Ashmita ChaudhuriM.Pharm (Pharmacology), 1st YearNSHM College of Pharmaceutical

Technology

ANTISENSE THERAPY

INTRODUCTION:

Antisense therapy is a form of treatment for genetic disorders or infections.

When the genetic sequence of a particular gene is known to be causative of aparticular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNAor a chemical analogue) that will bind to the messenger RNA (mRNA) producedby that gene and inactivate it, effectively turning that gene "off".

This is because mRNA has to be single stranded for it to be translated.Alternatively, the strand might be targeted to bind a splicing site on pre-mRNAand modify the exon content of an mRNA.

Antisense drugs are being researched to treat a variety of diseases such ascancers (including lung cancer, colorectal carcinoma, pancreatic carcinoma,malignant glioma and malignant melanoma), diabetes, Amyotrophic lateralsclerosis (ALS), Duchenne muscular dystrophy and diseases such as asthma,arthritis and pouchitis with an inflammatory component.

BASIC CONCEPT:

The two complementary strands of double-stranded DNA (dsDNA) are usuallydifferentiated as the "sense" strand and the "antisense" strand. The DNA sense strand lookslike the messenger RNA (mRNA) the DNA sense strand itself is not used to make proteinby the cell. It is the DNA antisense strand which serves as the source for the protein code,because, with bases complementary to the DNA sense strand, it is used as a template for themRNA. Since transcription results in an RNA product complementary to the DNAtemplate strand, the mRNA is complementary to the DNA antisense strand. The mRNA iswhat is used for translation (protein synthesis). Hence, a base triplet 3'-TAC-5' in the DNAantisense strand can be used as a template which will result in an 5'-AUG-3' base triplet inmRNA.

ANTISENSE OLIGONUCLEOTIDE:

The concept of antisense oligonucleotide gene silencing was first introduced in 1978 when Stephenson and Zamecnik (1978) used an antisense oligonucleotide to stop viral replication in cell culture.

An antisense oligonucleotide is a single strand of nucleic acid or nucleic acid analogs, most often an oligodeoxyribonucleotide, usually 15–20 nucleotides in length with sequence complementary to a specific target mRNA.

The antisense oligonucleotide and target mRNA bind together via Watson– Crick base pairing, and this hybridization leads to reduced levels of translation of the target transcript.

Antisense oligonucleotide-induced mechanisms of gene silencing include steric hindrance and interference with ribosomal function, as well as inhibition of mRNA splicing, which prevents mRNA maturation and destabilizes the pre-mRNA in the nucleus (Kurreck, 2003).

However, the most prominent mechanism of antisense oligonucleotide-induced gene silencing is induction of RNase H endonuclease activity resulting in hydrolysis of the mRNA in the antisense oligonucleotide-target transcript duplex.

MECHANISM OF ANTISENSE THERAPY

There are several aspects of antisense therapy utilizing

oligonucleotides that are potentially advantageous over

traditional drug mechanisms. Such as:

1) Oligonucleotides may be manufactured quickly, some times

within one week, or two and only the sequence of a gene is

all that is needed.

2) Potential sensitivity to therapy may be easily measured,

as the target is often one-dimensional versus multiple-

dimensional domains often targeted within proteins.

Sensitivity can be measured through database scanning

or Northern/Southern blotting for unknown genes.

3) Potential to produce longer lasting responses, as clonal

expansion may require more time to produce clinical

disease once mRNA is inhibited, versus just inhibition of

protein typical with conventional therapies.

POTENTIAL ADVANTAGES:

RNase H appears to be a

ubiquitous enzyme in eukaryotes and

bacteria.

The antisense oligonucleotide

(typically a deoxyribonucleotide)

binds the target RNA to form the

heteroduplex substrate.

RNase H binds via its binding

domain at the 3′ antisense

oligonucleotide/5′ RNA pole and

cleaves the target RNA

approximately 7 base pairs from its

binding site.

The target mRNA is degraded,

whereas the antisense

oligonucleotide remains intact,

allowing it to form another

heteroduplex substrate for induction

of RNase H cleavage.

TREATMENT OF VARIOUS DISEASES BY ANTISENSE THERAPY:

Antisense compounds are designed to have the right nucleotide sequence to bind specifically to and interfere with its associated mRNA, the instructions for the production of a particular protein.

Critical characteristics of antisense oligonucleotide design include:(1) the portion of the mRNA transcript to be targeted; (2) the nucleotide makeup of the antisense oligonucleotide; and (3) the binding energy between the antisense oligonucleotide and target.

The secondary structure of the mRNA target is also an important consideration for antisense potency, as the target region on the mRNA must be physically accessible for hybridization by the antisense oligonucleotide.

Computer algorithms are now available that allow determination of mRNA secondary structure folding energy for selection of target sites with minimal overall free energy.

Regions of mRNA transcripts amenable to oligonucleotide binding include their 5′ and 3′ terminal ends, internal loops, joint sequences, hairpins, and bulges .

TREATMENT OF CANCER:

TREATMENT FOR HUNTINGTON’S DISEASE:

Antisense oligonucleotides werefirst used to restore normalsplicing of the -globin gene in-thalassemia patients. In thiscontext, the antisensesequences were targeted toblock the use of cryptic splicesites in -thalassemia patients.Splicing of the cystic fibrosistransmembrane conductanceregulator (CFTR), a proteinwhich is spliced incorrectly insome cystic fibrosis patients,could likewise be re-establishedvia AONs. Both approaches dooffer therapeutic potential bythe redirection of normal splicingin patients.

TREATMENT FOR -THALASSEMIA AND CYSTIC FIBROSIS:

TREATMENT FOR HIV:

TREATMENT FOR CARDIOVASCULAR AND RENAL DISEASES:

TREATMENT FOR ALS:

TREATMENT OF ASTHMA:

In allergic asthma experimental models, antisense oligonucleotides (ASO)

are administered by inhalation or systemically.

ASO can be used for a large number of molecular targets: Cell

membrane receptors (G-protein coupled receptors, cytokine and chemokine

receptors), membrane proteins, ion channels, cytokines and related factors,

signaling non-receptor protein kinases (tyrosine kinases, and

serine/threonine kinases).

A respirable ASO against the adenosine A1 receptor was investigated in

human trials. RNase P- associated external guide sequence (EGS)

delivered into pulmonary tissues represents a potentially new therapeutic

approach in asthma as well as ribozyme strategies.

Small interfering RNA (siRNA) targeting key molecules involved in the

patho-physiology of allergic asthma are expected to be of benefit as

RNAi immunotherapy.

TREATMENT FOR DUCHENNE MUSCULAR DYSTROPHY:

TREATMENT OF DIABETES:

Mipomersen, which is a first-in-class antisense drug, wasdesigned to reduce LDL (low density lipoprotein)-cholesterol.Also, it is an antisense drug used to inhibit PTP-1B, and itis an insulin sensitizer for the treatment of Type 2 diabetes.In addition to lowering glucose, ISIS 113715 has anti-obesityand lipid-lowering potential.

Targeting the molecules and other factors important for thedevelopment of DR may result in new effective treatments forretinal neovascularization and leakage in patients with DiabeticRetinopathy (DR), including second-generation antisenseoligonucleotides, such as iCo-007, targeting c-Raf kinase.Defects in cell signaling pathways have been associated withmany human diseases, including ocular diseases as DR.

A FEW EXAMPLES OF ANTISENSE AGENTS AND THEIR TARGETS:

SERIAL NO. AGENTS TARGET

01 Genasense (oblimersen) Bcl-2

02 Affinitak PKC-alpha

03 ISIS 112989 (OGX 011) Secretory protein clusterin

04 ISIS 23722 Survivin

05 AP 12009 TGF-Beta2

06 GEM 231 Protein kinase A

07 Mipomersen apoB-100

08 MG98 DNA methyltransferase

09 Alicaforsen ICAM-1

10 EXC 001 Fibrosis

Isis Pharmaceuticals has taken the lead in the arena of antisense gene therapy technology. Though they have not yet engineered an antisense drug for HD, they have already been successful in getting one antisense drug, Vitravene©, on the market. This drug uses antisense technology to treat an eye disease associated with AIDS.

In addition, Isis has made a number of promising advances in scientific trials of antisense technology to treat other conditions including high cholesterol and Parkinson’s disease.

Isis has successfully used antisense technology to inhibit the action of the Huntington gene in mouse cells, mouse brains and human cells.

Isis is currently testing the technology on transgenic mice (mice that are genetically modified to have the mutated HD allele). If that goes well, the next step will be to try it in monkeys, and then eventually humans.

CURRENT DEVELOPMENT OF ANTISENSE GENE THERAPY:

CONCLUSION:

Antisense drugs have the potential therapeutic efficacy in the

treatment of various diseases than the conventional drug. Antisense

therapeutics is well positioned to play a significant role in the

progression of antisense technology for drug development in human

diseases.

However, proof of clinical efficacy, of any of the antisense

oligonucleotides in the field of oncology, is still missing. Large controlled

trials are needed to show that antisense oligonucleotides are better than

other treatment approaches. If this goal is achieved without a clear proof

of principle, the major dilemma of antisense is still unresolved.

The beauty and future potential of antisense depends on the design of

multiple drugs based on our increasing knowledge of genes and their

functions.

Only if the therapeutic activity of an antisense oligonucleotide is

defined by the antisense sequence, the future for antisense-based drugs

will become bright.

REFERENCES:http://www.biotecharticles.com/Healthcare-Article/Treatment-of-Cancer-With-Antisense-Therapy-141.html

http://www.ncbi.nlm.nih.gov/pubmed/12867066

http://en.wikipedia.org/wiki/Antisense_therapy

http://www.academicjournals.org/article/article1379934399_Saonere.pdf

http://web.stanford.edu/group/hopes/cgi-bin/wordpress/2010/06/antisense-gene-therapy/

www.isispharm.com/Antisense.../Antisense-Drug.../Antisense-Drugs.htm

https://www5.medicine.wisc.edu/~williams/antisense.pdf

http://www.genengnews.com/gen-articles/antisense-drugs-finally-turning-the-corner/5025/

http://www.nytimes.com/2013/01/30/business/fda-approves-genetic-drug-to-treat-rare-disease.html?_r=0

http://rna.dk/2013/rna-antisense-drugs-in-the-pipelines/

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