A presentation-on-nucleic-acid-therapeutics

Nucleic acid therapeutics are based on nucleic acids or closely related chemical compounds. They include antisense oligonucleotides, aptamers and small interfering RNAs, and are typically considered in cases where specific inhibition of the function of a particular gene involved in disease is thought to be therapeutically desirable.

1. A presentation on Nucleic Acid Therapeutics Tasmina Ferdous Susmi

2. Outline Therapeutic agent & Nucleic acid therapeutic agent. Types of Nucleic acid therapeutic agent. Mechanism of Nucleic acid therapeutic agent. Application of Nucleic acid therapeutic agent. Advantages & disadvantages of Nucleic acid therapeutic agent. Future aspects of Nucleic acid therapeutic agent.

3. Therapeutic agents The term comes from the Greek therapeutikos word which means “inclined to serve”. Therapeutics means serving and caring for the patient in a comprehensive manner, preventing disease as well as managing secific problems. Relating to therapeutics, the branch of medicine that is concernsed specially with the treatment of disease. Therapeutic dose of a drug is the amount needed to treat a disease

4. Nucleic acid as therapeutic agents Therapeutic system using nucleotide sequences are known as nucleic acid therapeutic agents. The delivery of nucleic acid molecules into cells to alter physiological functions at the genetic level is a powerful approach to treat a wide range of inherited and acquired disorders.

5. Nucleic acid as therapeutic agents Insertion of new genetic material into the cells of an individual can bring intentional therapeutic benefit Numerous nucleic acid-based therapy are under development. Some of which is used to inhibit gene expression at either the transcriptional or post transcriptional level.

6. Why Nucleic acid therapeutics ? To treat human diseases, researchers must be able to safely and effectively deliver nucleic acids to their targets. Nucleic acids interference therapies have the potential to treat many human diseases. P articularly those stemming from the gastro-intestinal (GI) tract, by transferring genetic materials to diseased cells to inhibit abnormal gene expression.

7. Nucleic acid as therapeutic agents Delivery vectors must protect the encapsulated material while traveling through the GI tract, and also be safe and easy to tolerate. Orally delivering nucleic acid therapy is a particularly promising option because it is simple to administer, non-invasive, and cost-effective. However , only a small percentage of these doses travel successfully to the disease site. They are impeded by anatomical and physiological barriers, including rapid degradation at low gastric pH and enzymatic degradation in the intestines.

8. Types of Nucleic Acid Therapeutics RNA based therapeutics: Antisense RNA Ribozymes RNA aptamers Interfering RNAs DNA based therapeutics: Plasmid DNA Antisense oligonucleotide DNA aptamer Deoxyribozymes Chimeric DNA-RNA molecules

9. RNA based therapeutics Antisense RNA Single stranded RNA molecule complementary to specific mRNA. 15-20 nucleotides Technology for prevention of expression of a gene translation level. Bind mRNA then take the help of RNAse H to breakdown the mRNA selectively.

10. RNA based therapeutics Ribozymes : Naturally occurring catalytic molecules that are 40-50 nucleotides in length. Have separate catalytic and substrate binding domains. Important advantages of ribozymes is that they are unlikely to evoke an immune response in a treated animal or human. Example : Self s plicing intron,SnRNAs,RNAse P, Hairpin,Hammerhead .

11. RNA based therapeutics RNA Aptamer : Single stranded nucleic acid segments, can directly interact with protein. The Word aptamer , derived from the latin " aptus "-fit and greek " meros "-part. Recognize their targets on the basis of shape complimentary. Binding specificity and affinity for target is high. Often identified using a technology called SELEX. Clinical study on humans with injection of anti VEGF aptamers in eye.

12. RNA based Therapeutics Interferring RNA: Part of transcriptional mechanism of gene silencing through chromatin remodeling,inhibition of protein or direct mRNA degradation. Used for down regulation of disease causing gene through RNA interference. Double stranded RNA molecules are cleaved by RNase lll like enzymes into single stranded RNA(21-23 nucleotides) called small interfering RNA. Inhibit HIV,Hepatitis,Influenza infection.

13. DNA based therapeutics Plasmid DNA: High molecular weight,double stranded DNA constructs containing transgene encode specific protein. Used in gene therapy . U sed to correct genetic errors that produce funtionally incompetent copies of a given protein. Antisense oligonucleotide : Oligonucleotide which is short single stranded segments of DNA that upon intematization can selectively inhibit the expression of single protein. Form duplex with mRNA and inhibit protein biosynthesis.

14. DNA based therapeutics DNA Aptamers : ds nucleic acid segments that can directly interact with proteins. Interfere with molecular function of disease implicated protein or those that participate in transcription or translation processes. Preferred over antibodies in protein inhibition owing to their specificity,non - immunogenecity and stability. Deoxyribozymes : Analogues of ribozymes with greater biological activity. Used against cardiovascular disease,cancer .

15. Chimeric RNA-DNA molecules Includes single mixed oligonucleotides with ribonucleotide and deoxyribonucleotides in a duplex conformation. In organization of nucleotides chimeric oligonucleotide is important . Protect the molecules from exonuclease . Shields the molecules from degradation of RNase . Helps in basepair correction.

16. Mechanism of Antisense RNA therapeutics Single stranded RNA. Complementary to mRNA. Introduced in a cell to inhibit it's translation machinery. Binds with mRNA & activates RNAse H enzyme. RNA duplex is unable of translation. Thus stops the production or activity of a specific protein.

17. Mechanism of Antisense RNA therapeutics(cont.) AUGAAA - Sense RNA/mRNA UACUUU - Antisense RNA

18. Mechanism of Antisense oligonucleotide Hybridizes with complementary nucleotide sequence/mRNA. Blocks the production of specific protein.

19. Mechanism of Ribozyme

20. Mechanism of Ribozyme

21. Mechanism of Ribozyme

22. Mechanism of Aptamer

23. Mechanism of Aptamer

24. Mechanism of RNAi Double stranded RNA containing complementary sequence of target mRNA is used. Dicer enzyme cuts the RNAi into siRNA's. Antisesne siRNA strand incorporated into RISC(RNA induced silencing complex). RISC binds to the mRNA and cuts it.

25. Mechanism of RNAi

26. Applications For treatment of any human disorder caused by over production of normal protein e.g , Cancer Inflammatory conditions Viral and parasitic infections

27. Applications Antisense RNA For treatment of malignant glioma (Human brain tumor) For treatment of prostate carcinoma Antisense oligonucleotide For treatment of cytomegalovirus infection To prevent restenosis after angioplasty Smooth muscle cell proliferation implicated in atherosclerosis, hypertension, diabetes mellitus, and the failure of coronary bypassed grafted are presumably controlled by antisense therapeutics. To control psoriasis(Uncontrolled epidermal growth) For treatment of tuberculosis

28. Applications Correction of a mutant splice site with an antisense oligonucleotide can be used to treat β - thalassemia .

29. Applications Ribozymes To prevent viral infections To prevent the accumulation of chemical/antibiotic resistance RNA Interference ( RNAi ) Gene silencing which plant and animals use to protect against viruses. To treat human neurodegenerative disease spinocerebellar ataxia 1 Scientists are optimistic about using RNAi to treat other neurological diseases such as Alzheimer’s and Hunting’s disease.

30. Applications DNA vaccine Antibody genes

31. Advantages Highly specific/selective Functional diversity Limited toxicity High efficiency

32. Limitations Susceptible to degradation by cellular nuclease enzyme. Less stability inside cell Need to be modified which may cause off target Determination of appropriate concentration of nucleic acid Viral vector may pose danger To deliver nucleic acid in target sites is difficult

33. Present situation of nucleic acid therapeutic agents Nucleic acid therapeutic agents have been researched as potential drug for diseases such as – Lung cancer Colorectal carcinoma Pancreatic carcinoma Malignant glioma Malignant melanoma Diabetes Amyotrophic lateral sclerosis Duchenne muscular dystrophy Spinal muscular atropy Asthma

34. Cancer To treate gliomas AP12009- A phosphorothioate antisens oligodeoxynucleotide specific for the mRNA of human transforming growth factor TGF-beta2 Experiment on 145 people -38 anaplastic astrocytoma patients treated with Karnofsky -89 anaplastic astrocytoma and glioblastoma patients treated with AP 12009 - 12 patients were treated with TMZ or PCV

35. Cytomegalovirus retinitis Also known as CMV retinitis Is an inflammation of the retina of the eye that can lead to blindness Caused by a type of herpes virus called Cytomegalovirus Treated with -Fomivirsen ( V itravene ) Approved by Food and Drug Administration

36. Familial hypercholesterolemia Known as homozygous familial hypercholesterolemia, or HoFH Causes extremely high cholesterol level and heart attacks Treated with Mipomersen ( kynamro ) Approved by Food and Drug Administration

37. Hemorrhagic fever In 2006 – 75% recovery rate Ebola hemorrhagic fever virus Treated with Morpholino Developed by sarepta therapeutics HIV/AIDS Starting 2004 Virus escape mutants pVRX494 and pVRX496 plasmid –derived from pUC18 backbone

38. Spinal muscular atrophy In 2004 Antisense oligonucleotide –( nusinersen ), developed by Ionis Pharmaceuticals FDA approval 2016 Duchenne muscular dystrophy A severe types of muscular dystrophy X-linked recessive Mutation of the gene for the protein dystrophin Treated with eteplirsen (a Morpholino oligo ) approved by FDA September 2016

39. Hypertriglyceridemia December 2016 Treated by volanesorsen Volanesorsen is in phase 3 clinical trials

40. Future aspects Antisense RNA and oligonucleotides : L ung cancer,  C olorectal carcinoma,  P ancreatic carcinoma, M alignant   melanoma,  D iabetes, Asthma Arthritis

41. Future aspects Antisense RNA and oligonucleotides : L ung cancer,  C olorectal carcinoma,  P ancreatic carcinoma, M alignant   melanoma,  D iabetes, Asthma Arthritis

42. Future aspects Aptamers : Therapeutic application of aptamers in lymphoma Aptamers “small bomb” mediates delivery of drug to cancer cells Potential next generation of diagnostic tools New drug development Bio-imaging Drug discovery

43. Future aspects Interfering RNAs: Used in treating cancer Defense against infection by viruses Suppress the transcription of viral genome Used to treat  bacterial diseases P otential for treating   neurodegenerative disease( Parkinson's disease   and   Polyglutamine disease)

44. Thank's to everybody for your kind attention