Gene therapy

Definition, Introduction, methods Types, Targets, approaches, Target Sites

1. GENE THERAPY Mrs. Jagtap P.N. HOD of Pharmacology PDEA′S SGRS college of Pharmacy , Saswad Ms. Sayali S. Chavan & Mr. Pranesh P. Memane 1 ST Year M. Pharmacy PDEA′S SGRS college of Pharmacy,Saswad

2. Introduction : Gene therapy is a novel method of treatment of disease. Genes or short oligonucleotide sequence is used as therapeutic molecule instead of conventional drug compound in the gene therapy. It is widely used to treat defective genes that contribute in the developement of disease. It is used to treat hereditary or acquired genetic defects. A single gene or more foreign genes are introduced in the organism in this technique. In gene therapy, DNA encoding a therapeutic protein is packed within a “vector”, which transports the DNA inside cell within the body. Advantage of gene therapy is the DNA inserted is expressed by the cell machinery & the disease is treated with minimal toxicity

3. History ( Gregory Mendel-Genetics ) He used pollen from some plants to carefully fertilize other plants. He was able to observe that characteristics are inherited in a predictable manner. He determine that alleles of a single trait segregate independently. He observe that the allels for different traits assort independently. This gave rise to a new discipline that we now call genetics .

4. Factors to be considered in gene therapy : How to deliver genes to specific cell and tissues and whole animals? How much and how long the introduced gene will be expressed. The site and dose of gene delivery. Any toxic effects. Is there any adverse immunological consequences of both vehicle and gene?

5. Types of gene Therapy :

6. Somatic gene therapy The technique of somatic gene therapy involves inserting a normal gene into the appropriate cell of an individual affected with a genetic disease, thereby permanently correcting the disorder. Following figure outlines the simplest method of getting genes into the person′s cells either viruses or liposomes .

7. Somatic cell therapy is viewed as a more conservative, safer approach because it affects only the targeted cells in the patients, & is not passed on to future generations. In other words, the therapeutic effect ends with the individual who receives the therapy. However, this types of therapy presents unique problems of its own. Often the effects of somatic cell therapy are short-lived. Because the cells of most tissues ultimately die and are replaced by new cells. Transporting the gene to the target cell or tissue is also problematic. Regardless of these difficulties, however somatic cell gene therapy is appropriate and acceptable for many disorders, including cystic fibrosis, muscular dystrophy, cancer & certain infectious disease, life-threatening disorders.

8. Germ line gene therapy : In this type, the functional genes, which are to be integrated in the genomes, are inserted in the germ cells, i.e., sperm or eggs. Targeting of germ cells makes the therapy heritable. Gene therapy using the germ line cells results in permanent changes that are passed down to subsequent generations. If done early in embryologic development, such as during pre-implantation, diagnosis & in-vitro fertilization. The gene transfer could also occur in all cells of the developing embryo.

9. The appeal of germ line gene therapy is its potential for offering a permanent therapeutic effect for all who inherit the target gene. Successful germ line therapies introduce the possibilities of eliminating some diseases from a particular family, & ultimately from the population, forever. The genetic change propagated by germ line gene therapy may actually be deleterious & harmful, with the potential for unforeseen effects on future generation.

10. DIFFERENCE SOMATIC THERAPY GERM LINE THERAPY 1.Gens are introduced into somatic cell. 1.Genes are introduced into germ line cells & will get distributed in both germ cells and somatic cells. 2. Changes are confirmed to the recipient. 2. Changes will be passed to the future generations. 3. Genes are tissue specific in most instances although not location specific in many. 3. High frequency of insertional mutations are observed in these process and cause teratogenic consequences. 4. Technical expertise for somatic cells manipulations in-vitro introduction of gene of interest & replanting somatic cells in body to make them functional is developed. 4. There are still many technical difficulties in introduction of gene into germ cells. 5. No ethical issues attached. 5. Ethical problems to be answered & precludes its use.

11. Strategies in gene therapy

12. Gene augmentation therapy (gat) : In GAT, simple addition of functional alleles is used to treat inherited disorders caused by the genetic deficiency of a gene product.

13. TARGET KILLING OF SPECIFIC CELLS : It involves utilizing gene encoding toxic compounds (suicidal genes), or prodrugs (reagents which confer sensitivity to subsequent treatment with a drug) to kill the transfected /transformed cells. This general approach is popular in cancer gene therapies.

14. Targeted inhibition of gene expression : This is to block the expression of any diseased gene or a new gene expressing a protein which is harmful for a cell. This is particularly suitable for treating infectious diseases & some cancers.

15. Targeted gene correction : It is used to correct a defective gene to restore its function which can be done at genetic level by homologous recombination or at mRNA level by using ribozymes or therapeutic RNA editing.

16. Approaches to gene therapy Classical gene therapy Produce a product that the patient lacks. Produce toxins so that diseased gene is killed. Activates cells of the immune system so as to help in killing of diseased cells. Non-classical gene therapy It involves the expression of gene associated with the pathogenesis or to correct a genetic defect and restore the normal gene expression.

17. Methods of gene therapy There are mainly two methods of for the transfer of gene in gene therapy: 1.Transfer of genes into patient cells outside the body (ex-vivo gene therapy). 2.Transfer of gene directly to cells inside the body (in-vivo).

18. Ex-vivo gene therapy : In this mode of gene therapy genes are transferred to the cells grow in culture, transformed cells are selected multiply and then introduced in to the patients. This cells are sourced initially from the patients to be treated and grown in culture before being reintroduced into the same individual. This approach can be apply to the tissues like hematopoietic cells and skin cells which can be removed from the body, genetically corrected out side the body & reintroduced into the patient body where they become engrafted & survive for a long period of time.

19. In vivo gene therapy : In vivo method of gene transfer of cloned genes directly into the tissue of patient. This is done in case of tissues whose individual cells cannot be cultured in vitro in sufficient numbers where re-implantation of the cultured cells in the patient is not efficient. Liposomes & certain viral vectors are employed for this purpose because of lack of any other mode of cellection .

20. Target site for gene therapy :

21. V ectors involved in gene therapy : To transfer the desired gene into a targeted cell a carrier is required. such vehicle of gene delivery are known as vectors .

22. Viral vectors : Viral vectors are mostly derivatives of viruses that infects animals. Viruses have evolved a way of encapsulating and delivering their genes to human cells to remove disease-causing genes and insert therapeutic ones. Virus bind to their hosts and introduce their genetic material into the host cell. Free replicating viruses- that multiply within the cell, but do not integrated into the genome of the host. Integrating viruses- that can integrates into the host genome. They enters the cell, copy their RNA genome into DNA.

23. T ype of viral vectors : a)Retrovirus vector system They uses the enzyme reverse transcriptase to convert the RNA into DNA which is integrated into genome further enters the host cell. It have the ability to infect a wide variety of cell type with high efficiency.

24. b)Adenovirus vector system The AAV genome comprises of inverted terminal repeats (ITR) as both of the DNA stand and open reading frames.(ORFs). This type of viruses is being uses, because it is non pathogenic and do not build an immune response. AVV vectors are used to deliver genes to the brain, this is possible because AAV viruses can infect non-dividing cells such as neurons.

25. c) Herpes simplex viruses Herpes viruses includes herpes simplex viruses that rarely can cause encephalitis and infect the non dividing cells so it have the ability to transduce neurons. Its advantage is being able to infect non dividing cells that help in treating neurological disorders.

26. Advantages of viral vectors-

27. Non viral vector system : Physical methods- Electroporation Microinjection Gene gun or a biollistic particle delivery system Magnetofection Chemical methods- DNA transfer by calcium phosphate method Liposome medicated transfer

28. Electroporation Electroporation is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane allowing chemicals, drugs, or DNA to be introduced into the cell. Short pulses of high voltage carry DNA across the cell membrane This cause temporary formation of pores and thus allow DNA molecules to pass.

29. Microinjection The microinjection is the process of transferring the desirable DNA into the living cell, through the use of glass micropipette. Glass micropipette is usually of 0.5 to 5 micrometer. It easily get penetrates into the cell membrane and nuclear envelope. The desired gene is then injected into the sub cellular compartment and needle is removed. Limitation of microinjection - costly - skilled person required - more useful for animal cells.

30. Gene Gun Or A Biollistic Particle Delivery System 1. Biollistics or particle bombardment also known as gene gun technique is a physical method that uses accelerated micro projectiles to deliver DNA or a other molecule into intact tissue and cells. 2. The gene gun is a device that literally fit DNA into target cells. 3. The DNA to be transformed into the cell which are coated onto microscopic beads made of either gold or tungsten. 4. The coated beads are then attached to the end of plastic bullets and loaded into the firing chamber of the gene gun.

31. 5. An explosive force fires the bullet with DNA coated beads towards the target cells that lie just beyond the end of the barrel. 6. Some of the beads pass through the cell wall into the cytoplasm of the target cells.

32. Magnetofection Magnetofection is a simple and highly efficient transfection method that uses magnetic fields to concentrate particles containing nucleic acid into the target cells.

33. Chemical Method 1. DNA transfer by calcium phosphate method- The process of transfection involves the mixture of isolate DNA with solution of calcium chloride and potassium phosphate. Cell are then incubated with precipitated DNA either in solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA precipitate by endocytosis .

34. 2. Liposome medicated transfer- Liposome are spheres of lipids which can be used to transport molecules into cells. These are artificial vesicles that can act as delivery agent for exogenous materials including trans genes. Promote transport after fusing with the cell membrane. Cationic lipids are those having a positive charge are used for the transfer of nucleic acid.

35. First Approved gene Therapy On September 14, 1990 at the U.S. National Institutes of Health, W. French Anderson M.D. and his colleagues R. Michael Blaese , M.D., C. Bouzaid , M.D., and Kenneth Culver, M.D., performed the first approved gene therapy procedure on four-year old Ashanthi DeSilva , Born with a rare genetic disease called severe combined immunodeficiency (SCID) What did they do In Ashanthi's gene therapy procedure, doctors removed white blood cells from the child's body, let the cells grow in the laboratory, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patient's bloodstream.

36. A success Story As of early 2007, she was still in good health, and she was attending college. Some would state that the study is of great importance despite its indefinite results, if only because it demonstrated that gene therapy could be practically attempted without adverse consequences

37. 'mending broken hearts' by using gene therapy Novel techniques to “mend broken hearts” using gene therapy and stem cells represent a major new frontier in the treatment of heart disease It was achieved by the researchers at Gladstone Institute of Cardiovascular Disease in California They were able to re- programme scar-forming cells into heart muscle cells, some of which were capable of transmitting the kind of electrical signals that make the heart beat They performed on a live mice, transforming scar-forming cells, called fibroblasts into beating heart muscle cells They injected three genes (cocktail of genes) into the heart of live mice that had been damaged by heart attack, fibroblasts could be turned into working heart cells. Researchers said that the “cocktail of genes” used to regenerate cells could one day be replaced with “small drug-like molecules” that would offer safer and easier delivery

38. First Real-Time MRI-Guided Gene Therapy for Brain Cancer Neurosurgeons at the University of California, San Diego School of Medicine and UC San Diego Moores Cancer Center are among the first in the world to utilize real-time magnetic resonance imaging (MRI) guidance for delivery of gene therapy as a potential treatment for brain tumors Using MRI navigational technology, neurosurgeons can inject Toca 511 ( vocimagene amiretrorepvec ), a novel investigational gene therapy, directly into a brain malignancy The new approach offers a precise way to deliver a therapeutic virus designed to make the tumor susceptible to cancer-killing drugs .

39. Toca 511 is a retrovirus engineered to selectively replicate in cancer cells, such as glioblastomas . Toca 511 produces an enzyme that converts an anti-fungal drug, flucytosine (5-FC), into the anti-cancer drug 5-fluorouracil (5-FU). After the injection of Toca 511, the patients are treated with an investigational extended- release oral formulation of 5-FC called Toca FC. Cancer cell killing takes place when 5-FC comes into contact with cells infected with Toca 511. .

40. UCLA researchers combine cellular and gene therapies to develop treatment for breast cancer Carol Kruse, a professor of neurosurgery and member of the Jonsson Cancer Center and the UCLA Brain Research Institute led the research on breast cancer Breast cancer is the most common form of cancer in women, and metastasis is a major cause of health deterioration and death from the disease Cellular therapy and gene therapy were used together to treat breast cancer Cellular therapy is a type of immunotherapy that uses T cells, the foot soldiers of the immune system, that have been sensitized in the laboratory to kill breast cancer cells. These sensitized T cells are injected into the parts of the brain to which cancer has spread. The research shows that the T cells can move through tissue and recognize and directly kill the tumor cells

41. stem cell gene therapy gives hope to prevent inherited neurological disease Scientists from The University of Manchester have used stem cell gene therapy to treat a fatal genetic brain disease It was used to treat Sanfilippo – a fatal inherited condition which causes progressive dementia in children Sanfilippo , is currently untreatable mucopolysaccharide (MPS) disease It is caused by the lack of SGSH enzyme in the body which helps to breakdown and recycle long chain sugars, such as heparan sulphate (HS) Children with the condition build up and store excess HS throughout their body from birth which affects their brain and results in progressive dementia and hyperactivity, followed by losing the ability to walk and swallow

42. Researchers have developed a stem cell gene therapy which overproduces the SGSH enzyme specifically in bone marrow white blood cells to increase SGSH enzyme from bone marrow transplants, and to target it to the cells that traffic into the brain It was seen that mice treated by this method produce five times the normal SGSH enzyme levels in the bone marrow and and 11 per cent of normal levels in the brain The enzyme is taken up by affected brain cells and is enough to correct brain HS storage and neuro inflammation to near normal levels and completely corrects the hyperactive behaviour in mice with Sanfilippo

43. Mucopolysaccharidosis Type IIIA potential gene therapy Mucopolysaccharidosis Type IIIA (MPSIIIA) is a metabolic disorder in which the body is missing an enzyme that is required to break down long chains of sugars known as glycosaminoglycans The glycosaminoglycans collect in the body and cause damage, particularly in the brain if not broken Fàtima Bosch and colleagues at Universitat Autònoma de Barcelona in Spain developed a form of gene therapy to replace the enzyme that is missing in MPSIIIA They injected the replacement gene into the cerebrospinal fluid that surrounds the brain and spinal cord This study demonstrates that gene therapy can be delivered to the brain through the cerebrospinal fluid and suggests that this approach could potentially be used as a therapy for MPSIIIA