Image: Flickr - shikeroku
By far the majority of marketed drugs modulate the activity of proteins. Since the discovery of RNA interference (RNAi) over 10 years ago, a major goal has been to develop agents which will selectively block the synthesis of target proteins. Small interfering RNAs (siRNAs) occur naturally and are believed to have evolved as a defence mechanism against viral infection but synthetic siRNAs could potentially be used to silence any target gene. Although the idea is not conceptually difficult and many cancer-associated genes have been silenced in laboratory experiments, there have been a number of obstacles in developing RNAi for clinical use, not least of which is how to deliver the siRNAs to the target cells.
A team led by scientists and clinicians at the California Institute of Technology (Caltech) has now published the results of a small phase I clinical trial in patients with skin cancer showing that targeted nanoparticles can traffic into tumours and deliver siRNAs in a dose-dependent fashion when administered intravenously. The siRNA-containing nanoparticles used in the study are being developed by Calando Pharmaceuticals, Inc. and the technique used for detecting and imaging the nanoparticles inside cells from tumour biopsies was developed at Caltech. Although phase I studies are primarily designed as safety studies, the team was able to demonstrate that, in one patient who received the highest dose of nanoparticles, the target mRNA (M2 subunit of ribonucleotide reductase (RRM2)) had been cleaved at the predicted position and that protein levels had also been reduced.
Although more trials will be needed to show that such treatment is safe and effective, the study provides the first evidence that nanoparticles and RNAi can be combined to reduce expression of cancer-associated genes in human patients.
The results are published in the journal Nature.
Although many studies have shown the potential for gene silencing using short interfering RNA (siRNA), a major hurdle to the therapeutic use of the technique has been the lack of effective delivery systems. Writing in the journal Nature, researchers at the University of Massachusetts Medical School now report a method of delivering siRNA to specific cell types following oral administration. The researchers exploited a characteristic of macrophages – the ability to engulf yeast particles – to deliver the siRNA. Macrophages are attractive targets for RNA interference therapy since they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes. Yeast particles were treated to remove components that would elicit an immune response to give β-1,3-D glucan, a polysaccharide formed from D-glucose, as the delivery vehicle. Oral delivery of β-1,3-D glucan-encapsulated siRNA particles containing as little as 20 µg kg-1 siRNA directed against tumour necrosis factor α (TNF-α) depleted messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum TNF-α levels. The technique was also used to identify the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) as a previously unknown mediator of cytokine expression. Silencing Map4k4 in macrophages protected mice from lipopolysaccharide-induced lethality by inhibiting TNF-α and interleukin-1β production. The siRNA-carrying particles were engulfed by macrophages in the gut and, over time, a large proportion of macrophages exhibited gene silencing, resulting in systemic immune suppression.
Quark Pharmaceuticals has recently announced that its partner Pfizer has begun a phase II clinical trial of a chemically modified small interfering RNA (siRNA) molecule in patients with diabetic macular oedema. PF-4523655 inhibits abnormal blood vessel growth and leakage independently of the VEGF pathway, reduces inflammation and suppresses apoptosis. Results from a Phase I/II trial completed by Quark on Pfizer’s behalf showed that PF-4523655 was safe and well tolerated in patients with wet age-related macular degeneration who failed to respond to currently approved therapies. The new trial will test the safety and efficacy of PF-4523655 compared with laser surgery.
siRNA molecules interfere with the expression of specific genes and there is currently much interest in using the technology to treat a wide range of diseases, although there are several challenges that must be overcome if exogenous siRNA is to become widely used.
Despite the challenges, several siRNA molecules have entered clinical trials including TD101 for pachyonychia congenita (PC Project / TransDerm), Sirna-027/AGN-745 for age-related macular degeneration (Allergan), metastatic melanoma (Duke University), CALAA-01 for solid tumours (Calando Pharmaceuticals), Cand5 for diabetic macular oedema (Opko Health) and I5NP for acute kidney injury following cardiac bypass surgery (Quark Pharmaceuticals).
Polo-like kinase 1 (Plk1) has received attention in recent years as a potential target for intervention in cancer. It is known to be important in regulation of cell cycle progression during M-phase and has been shown to be overexpressed in certain tumours. Now scientists at the NYU Cancer Institute and Howard Hughes Medical Institute have shown that Plk1 is involved in a new pathway associated with the cellular response to DNA damage. In the July 25th issue of Cell, the authors describe targeting of the phosphatase Cdc14B to APC/C, a protein that marks other proteins for destruction. The resultant activated APC/C then tags Plk1 for destruction. If Plk1 remains active, the cell continues to divide despite the DNA damage.
Plk1 crystal structure
Tekmira Pharmaceuticals, a specialist in delivery of RNA interference molecules, has just announced plans
to advance an siRNA product targeting Plk1 into Phase 1 clinical trials in the second half of 2009. Meanwhile, the search for small molecule inhibitors of Plk1 continues. Scientists at Pfizer deposited x-ray coordinates of the catalytic domain of a mutant Plk1(complexed with BI2536) in the protein data bank earlier this year. Additional structure factors have been deposited by Sunesis scientists, although the coordinates have not yet been released.