GENE THERAPY IN GENETIC DISORDERS LUKE SCID

Management of genetic disease with gene therapy

1. GENE THERAPY Presenter – Dr Vithal Patil Moderator- Dr Nagaraj Kotli Sir

2. OVERVIEW: DEFINITION INTRODUCTION HISTORY APPROACHES IN GENE THERAPY VECTORS IN GENE THERAPY STRATEGIES FOR GENE THERAPY ADVANTAGES DISADVANTAGES ETHICAL ISSUES CONCLUSIONS

3. WHAT IS A GENE ? Previous definition , GENE - a segment of DNA that carries the code for a particular protein Body has millions of proteins but only 30,000 genes Small percent of genes only produce RNA molecules Current Definition : GENE - An information containing segment of DNA that codes for the production molecule of RNA that plays a role in synthesis of one or more proteins

4. DEFINITION Gene therapy is the introduction of genes into existing cells to prevent or cure a wide range of diseases. It is a technique for correcting defective genes responsible for disease development.

5. There are more than 3000 genetic disorders caused by single gene defects. There are many other multifactorial disorders where genetic variation plays a significant role in the etiology of the disease. The possibility of curing the genetic disorder by replacing the defective gene or repairing the mutation insitu . Gene therapy, is an experimental modality of treatment where a functional gene is introduced into a cell to take over the function of the defective gene

6. HISTORY The first gene therapy trial in humans was approved in 1989. The first approved gene therapy experiment occurred on September 14, 1990 in US, when Ashanti DeSilva was treated for ADA-SCID Since then more than 1500 trials in various conditions have been carried out. Studies have provided the ‘proof of principle’ but safe and effective gene therapy for genetic disorders is still a long way to go. clinical trials using gene therapy are also being carried out for disorders like cardiovascular disease, diabetes mellitus, cancer and infectious diseases like human immunodeficiency virus (HIV).

7. REQUIREMENTS FOR GENE THERAPY Therapeutically suitable gene with a proven role in the pathophysiology of the disease Gene delivery system viral and non-viral vectors to transfer the gene into the cells Efficacy and safety studies Suitable manufacturing and analytical processes

8. APPROACHES IN GENE THERAPY In vivo gene therapy: direct delivery of genes into the cells of a particular tissue in the body. Ex vivo gene therapy :- Transfer of genes to cultured cells and reinsertion.

9. 1 st gene therapy – to correct deficiency of enzyme, Adenosine deaminase (ADA).

10. INVIVO GENE THERAPY Direct delivery of therapeutic gene into target cell into patients body Carried out by viral or non viral vector systems. It can be the only possible option in patients where individual cells cannot be cultured in vitro in sufficient numbers (e.g. brain cells). In vivo gene transfer is necessary when cultured cells cannot be re-implanted in patients effectively.

11. VECTORS IN GENE THERAPY To transfer the desired gene into a target cell, a carrier is required. Such vehicles of gene delivery are known as vectors. 2 Main classes ; 1] Viral vectors 2] Non Viral vectors

12. VIRAL VECTORS ADENO VIRUS VECTOR SYSTEM Adeno virus with a DNA genome – good vectors. Target- non dividing human cell Eg. Common cold adenoviru s.

13. RETROVIRUS VECTOR SYSTEM The recombinant retroviruses have the ability to integrate into the host genome in a stable fashion. Can carry a DNA of size – less than 3.4kb. Replication defective virus particles Target cell - dividing

14. C .ADENO ASSOCIATED VIRUS VECTOR. It is a human virus that can integrate into chromosome 19 It is a single stranded, non pathogenic small DNA virus AAV enters host cell, becomes double stranded and gets integrated into chromosome . D .HERPES SIMPLEX VIRUS VECTOR. Viruses which have natural tendency to infect a particular type of cell. They infect and persist in nervous cells

15. NON VIRAL VECTORS PURE DNA CONSTRUCT Direct introduction of pure DNA construct into target tissue. Efficiency of DNA uptake by cells and expression rather low. Consequently, large quantities of DNA have to be injected periodically

16. 2 .LIPOPLEXES LIPID DNA Complexes; DNA Construct surrounded by artificial lipid membrane. Most of it degraded by lysosomes

17. 3 . DNA MOLECULAR CONJUGATES Commonly used synthetic conjugate is poly- L- lysine bound to specific target cell receptor. Therapeutic DNA is then made to combine with the conjugate to form a complex. It avoids lysosomal breakdown of DNA 4 . HUMAN ARTIFICIAL CHROMOSOME Can carry a large DNA i.e , with one or more therapeutic genes with regulatory elements.

18. METHODS OF GENE DELIVERY 1.PHYSICAL METHODS Gene Gun Employs a high-pressure delivery system to shoot tissue with gold or tungsten particles that are coated with DNA. Microinjection -Process of using a glass micropipette to insert microscopic substances into a single living cell. -Normally performed under a specialized optical microscope setup called a micromanipulator

19. 2 . CHEMICAL METHODS A. USING DETERGENT MIXTURES Certain charged chemical compounds like Calcium phosphates are mixed with functional cDNA of desired function. The mixture is introduced near the vicinity of recipient cells. The chemicals disturbs the cell membrane, widens the pore size and allows cDNA to pass through the cell.

20. 2 LIPOFECTION It is a technique used to inject genetic materials into a cell by means of liposomes. Liposomes are artificial phospholipid vesicles used to deliver a variety of molecules including DNA into the cells.

21. STRATEGIES FOR GENE THERAPY GENE AUGMENTATION THERAPY TARGETED MUTATION CORRECTION RNA INTERFERENCE FOR INHIBITING GENE EXPRESSION

22. Gene Augmentation Therapy Most common form of gene therapy. Foreign gene replaces missing or defective gene. Eg. Replacement of defective p53 gene by a normal one in liver cancer.

23. Targeted Mutation Correction If a mutated gene produces a defective protein which by itself has the harmful effect. Dominant negative effect, then the mutation has to be repaired. For this purpose, a normal copy of the gene is required to be targeted to the same location as the defective gene by homologous recombination. This approach has not yet been possible, although much research is continuing in this area.

24. RNA Interference (RNAi) for Inhibiting Gene Expression RNA interference is the technology by which expression of the gene can be inhibited, so that the protein it codes for is not made. system depends on introduction of double stranded RNA (dsRNA) inside the cell which activates a cascade of events leading to destruction of the specific RNA molecule. The dsRNA may be in the form of silencing RNA (siRNA) short hairpin RNA, (shRNA)

25. ADVANTAGES: Gene therapy has the potential to eliminate and prevent hereditary diseases such as cystic fibrosis, ADA- SCID etc It gives someone born with a genetic disease a chance to life It can be used to eradicate diseases from the future generations.

26. DISADVANTAGES: Immune response to the transferred gene stimulates a potential risk to gene therapy. Viruses used as vectors for gene transfer may cause toxicity, immune responses, and inflammatory reactions in the host. Disorders caused by defects in multiple genes cannot be treated effectively using gene therapy

27. Gene silencing —repression of promoter Genotoxicity —complications arising from insertional mutagenesis. Phenotoxicity —complications arising from overexpression or ectopic expression of the transgene Immunotoxicity —harmful immune response to either the vector or transgene; or a harmful immune response of the vector (e.g. CAR T cells) Risks of horizontal transmission —shedding of infectious vector into environment Risks of vertical transmission —germline transmission of donated DNA

28. IMMUNODEFICIENCY DISORDERS: PROOF OF PRINCIPLE: The X-linked severe combined immunodeficiency disease (SCID), which from mutations in the gene (IL2RG) encoding the cytokine receptors required for normal development of T and natural killer (NK) cells . children treated in the five eventually developed a syndrome similar to T cell acute lymphocytic leukemia. The retroviral vector had integrated within a gene, LMO-2 (LIM only-2), which encodes a component of a transcription factor complex involved in hematopoietic development.

29. “suicide” gene cassette in the vector. Errant clones can be quickly ablated, or using “insulator” elements in the cassette, which can limit the activation of genes surrounding the insertion site. More clear-cut success has been achieved in a gene therapy trial for another form of SCID, adenosine deaminase (ADA) deficiency .

30. NEURODEGENERATIVE DISEASES: EXTENSION OF PRINCIPLE Use of genetically modified autologous cells carried several advantages including no risk of graft-versus-host disease, guaranteed availability of a “donor. X-linked adrenoleukodystrophy [ALD] Metachromatic leukodystrophy

31. Mutations in the gene encoding an adenosine triphosphate–binding cassette transporter. Affected boys present with clinical and neuroradiographic evidence of disease at age 6–8 and usually die before adolescence. Metachromatic leukodystrophy lysosomal storage disorder caused by mutations in the gene encoding arylsulfatase A (ARSA) progressive motor and cognitive impairment, and death within a few years of onset, due to accumulation of the ARSA substrate sulfatide in oligodendrocytes, microglia, and some neurons.

32. Lentiviral vector that directed supraphysiologic levels of ARSA expression in transduced cells. Transduction of autologous HSCs from children born with the disease, at a point when they were still presymptomatic, led to preservation and continued acquisition of motor and cognitive milestones . Trials are now under way for thalassemia and sickle cell disease, and for a number of other hematologic disorders, including Wiskott -Aldrich syndrome, and chronic granulomatous disease.

33. GENE THERAPY FOR CANCER MODIFYING THE CANCER TUMOR CORRECTION PRO-DRUG METABOLIZING GENES MODIFYING THE HOST Recruiting the Immune System Vaccination Adoptive Cell Transfer NON-IMMUNOLOGICAL MODIFICATIONS TO HOST

34. MODIFYING THE CANCER TUMOR CORRECTION Direct intratumoral approach was adenoviral-mediated expression of the tumor suppressor p53, which is mutated in many different cancers. Squamous cell carcinoma of the head and neck, esophageal cancer, and non-small cell lung cancer

35. Pro-Drug Metabolizing Genes I ntroduction of a prodrug or a suicide gene that would increase sensitivity of tumor cells to cytotoxic drugs. A frequently used strategy has been intratumoral injection of an adenoviral vector expressing the thymidine kinase Cells that take up and express the TK gene can be killed after the administration of gancyclovir , which is phosphorylated to a toxic nucleoside by TK T ransducing even a limited number of tumor cells are amplified by the spread of active drug to adjacent tumor cells. Brain tumors and locally recurrent prostate, breast, and colon tumors,

36. MODIFYING THE HOST Recruiting the Immune System monoclonal antibodies that produce anti-tumor activity by activating the immune response Immune cells are capable of almost unlimited expansion and persistence and can provide long-term tumor control. Vaccination more efficient recognition of tumor cells by the immune system. transduction of tumor cells with immune-enhancing genes encoding cytokines, chemokines, or co-stimulatory molecules, and the ex vivo manipulation of dendritic cells to enhance the presentation of tumor antigen

37. Adoptive Cell Transfer Host immune cells such as T cells, NK cells, and others can be modified to express new transgenic receptors intended to recognize tumor cells and their microenvironment Retargeting may use a modification of the cells’ own receptor or a synthetic chimeric antigen receptor (CAR) Significant responses reported with native receptors targeted to melanoma and synovial cell sarcoma and—most dramatically—with CARs targeted to CD19, an antigen expressed at high levels on normal and many malignant B cells

38. Non-Immunological Modifications to Host Gene transfer can be used to protect normal cells from the toxicities of chemotherapy Transduce hematopoietic cells with genes encoding resistance to chemotherapeutic agents, including the multidrug resistance gene MDRI or the gene encoding O6 -methylguanine DNA methyltransferase (MGMT). Gene transfer can be used to inhibit the host angiogenesis required for tumor support, for example by constitutive expression of inhibitors such as angiostatin and endostatin,

39. COMBINATION APPROACHES— MODIFICATION OF HOST AND TUMOR BY VIROTHERAPY Immuno Oncolytic Viruses These viruses are genetically modified to replicate in malignant but not normal cell. The replicating vectors thus proliferate and spread within the tumor, facilitating eventual tumor clearance. Physical limitations to viral spread, including fibrosis, intermixed normal cells, basement membranes, and necrotic areas within the tumor, may reduce clinical efficacy, and their activity against metastatic disease

40. CONCLUSION Theoretically, gene therapy is the permanent solution for genetic diseases. But it has several complexities. At its current stage, it is not accessible to most people due to its huge cost. A breakthrough may come anytime and a day may come when almost every disease will have a gene therapy. Gene therapy have the potential to revolutionize the practice of medicine

41. REFERENCES 1 .HARRISONS PRINCIPLES OF INTERNAL MEDICINE ,22 TH EDITION CHAPTER 458 2. API TEXTBOOK OF MEDICINE 11 TH EDITION

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