ASO drugs, Si-RNA, Delivery of si-RNA based drugs, CRISPR-Cas gene editing, mRNA based drugs, Aptamer based therapeutics, RNAI PATHWAY, Aptamer based delivery.

1. TAPPING THE RNA
WORLD FOR
THERAPEUTICS
By Hasnat Tariq
And
Hadiba

2. CONTENTS
•ABSTRACT
•BACKGROUND
•RNA-based strategies
•ASO drugs
•Si-RNA based drugs
•Delivery of si-RNA based drugs
•Advantages of RNA-based drugs
•CRISPR-Cas gene editing
•MRNA based drugs
•Aptamer based therapeutics
•conclusion

3. ABSTRACT
•REVOLUTIONIN RNA BIOLOGY:
• Transcription of extragenic DNA
• Identification of new RNA classes with unexpected functions andmodifications
• Use of RNA for therapeutics as RNA targets the nucleic acid so extends the domain of
targets.
•PAPER REVIEW:
• RNA based drug designs
• Discussion
• Limitations
• Applications

4. BACKGROUND
CENTRAL DOGMA:
DNA -> RNA -> PROTEIN
DNA:
CODING REGION AS EXONS
NON CODING REGION AS INTRONS
ETC

5. DNA TRANSCRIPTION
NON CODING RNA TRANSCRIPTS
ARE NOT TRANSLATED TO PROTEINS BUT DO ACQUIRE SOME FUNCTIONS
FUNCTIONALNON-CODING RNAINCLUDES:
◦ TRANSFER RNA
◦ RIBOSOMAL RNA
◦ REGULATORY RNA.
FUNCTIONOF NCRNA:
◦ TRANSCRIPTIONALAND TRANSLATIONAL MODIFICATION OF CODING SEQUENCES AS CHROMATIN MODIFICATION
MRNA STABILITY AND TRANSLATION.
◦ EPIGENETIC ACTIVITY
TYPES:
◦ SMALL NON-CODING RNA
◦ LONG NON-CODING RNA(LNCRNA)

6. Human genome:

7. FUNCTIONS OFRNA
Riboswitches:
a regulatory segment of
a mRNA-binds a small
molecule, resulting in a
change in protein.
Ribozymes:
it catalyzes specific
biochemical reactions,
as RNA splicing in gene
expression

8. SEQUENCING METHODS FOR RNAS HAVE REVEALED A COUNTLESS NUMBER OF NCRNA TRANSCRIBED IN BOTH SENSE AND ANTI-
SENSE DIRECTIONS FROM CODING GENES, AND JUNK DNA.
LNCRNA:
 LENGTHS EXCEEDING 200 NUCLEOTIDES.
 CAPPED, POLYADENYLATED AND ARE EVOLUTIONARY CONSERVED.
 PREDICTED TO BE TRANSLATED INTOPROTEINS.
 FUNCTION AND MECHANISM OF ACTION ARE UNDERSTOOD FOR FEWER LNCRNAS.
BINDING TO DNA MOLECULE:
 Triplexformation
 Regulation ofcis and transregions.
FUNCTIONS:
 RECRUIT OF EVICT TRANSCRIPTIONAL ACTIVATORS ORREPRESSORS.
 ASSEMBLE PROTEIN INTO COMPLEXES IN CYTOSOL OR NUCLEUS
 ACT AS RIBOZYMES
EXAMPLE:
XIST-RESPONSIBLE FOR INACTIVATIONOF X CHROMOSOME IN FEMALES

9. SncRNA
MIRNA:
A SMALL REGULATORY RNA, HAVE THE ABILITY TO REGULATE GENE EXPRESSION. IT CAN ACTIVATE OR INHIBIT GENE
EXPRESSION. THEY BIND TO A SPECIFIC LOCATION ON MRNA, PREVENTING THE MOLECULE FROM BEING TRANSLATED.
IT ALSO ENHANCES MRNA DECAY
EXAMPLE:
LIN-4 , A 22 NUCLEOTIDE DOUBLE STRANDED RNA ACT AS AN IMPORTANT REGULATOR IN
DEVELOPMENT OF C.ELEGANS. IT SUPPRESSES THE EXPRESSION OF A DEVELOPM ENTAL
GENE.
SI-RNA AND PI-RNA:
THEY REGULATE TRANSCRIPTION BY FORM ATION OF HETEROCHROM ATIN AND HALT
TRANSLATION

10. 3DFOLDING OF RNA
3D FOLDING IS MEDIATED BY :
1. BY WATSON- CRICK PAIRING OF SHORT STRETCHES
OF COMPLEMENTARY BASES
2. BY HOOGSTEEN BASE PAIRING (NON-CANONICAL)
3. BY HYDROGEN BONDS BETWEEN RIBOSE PHOSPHATE BACKBONE MOIETIES
TERTIARY SRUCTURE OF RNA:
APTAMER: ARE PEPTIDES OR OLIGONUCLEOTIDES BINDS TO A SPECIFIC TARGET
MOLECULE . UPON FOLDING ATTAINS A UNIQUE 3D STRUCTURE. BINDING
OF LIGAND TO APTAMER RESULTS IN ITS EXPRESSION AS REGULATING TRANSLATION,
SPLICING OR TRANSCRIPTION. IT IS USED IN THERAPEUTIC PROCESSES.

11. RNA BASED THERAPEUTIC STRATEGIES
NSMB LAUNCHED – ALL DRUGS BINDS TO THE ACTIVE SITES OF PROTEIN
RECEPTORS TO INHIBIT/ENHANCE THEIR FUNCTION. HOWEVER, ONLY 1/3 OF
20,000 PROTEINS ARE POTENTIAL TARGET TO THE DRUGS.
NOW BIOLOGICALS AS MONOCLONALANTIBODIES, REPLACEMENT ENZYMES
TARGETS THESE DRUGGABLE PROTEINS TO ENHANCE OR SUPPRESS THEIR
FUNCTIONS.
MOST OF THE RNA DRUGS USE NUCLEIC ACID ANALOGS AND TAKE ADVANTAGES OF COMPLEMENTARY
BASE PAIRING TO MIMIC ENDOGENOUS RNA PROCESSES
THEY ARE MODIFIED TO SHOW RESISTANCE TO RNASES
AND TO INCREASE BINDING AFFINITY TO THEIR TARGET I-E NUCLEIC ACID OR PROTEIN.
THE MAIN HURDLES ARE THE DELIVERY TO THE TARGET SITES AND ITS STABILITY.

12. EXAMPLES OF RNA BASEDDRUGS
 13-MER DNA OLIGONUCLEOTIDE:
INHIBIT TRANSLATION OF ROUS SARCOMA VIRUS IN A SEQUENCE SPECIFIC MANNER.
 ANTI-CD3:
THE 1ST MAB DRUG, ANTI-CD3 IS USED FOR TRANSPLANT REJECTION TREATMENT.
 IN 2016, FDA APPROVED 22 NEWS DRUGS OF WHICH 7 ARE MAB DRUGS AND 2 ARE ASO USED TO TREAT
NEURODEGENERATIVE DISORDERS

13. CLASSIFICATION
 DRUGS WHOSE SPECIFIC ACTIVITY IS MEDIATED BY AN ANTISENSE MECHANISM & MODULATING THE PROCESSING AND FUNCTION OF AGENE
 SENSE RNA THAT ENCODE A PROTEIN
 AND APTAMERS AND ITS CONJUGATES ARE UTILIZED AS DRUGS THAT TARGET PROTEINS.
 ASO DRUGS:
SINGLE STRANDED, MODIFIED AND STABILIZED NUCLEIC ACID ANALOG
ACTION OF ASO ON PRE-MRNA
 ACT ON PRE-MRNA TO CONTROL/MODULATESPLICING
 THEY CAN CAUSE SEQUENCE SPECIFIC DEGRADATION BY RECRUITING RNASEH.
 THEY CAN BIND TO THE POLY A RECOGNITION SITES AND BLOCKS POLYADENYLATION WHICH RESULTS IN MRNADECAY
ACTION OF ASO ON MRNA:
 ASO BINDS TO THE TRANSLATION SITE TO BLOCK TRANSLATION.

15. DESIGN OFASO
• CHANGES IN THE OLIGONUCLEOTIDES OF ASO ARE LEAD TO MORE STABLE NUCLEIC ACID ANALOG THAT SPECIFICALLY
BIND TO THEIR TARGET.
• ASO ARE DESIGNED TO INHIBIT GENE EXPRESSION AND TO INCREASE TRANSLATION POTENTIALLY PROVIDING A WAYTO
TREAT DISEASES CAUSED BY INADEQUATE GENE EXPRESSION.
• IT IS POSSIBLE TO DESIGN ASO TO CLEAVE MRNA AT SPECIFIC SITES BY INSERTING AN RNA- CLEAVING RIBOZYME
SEQUENCE BETWEEN SEQUENCES COMPLEMENTARY TO THE TARGETED CLEAVAGE SITES. BUT IT IS NOT DEVELOPED
BECAUSE OF POOR ACTIVITY IN VIVO.

16. EXAMPLESOFASODRUG
First approved ASO drug
is Fomivirsen in 1998- to
treat cytomegalovirus retinitis
Mipomersen- a recent ASO drug to
treat familial hypercholesterolemia.
Miravirsen-binds to hepatitis C virus
genomic RNA and block its
replication.

17. RNAI PATHWAY
•Group of mechanism that uses small RNA molecules to direct gene silencing.
•RNAi includes two imp types of RNA molecules miRNA and siRNA
•The siRNA are derived from longer double stranded RNAs, either produced in the cell itself or
delivered through experiments.
•MiRNA, comes from RNA that are transcribed in the nucleus, fold and processed before exposed into
cytoplasm as double stranded precursor miRNA.
•The precursors of miRNA and siRNA then binds to DICER which cuts the RNA into short segments.
•The short segments of RNA then binds Argonaut protein. One strand is selected and remain bounded
to the argonaut.
•This combination then binds to other proteins forming a complex called RISC.
•Si-RNA directs RISC to bind to specific mRNA sequence complementary to its sequences.
•Once bound argonaut catalyzes cleavage of mRNA which then degrade.
•MiRNA also guide RISC to mRNA. A segment of miRNA called seed pairs with the sequence on mRNA.
•The imprecise matching allows miRNA to target hundreds if mRNAs.

19. SI-RNA BASED DRUGS
 SILENCING RNA, SMALL INTERFERING RNA,SHORT INTERFERING RNA.
 OPERATE WITHIN RNAI PATHWAY(GENESILENCING)
 A CLASS OF DOUBLE STRANDED NON CODING RNA MOLECULES, 20-25 BP, SIMILAR TO MIRNA.
 DEGRADE THE TARGET MRNA
 HIGHLY SPECIFIC
 IDENTIFICATION OR IMPORTANCE OF A GENE- USING SIRNAOR SHRNA
RESULTS IN IDENTIFICATION OF NOVEL DRUG TARGETS.
DEVELOPMENT OF SIRNA:
 IN VITRO-INHIBITION OF HIV REPLICATION.
 IN-VIVO:INJECTION OF FAS SIRNA
SIRNA /MIRNA ARE STABLE WHEN TAKEN UP BY RISC

21. DELIVERY OF SI-RNA BASED DRUGS
 DEVELOPMENT OF LIPID NANOPARTICLES(LNPS) TO INTRODUCE SIRNAINTO HEPATOCYTES
 COMPLETED PHASE 3 STUDY SHOWED THE KNOCKDOWN OF THE TTR(TRANSTHYRETIN) GENE AND SHOWED IMPRESSIVE NEUROLOGICAL SYMPTOMS.
IT IS LIKELY TO BE APPROVED AS FIRST SIRNA DRUG.
LIMITATIONS OF LNP-SIRNA DRUGS:
 IMMUNE STIMULATORY SIDE EFFECTS.
 PRODUCTION
CONJUGATED DRUGS(NEXT GENERATION DRUGS):
 DUGS AS ASO, APTAMERS, SIRNA ARE TARGETED TO THE SITE BY CONJUGATING TO GALNAC WHICH IS RECOGNIZED BY RECEPTORASGPR
OF HEPATOCYTES.
 HIGHER SPECIFICITY, EXPRESSION OF THE RECEPTOR, AND RECEPTORRECYCLING.
 MODIFICATION CAN BE MADE FOR BETTER STABILITY AND RESULTS
 KNOCKDOWN OF THE GENES LASTING FOR 3-6 MONTHS.

23. TREATMENT OF GENETIC ORPHAN
DISEASES
USING SIRNA-CONJUGATION DRUGS, SUCH DISEASES CAN BE TREATED AS KNOCKDOWN OF GENES AS;





PCSK9
ANTI-THROMBIN 3
ALAS1
THE COMPLEMENT COMPONENTC5
GLYCOLATE OXIDASE
FUTURE PROSPECTS:
 DELIVERY OF ASO AND ANTAGOMIRS(EARLY PHASETRIALS)
APPROACHES IN MOUSE MODELS INCLUDE DIFFERENT SIRNA CONJUGATION TO TREAT CANCER CELLS OR KNOCKDOWN THE GENES IN IMMUNE CELLS. SIRNA CONJUGATES
TO:
1. TO RNA/DNA
2. CPG NUCLEOTIDES
3. FUSION WITH ANTIBODIES
4. TO FATTY ACID CONSTITUENT OF NEURONAL MEMBRANE

24. DIFFERENCES BETWEEN ASO ANDRNAI
SiRNA used over
and over again to
degrade many
mRNAs.
ASO acts on aone
to one basis.

25. CHALLENGES FOR DEVELOPING RNA-
BASED DRUGS
•Intracellular delivery
•Poor pharmacokinetic properties
•Activation of immune response.
•Off target effectsinclude;
• Activation of DNA repair system
• Translocation or imprecise gene editing
• Suppression of homologous genes
•MODIFICATIONS:
•Chemical modification of the 2' position of the ribose
•Substitution of the DNAbase

26. ADVANTAGES OF RNA BASEDDRUGS
COMPARED TOANTIBODIES
 CHEMICAL SYNTHESIS
 CHEAPER AND EASY TO PRODUCE
 STABLE AND LOW DOSE
 REDUCED BYSTANDER TOXICITY
 LOW IMMUNOGENITY
 EASY TO COMBINE WITH OTHER DRUGS
 EASY TO INACTIVATE THE DRUG BY DESIGNING ITS COMPLEMENTARY SEQUENCE.

27. CRISPR-CAS GENE EDITING
 CUT AND PASTE METHOD DEVELOPED FOR GENOMIC DNA EDITING INCLUDING GENE EXPRESSION
, SUPRESSION OR GENOME WIDE SCREENING.
 CORRECT MUTATIONS IN SOMATIC AND GERMLINE CELLS.
COMPONENTS
1. CAS 9- A DNA CUTTING ENZYME
2. A GUIDE RNA THAT RECOGNIZE THE SEQUENCE TO BE EDITED EITHER BY MODIFYING, DELETING OR
INTRODUCING NEW SEQUENCE.
 ANTISENSE PAIRING OF SGRNA TARGETING SPECIFIC GENES ON CHROMOSOMAL DNA.
 SGRNA INDUCE STABLE CHANGES.
 FIRST APPROVED DRUG IS TO PRODUCE CAR-T CELLS.

29. Gene knockout or knock in
Identification of genes
Synthetic lethal interaction
Identification of novelgenes.
APPLICATIONS:
Ex-vivo editing of differentiated cells controlled for gene modification, that will be infused
into patients
FUTURE
PROSPECTS:
On and off target toxicity
Oncogenic gene translocation
Cell death
LIMITATIONS:

30. mRNA BASEDDRUGS
INTRODUCTION OF CHEMICALLY MODIFIED, STABILIZED MRNA INTO CELLS TO BE TRANSLATED TO PROTEIN.
NO MODIFICATION OF THE GENOME
APPLICATIONS:
STABILIZED MRNA FOR VACCINATION
LIMITATION:
1. TRANSIENT EXPRESSION
2. INTRACELLULAR DELIVERY
3. REQUIREMENT OF NANOPARTICLES FOR DELIVERY OF LARGER MRNA & PREVENTION AGAINST RNASES.

32. APTAMER
BASED
THERAPEUTICS
FOLDED RNA MOLECULE THAT BEHAVE LIKE
NUCLEIC ACID ANTIBODIES
ARE SELECTED USING A TECHNIQUE CALLED SELEX.
HIGH AFFINITY AND SPECIFIC BINDING TO
TARGET MOLECULES.
INHIBIT OR ACTIVATE THE FUNCTIONS OF PROTEIN TARGETS
LOW IMMUNOGENOCITY
EASIER LARGE SCALE PRODUCTION
COST EFFECTIVE
DIAGNOSTIC APPLICATIONS ARE STILL
DEVELOPING.

33. APTAMERS ARE MODIFIED TO INCREASE NUCLEASE
RESISTANCE.
THE FIRST APTAMER APPROVED- PEGAPTANIB SODIUM FOR MACULAR DEGENERATION
EIGHT OTHER APTAMERS ARE CURRENTLY UNDERGOING CLINICAL EVALUATION FOR VARIOUS HEM ATOLOGY,
ONCOLOGY, OCULAR AND INFLAMMATORY INDICATIONS.
APPLICATIONS:
 DIAGNOSTICS
 BIOMARKER DISCOVERY
 AND DRUG DELIVERY
 TIME SAVING
EASY PRODUCTION. LIMITATIONS:
Laborious
Do not bind to some targetmolecules.

34. APTAMER BASED DELIVERY
APTAMERS ARE USED AS VECTOR TO DELIVER THERAPEUTIC GENES AS SI -
RNA TO THEIR TARGET SITES.

35. CONCLUSION
RNA-BASED DRUGS CAN BE USED
 INHIBITION OF GENE EXPRESSION OR PRODUCTION OF FUNCTIONAL PROTEINS
 EDITING MUTATED DNA
 FIRST RNA BASED DRUG FOR SPLICING IS JUST APPROVED, AND SI-RNA BASED DRUG IS LIKELY TO BE
APPROVED
 HOWEVER, RNA IS INHERENTLY UNSTABLE, POTENTIALLY IMMUNOGENIC, TYPICALLY REQUIRES A DELIVERY
VEHICLE FOR EFFICIENT TRANSPORT TO THE TARGETED CELLS TO BE USED AS DRUG. THESE ISSUES HAVE
HINDERED THE CLINICAL PROGRESS OF SOME RNA-BASED DRUGS AND HAVE CONTRIBUTED TO MIXED
RESULTS IN CLINICAL TESTING.
 RESULTS FROM RECENT CLINICAL TRIALS SUGGEST THAT THESE BARRIERS MAY BE OVERCOME WITH
IMPROVED SYNTHETIC DELIVERY CARRIERS AND CHEMICAL MODIFICATIONS OF THE RNA TO MAXIMIZE
DRUG POTENCY WHILE MINIMIZING OFF TARGET TOXICITY AND IMMUNOGENICITY.

36. REFERENCE
Lieberman, J. (2018). Tapping the RNA world for therapeutics. Nature structural
& molecular biology, 25(5), 357-364.

37. THANK YOU