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
Oligo Company Status / Remarks
Resten NGE2F Decoy
AVI BioPharmaCorgentech
Target Disease: Cancer
Target Disease: Cardiovascular disorder
Oligo Company Status / Remarks
ISIS 2302
ISIS 104838AVI 4014
ISIS Pharma
ISIS PharmaAVI BIoPharma
Crohn’s D (PII/III)
Target Disease: Autoimmune & Inflammatory
Oligo Company Status / Remarks
Durason EpiGenesis
Target Disease: Respiratory disorder
Oligo Company Status / Remarks
AVI 4126 AVI BioPharma
Target Disease: Urological Disease
Oligo Company Status / Remarks
ISIS 14803HEPTAZYMEHepBzymePNAbioticsNeuBiotics
ISIS PharmaRibozyme PharmaRibozyme PharmaPanthecoAVI Biopharma
Target Disease: Infectious Disease
Oligo Company Status / Remarks
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