The advent of RNA as major scientific hobnob is a spectacular. Before 1980s RNA were just ignored as a molecule of assistant role of DNA and they helped in protein synthesis. In early 1980s Tom Cech and Sydney Altman discovered the catalytic role of RNA, for which they were awarded the noble prize. The growing interest in RNA and mammoth information gathered in the process made Sir Walter Gilbert to coin word as “RNA WORLD”. In 1999, it was discovered that siRNA successfully silenced gene expression. It was key discovery as it promised manipulation of protein synthesis without killing the cell. The post transcriptional inhibition that was the high way of most of the diseases could be halted in a sequence specific manner. This had many promising therapeutic applications as many pathological events of diseases being mediated by protein synthesis. The RNA interference is got a thing in biotech, which has scope in all human diseases. It has hoped to resolve almost all major disease, which are haunting mankind today. RNAi application range from mundane diseases like hypertension, diabetes to dreadful diseases like AIDS, cancer and hepatitis. The demonstration of RNAi in plants and earthworms fallowed mammalian cells has raised hopes of scientists of successful RNAi based therapeutics.
Further research indicated that RNAi is a ubiquitous phenomenon in all living organisms by which the fine tuning of gene expression happens. RNAi is a highly conserved mechanisms which might have originally evolved to combat routine viral infections. This has a rational to believe because RNAi can prevent viral infections, inhibits viral antigen expression, known to suppress viral replication, known to silence the accessory viral genes, hampers assembly of viral particles and displays role of viral host interactions.
The RNAi appears to be most promising effective bio therapeutics as it happens to act at the stage of post translational. It also appears to be safe as it does not harm other cells by interfering with its function. RNAi is to interfere with intended protein synthesis without effecting the normal cell functional. It may not kill the cell it affects, but may prevent the identified protein synthesis through which the pathogenesis of the disease in question is mediated. Thus RNAi become the prominent technologies to treat all those diseases mediated via protein synthesis. The range of application for RNAi has a wide spectrum of diseases like cancer, HIV, hepatitis, type II diabetes mellitus, obesity, hypercholestremia, Rheumatoid Arthritis. The scope for RNAi is ever expanding due to efforts world wide and novel applications of technology to combat the disease state in a novel way.
The current clinical trials based on RNAi technology include a range of products for diversified disease conditions. BevaSirnib is undergoing phase 3 clinical trials for wet age macular degeneration and in phase 2 for diabetes macular oedema. siRNA 027 is phase 2 clinical trials for wet age macular degeneration. ALNRSVOL is another siRNA in phase 2 trial for respiratory synctitum viral infections. For hepatits B, NUCB 1000 is tested in phase 1 clinical trials. For AIDS, Anti tat/rev shRNA is undergoing pilot feasibility studies. CALAA01 is tested for solid tumours in phase 1 clinical studies. TD101, novel Sirna is under phase 1 studies for pachyonyctia congenita.
The major hurdle in development of Sirna-based products is the delivery of the Sirna to the effected targets, which are located inside the cell membrane. The Sirna is comparatively large molecule than conventional chemically based drug. The Sirna is negatively charged which makes the delivery a challenge. The site of action of Sirna being inside the cell, it becomes effective only if Sirna gets delivered to its site of action in intact form. To achieve the inter cellular delivery of Sirna, a novel technology has been developed. These can be broadly classified as viral based and non-viral delivery system. The main advantage of viral delivery system triggers long term sustained therapeutic outcomes that are highly optimized for chronic diseases like HIV and hepatitis. For e.g., Lentiviral vector being successfully tried in AIDS and hepatitis. However, Adenovirus vector is useful in targeting the Sirna to brain.
The non-viral technologies involve chemical modification of Sirna or encapsulating the Sirna in a liposome. The challenge here is to deliver a functional Sirna into the cytoplasm by crossing the cell membrane, which repels the Sirna being negatively charged. The linking of a cholesterol molecule to Sirna has not only enhanced the delivery of Sirna to cross the cell membrane but also enhanced the stability of Sirna.
Sirna has been formulated as transferrin-cyclodexterin poly-cation nano particles, which has targeted delivery characteristics. Sirna has been successfully encapsulated as stable nucleic acid lipid particles with lipid bilayers and polyethylene glycol coats. Masked endosomolytic agents, which are dynamic poly conjugate particles, are not only small particles, which can be useful for cell specific targeted delivery. The receptor mediated uptake with a view on targeting the Sirna has been successfully attempted with positively charged antibody conjugates of protamine and other similar proteins. RNA aptamers allow the targeting to appropriate receptors.
Limitations of RNAi based therapeutics
The major limitations of RNAi based therapeutics are the fear of adverse effects that may arise due to intervention in highly conserved nature mechanisms. Recent advances indicate involvement of variety of Sirna like molecules which regulate the protein synthesis in a desired fashion. This involves manipulation of protein synthesis and gene silencing The other endogenous small rna are endo siRNAs, miRNAs, piRNAs, ta siRNA, natRNAs, re siRNA, SCN RNAs and tncRNA. Meddling with artificial siRNA would result in hampering of normal protein, which could be deleterious to the patient. It is also observed in animal studies the treatments of siRNA would provoke the immune responses resulting in production of pro inflammatory cytokines involving type 1 interferon responses in mice. The death of mice in a Sirna treatments for hepatitis B is an alarming signal. Micro array techniques revealed that there can be non targeted gene silencing following Sirna treatments.
However, it is possible to contain above adverse effects by treating with lower dose and modifying siRNA with 2’-O-methoxy modifications.
(The author is with MCOPS, Manipal 576 104)