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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.

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Antisense General

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  1. 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

  2. Relation of antisense technologies to other segments of biopharmaceutical industry PHARMACEUTICALS ANTISENSE TECHNOLOGIES MOLECULAR DIAGNOSTICS GENOMICS BASED DRUG DISCOVERY DRUG DELIVERY SYSTEMS GENE THERAPY

  3. 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)

  4. Antisense Drugs and Companies

  5. 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)

  6. 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)

  7. Antisense Drugs in Developments Target Disease: Cardiovascular disorder Target Disease: Cancer Target Disease: Autoimmune & Inflammatory Target Disease: Respiratory disorder

  8. Target Disease: AIDS & Related disease Target Disease: Urological Disease Target Disease: Infectious Disease

  9. Antisense Companies appeared in Theta Reports, April 2002 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)

  10. ISIS Pharmaceuticals

  11. Genta

  12. 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

  13. Functional Genomics General • - Target identification • Target validation study • Drug candidate

  14. 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 • Proteinresponsible 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

  15. Alteration of Splicing

  16. Inhibition of Ribonucleoprotein

  17. Cellular uptake of antisense Nuclease action: Degradation by nuclease: Full degradation of 18 mer natural oligomucleotide within 30 minutes

  18. Basic of Cellular Uptake

  19. 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

  20. Antisense Action Mechanism • RNase H independent (Inhibition of Translation) • - By steric hindrance upon binding of oligonucleotides • - 5’UTR • - AUG start codon • - ORF • RNase H dependent • - By RNase H activity to digest RNA-oligo duplex

  21. 5’UTR

  22. AUG start codon

  23. ORF

  24. RNase H dependent

  25. Current Antisense Material - 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

  26. 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)

  27. Demands for New Antisense Material • Obstacles for Oligonucleotide Mediated Inhibition • RNA의 2차 구조 때문에 좋은 inhibitory sequence를 찾기에 어려움  2차 구조도 인지할 수 있는 새로운 material의 필요성 • 2. Cell death는 아니고 inhibition만 하는 적정 dose를 찾기가 어려움 • 3. Protein에 비특이적으로 결합하여 비특이적 표현형 나타냄 •  Need to improve binding and selectivity by modifying oligonucleotide

  28. 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

  29. New Antisense Material – Locked Nucleic Acid • - 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

  30. Chimeric Gapmers: LNA-DNA-LNA

  31. New Antisense Material – siRNA 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

  32. Action mechanism of RNAi

  33. New Antisense Material – Peptide Nucleic Acid 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

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

  35. 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)

  36. Antisense and Antigene by PNA binding

  37. Inhibition of transcription factor binding

  38. Activation of transcription

  39. 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

  40. Cellular uptake of PNA 2. Modified PNA Needs for more efficient and more general delivery tools for antisense use 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

  41. 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

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