The RNAi phenomenon was reported as early as 1993: the gene that produces the purple pigment was transferred to the purple petunia, and it was hoped that the purple flower would be deeper, but it was counterproductive, but it did not deepen the purple color, but turned white. At that time, it was thought that this was the purple pigment gene originally contained in Petunia and the foreign purple pigment gene transferred into it, which was called "co-suppression". In 1995, Dr. Su Guo of Cornell University used antisense RNA to block the expression of nematode genes. Antisense RNA (anti-sensing RNA and sense RNA) blocked gene expression. The results were puzzling. Until 1998, Andrew Fire's research demonstrated that in experiments where sense RNA blocks gene expression, what really works is double-stranded RNA. These double-stranded RNAs are generated when in vitro transcription of sense RNA. So I put forward the word RNAi.
At present, the mechanism of action of RNAi is mainly elucidated in living organisms such as nematodes, fruit flies and zebrafish. Double-stranded RNA in an organism can be derived from an RNA virus infection, a transposon transcript, and an exogenously introduced gene. The double-stranded RNA from these sources induces an intracellular RNAi mechanism. As a result, the virus is cleared, the expression of the transposon is blocked, the expression of the exogenously introduced gene is blocked, and the expression of the gene in the genome of the cell homologous thereto is also Blocked.
After the double-stranded RNA enters the cell, it is cleaved into siRNA under the action of Dicer enzyme, and on the other hand, it is amplified by RdRP (RNA-directed RNA polymerase). It is then cleaved into siRNA by Dicer enzyme. The double strands of siRNA are unfolded into single strands and form complexes with certain proteins. Argonaute 2 is currently the only known protein involved in complex formation. This complex binds to mRNA complementary to the siRNA, causing the mRNA to be cleaved by RNase.
On the other hand, the combined product uses SiRNA as a primer and mRNA as a template to synthesize a complementary strand of mRNA under the action of RdRP. As a result, mRNA also became double-stranded RNA, which was also cleaved into siRNA under the action of Dicer. These newly generated siRNAs also have an effect of inducing RNAi, and by this polymerase chain reaction, intracellular siRNA is greatly increased, and the inhibition of gene expression is remarkably increased. RNAi can be induced from 21 to 23 nucleotide siRNA to a few hundred nucleotides of double-stranded RNA, but long double-stranded RNA blocks gene expression significantly better than short double-stranded RNA.
More and more researchers have begun to use RNAi technology to study gene function aids, drug target screening, and cell signaling pathway analysis. Several commonly used methods for preparing siRNA include:
Chemical synthesis
In vitro transcription
siRNA expression vector
PCR expression cassette
Synthetic material
21mer RNA oligo
29mer DNA oligo
55-60mer DNA oligo
50mer DNA oligo
Preparation time
4-7 days
More than 10 days
More than 10 days
More than 8 days
Personal operation
No need
medium
many
medium
Can you mark
can
can
Can not
Can not
Can it be modified?
can
Can not
Can not
Can not
Easy to transfect
easy
easy
difficult
difficult
Large scale synthesis
can
limited
can
Can not
Large-scale screening
Suitable for
Not suitable
Not suitable
Not suitable
Usage control
can
No
No
No
Toxin effect
small
Big
Big
Big
cost
Â¥589/pair
expensive
expensive
expensive
Progress in RNAi drug development
RNAi has become the basis of current gene function research, drug target discovery and drug development. RNAi drugs are expected to become a more efficient and rapid disease treatment. The clinical application of siRNA must overcome technical difficulties, including improving the pharmacokinetic properties of siRNA, avoiding off-target effects and interferon response. Great progress has been made in nucleic acid chemistry and introduction, making RNAi treatment more promising and potential. RNAi drugs are bound to greatly promote the development of clinical drugs and bring about a revolutionary change in clinical treatment.
In the past few years, many companies have made great achievements in the field of RNAi. After establishing the RNAi research platform, these companies successfully established the RNAi drug research platform. Some companies have made great progress in drug delivery systems.
According to preliminary statistics, about 19 companies are developing 85 siRNA drugs, and 8 companies are developing 64 antisense nucleic acid drugs. These drugs are mainly concentrated in cancer, cancer, cardiovascular, eye, anti-virus, respiratory, antibacterial, diabetes and so on. At present, Sirna, Merck and Roche are in the first camp of RNAi drug research, and other large companies are also conducting research cooperation or independent research on siRNA drugs.
In June 2006, the annual meeting of the American Society of Gene Therapy announced the exciting news of the success of the world's first siRNA clinical trial. This clinical trial showed that siRNA targeting the vascular endothelial growth factor (VEGF) gene is effective in the treatment of age-related macular degeneration (AMD). The results of this trial are a milestone in the development of RNA therapy, and siRNA will soon become a highly effective molecular drug for clinical treatment. In recent years, siRNA drugs for various diseases have entered the different stages of clinical trials. You may notice that most RNAi drugs are chemically synthesized siRNA for a variety of reasons: easy to operate, controllable; high transfection efficiency; small side effects on cells; various modifications and connections; Scale screening; large-scale preparation and the like.
disease
RNAi reagent
Import method
Clinical trial phase
Company or institution
Eye disease
Wet type old age macular degeneration (AMD)
siRNA
Vitreous injection
(Intravitreal)
Preclinical trial
Quark Biotech
Http://
Modified siRNA (VEGF)
Vitreous injection
(Intravitreal)
Phase I/II clinical trial
Sirna
Http://
Modified siRNA (VEGFR)
Vitreous injection
(Intravitreal)
Phase I clinical trial
Acuity
Http://
Viral infection
HBV and HCV
siRNA
Cationic liposome
(Liganded nanoparticle:cationic-lipid delivery system)
Preclinical trial
Nucleonics/Intradigm
Http://
Http://
Respiratory Syndrome (RSV)
siRNA
Nasal drops, sprayer
(Intranasal, Aerosol)
Phase I clinical trial
Alnylam
Http://
AIDS
(HIV)
shRNA
Lentivirus-mediated
(Lentivirus)
Phase I clinical trial
Benitec/City of Hope
Http://
cancer
Liver cancer
(Hepatic cancer)
siRNA
Adamantane-PEG-cyclodextrin cationic polymer
(Liganded nanoparticle: cyclodextrin-containing polymers, adamantane-PEG modifier)
Preclinical trial
Calando
Http://
Solid tumor
(Solid tumour cancers)
siRNA (VEGF)
PEG-cationic polymer
(Liganded nanoparticle: PEG-PEI)
Preclinical trial
Intradigm
Http://
other illnesses
Amyotrophic lateral sclerosis (ALS)
siRNA
N/A
Preclinical trial
CytRx
Http://
Inflammation
(Inflammatory diseases)
siRNA
Peptide
(Peptide)
Preclinical trial
Nastech
Http://
Note: Some websites are temporarily unavailable.
In fact, many pharmaceutical companies have invested a lot of resources in RNAi, and the prospect of using siRNA as a treatment is attractive. From the discovery of siRNA to the present, the first RNAi drug Bevasiranib (Acuity) has been born. For the treatment of wet age-related macular degeneration (AMD).
According to reports, the FDA has approved Quark's new clinical trial application for the siRNA drug DGFi for kidney transplant patients. In the second half of 2008, Phase I or Phase II clinical trials have been initiated.
Currently, Alnylam is the leader in RNAi drug development. Its siRNA research field is relatively wide. Some RNAi drugs have been in clinical stage, especially in the anti-virus, and it has been clinically staged. It is expected that RNAi drugs will be available in five years. It is. Alnylam's RNAi drugs are used to treat respiratory syncytial virus, influenza, Parkinson's disease, and hypercholesterolemia. As a drug, siRNA will develop faster than other small molecule drugs, and the selectivity will be better. If it can better solve the target cell delivery, it is believed that it can treat many diseases that are not satisfactory in current drug treatment.
China's research on innovative drugs is still far behind Western countries. The development history of RNAi technology is very short. It is a biotechnology that has just emerged. Although we have a certain gap with developed countries, the gap is only a few years. If we accelerate development, we are fully qualified to catch up with the developed countries. The country may make breakthroughs in the development of innovative drugs in a certain period of time. In particular, the use of this technology can make up for the huge gap between China's chemical and pharmaceutical industries and developed countries. This new technology has brought us a once-in-a-lifetime development opportunity.

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