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Mastering the gene

Preventing the expression of faulty genes has the potential to treat many diseases

A breakthrough in the biology of RNA interference (RNAi) - a collection of processes involved in gene expression - for which Andrew Z Fire and Craig C Mello were awarded the Nobel Prize in Physiology or Medicine in 2006, is paving the way for a broad new class of human therapeutics.

This recognition of the importance of Fire and Mello's paper, boosted interest within the scientific community in this area of genetics and has led to significant progress in the development of treatments for both age-related macular degeneration (AMD), cancer and neurological indications. Gene expression is the major factor in the process that results in genes becoming specialised to perform a particular task.

It is guided by RNA (a macromolecule with many forms and functions). RNA interference (RNAi) refers to the ways in which this RNA-guided regulation of gene expression is mediated. The natural RNAi process begins within the cytoplasm of cells and drives fragments of RNA known as microRNAs (miRNAs) towards messenger RNA (mRNA) - ie, the RNA that carries information. One promising therapeutic strategy involves introducing synthetic versions of miRNAs into this process, to alter the messages being delivered and therefore prevent the expression of faulty or microbial genes. These synthetic miRNAs are known as short interfering RNAs (siRNA).

Commercial value
In October 2004, Acuity Pharmaceuticals initiated a phase I trial for bevasiranib sodium, a potential therapy for the eye condition neovascular AMD. This was the first therapeutic application of RNAi technology and marked the start of a spate of activity for siRNA therapies for AMD.

Alnylam Pharmaceuticals has completed two phase I trials of ALN-RSV01, a potential intranasal treatment for respiratory syncytial virus (RSV), and began trials of an inhalable formulation late last year. This highlights the broadening therapeutic scope of RNAi-based therapeutics, which looks set to encompass other viral infections, such as influenza and hepatitis, as well as hypercholesterolaemia and cancer.

Merck & Co's acquisition of specialist company, Sirna Therapeutics, for a reported USD 1.1bn in December 2006 is indicative of the significance and size of the potential market for RNAi-based therapeutics. Alnylam, regarded by many as the leader in the field, enjoyed steady increases in share prices over 2006, and share value peaked at USD 24 at the time of the Sirna acquisition. CytRx has steadily gained ground in the RNAi field over the past few years. The company established a subsidiary, RXi Pharmaceuticals, earlier this year to deal with its RNAi interests.

AMD is a degenerative condition of the macula (central retina) affecting about two per cent of people aged over 50 years in developed countries. Neovascular AMD (also known as wet AMD) is much less common than the other form (atrophic or dry AMD), but is more of a threat to sight. In neovascular AMD new, fragile blood vessels leak blood and cause scarring. This is known as choroidal neovascularisation (CNV) and studies have suggested that VEGF (vascular endothelial growth factor) plays a part in this condition. Acuity's bevasiranib sodium (previously known as Cand5) is designed to silence the gene that encodes VEGF. Phase II data included evidence of a dose-dependent reduction of CNV lesions over 12 weeks and an improvement in visual acuity in one-third of patients.

Acuity plans to initiate its phase III programme in 2007 and in March the company agreed a merger with several partners to establish a publicly-traded company, Opko. This transaction reportedly generated USD 28m to facilitate the start of a phase III trial to test bevasiranib sodium in combination with ranibizumab (Lucentis), a high-affinity variant of Genentech's anti-VEGF monoclonal antibody, bevacizumab. If the trial goes as hoped, Opko may lead the race to reach the market with a siRNA drug. Its main opponents are other firms exploring the use of siRNAs for AMD, including Alnylam, which, in collaboration with Merck & Co, was exploring VEGF as a target but the programme was put on hold in 2005 amid increased competition. Opko's main competitor now appears to be Allergan, which recently launched a phase II trial of AGN-211745, the VEGF receptor-1 gene-targeting candidate that it acquired from Sirna.

An alternative prospect in this field may be Pfizer's RTP801i-14, which emerged through work by Atugen and Quark Biotech, both hot prospects for future RNAi-based activity. RTP801i-14 targets Quark/Atugen's proprietary hypoxia-inducible gene RTP801 - shown in vitro to promote neuronal cell apoptosis (programmed cell death) and the generation of reactive oxygen species (which also have a role in cell death). It represents a mechanism independent of VEGF. A Pfizer-funded phase I study was initiated in February 2007.

Viral infections
The specificity of RNAi and a diverse selection of established viral targets, suggests that siRNA-targeting antivirals may also be a promising application of RNAi technology. Alnylam is leading the way with inhaled and intranasal formulations of ALN-RSV01, which suppress the nucleocapsid N gene of respiratory synctial virus (RSV).

In February 2007, Alnylam reiterated its intention to start a phase II trial for the RSV infection candidate in the second half of the year and forecast that data from its ongoing inhalation study would become available at that time. Phase I data from a study of the intranasal formulation were made available during 2006 and confirmed that investigational doses were safe and well tolerated.

Before its acquisition by Merck, Sirna had reported that it was actively involved in siRNA research for RSV and was generating drug candidates against proprietary targets, as well as against those of its strategic partner GlaxoSmithKline (GSK). It had reported advances in its preclinical programmes for hepatitis B and C (HBV and HCV) and had even disclosed Sirna-034 as the lead HCV compound set to enter the clinic by the end of 2006.

Alnylam Pharmaceuticals is also working in the influenza virus arena and, despite competition from Nucleonics and Nastech, among others, finds itself placed ahead of the pack. It has identified a candidate (ALN-FLU01) with activity against NP and PA genes of the virus, including the H5N1 strain, and expects to file an IND application in 2007.

Cancer treatments
The siRNA landscape for chemotherapeutics appears more open with no big pharma or high-profile specialist biotechs operating in this indication. Atugen is a subsidiary of SR Pharma and is noteworthy for its effort in the Quark/Pfizer AMD programme. It has established a promising portfolio of early-stage therapies, including Atu-027, which mediates its effects on the phosphatidylinositol 3-OH kinase (PI 3-K) signalling pathway.

Recently reported preclinical data, in which a reduction in tumour size and metastases were observed in a pancreatic cancer model, support its clinical evaluation for gastrointestinal cancers. Another of its candidates, Atu-093 is also predicted to enter the clinic sometime in 2007 for non-small cell lung cancer (NSCLC). Candidates for prostate cancer and hepatocellular carcinoma (HCC) are also in the pipeline. Studies have shown that the expression of the R2 subunit of ribonucleotide reductase has a role in cancer progression and metastatis.

Lorus Therapeutics recently disclosed, siRNA-1284, which has been shown to decrease the expression of R2. The candidate has shown promising results in a range of in vitro cell lines and in several in vivo models. Calando Pharmaceuticals is targeting the M2 subunit of the same target witrh its siRNA, CALAA-01, and recently reported data from a pilot safety study.

Neurological indications
A number of neurological disorders top the list of other potential indications for siRNA therapies. Unsuprisingly, Alnylam features heavily in this area with programmes targeted towards Nogo genes and the Huntingtin (htt) gene, for the potential treatment of spinal cord injuries and Huntington's disease, respectively. It is also exploring the suppression of alpha-synuclein (SNCA) expression for the potential treatment of Parkinson's disease.

Targeted Genetics is also active in Huntington's disease research and was working with Sirna to develop an adeno-associated virus (AAV) vector to deliver siRNAs with that could suppress the expression of a toxic Huntington's disease-associated protein. RXi is evaluating mutant superoxide dismutase-1 (SOD-1) as a target for amyotrophic lateral sclerosis (ALS). Outside the established disease classes, several other siRNA programmes of significance are advancing. Alnylam's proprotein convertase subtilisn/kexin type 9 (PCSK9) gene-silencing programme has already yielded a candidate (ALN-PCS01) with significant gene suppression capabilities in murine and human models.

It is expected to be the subject of an IND application in 2007 with a view to initiating a trial for hypercholesterolaemia. RXi, in addition to its ALS interests, is considering the receptor interacting protein 140 (RIP140) gene as a target for metabolic therapies such as obesity and diabetes, and is also exploring the suppression of cytomegalovirus (CMV) genes for the treatment of CMV retinitis. Quark, meanwhile, is focusing its non-RTP-801i-14 resources on a number of internal programmes for acute and chronic renal failure, acute hearing loss, chronic obstructive pulmonary disorder (COPD) and pressure sores ototoxicity.

Future prospects
Regulation of genes by the RNAi system is an innate process and manipulation of it ought to be well tolerated. This has been observed in clinical investigations so far. Dealing with off-target effects, and ensuring effective delivery, stability and precision remain the major challenges for developing future siRNAs. Clinical trials in AMD have not needed to address these concerns because they involve local delivery to the eye. Thus siRNAs used in this therapy are not required to be encapsulated, and reach their target with less likelihood of causing off-target effects in other organs.

However, the development of a systemic delivery technology appears to be the next big challenge. Most recently, Alnylam and Inex Pharmaceuticals published claims relating to lipid compositions, including cationic liposomes, for delivering siRNA drugs. Sirna, which reported encouraging data for lipid nanoparticle-formulated versions of its hepatitis virus candidates, Atugen, which formulates its candidates with proprietary atuPlex technology, and Calando are among others that have established an understanding of systemic delivery.

Beyond siRNA therapy, other parts of the RNAi mechanism are seen as therapeutic targets. Alnylam has published data supporting chemically-engineered oligonucleotides called antagomirs that could silence miRNAs and RXi has supplemented its patent portfolio with the addition of technology from Cold Spring Harbor Laboratory that could see it use short hairpin RNA to trigger RNAi.

3rd May 2007


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