THERAPEUTIC PROMISE & CHALLENGE

Presented by:

Jyoti Lakshmi Hati Boruah

PhD Scholar,P1706

BTY

MAJOR CREDIT SEMINAR

Recent advances in antisense drugs has

broadened the scope of therapeutic targets for a

variety of human diseases.

Currently under clinical investigation ranging

from genetic disorders to viral infection to

various cancers.

1985 : James E. Summerton in collaboration with Dwight D. Weller

deviced Morpholino

Morpholine ring

Phosphorodiamidatelinkage

Phosphorodiamidate

morpholino oligo

Summerton, 2007

25-base Morpholino oligos strongly

and specifically binds to its

complementary 25-base target site

in a strand of RNA via Watson-Crick pairing

WHY CHOOSE MORPHOLINOS

WATER

SOLUBILTY

STABILITY

BINDING

AFFINITY

EFFICACY

SPECIFICITY

Used as a tool for

reverse genetics

by knocking down

gene functions.

This property is

exploited to block

translation, block

splicing, block

miRNAs or their

targets

Specificity: Dependent on MIL, TPS &

Length

Calculate

expected number of inadvertant targets =

(TPS/4MIL) (Length – MIL+1)MIL- Minimum inhibitary length

TPS- Targetable pool size

Length- No. of bases in antisense oligos which

contribute to target recognition

Target success rate:

S-DNA- 10-20%

siRNA – 50%

Morpholinos- about 80%

Reason= 1. extended length

2. high affinity

For morpholino:

MIL= 14-15 bases

TPS= 3 billion bp of DNA in cells

4% DNA transcribed

120 billion unique sequence RNA

in cell pool of pre spliced RNA transcript

MO- Targetable sequence in human cells

about 8.6 million bases of unique RNA

transcripts

For 25 mer MO

Expected No. of inadvertant targets=

(8,600,000/414)(25-14+1)= 0.4 (Summerton, 2007)

• Steric blocking of

translation initiation

complex.

• Target protein band

disappears from Western

blots.

•Do not cause degradation

of their RNA targets

• Blocks the biological

activity of the target RNA

until that RNA is degraded

naturally, which releases

the Morpholino.

• Useful to know function of

particular protein

Moultan and Jiang,2009

Blocks sites involved in pre-

mRNA splicing

Modify and control normal

splicing events.

This activity can be

conveniently assayed by RT-

PCR, with successful splice

modification appearing as

changes in the RT-PCR product

band on an electrophoretic gel.

Draper et. al.,2001

Moultan and Jiang,2009

Morpholinos are

usually targeted

to one of these

sites of primary

miRNA TARGET

SITE ON miRNA

:

Guide

Star

Guide Drosha

Guide Dicer

Star Drosha

Star DicerMoultan and Jiang,2009

In most tissues, bare Morpholino oligosenter cells poorly.

Methods for enhancing delivery into the cytosol

Method Application

Endo-Porter Cell culture

Peptide conjugate Cell Cultures & invivo

Microinjection Embryos

Scrape-loading Cell culture

Modified Morpholinos

Peptide conjugated

PMO (PPMO)

Eg. PMO linked to

(RXR)4XB called P7 , R=

L-arginine, X= 6-

aminohexanoic acid and

B= β-alanine(Delcroix

and Riley,2010)

Vivo morpholinos

PMO linked to octa-

guanidium dendrimer(Paul

et al, 2008)

Injection Site

0 hr

1.5 hrs 4 hrs

28 hrs

Rosen et al, 2009)

ENDOPORTER SYSTEM SCARPE DELIVERY

ELECTROPORATIONMICROINJECTION

Mellert et al., 2012

HeLa cells with red dextran

Patridge et al., 1996

Cerda et al., 2005

Incorporate a photo-sensitive subunit

cleaved by 365 nm light

Allows to look

at the effects

of genes in

specific

tissues of an

organism or

during

different

periods of

development

Structure of an unmodified Morpholino oligo and a Photo-Morpholino compared

Tallafuss et al., 2012

Antisense photo morpholino strategySense photo morpholino strategy

GIVES

VIRTUAL

ON & OFF

SWITCH

In 2003 the ‘progerin’ was discovered by Nicolas Lévy’s

teamNuclear membrane: normal

Nuclear membrane: HGPS

Carlos López-Otín

The research, published

October 26, 2011

in Science Translational

Medicine

Osorip et al., 2011

"vivo-morpholino"

antisense oligonucleotide

technology.

Production of progerin

and lamin A were

reduced.

Highly significant

increase in life

expectancy of in

treated, mice to a

maximum of 190 days

caused by

mutations in

dystrophin gene

(large and

mutation-prone

gene, comprised

of 79 exons)

Common

mutations

causing DMD

are deletions,

insertions, and

nonsense

mutations

Dual Myostatin and

Dystrophin Exon

Skipping by Morpholino

Nucleic Acid Oligomers

Conjugated to a Cell-

penetrating Peptide Is a

Promising Therapeutic

Strategy for the

Treatment of Duchenne

Muscular Dystrophy

Eteplirsen, antisense drug , Sarepta

Therapeutics, under clinical trial

Malerba et al., 2012

Du et et al.,

AMO-mediated

splicing modulation

represents an

attractive therapeutic

strategy

Restore gene function by correcting aberrant

splicing (caused by mutations)

Removing nonsense mutations from mRNA by

exon skipping

Regulating alternative splicing process

(Du and Gatti, 2009)2012

MORPHOLINOS IN NEURODEGERATIVE DISORDERS AND PIDS

MRSA

Bacillus anthracis

E. coli

Mycobacterium tuberculosis

Enterococcus casseliflavusEndophthalmitis

klebsiella pneumoniae

Campylobacter jejuni

Salmonella typhimurium

Burkholderia cepacia

Entamoeba histolytica

Acinetobacter

Multi drug resistance

Conventional antibiotics

Antisense antibiotics

Targets proteins or

macromolecular

complexes

Reversal of resistance

Specific genes, inhibit

expression of the targeted

sequence

Pseudomonas aeruginosa

MRSA

Enterococci ( VRE)

Mycobacterium tuberculosis

Escherichia coli

Klebsiella pneumoniae

Actinobacter baumannii

Enterococcus faecalis

Campylobacter jejuni

Bacillus anthracis

Mycobacterium avium

Entamoeba histolytica

Streptococcus mutans

Burkholderia cepacia

Salmonella entericaserovaTyphimurium

Pseudomonas aeruginosa

Bacillus subtilis

Corynebacterium efficiens

Peptide Conjugated Phosphorodiamidate Morpholino Oligomers (PPMOs) targets two essential bacterial gene : acyl carrier protein & gyrase A .

(Geller 2010)

CLINICAL TRIALS GOING ON :

Plasmodium vivax

GiardialambliaToxoplasma gondii

Trypanosomagambiensie

Leishmaniadonoveneii

PPMO ---cell

membranes into

parasites--disrupt

genetic signals

Block production of

enzyme dihydrofolate

reductase

.

Anti-DHFR PPMO

were used against

infected mice ,within 96

hrs number of parasite

will reduced by 83% to 97%. CLINICAL TRIAL GOING

ON: Trypanosoma cruzi

Plasmodium vivaxGiardia lamblia

Toxoplasma gondii

Leishmania donoveneii

(McLoed Lab 2012 and Oreggo et al., 2014)

IN CELL CULTURE AND

MOUSE

Coxsackievirus B3

Venezuelan equine

encephalitis virus

Ebola virus

Influenza A virus

Japanese encephalitis

virus

Mouse hepatitis virus

Equine arteritis virus

MORPHOLINOS AS ANTIVIRAL DRUGSAntiviral cells successfully

delivered in virus infected

cell via CPP conjugated

PMO

IN CELL CULTURE

West nile virus

St. Louis encephalitis

virus

Dengue virus

SARS coronavirus

Porcine reproductive and

respiratory syndrome virus

Respiratory syncytial virus

Measles virus

Human rhinovirus

Coxsackie B2

Foot and mouth disease

virus

Sindbis virusDelcroix and Riley, 2010

Ebola virus

Influenza A virus

Japanese

encephalitis virus

Dengue virus

fungal strains of Aspergillusniger

Candida albicans- Candida 6 and Candida 51

Novel morpholino

pyrimidinyl acetamides

are used as antisense

antifungal drug

Kanagarajan et al.,2011

.

In 2000, the field of

developmental biology was

revolutionized by

morpholinos

provided specific, reliable

tools for blocking the

expression of any selected

gene throughout the course

of embryogenesis in model

organisms such as zebra

fish, frog, sea urchin, and

chick.

(Ekker and

MOs are valuable tool for discovery

screening as well as individual gene

knockdown analysis

Affinity purification of RNA ( Wages et al., 2oo7)

Morpholino monolayer (Tercero et al.,2009)

Morpholino-functionalized silicon nanowire biosensor

for sequence-specific label-free detection of DNA

Uncharged nature of MOs has brought advantages to surface hybridization technologies such as DNA MICROARRAYS using MOs as probe

Further development is required for use of morpholinos against multiple

drug resistant bacteria, viruses and as broad spectrum antisense

antimicrobials and antiviral drugs.

Treatment with combination of morpholinos targeting multiple sequence

would be fruitful in clinical application

Improvement in design of cell penetrating peptides to tackle the issue of

in-vivo toxicity and make conjugated drug safer.

To Check toxicity level by treatment with doses several times higher

than expected therapeutic dose

In near future, antisense drugs will likely become a critical member of

our arsenal in the defense against highly pathogenic, emerging or

genetically engineered pathogens.

Area of research should be to isolate most critical target and identify the feasibility of using such genes as targets for therapeutic purposes