GENE THERAPY

GENE THERPAY PPT

1. Presented by N .Narmatha Gene therapy

2. c ontents Introduction History Gene therapy Genes Function of genes General principles of gene therapy Mode of transmission – vectors - types Types of gene therapy Techniques of gene therapy for oral cancer Clinical application in dentistry

3. INTRODUCTION: Gene therapy is an experimental technique that uses genes to treat or prevent disease Consists of introducing specific genetic material into target cells to compensate for abnormal genes or to make a beneficial protein Replace a faulty gene to introduce a new gene - cure or to favourably modify the clinical course of the condition.

4. Transferred genes – used for reparative or pharmacological process Genes are altered Encoded proteins Unable to carry out their normal function Genetic disorders result

5. 1995- potential impact of gene therapy on dentistry has been described On the basis of initial studies of gene transfer applications to salivary glands, keratinocytes and cancer cells Gene therapy in seven areas relevant to dental practice are Bone repair Salivary glands Autoimmune disease Pain DNA vaccinations Keratinocytes C ancer

6. GENE THERAPY: It is a technique to deliver small DNA or RNA sequences to cells or tissues to correct a genetic defect or treat a disease. E arly 1980’s – genetic replacement therapy GENES: Genes are the smallest functional units of the genetic system specific sequences of bases that encode instructions to make proteins linear sequence of DNA that codes for particular protein.

7. FUNCTON OF GENES: Determine the structure of proteins Controlling where, when and in what quantity each protein is made.

8. GENERAL PRINCIPLES OF GENE THERAPY: I ntroduction of exogenous genes into somatic cells – form organs with desired therapeutic effect

9. Vector constructs- basis of recombinant DNA techniques S econd step entry of modified vector to the target human cells Releasing DNA sequence becomes integrated within the chromosome. As the gene is “switched on” in its correct location, the cells with the new genetic design start forming the required therapeutic proteins

10. MODE OF TRANSMISSION: VECTOR: Carrier called VECTOR is used to introduce therapeutic gene into patient’s target cells. Most common vector – VIRUS that has been genetically altered to carry normal human DNA Types of vectors Viral Non viral Physical

11. VIRAL VECTORS Adenovirus Retrovirus Adenovirus –associated virus Lentivirus Vaccinia virus Herpes simplex virus NON VIRAL VECTORS: Lipid complex Liposomes Peptide/protein Polymers PHYSICAL VECTORS Electroporation Microinjection Gene gun Ballistic particles

12. TYPES OF GENE THERAPY: GERM LINE THERAPY SOMATIC THERAPY Gene insertion Into germ cells Into body cells Modification Inherited by offspring Restricted to individual Safety Not ethically safe Ethically sound and safe TYPES OF GENE THERAPY

13. TECHNIQUES OF GENE THERAPY FOR ORAL CANCER Gene addition therapy Gene excision therapy Antisense RNA therapy Suicide gene therapy Gene therapy by oncolytic virus Immunotherapy

14. Gene addition therapy Regulates tumor growth Addition of tumor suppressor gene P53 Induce apoptosis

15. Gene exicision therapy Inhibits tumor growth Removes defective oncogene Egr-1 (early growth response factor) Inhibits cell division

16. ANTISENSE RNA THERAPY Gene expression inhibited by RNA Prevents activity of oncogenes – myc , ras and fos Inhibits virus- HPV 1, HTLV, HSV -1

17. SUICIDE GENE THERAPY Genes introduced stimulates generation of products toxic to tumour cells Enable prodrug to active cytotoxic drug

18. IMMUNE THERAPY Increases patient immune response Increases efficacy of NK cells,T cells and cytokines TYPES: Therapeutic cancer vaccines Monoclonal antibodies Checkpoint inhibitors Cytokines Administration of IL -2 activates T cells & NK cells activates TNF

19. GENE THERAPY BY ONCOLYTIC VIRUS: Viruses are genetically modified Replicates and lyses tumor cells Virus – HSV , cheifly adenovirus Adeno ONYX-015 Adeno ONYX15 + 5 – fluoracil Increase in cytokines- IL – 6 ,TNF Tumor regression

20. CLINICAL APPLICATION: Accomplished in either of the two ways Invivo exvivo Invivo gene transfer: Foreign gene introduced into patient by viral or non viral methods Ex vivo gene transfer: Foreign gene transduced into the cells of a tissue biopsy Results in genetically modified cells Transplanted back into patient

21. CLINICAL APPLICATION IN DENTISTRY

22. GENE THERAPY FOR PAIN MANAGEMENT M anagement of chronic pain by sparing the use of drugs modified adenovirus , adeno associated virus (AAV) lipid encapsulated plasmids coding for Interleukin-10 (therapeutic protein) sub arachnoid space to transduce the pia mater cells production and secretion of anti-nociceptive proteins in or near spinal dorsal horns

23. Modified herpes virus intradermal injection to the skin nerves of Dorsal Root Ganglia (DRG) rationale for using herpes virus - infects nerves, ability to travel to the DRG via nerve endings in the skin codes for inhibitory neurotransmitter, an anti-inflammatory peptide or decreases the synthesis of an endogenous nociceptive molecule alleviation of pain

24. CARCINOMAS R esponse rate is higher in combination of gene therapy with other conventional treatment modalities. Carcinogenesis occurs by either overexpression of oncogenes such as Ras , Myc , Bcl‐2, ErbB2 underexpression mutation of tumour suppressor genes such as p53, p16, p21, Rb genes

25. Injection into the localized tumor mass precludes unwanted side effects on the body and premature degradation of the gene before it reaches the target cells

26. CORRECTIVE GENE THERAPY (GENE REPLACEMENT THERAPY) correction of underlying genetic defect - control unrestricted multiplication of tumour cells. over-expressed oncogenes are blocked or silenced by inclusion of DNA into the cell - disrupting transcription and translation. With gene therapy correct copy of p53 gene introduced into the tumor cells apoptosis .

27. In 2003 - the first gene therapy drug Gendicine , a recombinant human adenovirus p53 - formulated for the treatment of SCCHN and other cancerous lesions.

28. CYTOREDUCTIVE GENE THERAPY A ims at destruction of tumour cells I nsertion of suicide genes - tumour cells - encode for enzymes – convert chemotherapeutic drugs into their toxic form Limit angiogenesis apoptosis in tumour cells S electively multiply in tumour cells and kill them- size of the tumour after its surgical removal and prevent metastasis In 2005 - first genetically engineered oncolytic virus, H101 Adenovirus, - approved for treatment of SCC

29. MODIFICATION OF IMMUNE SYSTEM: I njecting genetically modified hematopoietic stem cells & T cells boost up host’s immune system I dentify and kill tumor cells. Insertion of a gene in tumor cells U pregulate their antigen markers S usceptible to destruction by the body’s own immune system.

30. Insertion of a gene encoding for cytokines. concentration of cytokines in tumor cells Immunotherapy beneficial in treatment of SCC, melanoma, lymphoma and some virus induced malignancies

31. BONE REGENERATION At least four imperative elements are required for successful bone regeneration osteoinduction , differentiation of osteoblasts osteoconduction and mechanical stimulation . Gene therapy enhances the first three conditions

32. Osteoinductive potential of Bone morphogenetic proteins (BMP-2, 4 and 7) - induce de novo bone formation delivering the BMP-2 genes directly to the tissues via an adenoviral vector healing of mandibular osseous defects

33. Non-osteogenic fibroblasts (from human gingiva and dental pulp) and myoblasts, osteoblasts - express BMP-7 gene after infected with an adenoviral vector – differentiate into bone forming cells when placed in an osseous defect in vivo

34. Growth arrest specific (gas) gene encodes the PDGF factor – down regulation of PDGF activity – transient biological activity B ioactive PDGF gene - inhibitory effects of growth arrest gene - wound healing bone sialoprotein - involved in bone repair along with the CBFA1 gene- involved in cell differentiation and gene expression of bone sialoprotein

35. Salivary glands exhibit S elf-containment due to a surrounding capsule - minimize undesirable access of administered vectors and transgenes to other tissues H ighly efficient protein production S ecrete proteins into bloodstream - potentially useful target sites for gene transfer in a minimally invasive manner with the help of intraductal cannulation.

36. IONIZED RADIATION Damage to fluid secretory portion (acinar cells) of the salivary gland Hypofunctional salivation aquaporin 1 gene Aquaporin 1 (aq1) is a water channel protein that counter balances this detriment by a constitutively activated water channel.

37. H uman trial in 2012 - promising results for using aquaporin-1 cDNA for the management of radiation-related salivary hypofunctions AQP1 gene therapy for the management of xerostomia patients

38. Osteoprotegerin (OPG )+ receptor activator of nuclear factor kappa-B ligand (RANKL ) - inhibits osteoclastogenesis jamming the process of bone resorption . Local RANKL gene - transferred to periodontal tissue - accelerated orthodontic tooth movement (150 % after 21 days) - reducing the time of treatment.

39. DNA VACCINATION Immunization of salivary gland plasmid DNA encoding P.gingivalis fimbrial gene fimbrial protein in salivary gland tissue specific salivary immunoglobulin A, or IgA, G , or IgG, fimbrial protein in saliva bind to pellicle components inhibit the attachment of P. gingivalis to the developing plaque .

40. Prevent periodontal diseases and dental caries. plasmid pCIA -P encoding pac gene of S.mutans - induce anticaries immune responses

41. TOOTH REPAIR AND REGENERATION Gene therapy presents an attractive concept of restoring the oral tissues lost due to caries, periodontal diseases and trauma. W iden the scope for development of new teeth—the biological implants for missing teeth.

42. Two basic approaches In vivo gene therapy , Healing potential of tissues such as dentine pulp complex - enhanced by genes - dentine formation - on the exposed dental pulp Third dentition - induced to form teeth - turning on or activating genes - code for proteins and signaling molecules making up the basic structure of teeth

43. E x vivo gene therapy - based on multipotent dental stem cells - potential to differentiate into any tissues including dental tissues. sources - dental pulp, apical papilla, dental follicles, deciduous teeth, and periodontal ligaments .

44. Stem cells - cultured, modified or transfected - re-implanted back into the recipient. stem cells engineered by the adenovirus to express the BMP-2 gene regeneration of the periodontal attachment apparatus including alveolar bone and cementum

45. CONCLUSIONS Considering the exponential rise in reported cases of oral squamous cell carcinoma and periodontal diseases, gene therapy is expected to be a very useful tool for the management of oral diseases and improving the prognosis and quality of life.

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