Genetic screening & gene therapy

1. GENETIC SCREENING & GENE THERAPY Dr. Dinesh T Junior resident, Department of Physiology, JIPMER Genetic screening & Gene therapy Dr sclero dinesh

2. Introduction

3. History Technology to detect and treat inborn diseases - 1961. 1972 Friedmann and Roblin authored a paper in Science titled " Gene therapy for human genetic disease?“ The late 1980's, an international team of scientists began the project to map the human genome. September 14, 1990 - first approved gene therapy case in the United States took place.

4. 1992 - Doctor Claudio Bordignon , Milan, Italy performed the first procedure of gene therapy using hematopoietic stem cells. 1995 DNA testing in forensic cases gains fame in the O.J. Simpson trial . 2002 - first successful gene therapy treatment for adenosine deaminase -deficiency (SCID) 2003 – at University of California, Los Angeles research team inserted genes into the brain using liposome coated in a polymer called polyethylene glycol

5. 2006 - Preston Nix from the University of Pennsylvania School of Medicine reported on VRX496, a gene-based immunotherapy for the treatment of human immunodeficiency virus (HIV) that uses a lentiviral vector for delivery of an antisense gene against the HIV envelope 2007 – Moorfields Eye Hospital and University College London's Institute of Ophthalmology announced the world's first gene therapy trial for inherited retinal disease 2008 there were more than 1,200 clinically applicable genetic tests available.

6. Genetic screening

7. What is genetic screening? The newest and most sophisticated of the techniques used to test for genetic disorders. One of the fastest moving fields in medical science. A technique to determine the genotype or phenotype of an organism . Determines risk of having or passing on a genetic disorder.

8. Genetic screening Genetic screening is often used to detect faulty or abnormal genes in an organism Can detect some genes related to an increased risk of cancer Can detect some genes known to cause genetic disorders

9. Genetic tests The analysis of chromosomes (DNA), proteins, and certain metabolites in order to detect heritable disease-related genotypes, mutations, phenotypes, or karyotype for clinical purposes.

10. Gene tests (also called DNA-based tests), in a broader sense Direct examination of the DNA molecule Biochemical tests for such gene products as enzymes and other proteins Microscopic examination of stained or fluorescent chromosomes

11. Genetic tests Who can order? What are the samples needed? How to interpret the tests? What are all the risks? Ethical considerations?

12. Types of screening tests Carrier screening Prenatal diagnostic testing Newborn screening Pre symptomatic testing for predicting adult-onset disorders such as Huntington's disease Pre symptomatic testing for estimating the risk of developing Adult-onset cancers and Alzheimer's disease. Conformational diagnosis of a symptomatic individual Pre implantation genetic diagnosis Forensic/ identity testing Research Pharmacogenomics

13. Genetic screening Adult Polycystic Kidney Disease Alpha-1-antitrypsin deficiency Amyotrophic lateral sclerosis Alzheimer's disease Ataxia telangiectasia Central Core Disease Charcot-Marie-Tooth disease Congenital adrenal hyperplasia Cystic fibrosis Duchenne muscular dystrophy/Becker muscular dystrophy Dystonia Emanuel Syndrome Fanconi anemia , group C Factor V-Leiden Fragile X syndrome Gaucher disease Hereditary Hemochromatosis Huntington's disease Hereditary nonpolyposis colon cancer Hemophilia A and B Inherited breast and ovarian cancer Marfan Syndrome Mucopolysaccharidosi Myotonic dystrophy Neurofibromatosis type 1 Phenylketonuria Polycystic Kidney Disease Prader Willi / Angelman syndromes Sickle cell disease Spinocerebellar ataxia, type Spinal muscular atrophy Tay-Sachs Disease Thalassemias Timothy Syndrome Galactosemia

14. Methods for prenatal screening

15. Abnormal results in prenatal screening Previous child with a chromosome abnormality (probability of translocation carrier in parents) Family history of a chromosome abnormality Family history of a single gene disorder Family history of neural tube defect or other congenital abnormalities Indications for prenatal diagnosis

16. Maple Syrup Urine Disease Congenital Adrenal Hyperplasia Congenital Hypothyroidism Glactosemia Biotinidase Deficiency Homocystinuria Phylketonuria (PKU) Sickle cell and Other Hemoglobinopathies Newborn Screening Tests:

17. Pre implantation Genetic Diagnosis (PGD) Pre implantation Genetic Diagnosis (PGD) uses in vitro fertilisation (IVF) to create embryos. Tests one or two cells from each embryo for a specific genetic abnormality. Identifies unaffected embryos for transfer to the uterus. The approach through PGD assists couples at risk of an inherited disorder to avoid the birth of an affected child without going through selective pregnancy termination.

18. Pros and cones of gene testing To clarify a diagnosis and direct a physician To avoid having children with devastating diseases Identify people at high risk P rovide doctors with a simple diagnostic test Transforming it from a usually fatal condition to a treatable one Possibility of laboratory errors Potential for provoking anxiety, and risks for discrimination social stigmatization could outweigh the benefits of testing

19. Genetic counseling

20. The process by which patients or relatives, at risk of an inherited disorder, are advised of the consequences and nature of the disorder, the probability of developing or transmitting it, This complex process can be seen from diagnostic (the actual estimation of risk) and supportive aspects. Genetic counseling

21. Conception (i.e. when one or two of the parents are carriers of a certain trait ) During pregnancy (i.e. if an abnormality is noted on an ultrasound or if the woman will be over 35 at delivery ) After birth (if a birth defect is seen) During childhood (i.e. if the child has developmental delay) During adulthood (for adult onset genetic conditions such as Huntington’s disease or hereditary cancer syndromes ). When can we do counseling?

22. The couple should be counselled by a genetic counsellor to inform them of the test results and the risks to the foetus. Providing the options open to them in management and family planning in order to prevent, avoid or ameliorate it . Autonomy of decision is crucial. The ethical, legal, and religious issues should be respected.

23. A type of genetic test that is accessible directly to the consumer without having to go through a health care professional. A variety of DTC tests, ranging from testing for breast cancer alleles to mutations linked to cystic fibrosis. Benefits of DTC testing are the accessibility of tests to consumers, promotion of proactive healthcare and the privacy of genetic information. Risks of DTC testing are the lack of governmental regulation and the potential misinterpretation of genetic information . Direct-to-Consumer (DTC) genetic testing

24. Gene therapy

25. Gene therapy is the replacement of faulty genes. Introduction of functional genetic material into target cells to replace or supplement defective genes, or to modify target cells so as to achieve therapeutic goals.

26. In theory it is possible to transform either somatic cells (most cells of the body) or cells of the germ line (such as sperm cells , ova, and their stem cell precursors).

27. All gene therapy so far in people has been directed at somatic cells. Germ line engineering in humans remains only a highly controversial prospect.

28. For the introduced gene to be transmitted normally to offspring, it needs not only to be inserted into the cell, but also to be incorporated into the chromosomes by genetic recombination

29. Somatic Cell Therapy This is when a gene is introduced into a patient to help them recover from a disease.

30. Germ Line Therapy Changes are made to genes that will affect subsequent generations.

31. Applications of Gene Therapy Radical cure of single gene diseases e.g. cystic fibrosis, haemoglobinopathies. Amelioration of diseases with or without a genetic component e.g. malignancies, neurodegenerative diseases, infectious diseases.

32. Gene therapy concerns

33. Vectors in gene therapy: Viruses Retroviruses Adenoviruses Adeno -associated viruses Envelope protein pseudotyping of viral vectors Non-viral methods Naked DNA Oligonucleotides Lipoplexes and polyplexes Hybrid methods

34. A new gene is inserted into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein. Gene therapy using an adenovirus vector.

35. Non viral vectors Un complexed plasmid DNA DNA coated gold particles Liposomes DNA – protein conjugates

36. Modes of introducing genetic material

37. Un complexed Plasmid DNA Purified DNA or mRNA injected directly into tissues Injected into muscle and skin • Utility in immunization/ vaccination against Infectious diseases • Ectopic synthesis of therapeutic proteins as erythropoietin.

38. DNA coated Gold particles Plasmid DNA + Gold particles ( 1 micron india ) “shot” into cells using electric spark or pressurized gas – “gene gun” Epidermis Skin tumours (melanomas) Gene mediated immunization

39. Liposomes • DNA surrounded by hydrophobic molecules • Anionic – given i.v . Targets reticuloendothelial cells of liver • Cationic – transgene expression in most tissues if given in afferent blood supply • Intra airway injection or aerosol to target -lung epithelium

40. DNA- Protein conjugates • Cell- specific DNA delivery systems • Utilize unique cell surface receptors on target cells • Chemical cross linking methods used

41. A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. An abnormal gene could be swapped for a normal gene through homologous recombination. The abnormal gene could be repaired through selective reverse mutation , which returns the gene to its normal function . The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered. Methods

42. • In vivo Suspension containing vector is injected directly into the patient either systemically ( i.v .) or directly into target tissue (e.g. malignant tumour) • Ex vivo Target cells (stem cells,myoblasts,fibroblasts etc) removed from the patient, treated with vector and injected back into the patient Gene Transfer techniques

43. Spectrum of gene expression Gene replacement for single gene disorders Gene repair Gene inactivation Ectopic synthesis of therapeutic proteins Cancer gene therapy

44. A) Immunodeficiency Disorders Adenosine Deaminase Deficiency X- linked SCID Chronic Granulomatous disease B) Liver Disease Familial Hypercholesterolemia Haemophilia A Target diseases

45. Hemoglobinopathies D) Lung Diseases • Cystic Fibrosis • α- 1 Antitrypsin Deficiency E) Skeletal Muscle • Duchene Muscular Dystrophy • Limb Girdle Muscular Dystrophy

46. Challenges Short-lived nature of gene therapy Difficult to treat multi gene or multi factorial disease Inserting gene into correct cells. Controlling gene expression. Possibility of over expression Damage to the host gene Acquirement of virulence Chance of inducing a tumour ( insertional mutagenesis)

47. Conclusion

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