1 Molecular Cancer Therapeutics

1. 1Molecular Cancer Therapeutics Antisense Oligonucleotides RNA interference (RNAi) Vũ Mạnh Huỳnh Tiến Sĩ Hóa Học

2. Concepts Mechanisms Progresses • Developments in the sequencing of humangenome led to the use of short fragments of nucleic acid,antisense. • Antisense technology is the use of a complementary seq. of Watson-Crick bp hybridization, to a specific mRNA can inhibit its expression and then induce a blockade in the transfer of genetic information from DNA to protein. • The first antisense,Vitravene, for cytomegalovirus (CMV) retinitis, isapproved by FDA in 1998. • Many others are in clinical trials for disease treatments. • Antisense companies merged to major Pharmaceutical companies: ISIS/Ely Lilly, Coley, Idera, Genentech,… • Although antisense are commonly in use in laboratory and clinic, Scientists believed there are many questions concerning the molecular mechanism of action of these compounds.

3. Purines & Pyrimidines Hybridization

4. DNA Structure:

5. Blockade of Translation by Antisense: Antisense complementary to a mRNA bind mRNA, preventing translation by steric effect or by inducing degradation of mRNA by RNase

6. Antisense Directed against HIV

7. Antisense Designs for HIV • Oligonucleotides have been designed to bind at various sitesalong the genome of the human immunodeficiency virus (HIV). • Antisense nucleotides can bind to the proviral long terminalrepeats, which are regulatory regions that have an importantrole in integrating the proviral DNA into the human genome (anecessary step in the life cycle of HIV). • Several groups arestudying antisense oligonucleotides against HIV mRNA, especiallythe mRNAs for proteins that regulate viral expression, suchas tat and rev. • It has been possible to inhibit viral replicationin vitro with such antisense oligonucleotides.

8. Inhibition of Angiotensinogen by Antisense Oligonucleotides • DNA transcription is followed by translation of mRNA to form angiotensinogen, which in turn is converted • To angiotensin I by renin and • To angiotensin II by angiotensin-converting enzyme (ACE). • This cascade results in increased blood pressure. • ACE inhibitors are one form of pharmaceutical therapy that interrupts this cascade and lowers blood pressure. • Antisense oligonucleotides have been used in animal models to prevent the translation of mRNA into angiotensinogen, with decreases in blood pressure.

9. Inhibition of Angiotensinogen by Antisense Oligonucleotides

10. Antisense applications of DNA/RNA • Antisense therapy could be used in Cardiovascular disease. • Another possibility for the use of oligonucleotides is in thetreatment of leukemia. • DNA/RNA to block protein function to prevent the translationof messenger RNA (mRNA) into protein. • DNA or RNA that contains the information for the amino acidsequence of the protein is called the “sense” strand. The other sequence is complementary to the sensestrand and is called the “antisense” strand.

11. Technical issues of Antisense • In an attempt to overcome the various nonspecificproblems, new chemical modifications have been developed. • These"second-generation" oligonucleotides are resistant to degradationby cellular nucleases • Hybridize specifically to their targetmRNA • Higher affinity than phosphodiesteror phosphorothioate.

12. Antisense with new backbone

13. TETD (Tetraethylthiuram disulfide)Sulfurization1 TETD converts cyanoethyl phosphite to the phosphorothioate triester. • TETD in ACN is available for synthesizing Phosphorothioate Oligo. • Enzymatic Digestion shows no detectable base modification. 1 Vu, H. and Hirschbein, L. B. "Internucleotide Phosphite Sulfurization WithTetraethylthiuram Disulfide. Phosphorothioate Oligonucleotide Synthesis via Phosphoramidite Chemistry.", Tetrahedron Lett.,1991, 32, 3005-3008.

14. Synthesis of (N3’P5’) Phosphoramidate Vu, H., Rao, T. S., Akiyama, T., Hogan, M. E., Ojwang, J. O., Rando, R. F., Revankar, G. R. “Automated synthesis of oligonucleotide (N3P5) phosphoramidates using 3-amino-2,3-dideoxynucleoside phosphoramidites” presented at 213th ACS National Meeting, April 13-17, 1997, San Francisco, CA.

15. Phosphoramidate Antisense • Another example of a "second-generation" oligonucleotide isthe N3'P5' PN. • Oxygen of 3’-position of ribose is replaced by an Amine. • Can form very stable complexes with RNA, and single or double stranded DNA. • Can exhibit highly selectiveand specific antisense activity in vitro and in vivo. • Inhibited efficiently the growth of treatedBV173 cells. • Inhibited selectivelythe c-myc protein expression and the proliferation of HL-60cells.

20. Phosphoramidate Antisense • Another example of a "second-generation" oligonucleotide isthe N3'P5' PN. • Oxygen of 3’-position of ribose is replaced by an Amine. • Can form very stable complexes with RNA, and single or double stranded DNA. • Melting Temperature shows significant increase (10.9 °C). • Can exhibit highly selectiveand specific antisense activity in vitro and in vivo. • Inhibited TNF production in phorbol myristate acetate andinterferon gamma (PMA/IFN) stimulated THP-1 cells. • Inhibited efficiently the growth of treatedBV173 cells. • Inhibited selectivelythe c-myc protein expression and the proliferation of HL-60cells.

21. Locked Nucleic Acids - LNAs • LNAs a class of restricted nucleotide analogs. • LNA increases the affinity of RNA or DNA. • LNA increases the melting temperature (Tm) of duplex. • Differentiate effectively between a perfect matched target and a mismatched target • Highly sensitive, and discriminatory in miRNAs

22. Advantages of Antisense • Antisense oligonucleotides have potential as a unique way totreat a variety of diseases. • There is concern about the mechanismof action of the oligonucleotides, drug-delivery systems, cellular-uptake systems, andlong-term effects. • Oligonucleotide therapy does not havethe safety and efficacy issues associated with expressed-vectorgene therapy, • Its use in some applications is advancingon the road to approval by the Food and Drug Administration.