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Antisense RNA Technology and its Applications
#1
There are certain disorders like cancer, parasitic and viral infections, which cause excessive production of specific proteins in day to day life. An alternative treatment for these disorders is known as Antisense Technology. A single stranded RNA that is complementary to messenger RNA (mRNA) is referred to as Antisense RNA. The antisense therapy includes inhibition of translation by using single stranded nucleotide, any DNA or RNA sequences or even synthetic ones. From the practical point of view, most of the antisense therapies work efficiently and produce best results if used with RNA since RNA specifically binds to target mRNA and blocks protein synthesis.

Antisense technology was also referred as Gene Subtraction, but it is proven to be a misnomer as this technology does not remove gene, rather it just involves inactivation of the gene. Naturally occurring mRNA antisense mechanism is the hok/sok system in E.Coli R1 plasmid.

The antisense technology is carried out on the basis of the principle that the cloned gene is ligated into the vector in reverse orientation. Now, as the antisense technology obstructs the mechanism of translation it is stoichiometric in nature, and it can prevent synthesis of the product of the gene that it directs against. The antisense RNA mechanism involves hybridization of the antisense and sense copies of RNA. Now, as the ds-RNA molecule is formed, it rapidly degrades by ribonucleases and the expression is blocked. Another reason could also be the antisense RNA preventing ribosomes to bind to the sense strand. In simpler words, if an oligonucleotide is introduced into the cell, it binds to specific mRNA which forms an RNA dimer in the cytoplasm and halts the translation mechanism; this is because the mRNA no longer has access to ribosome and dimeric RNA is rapidly degraded by ribonuclease which in turn on introduction of oligonucleotide complementary to mRNA leads to blockage of translation by particular gene, turning off the gene.

RNAi (RNA interference) and Antisense RNA technology though has the same effect but their mechanisms are quite different. Firstly, RNAi technology involves degradation of mRNA by small interfering RNAs triggering catalytic gene silencing; whereas in Antisense RNA technology mRNA is degraded by RNase H. Secondly, in comparison to antisense RNA, RNAi are twice larger.

The application of Antisense RNA technology is in many sectors. This technology was used in Flavr Savr, for tomato ripening; ripening in tomato produces enzyme Polygalactourodase (PG) which softens tomatoes and finally rotten them quickly. Two biotechnology companies: Calgene, USA and ICI Seeds, UK introduced a gene in plant which synthesizes a complementary mRNA to PG gene and inhibites the synthesis of PG enzyme delaying over ripening and rotting. Antisense therapy is considered for treating certain genetic disorders and infections. This is also referred to as Gene Therapy. It includes: isolation of specific gene; it’s cloning and inserting it into target tissue cells to make desired protein. It has to be ensured that the gene is expressed correctly and sufficiently without causing harm to patient in context with the immune response. Antisense RNA technology is also used for cancer therapy. This therapy is used for treating brain cancer – malignant glioma and cancer of prostate gland, in malignant glioma IGF-I was over produced and in the prostate cancer IGF-IR was synthesized more. These two were used to block the translation. Research is carried on in regard with Antisense RNA drugs for treatment of CMV, HIV, cancer, etc. Antisense antiviral drug named Formivirsen is developed to treat CMV, which was licensed by FDA in August 1998. In 2010, scientist at NIPGR by using Antisense technology developed tomato which could last longer for more than 30 days by silencing two genes (alpha - man and beta – hex) which causes softening and wrinkling in tomatoes during ripening process.

Despite of achieving some success, Antisense technology has few challenges. Like: delivery into the patients body, then possibility of toxic effects due to it’s regulation on both the normal and mutant alleles. Antisense RNA technology is also used to study certain gene functions.

Looking at the advancements in the Antisense RNA technology, it has potentials for development of pharmacological agents, studying physiological and pathological processes as well as it’s use in effective treatment as gene therapy.
#2
Another unexpected but more than useful feature of antisense RNA

Antisense RNA is showing great potential for future treatment of severe diseases such as cancer, HIV, CMV… Recently, another unexpected feature of antisense RNA is discovered - it actually can stimulate protein synthesis!

All proteins in the human body are produced as a result of complex and well controlled machinery that include information written in the DNA, mRNA molecule that transfer genetic information from DNA to the ribosome, and ribosomes, which act like a protein factory where nucleotide chain will be converted in the amino acids chain in the process called translation. All these cascade events are supported by numerous enzymes. Human organism produces around 25 000 coding mRNA molecules which means that each one of them can be turned into protein. This set of mRNA is known as transcriptome. Each mRNA has its own antisense matching pair. Antisense RNAs were considered to be a junk RNA because they don't produce valid proteins. They are implicated in negative regulation of the transcription through binding to their complementary (coding) mRNA. Result of binding is a double stranded RNA that will be recognized as abnormal and harmful for the cells and thus it will be eliminated as soon as possible via enzymatic degradation. 70% of all RNA in the cell is antisense RNA, which tells us that this RNA must be very important for normal cell functioning. Scientists believed that all details and tricky parts associated with genetic expression and protein synthesis are well known, but as it usual happen in the science, new information and latest discoveries are revealed by accident.

Group of Italian scientists studied a gene associated with Parkinson disease in its mutated form. This gene has same structure and function in human and mouse. It is called Uchl1 and it codes ubiquitin carboxyl-terminal esterase L1, which is associated with both normal brain function and neurodegeneration. Using a mouse model, scientists discovered that Uchl1 has more than one class of antisense RNA and even a bigger surprise came later when they discover that these antisense molecules doesn’t work the way they expected them to do - instead being focused on suppression of mRNA translation, they stimulate it. This was the first time that one antisense molecule became recognized as stimulator of translation that could regulate protein synthesis positively. Mechanism of this stimulation is really simple: antisense RNA increases association between mRNA and ribosome through additional set of nucleotides called SINEB2 element. Antisense molecules are longer than mRNA because they contain part that doesn’t match with sense RNA but accelerate protein synthesis through enhancing the binding of double stranded RNA with ribosome. Other part of the molecule is classical antisense RNA complementary with mRNA. Once mRNA and antisense RNA join together, this complex will bind to the ribosome and translation will start.

How this discovery can be exploited further? Binding of the SINEB2 element with ribosome is unrelated to the remaining part of the antisense molecule, which means that these elements can be applied universally to enhance protein synthesis of each therapeutically or industrially needed protein simply by adding SINEB2 element to the antisense RNA of interest. They are already available commercially under the name SINEUPs (SINE for SINE sequence they contain, and UP because they up-regulate protein production). Their main advantages are fast and efficient protein production (they increase protein synthesis 5-10 times) and lower cost compared to standard methods used. Also, work with these molecules doesn't demand too much effort. Kit contains universal SINEUP in combination with SINEUP-based protein-coding cloning vector. SINEUP can be transfected into desired cell line or in vivo with gene of interest, or it can be inserted alone to increase endogenous protein levels.

SINEUPs are the only known antisense molecules that regulate protein synthesis by enhancing translation directly. Discovery of SINEUPs reduced the cost of protein manufacture and opened some new fields of research. Modern medicine pays a lot of attention to recombinant proteins and SINEUPS would allow faster and cheaper production of already known medically valuable proteins. It will also enhance research and provide even more terapeutics that could improve human health. Since SINEUPs can increase production of numerous mammalian proteins, they can also be applied beyond medical and pharmaceutical field. Proteins are important part of agriculture, animal food, play important role in detergent production, in brewing industry. As with any other high tech - possibilities are endless.
#3
RNA has long been thought of as a promising technique for disease therapy; the only such case to have reached the market is the drug fomivirsen. One commentator has characterized antisense RNA as one of "dozens of technologies that are gorgeous in concept,
#4
RNA interference and Cystic Fibrosis

Part of the challenge of targeting antisense RNA and small interfering RNA is the individual cell and tissue environments which need to be targeted in different diseases. Cystic fibrosis is a devastating autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. It affects most notably the lungs, but also the liver, pancreas and intestines. It involves a defect in epithelial transport of sodium and chlorine. In this context, hyperactivity of epithelial sodium channels (ENaC) has been implicated in CF pathogenesis due to dysregulation of fluid and electrolytes in the airways.

A study from Erasmus MC, Rotterdam in The Netherlands addressed the possibility of targeting ENaC by RNA interference techniques as a possible addition to the armoury of CF therapeutic agents. A proof-of-principle study was performed using lentiviral-mediated RNA interference cells including immortalised normal (H441) and CF mutant (CFBE) airway cells, and differentiated human bronchial epithelial cells in air-liquid interface culture (HBEC-ALI). Air-liquid interface culture more accurately mimics in vivo conditions than solid phase culture. The cells were transduced with a vesicular stomatitis virus G glycoprotein pseudotyped lentiviral (LV) vector which was engineered to express a short hairpin RNA (shRNA) designed to interfere with the α subunit of ENaC (ENaCα). A marker gene was included to allow verification of transduction efficiency, which was confirmed at close to 100% efficiency of H441, CFBE and HBEC-ALI before differentiation and polarization. The study confirmed that ENaCα mRNA was inhibited by shRNA transduction, as was antigen expression. ENaC-dependent short circuit current and fluid transport was proportionally decreased while transepithelial resistance or cAMP-induced secretion responses were unaffected in HBEC-ALI. Off-target effects mediated by Toll-like receptor 3 or RNA-induced silencing complex were ruled out. The study concluded that the generic method to down-regulate ENaCα by lentiviral shRNA expression vectors has potential therapeutic value in CF treatment.

Source

AARBIOU, J. et al., 2012. Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells. The journal of gene medicine, 14(12), pp. 733-745
  

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