gene therapy : recent advances in gene therapy and clinical application

it is a topic related gene mutation , disease undertaking ,classification ,curable or not main application.

1. GENE THERAPY PRESENTED BY , Pooja .g. anil M-PHARM pharmaceutical chemistry Amrita school of pharmacy , kochi

2. EX VIVO GENE THERAPY STEPS: Isolate cells with genetic defects from a patient Grow the cells in cu l ture Introduce the therapeutic genes Select genetically corrected cells and grow. 5. Transplant the modified cells to the patient.

3. EXAMPLE OF EX VIVO GENE THERAPY 1 st gene therapy: to correct deficiency of enzyme, Adenosine deaminase(ADA) Performed on a 4 year old girl She was st1ffering from SCID- Severe combined imm unodeficiency Caused due to defect in gene coding for ADA

4. IN VIVO GENE THERAPY Di rect delivery of the therapeutic gene into target cell into patients body. Carried out by viral and non viral vector systems It can be only possible option in patients where individual cells cannot be cu l tured in v i tro in suffic ie nt numbers.( eg - bra in cells.)

5. EXAMPLE OF IN VIVO GENE THERAPY Therapy for cystic fibrosis - In patients with cystic fibrosis , a protein called cystic fibrosis transmembrane regulator(CFTR) is absent. In the absence of C FT R chloride ions concentrate with i n the cells and it draws water from surrounding . This leads to the accumulation of sticky mucous ii1 respiratory tract and lungs. Treated by in vivo replacement of defective gene by adenovirus vector.

6. VECTORS FOR GENE THERAPY To transfer the desired gene into a target cell , a carrier is required. Such vehicles of gene de l ivery are known as vectors. T wo mai n c l ass e s Viral vectors Non viral vectors

7. VIRAL VECTORS 1) retrovirus vector system the recombinant retrovirus have the ability to integrate into the host genome is a stable fashion. Target cell-dividing. 2) adeno virus vector system- Adeno virus with a DNA genome – good vector Target- non dividing human cell.

8. 3) Adeno associated virus vector- It is a single stranded, non pathogenic small DNA virus. AAV enters host cell, becomes double stranded and gets integrated into chromosome. 4) Herpex simplex virus vector- Viruses which have natural tendency to infect a particular type of cell .

9. NON VIRAL VECTORS 1 ) PURE DNA CONSTRUCT Direct introduction of pure DNA construct into target tissue. Efficiency of DNA uptake by cells and expression rather low. Consequent l y Large quantities of DNA have to be injected periodically. 2) LIPOPLEXES Liquid DNA con1plexes;DNA construct surrounded by artificial lipid layer.

10. 3) DNA MOLECULAR CONJUGATES Commonly used synthetic conjugates 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 lisosomal breakdown of DNA. 4) HUMAN ARTIFICIAL CHROMOSOME : Can carry a large DNA ie , with one or more tlherapeutic genes with regulatory elements.

11. METHODS OF GENE DELIVERY PHYSICAL METHODS GENE GUN Employs a high- pressure delivery system to shoot tissue with gold or tungsten particles that are coated with DNA MICRO INJECTION 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

12. CHEMICAL METHODS 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 chemical disturbs the cell membrane, widens the pore size and allows the CDNA to pass through the cell.

13. LI POFECTION 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 .

14. OTHER TYPE OF GENE THERAPY GENE AUGMENTATION THERAPY Most common form of gene therapy. Foreign gene replaces missing or defective gene. o eg - replacement of defective p53 gene by a normal one in liver cancer GENE INHIBITION THERAPY- Done to block the over production of some proteins. 2 types- antigene and antisense therapy

15. ADVANTAGES Gene therapy has the potential to eliminate and prevent hereditary disease such as cystic fibrosis, ADA- SCID etc . It is a possible ct1re for heart disease , AIDS and cancer. It g i ves someone born with a genetic disease a chance to life. It can be used to eradicate diseases from the future generations.

16. DISADVANTAGES Long lasting therapy is not achieved by gene therapy; due to rapid dividing of cells benefits of gene therapy is short lived. 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.

17. GENE THERAPY FOR GENETIC DISORDERS SEVERE COMBINED IMMUNE DEFICIENCY ( ADA – SCID ) Affected children are born without an effective immune system and will succumb to infections . The disease is caused by a mutation in gene on chromosome 20 . The gene codes for the enzyme adenosine deaminase (ADA) The therapeutic gene called ADA was introduced into the bone marrow cells of such patients in the laboratory , followed by transplantation of the genetically corrected cells back to the same patients. The immune system was reconstituted.

18. HEMOPHILIA : Patients are not able to induce blood clots and suffer from external and internal bleeding that can be life threatening. In a clinical trail conducted in the United states ,the therapeutic gene was introduced into the liver of patients , who then acquired the ability to have normal blood clotting time. The therapeutic effect was transient because the genetically corrected liver cells were recognized as foreign and rejected by the healthy immune system in the patients and curative outcome by gene therapy .

19. GENE THERAPY TRAIL FOR INHERITED BLINDNESS : Choroideremia is a rare inherited cause of blindness that affects around 1 in 50,000 people. There is currently no cure. It is caused by defects in the CHM gene on the X chromosome. Without the protein produced by the CHM gene, pigment cells in the retina of the eye slowly stop working , then die off .

20. CANCER : Multiple gene therapy strategies have been developed to treat a wide variety of cancers , including suicide gene therapy , oncolytic and therapeutic gene vaccines . & gt ; Two-thirds of all gene therapy trials are for cancer and many of these are entering the advanced stage, including a Phase III trial. Additionally, numerous Phase I and Phase II clinical trials for cancers in the brain, skin, liver, colon, breast and kidney among others, are being conducted in academic medical centers and biotechnology companies, using novel technologies and therapeutics developed on-site.

21. What is CRISPR-Cas9? • CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats ). • Genome editing system- Manipulate genes in different organisms. • Major components- CRISPR (Small segment of RNA structure)Cas9 (Nuclease Protein) • The complex cleaves target DNA at specific location.

22. Origin of CRISPR-Cas9 System Concept – Bacterial defence against viral attack (acquired immunity)The phage genome- CRISPR kind of sequences. Bacteriophages attack the bacteria and destroys it during the growth of bacteriophage. When viral genome released for next time, the bacteria already has viral geneticmaterial as a copy or memory. Bacterial cell produces RNA copy (crRNA) along with Cas9 protein. The CRISPR-Cas9 complex interacts with the viral genome and cleaves the targetDNA . Scientists tried to replicate the process in in-vitro studies

23. Advantage of using CRISPR-Cas9 system: The total DNA content can be changed. 2. The function of the gene can be analyzed. 3.If tagged with fluorescent dye, the CRISPR-Cas9 can mark specific region or specific DNA in the whole genome of complicated organism. E.g. HumansApplications : 1.Food industryImmunization of bacteria used in food production against viruses (cheese/yogurt) 2. Medical TreatmentCRISPR therapy would allow silencing of defective genes (Huntington Disease) 3. Agriculture Development of disease resistant transgenic plants,Increased oil production in (Canola crop), etc.

24. REFERENCES Satyanarayana U, Biotechnology, 1 s t edition , Book and allied (P) Ltd, Kolkata. http: // www.rned indi a.net/articles / genetherapy _ treatmen t.Htm http://en.wikipedia.org/wiki/Gene _ therapy