ANTISENSE THERAPY

PRESENTED BY

POORANACHITHRA M

IST M.TECH BIOTECHNOLOGY

Introduction:Most human diseases are caused by production of abnormal proteins or malfunctioning proteins. Antisense therapy involves inhibiting production of these proteins.

Antisense gene therapy is a gene silencing technique in which, When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene "off"

Comparison Of Different Gene Silencing Strategies:

How Is Antisense Gene Therapy Different From RNA Interference?

Antisense technology destroys target mRNA by recruiting the enzyme RNase

H, while RNAi recruits a different RNase enzyme known as dicer

RNAi molecules are twice as large as antisense oligonucleotides (because they

are double stranded rather than single stranded)

Mechanism Of Action:

1. Antisense oligonucleotide binds to the mRNA.

2. This double stranded region can inhibit the production of protein two

mechanisms

* Stopping the ribosome from reading the message,

* Leading to the destruction of the mRNA by an enzyme already in the

cells called RNase H.

Other Proposed Mechanisms: Proposed mechanisms include

* Triplex formation,

* Blocking RNA splicing,

* Preventing transport of the mRNA antisense complex into the cytoplasm,

* Increasing RNA degradation,

* Blocking the initiation of translation.

Antisense Drug Therapy:

FDA Approved The Antisense Product

Drug : Mipomersen

Brand Name : Kynamro

Disease: Homozygous Familial Hypercholesterolemia

Drug: Formivirsen

Brand Name : Vitravene

Disease: Cytomegalovirus (CMV) Retinitis In AIDS Patients

Familial Hypercholesterolemia:A genetic disorder characterized by high cholesterol levels specifically very

high levels of low-density lipoprotein (LDL, "bad cholesterol"), in the blood

People have mutations in the LDLR gene that encodes the LDL receptor protein,

which normally removes LDL from the circulation, or apolipoprotein B (ApoB),

which is the part of LDL that binds with the receptor

Treatment:

High doses of statins, often in combination with other medications

 LDL apheresis which is similar to dialysis

 After all In January 2013, The US FDA also approved mipomersen, which

inhibits the action of the gene apolipoprotein B, for the treatment of homozygous

familial hypercholesterolemia

Mipomersen: Second-generation ASO, developed to inhibit synthesis of apob-100 in the liver.

Mipomersen sequence is made of 20 oligonucleotides linked with phosphorothioate linkages.

Red residues (2’-MOE) are 2’-o-(2-methoxyethyl) nucleosides. Blue residues are 2'-deoxynucleosides that are the site for RNase h -mediated degradation. Substitution at the 5-position of the cytosine (C) and uracil (U) bases with a methyl group is indicated by me.

Mechanism Of Action:

Mipomersen acts by hybridizing within the coding region of apoB-100 mRNA

and activating RNase H.

RNase H degrades the mRNA strand but leaves the ASO intact.

 Modifications to the ASO were shown to improve delivery of the drug, and

allow resistance to degradation by endonucleases and exonucleases in addition to

maintaining high affinity and specificity to the target mRNA

PD & PK: Absorption - Following subcutaneous injection, peak concentrations of mipomersen are reached in 3 to 4 hours, bioavailability is 54% to 78%.

Distribution - Mipomersen has a distribution plasma half-life of 2 to 5 hours.

Metabolism - It is metabolized in tissues by endonucleases to form shorter oligonucleotides that are then metabolized by exonucleases.

Excretion - Mipomersen eliminated in urine, Following subcutaneous administration, elimination half-life for mipomersen is 1 to 2 months.

Side Effects: Adverse events associated with the use of Kynamro may include, but are not limited to, the following:

injection site reactions (erythema, pain, hematoma, pruritus, swelling and discoloration)

flu-like symptoms

nausea

headache

elevations in serum transaminases

Cytomegalovirus Retinitis:CMV retinitis is an inflammation of the retina of the eye that can lead to blindness, Caused by human cytomegalovirus.

 It is usually treated by antivirals such as ganciclovir or foscarnet, which can be taken orally, intravenously, injected directly into the eye (intravitreal injection), or through an intravitreal implant.

Then Fomivirsen, the first antisense drug to be approved by the FDA, was approved in August 1998 as an intraocular injection for the treatment of cytomegalovirus retinitis

Fomivirsen:Fomivirsen is a phosphorothioate oligonucleotide that inhibits human cytomegalo virus (HCMV) replication through an antisense mechanism and it is given as an intravitreal injection.The nucleotide sequence of fomivirsen is complementary to a sequence in mRNA transcripts of the major immediate early region 2 (IE2) of HCMV.

This region of mRNA encodes several proteins responsible for regulation of viral gene expression that are essential for production of infectious CMV.

Binding of fomivirsen to the target mRNA, results in inhibition of IE2 protein synthesis, subsequently inhibiting virus replication.

 5'-GCG TTT GCT CTT CTT CTT GCG-3'

Antisense Molecules In Late-stage Preclinical Or Clinical Development:

Diseases: Antisense drugs are being researched to treat

* cancers including lung cancer, colorectal carcinoma, pancreatic -

carcinoma, malignant glioma and malignant melanoma,

* diabetes

* Duchenne muscular dystrophy

* asthma and

* arthritis

POTENTIAL ADVANTAGES:

Oligonucleotides may be manufactured

quickly

Potential sensitivity to therapy may be easily

measured

Potential to produce longer lasting responses

Potential for enhanced binding affinity to

target

PROBLEMS WITH ANTISENSE TECHNOLOGY

It is still difficult to express aRNA only in

targeted tissues

Furthermore, the uptake of aRNA is still

imprecise.

THANK YOU