Antisense General

Antisense General

- Human Genome project

- About 30,000 gene , At least 5, 000 disease related gene potential target for

antisense therapy

- Expression regulation on sense mRNA

- Direct therapeutic purpose including other functional genomics study

ANTISENSE TECHNOLOGIE

S

GENOMICS BASED DRUG DISCOVERY

PHARMACEUTICALS

MOLECULAR DIAGNOSTICS

GENE THERAPYDRUG DELIVERY SYSTEMS

Relation of antisense technologies to other segments of biopharmaceutical industry

Application of Antisense oligonucleotides

Antisense Drug-The First Antisense Drug

Fomivirsen: developed by ISIS for treatment of cytomegaloviral induced retinitis

-Two antisense drugs in Clinical test (Phase III)

- Genassense by Genta ; Bcl2 inhibition

- ISIS 3521 by ISIS (Elly Lilly);Inhibition of Protein kinase C-alpha,

Effective against non-small cell lung cancer

Functional Genomics-Antisense oligonucleotides for genetic knockouts

- Difficulties in predicting mRNA sequences of susceptible target for antisense

oligonucleotides

- 10 –20% of oligos tested block gene expression (20 oligos at least tested)

- Needs for HTS system (www.genetrove.com)

Antisense Drugs and Companies

Oligo Company Sequences Target Rt. Disease Rt.of Adm.

Status / Remarks

ISIS 3521ISIS 2503ISIS 5132Oncomyc-NGAP12009AVI 4557GenasenseGenasenseGenasenseGenasenseGEM 231GTI 2040GTI 2501MG98

LEafAONPAN 346HERZYMEANGIOZYMEANGIOZYME

ISISISISISISAVIAntisense AVIGentaGentaGentaGentaHybridonLorus LorusMethyl/lHybridon

NeoPharmPanacea RibozymeRibozymeRibozyme

GTTCTCGCTGGTGAGTTTCATCCGTCATCGCTCCTCAGGGTCCCGCCTGTGACATGCATTN/A

TCTCCCAGCGTGCGCCATTCTCCCAGCGTGCGCCATTCTCCCAGCGTGCGCCATTCTCCCAGCGTGCGCCATGCGUGCCTCCTCACUGGCN/A

N/A

RibozymeRibozymeRibozyme

PKC-aH-rasRafc-myc

Bcl-2Bcl-2Bcl-2Bcl-2PKA-RIaRNR

DNA methyl Transferase

HER2VEGFR-1VEGFR-1

PT

PT

PT

MO

PT

PT

PT

PT

CPT

PT

Mixed backbone

R/D

R/D

R/D

NSCLCPancreaticOvarianCancer

NHL MelanomaCCLAMLRefractory tumorcancer

Lung, colon, breast cancer

CancerBreastCololectal

I.VParenteralI.VI.V

SubcutaneI.VI.VI.VI.VI.V

I.V

I.VI.VI.V

PIII PIIPIIPI

PIIIPIIIPIIIPIIIPIIPII

PII

PIPIPII

Target Disease: Cancer

N/A: Not Available or Company will not disclose

PT: Phosphorothioate

CPT:Chimeric Phosphorothioate

R/D: RNA/DNA

MO: Morpholino

PTm: Phosphorothioate (all Cs are 5-methyl-C)

Oligo Company Sequences Target Mt. Disease Rt.of Adm. Status / Remarks

Resten NGE2F DecoyISIS 2302ISIS 2302ISIS 2302ISIS 104838ISIS 104838AVI 4014Durason(EPI-2010)

AVI 4126 ISIS 14803HEPTAZYMEHepBzymePNAbioticsNeuBioticsGEM 92HGTV43INX4437INX3001INX3280

ORI 1001R-95288

HGTV43Reticulose

AVICorgentechISIS ISISISISISIS/ElanISISAVIEpiGenesis

AVIISISRibozyme Ribozyme PanthecoAVIHybridonEnzoInexInexInex

OrigenixSankyo KK

EnzoAVRC

N/A

GCCCAAGCTGGCATCCGTCAGCCCAAGCTGGCATCCGTCAGCCCAAGCTGGCATCCGTCAGCTGATTAGAGAGAGGTCCCGCTGATTAGAGAGAGGTCCC

GATGGAGGGCGGCATGGCGGG

N/AGTGCTCATGGTGCACGGTCTRibozyme

UCGCACCCATCTCTCTCCUUC

GGACCCTCCTCCGGAGCCTATGCTGTGCCGGGGTCTTCGGGCAACGTTGAGGGGCAT

N/ADMT-TGGGAG

Stealth Vector,(gene transfer vector)Peptide Nucleic Acid

c-myc

ICAM-1ICAM-1ICAMTNF-aTNF-a

Adenosine A1 receptorC-myclHCV

Gag

IGF-IRC-mybC-myc

E1 HPVHIV-1

T-cellsCCR5

MO

PTPTPTCPTCPT

PT

MOPTmR/D

CPT

PTPTPT

N/AAptamer, P diester DNADNAPNA

Restenosis

Crohn’s Dis.Topical PsoriasisIllcerative ColitusArthritis/Crohn’sPsoriasis

Asthma

PKDHepatitis CHepatitis C

HIV/AIDS

Intra abdominal implantPurged bone marrowStent restinosis

Genital wartsAIDS

HIV/AIDSHIV

I.V

PareteralTopicalEnemaPar/OralTopical

Aerosol

I.VPareteralI.V

I.V

Ex-vivoEx-vivoIntra-coronaryTopicalI.V

Ex-vivoSubcute.

PII

PIII

PII

PII

PII

PII

PII

PI

PII

PI/II

PI

PI

PII

PI

PI

PI

PI

PIII

Target Disease: Others

N/A: Not Available or Company will not disclose

PT: Phosphorothioate

CPT:Chimeric Phosphorothioate

R/D: RNA/DNA

MO: Morpholino

PTm: Phosphorothioate (all Cs are 5-methyl-C)

Antisense Drugs in Developments

Oligo Company Status / Remarks

ISIS 3521ISIS 2503ISIS 5132AP12009Oncomyg NGAVI 4557GenasenseGEM 231GTI 2040GTI 2501LEafAONPAN 346HERZYMEANGIOZYME

ISIS PharmaISIS PharmaISIS PharmaAntisense PharmaAVI BIoPharmaAVI BIoPharmaGentaHybridonLorus TherapeuticsLorus TherapeuticsNeoPharmPanacea PharmaRibozyme PharmaRibozyme Pharma

PIII (Affinitak)

PIII

PIII


Resten NGE2F Decoy

AVI BioPharmaCorgentech

Target Disease: Cancer

Target Disease: Cardiovascular disorder


ISIS 2302

ISIS 104838AVI 4014

ISIS Pharma

ISIS PharmaAVI BIoPharma

Crohn’s D (PII/III)

Target Disease: Autoimmune & Inflammatory


Durason EpiGenesis

Target Disease: Respiratory disorder


AVI 4126 AVI BioPharma

Target Disease: Urological Disease


ISIS 14803HEPTAZYMEHepBzymePNAbioticsNeuBiotics

ISIS PharmaRibozyme PharmaRibozyme PharmaPanthecoAVI Biopharma

Target Disease: Infectious Disease


GEM 92HGTV43

HybridonEnzo

Target Disease: AIDS & Related disease

1. Antisense Pharma GmbH 2. Atugen 3. AVI BioPharma, Inc. 4. Corgentech Inc. 5. CytoGenix, Inc. 6. Enzo Biochem, Inc. 7. EpiGenesis Pharmaceuticals, Inc. 8. Genta Inc. 9. Hybridon, Inc. 10. Immusol Inc. 11. Isis Pharmaceuticals, Inc. 12. Lorus Therapeutics Inc. 13. NeoPharm, Inc. 14. Panacea Pharmaceuticals, Inc. 15. Pantheco 16. RiboTargets Ltd. 17. Ribozyme Pharmaceuticals, Inc. 18. Salus Therapeutics Inc. 19. Sequitur, Inc. 20. SomaGenics, Inc. (** Referred from Theta Reports, April 2002)

Antisense Companies appeared in Theta Reports, April 2002

ISIS Pharmaceuticals

Genta

- Bcl2: mitochondrial membrane protein regulate the release of Cytochrome C,

an activator of cascades which ultimately result in cell death.

- High levels of bcl2 in human cancer blocks release of cytochrome C triggered

by ordinary cancer therapy Antisense to block expression of bcl2

Functional Genomics General

- Target identification

- Target validation study

- Drug candidate

Alteration of Splicing- Alternative splicing for multiple protein synthesis from a single mRNA

- Increased expression of alternatively spliced protein variant by blocking one splice site

PNA, Morpholino, 2’-modified RNA: ideal tools for this application: non RNase H action

- Upregulation of Luciferase using 2’-O-methyl oligonucleotides against alternative splice

site good example showing function of oligonucleotides in cell

- Other examples of alternative splicing: Bcl-x, Dystrophin

Inhibition of Ribonucleoprotein- Telomerase: - Ribonucleoprotein containing RNA domain and protein domain

RNA responsible for binding to telomere ends

Proteinresponsible for maintaining telomere length from one

generation to the next.

- expressed only in cancer cells, not in normal cells

- PNA and 2’-O-alkyl RNA against RNA domain of telomerase telomere shortening

reduce cell proliferation

- Other ribonucleoproteins involved in signaling or enzyme activity potential target for

antisense inhibition

Alteration of Splicing

Inhibition of Ribonucleoprotein

Cellular uptake of antisense

Nuclease action:

Degradation by nuclease: Full degradation of 18 mer natural oligomucleotide within 30 minutes

Basic of Cellular Uptake

Cellular uptake of antisense oligonucleotide

- For cultured cells

- Many transfection reagents should be tested depending on each cell line

- For tissue

- IV injection: Most efficient delivery to liver and kidney

Evidence to enter tumor cells

- Oral bioavailability: useful route for future therapeutics

- CpG motif: immune stimulation,

but mislead to non-antisense effects and un-wanted results

Antisense Action Mechanism

1. RNase H independent (Inhibition of Translation)

- By steric hindrance upon binding of oligonucleotides

- 5’UTR

- AUG start codon

- ORF

2. RNase H dependent

- By RNase H activity to digest RNA-oligo duplex

5’UTR

AUG start codon

ORF

RNase H dependent

- Resistance to nuclease

- Increased in vivo half life by promoting binding to serum proteins

- But unintended interaction between oligo and protein

by use of mismatch and scrambled control oligonucleotides

by avoiding G-rich oligoemrs that can form quadruplex secondary structure

- 2’ modified RNA, 2’-O-Methyl or 2’-methoxyethyl RNA (viewed as 2nd

generation Vs PS DNA as first generation)

-To enhance the affinity of oligonucleotide binding

- Currently testing in clinical trials

Current Antisense Material

Advantages of Phosphorothioate Antisense ODNs (PTOs)

- Convenient application in vivo and in vitro

- Excellent stability against exo- and endonuclease (half life >48hours in serum)

- Easy uptake from most eukaryotic cell type by active transport mechanism

(Usually no transfection procedure required)

-Excellent solubility

- Highly specific hybridization characteristics

- Low toxicity

- Established synthesis procedure and fast production

- Numerous clinical trials worldwide

- Only PTOs approved as antisense drugs by the FDA

- Low price ($200 / g, over 1kg volume)

Demands for New Antisense Material

Obstacles for Oligonucleotide Mediated Inhibition

1. RNA 의 2 차 구조 때문에 좋은 inhibitory sequence 를 찾기에 어려움 2 차 구조도 인지할 수 있는 새로운 material 의 필요성

2. Cell death 는 아니고 inhibition 만 하는 적정 dose 를 찾기가 어려움

3. Protein 에 비특이적으로 결합하여 비특이적 표현형 나타냄

Need to improve binding and selectivity by modifying oligonucleotide

New Antisense Material - Morpholino

- Nonionic DNA analogue (Gene Tools LLC)

- RNA-Morpholino duplex; no RNase H activity, Less strong

affinity than PNA, need 25 mer for actual working, less

interaction with cellular proteins

-Inhibition of expression: 5’UTR through +20

- Application in early developmental stage of Zebrafish, Seaurchin,

Xenopus embryo (Journal Genesis; fully devoted to Morpholino

antisense)

- Why morpholino more effective than other antisense chemistry?

- Easier invasion to local RNA 2dary structure owing to neutral

backbone

- The more disruptive confirmation of the backbone

- LNA-RNA hybrid: Not a substrate for RNase H activity

- Higher affinity than PNA (by up 10 C per substitution)

- Introduction of LNA by standard DNA/RNA synthesis methods

- Available at Proligo or Cureon as Oligomer

- Chimeric “gapmers” : LNA-DNA-LNA for RNase H activity with more

specificity owing to LNA

- Few studies on antisense inhibition of genes except a couple of

research papers

- Highly expensive

New Antisense Material – Locked Nucleic Acid

Chimeric Gapmers: LNA-DNA-LNA

Known by Fire and co-workers in 1998: DS RNA has inhibitory activity of gene expression in C.elegans

Long DS RNA processed to 20-22 base oligomers inside cell and short synthetic RNA transferred to mammalian cells

Good candidate for the tools of gene expression study in future

New Antisense Material – siRNA

Action mechanism of RNAi

Characteristics: neutral backbone, stronger affinity to DNA or RNA,

Chemically and Biologically stable, No binding to cellular proteins (avoiding a major source of nonspecific interaction)

Compatibility with current peptide chemistry advantage to peptide conjugation to augment PNA functions

- Promotion of strand invasion

- Increase of cell permeability

- Solubility improvement

- Improvement of permeability

- PNA + negative charged oligomer

- PNA + anionic lipid

- PNA+ peptide sequences

- PNA-RNA duplex: not a substrate for RNase H

New Antisense Material – Peptide Nucleic Acid

Application of PNA as Antisense material(PubMed search result, August 1, 2002)

Keywords Results

Phosphorothioate + Antisense 1,273 hits

PNA + Antisense 129 hits

Morpholino + Antisense 124 hits

RNAi + Antisense 74 hits

Methoxyethyl + Antisense 29 hits

LNA + Antisense 9 hits

Methoxyethoxy + Antisense 5 hits

PNA as Antisense and Antigene material

1. Inhibition of Transcription

- Triplex invasion at homopurine region

- Triplex forming at a regulatory region

2. Inhibition of Post-transcriptional modification

- Inhibition of splicing of pre-mRNA

3. Inhibition of Translation

- AUG start codon

- 5’UTR

4. bis PNA

- Inhibition of transcription factor binding

- Activation of transcription

5. Inhibition of Reverse Transcription (PCR clamping / Enhanced PCR)

Antisense and Antigene by PNA binding

Inhibition of transcription factor binding

Activation of transcription

Cellular uptake of PNA 1. Unmodified PNA

1) Microinjection

- First study of PNA effect in cell

- T Ag of SV40, 1um of 15mer and 20 mer showed 40% and 50% inhibitory effect.

- Too laborious only for small scale experiment

2) Electroporation

- 60% inhibition of telomerase activity

- Interfering with pre-mRNA splicing of IL-5Ralpha

- More feasible than microinjection

3) Co-transfection with DNA

- by Corey group, PNA-DNA hybrid and transfer with cationic lipid

4) Permeabilised cells

- Cell permeabilization by streptolycin-O deliver to nucleus (point mutation study)

5) Direct delivery

- E.coli mutant AS19, High concentration of PNA in Eukaryotic cell

1) Conjugation to lipophilic moieties

- Conjugation to liphophilic (adamantyl:ada-PNA, triphenylphosphonium:ph-PNA)

- adaPNA: dependent on cell type and PNA sequence less efficient

- ph-PNA: uptake by mitochondria but not active

- biotin-PNA: uptake by nucleus but no further study

2) Conjugation to peptides

- Trojan peptides: a class of amphiphilic cationic/hydrophobic peptide that transport

molecules across biological membrane in a receptor independent way.

- Penetratin: 16 aa from Drosophila transcription factor (Antennapedia) still

contradictory results (same result for Transportan)

- NLS(PKKKKRKV): Transport PNA across membrane, but Lys effect?

Significant nuclear uptake of Lys-PNA

- cell wall/membrane active peptide (KFFKFFKFFK): very efficient in bacteria

Cellular uptake of PNA 2. Modified PNA

Needs for more efficient and more general delivery tools for antisense use

3) Conjugation to cell-specific receptor ligands

Object: Instead of general protocol or modification, delivery to a specific cells

avoiding side effect on non-targeted cells

- PNA-peptide specific to IGF1R only uptake by IGF1R expressing cells

: Not useful vesicular inclusion

- PNA-lactose conjugate: recognize by ASGP-R(asialoglycoprotein receptor)

: Very limited biological effect weak uptake and vesicular inclusion

- PNA-dihydrotestosteron (T): uptake by cell lines derived from prostate carcinoma

: Highly controversial result

Receptor mediated endocytosis of PNA the Most likely solution to deliver PNA to specific cells

Documents

Antisense General