miRNA expression, it is conceivable that normal levels could be achieved, either by targeting the specific miRNA if expression is too high or by delivering a miRNA mimic if expression is too low. However, the specificity and efficacy of delivery systems would need to be improved for this goal to be accomplished. Moreover, correct modulation of the targeted miRNA’s expression is not an easy task, and it is not clear whether one miRNA can be specifically targeted without affecting other miRNAs of the same family.
The regulatory complexities of miRNAs should also be taken into consideration when either ablation or restoration of miRNA function is being considered in a therapeutic setting. A single miRNA can regulate the levels of hundreds of proteins78,79
, raising cautionary flags about the consequences of downregulating or ectopically expressing even a single miRNA species.