morpholinos
TRANSCRIPT
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