Downloaded 74 times
More Related Content
Similar to Gene rx [autosaved]
Gene rx [autosaved]
- 1. GENE THERAPY - Dr.Chandini Moderator: Dr. Vinitha 1
- 2. OVERVIEW o Introduction o History oTypes of gene therapy o Techniques o Approaches o Methods of gene delivery (vectors) o Applications o Pros & Cons o Success stories in the recent past o Recent advances o Conclusion 2
- 3. INTRODUCTION • Genes, thebasic functional unit of heredity. • Variations in the DNA sequence or code of a gene = mutations. • Each human being carries normal as well as some defective genes. 3
- 4. Why Gene therapy?? • Faulty gene defective protein DISEASE. • Genetic disorder = Disease caused by a "variation or "mutation of a gene. • Passed on to family members • > 4,000 genetic disorders identified, 4
- 5. TYPES OF GENETICDISEASES 1. Single gene disorders Eg :- Sickle cell anemia, Cystic fibrosis, Marfan’s syndrome 2 . Chromosome disorders Aneuploidy, Deletion, Inversion, Translocation, Mosacism Eg. Down’s, syndrome, Turner’s syndrome 3 . Multifactorial inheritance disorders Eg. CVDs, Alzheimer's disease, Diabetes mellitus, AIDS, Most cancers 5
- 6. WHY IS GENETHERAPY SO ATTRACTIVE? Radical cure for single gene diseases Common acquired conditions –large genetic component Conventional treatment- far from ideal, Ability to control gene expression 6
- 7. WHAT IS GENETHERAPY? • Technique in which a functional gene replaces defective gene so that the body can make the functional protein & therefore eliminate the root cause of the disease, or transfer genetic material into cells or tissues to prevent or cure a disease. 7
- 8. HISTORY.. o 1971: keyscientific experiment (galactosemia) - DNA could be injected into human cells could fix the biological problem o 1972: formal proposal to use gene therapy as a means to treat human genetic disease 1st put forth. o late 1980s: 1st attempt to use gene therapy to treat live humans in clinical trials began 8
- 9. FIRST SUCCESSFUL CASE.. • Sep 14th 1990: Ashanthi DeSilva, ADA-deficient Severe Combined Immunodeficiency, 9
- 10. TYPES Somatic Genetherapy: • Transfer of genes into body cells other than germ cells ie somatic cells • Not passed on to offspring • affects only targeted cells • More conservative & safer approach 10
- 11. Germline genetherapy – • Functional genes introduced into germ cells (sperm or egg). Changes are permanent Passed on to future generations • Theoretically highly effective. Offer the possibility of removing an inherited disorder from a family line forever Controversial Existing gene therapy treatments & experiments are all somatic. 11
- 12. TECHNIQUES IN GENETHERAPY Ex vivo technique In vivo technique 12
- 13.
- 14. 1. Ex vivogene therapy : a)Isolate cells with genetic defect from patient b) Grow the cells in culture c) Introduce the therapeutic gene to correct gene defect d) Select the genetically corrected cells & grow e) Transplant the modified cells to the patient 14
- 15. 2. In vivogene therapy: • The direct delivery of the therapeutic gene into the target cells of a particular tissue. • Many tissues are the potential candidates for this approach. For eg. liver, muscle, skin, spleen, lung, brain and blood cells etc. 15
- 16.
- 17. APPROACHES FOR GENETHERAPY 1. Gene augmentation therapy. 2. Gene replacement therapy. 3. Gene inhibition therapy. 17
- 18. 1. GENE AUGMENTATIONTHERAPY • Corrects metabolic deficiencies caused by a missing or defective gene. • Does not substitute the flawed / absent gene. • Eg. Cystic fibrosis 18
- 19. 2. GENE REPLACEMENTTHERAPY • Replacement of mutant copy with a correctly functioning copy in situ. Eg. oncogenes 19
- 20. 3. GENE INHIBITIONTHERAPY • Aim: • Inhibition of expression of faulty gene • Inhibit activity of product of another gene. • Eg. oncogene 20
- 21. METHODS OF GENEDELIVERY VECTORS: • The carrier particles or molecules used to deliver genes. Viral vectors Non-viral vectors Physical methods Chemical methods Ideal vector - accommodate foreign genes of sufficient size -deliver gene to a specific cell type, -non-immunogenic and safe 21
- 22. VIRAL VECTORS • Mostpromising system of gene delivery 1. Gene transfer - more efficient & specific 2 . Multiple & repeated doses not required. - single dose is sufficient. 22
- 23. VIRUSES USED INGENE THERAPY 23
- 24.
- 25.
- 26. NON-VIRAL VECTOR SYSTEMS PHYSICALMETHODS • Microinjection • Electrophoration • Gene guns • Sonoporation • Magnetofection CHEMICAL METHODS • Liposomes • Oligonucleotides • Dendrimers • Amino acid polymers • Nanoparticles 26
- 27. Gene guns- • DNAparticle bombardment by gene gun • Gold or tungsten spherical particles (1–3 μm diameter) coated with plasmid DNA, 27
- 28.
- 29. Electroporation- • Disadvantage: A highrate of cell death following electroporation 29
- 30. Sonoporation- • Ultrasonic frequencies nanomeric pores in membrane 30
- 31. Magnetofection- • Magnetic fieldsused to concentrate particles containing nucleic acid into the target cells. 31
- 32. CHEMICAL METHODS OFGENE DELIVERY • More common than physical methods • Nanomeric complexes – compaction of negatively charged nucleic acid by polycationic nanomeric particles 32
- 33. Liposomes (lipoplexes) – •The nanomeric complex between a cationic liposome and nucleic acids = lipoplex 33
- 34. Oligonucleotides- • Aim ingene therapy: to inactivate the genes involved in the disease. • Approaches: o Antisense specific to target gene- to disrupt the transcription of the faulty gene. o Short interfering RNA (Si RNAs) cleave specific sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA. 34
- 35.
- 36. DISORDERS FOR WHICHGENE THERAPY HAS BEEN TRIED OR CONSIDERED Genetic diseases 1. Adenosine deaminase deficiency 2 . Cystic fibrosis 3 . Familial hypercholesterolemia 4 . Storage disorders - eg. Gaucher disease 5 . Coagulopathies - eg. Haemophilias A , B 6 . Haemoglobinopathies - Β thalassaemia , sickle cell disease 36
- 37. Acquired diseases 1 .Cancer - eg. Melanoma , brain & renal tumors 2 . AIDS 3 . Vascular disease 4 . Neurological disorders - Parkinson disease Alzheimer disease 37
- 38. GENE THERAPY INCANCER Strategies – 1. Immunogene therapy: Mking cancer cells immunogenic. 2. Suicide Gene therapy – • converts prodrugs of cytotoxic drugs to active compounds lethal to tumor cells. 38
- 39. 4. Gene therapywith Antisense oligonucleotide • Antisense RNA hybridizes with sense RNA. 5. Gene therapy with chemoprotection Genes • Transfer of drug resistance genes into hematopoietic stem cells 6. Gene therapy with tumor suppressor gene • Reintroduction & expression of wild-type p53 into p53 altered tumor cells 39
- 40. CARDIOVASCULAR DISEASE • Genetransfer into vascular cells (endothelial) & smooth muscle Prevent restenosis Thrombolysis Hypertension Ischemic heart disease 40
- 41. GENE THERAPY INCNS Gene-based therapies, Neurotrophic factors (NTFs), Nervous-system growth factors, Stem cells, Novel vaccines, • potential to prevent cell loss & degeneration in brain, 41
- 42. PROS AND CONSOF GENE THERAPY Pros: Gene therapy is a “medicine” for the future - can wipe out genetic diseases before they can begin & eliminate suffering. Cons: 1) Short-lived nature of gene therapy:- have to undergo multiple rounds of gene therapy 42
- 43. 2) Problems withviral vectors 3) Immune response 4) Multi-gene disorders 5) Insertional mutagenesis 6) Risk of procedure 7) Expensive. 43
- 44. 7) Ethical issues •Recombinant Advisory Committee (RAC) • Misuse of gene therapy - personality or physical enhancement (Human genetic engineering) • Religious issues ? - Somatic cell therapy >> germline therapy 44
- 45. SUCCESS STORIES INTHE RECENT PAST.. • world’s 1st gene therapy trial for Leber’s Congenital amaurosis, (mutation in RPE65 gene.) • Sub-retinal delivery of recombinant AAV carrying RPE65 gene - positive results 2007: London’s Institute of Ophthal- mology 45
- 46. • Approved Glybera(Adipogene tiparvovec) - lipoprotein lipase deficiency July 2012: EMEA • Gene Rx for Metachromatic Leukodystrophy & Wiskott- Aldrich Syndrome (7-32 months) July 2013: Italian San Raffaele Telethon Institute • 6 patients with choroidemia treated with genetically engineered AAV with a copy of REP1 gene – improvement in sight Jan 2014: Universit y Of Oxford 46
- 47. BREAKTHROUGH 2017-2018! CAR– T immunotherapies: FDA-approved (2017) - B-cell ALL (kids & young adults) - Non-HL (adults) • in those who don’t respond to std Rx. • Ongoing – multiple myeloma, glioblastoma 47
- 48. • novel genetherapy treatment of Leber’s congenital amaurosis. • targets mutated RPE65 gene. • subretinal injection • 1st in vivo gene therapy approved by the FDA. • not a cure. Improves vision. 48
- 49. CRISPR-CAS9 COMING TOA HUMAN NEAR YOU ! Clustered regularly interspaced short palindromic repeats (CRISPR) – Cas9 - used to edit genes within organisms - goes into the nucleus and directly cuts out faulty genes. 49
- 50. Trial tofix genetic defect in beta thalassemia (CRISPR therapeutics Cambridge) Human trial for sickle cell anemia (Stanford University) : FDA approved 50
- 51. CONCLUSION Gene therapy isthe permanent solution for genetic diseases. • Both beneficial & harmful. • Potential cure for deadly diseases (AIDS, cancer etc) • Ethical concerns – human genetic engineering 51
- 52. Future prospects: Identifymore efficient ways to deliver genes Develop more target-specific vectors, (insert genes on the precise location) Ensure transplanted genes are precisely controlled by the body’s normal physiologic signals 52
- 53. REFERENCES 1) Najmul Hasan,Dr. Savita Saini, Gene therapy: Current status and future perspectives / International Journal of Pharma Sciences and Research (IJPSR) 2) Biju mammen, Ramakrishnan T, Uma sudhakar . Principles of Gene therapy .Indian journal of pharmacology, 2010 ,18: 196-200 . 3) Yamamoto, M. & Tani, K. Current status and recent advances of gene therapy in hematological diseases. Int J Hematol (2016) 104: 4 4) https://singularityhub.com/2018/01/09/gene-therapy-had-a-breakthrough- 2017-2018-may-be-even better/#sm.0001zyc2phb9crg10gp1gf7sm3x91 5) Rang & Dale’s Pharmacology, 8th edition 6) Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 12th edition 53
Editor's Notes
- #4 Genes, the basic functional unit of heredity, are specific base sequences that carries information needed to make specific proteins. Variations in the DNA sequence or code of a gene are called mutations. Each human being carries normal as well as some defective genes.
- #5 Faulty gene defective protein DISEASE. Genetic disorder = Disease caused by a "variation" or "mutation“ of a gene. Passed on to family members About 10% of population likely to develop. More than 4,000 medical disorders caused by defective genes have been identified, each with varying degrees of seriousness. Usually, the individual does not become aware of the presence of a defective gene until a disease associated with the gene is manifested in him or her or in a relative. autosomal or X‑linked recessive single gene disorders (e.g. cystic fibrosis, haemophilia, muscular dystrophy, sickle cell anaemia etc.), acquired genetic diseases such as acquired immunodeficiency syndrome (AIDS) and cancer. Other conditions such as cardiovascular diseases, arthritis, diabetes mellitus, parkinson’s and alzheimer’s disease a
- #6 1. Single gene disorders caused by a mistake in a single gene. Example :- Sickle cell Cystic fibrosis 2 . Chromosome disorders Aneuploidy. More or fewer chromosomes than the normal number, including: Down syndrome (trisomy 21). Cells contain three #21 chromosomes. Turner syndrome. One of the two sex chromosomes is not transferred, leaving a single X chromosome, or 45 total. 3 . Multifactorial inheritance disorders caused by a combination of small variations in genes. Example :- Heart disease Alzheimer's disease Most cancers
- #9 In 1971, a key scientific experiment revealed that DNA could be injected into human cells and that this injected DNA could fix a biological problem in those cells (Merril, 1971). The injected DNA restored enzyme activity that was missing from human fibroblast cells. These cells had been extracted from patients suffering from a disease called galactosemia. In 1972, a formal proposal to use gene therapy as a means to treat human genetic disease was first put forth in this Science article. Inspired by the 1971 results, The first attempt to use gene therapy to treat live humans in clinical trials began in the late 1980s. These trials weren’t reported until the beginning of the 1990’s.
- #10 A four-year old girl became the first gene therapy patient on September 14, 1990 at the NIH Clinical Center. She has adenosine deaminase (ADA) deficiency, a genetic disease which leaves her defenseless against infections. White blood cells were taken from her, and the normal genes for making adenosine deaminase were inserted into them. The corrected cells were reinjected into her.
- #11 Somatic Gene therapy: Transfer of a gene or genes into body cells other than germ cells ie somatic cells, with effect only on the patient. New genetic material cannot be passed on to offspring More conservative & safer approach - affects only targeted cells & not passed on to future generations
- #12 In somatic gene therapy, the patient’s genome is changed, but the change is not passed along to the next generation In germline gene therapy, the patient’s egg or sperm cells are changed with the goal of passing on changes to their offspring
- #19 A procedure for correcting metabolic deficiencies caused by a missing or defective gene by having a healthy geneproduce the necessary product without actually substituting that gene for the flawed or absent gene in the DNA. Eg. For missing gene disorders – cystic fibrosis.
- #21 The aim is to introduce a gene whose product either: inhibits the expression of another gene interferes with the activity of the product of another gene. The basis of this therapy is to eliminate the activity of a gene that encourages the growth of disease-related cells. For example, cancer is sometimes the result of the over-activation of an oncogene? (gene which stimulates cell growth). So, by eliminating the activity of that oncogene through gene inhibition therapy, it is possible to prevent further cell growth and stop the cancer in its tracks
- #23 Viruses are actually genes wrapped in a protein coat. This coat contains special proteins that can bind to the surface of cells. 1. Gene transfer - more efficient & specific than physical & chemical method 2 . Multiple & repeated doses are required in case of physical and chemical method 3. In case of viral vector even a single dose is sufficient.
- #28 accelerated to high speed by pressurized gas to penetrate into target tissue cells
- #30 Electroporation- It is temporary destabilization of the cell membrane targeted tissue by insertion of a pair of electrodes into it. So that DNA molecules in the surrounding media of the destabilized membrane would be able to penetrate into cytoplasm and nucleoplasm of the cell Disadvantage: A high rate of cell death following electroporation
- #31 Ultrasonic frequencies are used to make nanomeric pores in membrane Facilitates intracellular delivery of DNA particles into cells of internal organs or tumors Size and concentration of plasmid DNA - role in efficiency of the system
- #32 In this method the magnetic fields are used to concentrate particles containing nucleic acid into the target cells.
- #33 generally are nanomeric complexes, which include compaction of negatively charged nucleic acid by polycationic nanomeric particles
- #34 In 2003, Los Angeles research team used liposome coated in a polymer to insert genes into brain. This method has potential for treating Parkinson’s disease. The transfer of genes into brain is a significant achievement because viral vectors are too big to get across the blood brain barrier
- #35 Aim of using synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease. One strategy uses antisense specific to target gene to disrupt the transcription of the faulty gene. Another approach uses small molecules of RNA called as short interfering RNA or Si RNAs which signal the cell to cleave specific sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA.
- #39 One of the first demonstrations of effectiveness of gene therapy in treating cancer comes from the success of the scientists at the National Institute of Health who successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells. Cytokine gene therapy: tranducing cytokine genes into tumor cells acquire the capability of protective immunity. ( cDNA of IL-1 & IL-4 & GM-CSF)
- #41 Gene transfer into vascular cells : Genes expressed in endothelial & vascular smooth muscle cells can produce local effects by their ability to influence the function & integrity of the arterial wall Endothelial cell gene transfer invitro, invivo & directly into vasculature has been accomplished Expression of direct intramyocardial injection of recombinant adenoviral vectors in pig has been successful
- #42 Neurodegeneration is often caused by misfolding of proteins in such a way that they can no longer perform their cellular functions and instead trigger equivalent modifications in normal proteins, thus creating a cascade of damage that eventually results in significant neuronal death Includes gene-based therapies, neurotrophic factors (NTFs), nervous-system growth factors, stem cells, novel vaccines, therapeutic manipulation of RNA interference (RNAi) and modulation of the immune system. Offer potential to prevent cell loss and degeneration in brain, rather than attempting to compensate for loss of neurons once it has occurred.
- #43 Pros: The positive aspect of gene therapy is apparent. Gene therapy is a “medicine” for the future since it can wipe out genetic diseases before they can begin and eliminate suffering for future. However, no therapy is without some associated risks
- #44 2) Problems with viral vectors :- present potential problems to the patient, like toxicity, immune and inflammatory responses, and gene control and targeting 3) Immune response :- stimulating immune system that reduces gene therapy effectiveness - potential risk 4) Multi-gene disorders :- most common disorders - heart disease, high blood pressure, Alzheimer's disease, arthritis and diabetes, are caused by the combined effects of variations in many genes. 5) Insertional mutagenesis- creation of muations in DNA by addition of 1 or more base pairs. If the DNA is integrated in wrong place in the genome, for example in a tumour suppressor gene, it could induce a tumour. 6) Expensive.
- #45 There are several ethical and legal issues associated with gene therapy. A review board, the Recombinant Advisory Committee (RAC) has been developed to address these concerns. The consequences of gene therapy are many. The first issue targets putting human fate in our own hands. Some people are concerned that gene therapy could be used for any genetically linked trait such as eternal appearance, personality or physical enhancement. Another great concern is religion. Some consider it sinful to manipulate DNA. If religion is a factor then somatic cell therapy should be applied which allows the next generation to make their own decision. In addition to ethical issues, one of the major concerns is the cost of gene therapy. However, scientists are optimistic that the cost will be much cheaper in future. The last but not the least is the risk of the procedure. Since gene therapy is still in its developmental stage, finding the precise location of the gene and replacing with a normal one is definitely a challenge. But it is true that with the invention of new and advanced techniques, researchers will soon be able to achieve a great success in this applied modern science
- #46 In 2007, Moorefield’s Eye Hospital and University College London’s Institute of Ophthalmology announced the world’s first gene therapy trial for a type of inherited retinal disease i.e. Leber’s Congenital amaurosis, which is caused by a mutation in the RPE65 gene. Sub-retinal delivery of recombinant AAV carrying RPE65 gene yielded positive results with no apparent side- effects
- #47 In July 2012, the European Medicines Agency recommended approval of a gene therapy treatment called Adipogene tiparvovec (Glybera) which compensates for the lipoprotein lipase deficiency. In July 2013, the Italian San Raffaele Telethon Institute for gene therapy reported that the treatment of two severe hereditary diseases i.e. Metachromatic Leukodystrophy and Wiskott- Aldrich Syndrome yielded positive results after 7-32 months of gene therapy. In January 2014, researchers at the University of Oxford reported an improvement in the sight of six people suffering from choroideremia, an inherited genetic eye disease. These patients had been treated with a genetically engineered AAV with a copy of REP1 gene.(33) In March 2014, researchers at the University of Pennsylvania reported that 12 patients with HIV had been treated since 2009 in a trial with a genetically engineered virus with a rare mutation known to protect against HIV (CCR5 deficiency)
- #48 FDA has not yet approved any human gene therapy product for sale Current gene therapy is experimental & has not proven very successful in clinical trials After decades of promises but failed deliveries, in 2017 – multiple breakthrough developments - But in 2017, the FDA approved a double whammy of CAR-T immunotherapies. The first, green-lighted in August, helps kids and young adults battle an especially nasty form of leukemia called B-cell acute lymphoblastic leukemia. Two months later, a therapy for adults with non-Hodgkin lymphoma hit the scene. Together, these approvals marked the long-anticipated debut of gene therapy in the US market. Previously, Europe has led the charge with its approval of Glybera in 2015, a gene therapy that reduces fatty acid buildup in the bloodstream. CAR-T is especially exciting for the cancer field because it helps people who don’t respond to other classic treatments, such as chemotherapy. Already in the works are treatments that target multiple myeloma, which causes multiple tumors in the bone or soft tissue, and glioblastoma, an aggressive brain tumor for which there is no cure. 9
- #49 Just before Christmas, the FDA dropped another bombshell with its approval of Luxturna (voretigene neparvovec) , novel gene therapy treatment of Leber's congenital amaurosis (rare form of inherited blindness) that targets mutated RPE65 gene Made by Spark Therapeutics, given as an subretinal injection. first in vivo gene therapy approved by the FDA. The gene therapy is not a cure for the condition, but substantially improves vision in those treated.
- #50 clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. CRISPR-Cas9 that can be used to edit genes within organisms.[2] CRISPR isn’t technically gene therapy - replacing a diseased gene with a good one
- #51 This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases. n its current form, CRISPR isn’t technically gene therapy. Rather than replacing a diseased gene with a good one, it goes into the nucleus and directly cuts out faulty genes. CRISPR Therapeutics, based in Cambridge, has already sought approval from European regulatory agencies to begin a trial to fix a genetic defect that causes beta thalassemia, an inherited blood disorder. Stanford University. Like CRISPR Therapeutics, the school seeks to start a human trial for sickle-cell disease in 2018. FDA given approval.
- #52 Conclusion Theoretically, gene therapy is the permanent solution for genetic diseases. But it is not as simple as it appears since gene therapy has several inbuilt complexities. Gene therapy is both beneficial and harmful depending on how it is applied. The advantage of gene therapy is to cure someone who is born with a genetic disorder or who develops deadly diseases like AIDS, cancer etc. The government, the public groups and the scientific society should cooperate and walk hand in hand to encourage gene therapy applications. A breakthrough may come anytime and a day may come when almost every disease will have a gene therapy, as one of the treatment modalities