1. Synthetic DNA as Drugs Supported by the NSF award 0647129 Simple Methods for Oligonucleotide Purification

2. Why Can DNA Be Used as Drugs? The Structure of DNA and RNA  Four bases in DNA and RNA  They are A, T (U in RNA), G and C  Genetic information is stored in the sequence of A, T, G, C in DNA  DNA double stranded  RNA double/single stranded  A pairs to T (U in RNA) only and G pairs to C only  DNA-DNA, RNA-RNA, DNA- RNA

3. DNA-DNA: A-T, G-C RNA-RNA: A-U, G-C DNA-RNA: A-U, G-C Why Can DNA Be Used as Drugs? The Base Pairs

4. Why Can DNA Be Used as Drugs? The Central Dogma of Molecular Biology  DNA: double stranded, contains genetic information  RNA: single stranded  Protein: the function molecule of life, function determined by the sequence of amino acids

5. Why Can DNA Be Used as Drugs? How People Get Disease? Mono-genetic disorder  Currently, a total of ~4,000 genetic disorders are known  Some are single genetic disorder  Changes (mutations) of the sequence of one gene (DNA) Point mutation Deletion And more  List of genetic disorders at http://en.wikipedia.org/wiki/List_of_genetic_disorders http://en.wikipedia.org/wiki/List_of_genetic_disorders  The mutated genes produce proteins that cannot function properly, diseases occur  Examples: Sickle-cell anemia, Cystic fibrosis ( 1/3900, most common, difficult breathing, die in 20s-30s, no cure ), Color blindness

6. Why Can DNA Be Used as Drugs? How People Get Disease? Poly-genetic disorder  Mutations occur in many genes  Do not have a clear cut of inheritance  But do “run in families”  The mutated genes produce proteins that cannot function properly, diseases occur  Difficult (not impossible) to study and treat because direct cause is unknown  Examples: Heart disease, hypertension, diabetes, obesity, cancers, low IQ

7. Why Can DNA Be Used as Drugs? How People Get Disease? Bacteria and virus infections Smallpox –300-500 million deaths in 20 th century –Eliminated by vaccination HIV/AIDS –Human immunodeficiency virus/acquire immunodeficiency syndrome –Still taking many lives each year, in 2000, 2.8 million Hepatitis B –0.1 million lives taken in 2000 –Liver inflammation, vomiting, and rarely death, but can lead to cancer Tuberculosis –Bacteria, mostly infect lung, but also other parts of body –In 2004, 1.7 million deaths

8. Why Can DNA Be Used as Drugs? How to stop genetic disorder using DNA drugs?  Design a short DNA sequence that matches the sequence of mRNA that is transcribed from the mutated gene (which causes diseases)  The DNA drug binds to the mRNA (A-U, G-C)  The mRNA cannot be translated to protein  Because no disease-causing protein, disease is cured

9. Why Can DNA Be Used as Drugs? How to stop diseases caused by microorganisms using DNA drugs?  Select one or more genes that are critical for the disease-causing bacteria or virus  Design DNAs that can stop the critical gene expressions  The bacteria or virus dies and diseases cured

10. Why Use Synthetic DNA?  Natural DNA will be digested by enzymes, and also can cause immune response  Synthetic DNA cannot be recognized by enzymes, so they are stable and may not cause immune response  So, synthetic DNA can selectively block gene expression

11. An Example of DNA Drug

12. Current Research At a company called ISIS

13. Current Status of Antisense Drugs Antisense drugs stop making sense? 18/12/2006 - The once hyped antisense sector of the pharmaceutical industry has again proved a disappointment after one drug in development was refused approval and another was scrapped. Genta,, US, has announced that the lead compound in their antisense programme failed to win approval from the US Food and Drug Administration (FDA). Genasense was to be given alongside chemotherapy in the treatment of chronic lymphocytic leukaemia (CLL). "We are keenly disappointed by this decision," said Dr Raymond Warrell, Genta's chief executive. "We believe that Genasense has amply demonstrated its efficacy and safety in patients with relapsed and refractory CLL in a carefully designed and executed randomized clinical trial. "As we decide on next steps with this application, we will continue working to seek worldwide approval of Genasense for patients who have advanced cancer." Genasense inhibits production of Bcl-2, a protein made by cancer cells that prevents cell death. By reducing the production of Bcl-2, it was hoped that Genasense would increase the effectiveness of other cancer treatments. This is not the first time Genta have suffered disappointment with

14. Current Status of Antisense Drugs First Antisense Drug Reaches Review Applied Genetics News, Sept 1, 1998 Applied Genetics NewsSept 1, 1998 The FDA's Ophthalmic Drugs Subcommittee has voted to recommend approval of trademarked Vitravene (fomivirsen) for the treatment of cCytomegalovirus (CMV) retinitis in AIDS patients. The product is an antisense drug invented by Isis Pharmaceuticals (2292 Faraday Ave., Carlsbad, CA 92007; Tel: 619/931-9200, Fax: 619/931-9639) and to be marketed by Ciba Vision Corp., the eye care unit of Novartis (59 Rte. 10, E. Hanover, NJ). Vitravene is the first antisense drug to be filed for commercial marketing review. It is an inhibitor of CMV replication, the virus that causes retinitis. CMV retinitis is a degenerative opportunistic infection that affects people with AIDS and results in blindness. The new drug application (NDA) for Vitravene was filed by Isis on April 9, 1998, and is being reviewed under the agency's priority review process. According to Daniel Kisner, president of Isis, the rising failure rate of protease inhibitor-based combination AIDS therapy due to resistance, toxicity, and non-compliance signals a potential resurgence in CMV retinitis and other opportunistic infections. Vitravene is administered locally into the eye by intravitreal injection. The most frequently observed ocular side effects were transient--increased intraocular pressure and generally mild to moderate, reversible intraocular inflammation. The retinal detachment rate overall in the Phase III program was considerably lower than the incidence of retinal detachment commonly observed in patients with CMV retinitis. There was no evidence of any systemic toxicity from the drug.

15. Current Status of Antisense Drugs 2007

16. Current Status of Antisense Drugs  Potency---Stability, Affinity  Delivery  Toxicity---Selectivity  Only two drugs are proved by FDA

17. Exercise Design a DNA drug that may cure sickle-cell anemia Genetic disease that can cause death (affects 0.4% African Americans, die at ~40s) Inherited Hemoglobin is a protein that carries oxygen from lung to cells, it is in red blood cells in the blood In sickle-cell Hemoglobin, Glu (negatively charged) is mutated to Val (hydrophobic) Val binds to hydrophobic pocket of another deoxygenated Hb S, form dimers and then polymeric fibers The polymerization cause sickled red blood cells Sickle cells cannot pass small capillaries of circulation system Because of insufficient oxygen supply, many organs such as bone and kidney can be damaged; short breath, paleness, pain, fatigue The amino acid change in sickle-cell Hemoglobin is resulted from the mutation of the Hemoglobin gene (following are antisense strands, templates for mRNA synthesis): –Normal hemoglobin gene, ……CTG ACT CCT GAG (codes Glu) GAG AAG TCT…… –Sickle-cell hemoglobin gene, ……CTG ACT CCT GAG (codes Val) GAG AAG TCT…… What is the sequence of DNA drug for blocking the expression of the mutated gene? What might be the problem after blocking the mutated gene?

18. Exercise Design a DNA drug that may cure cancer  Many cancer cells over express Bcl 2 protein, this protein prevents cell death  Block the synthesis of the protein will increase the chance of killing cancer cells by chemotherapy  The Bcl-2 gene is: ……TGT TCT CCC GGC, TTG, CGC, CAT….  What should be the sequence of the antisense DNA?

19. Antisense Sense strand, non-coding strand DNA Antisense strand, coding strand, template for mRNA synthesis mRNA Sense sequence DNA drug Antisense sequence