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GENES Are the basic unit of heredity carried on a chromosome. Specific sequences of bases that Encode instruction on how to make a protein. DNA RNA proteins Mutation Of Genes Results in- -Failure to synthesize a particular protein. -Synthesis of a defective Protein. These results in abnormalities Recognized as GENETIC DISORDERS. GENETIC DISORDERS can be treated by Gene Therapy by- -Add functional Gene to Cope with condition. -Replace Defective Gene with Therapeutic Gene.

The Beginning…… :

The Beginning…… The idea of GT was first Hypothesized by ANDERSON in Late Sixties. In the 1980s, Scientists began to look into gene therapy: They would insert human genes into a bacteria cell. Then the bacteria cell would transcribe and translate the information into a protein. Then they would introduce the protein into human cells.

What is Gene Therapy? :

What is Gene Therapy? Gene therapy is an experimental treatment that involves introducing genetic material into a person’s cells to fight or prevent disease. GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized  to improve a genetic disorder A gene can be delivered to a cell using a carrier k/a a “vector”. The most common types of vectors used in gene therapy are viruses. The viruses used in gene therapy are altered to make them safe, although some risks still exist with gene therapy. The technology is still in its infancy, but it has been used with some success.

Approaches Used in Gene therapy:

Approaches Used in Gene therapy These Includes the following- (A)Gene Modification In this expression of a defective Gene is modified to render it functionally normal. Patient with Genetic Disorder Isolation of Genomic DNA Construction Of Genomic Libraries Identification Of Defective Genes (1)Gene Replacement- -It is the Removal of a Mutant Gene sequence from the Host Genome and its replacement with a normal Functional Gene. -Used for Disorder marked by deficiency of ENZYME/PROTEIN. -DISADVANTAGE- Very difficult Process

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(2) Gene Correction- - Involves only defective portion of a mutant gene which is altered to provide the Functional Gene, without precisely changing the gene back to its natural form. - Advantage- Feasible, Dominant Disorders, Associated with production of an abnormal gene product. - Disadvantage- Beyond the Bounds of In vivo Technology. (3) Gene Augmentation- - Expression of mutant gene in defective gene is modified by introducing a normal genetic sequence into Host Genome without altering the defective one. - Advantage- Simplest of all (B) Gene Transfer Method- (1)Non-Viral Gene Transfer (2) Viral Mediated Gene Transfer

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Generally adopted for improvement of a specific disease. Ex- Introduction of GH gene to increase the Height. Genes can be introduced into cell by following method- (1) Physical Methods- *Electroporation- Short electric pulses of specific strength is applied which creates holes in cell membrane through which foreign DNA in suspension can enter inside the cell. Advantage- Simple and technically easy. Disadvantage- High risk of contamination. *Microinjection- Very fine glass capillaries are used for injecting DNA directly into Nucleus of target Cells. Advantage - High efficient Disadv - Only limited no of cell can transformed at a time. *Gene Gun- Simple, ideal for gene mediated immunization. - Simple for Large scale application. - Limited depth of DNA penetration (GE Last for few days)

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(2) Chemical Method- *Naked DNA Injection- DNA is introduce into cell by using chemicals EX- CaCl2 + DNA Solution + PBS(PO4 buffer Saline) - Ca(PO4)2 -DNA gets entrapped with ppt - Cells take up DNA *Liposome Mediated Transfer- Foreign DNA is incorporated into Liposomes by sonication of a lipid sol and DNA in Ether. - Lipid form liposome enclose negatively charged DNA within it - These are absorbed through the fat based cell membrane. - Advantage- Protection of DNA from Nuclease, non toxic nature. - Disadvantage- No of genes transferred are < than by viral vector. *Synthetic Carriers- Ex- Spermine (+ve charged molecule) - Principle is same as of Liposomes. Lipid + Spermine -- Lipospermine DNA(-ve charge) Lipospermine case around DNA - 4 times < efficient than Bio. Vector

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(3) Biological Method (Viral Method)- - Due to limitation of above method in gene transfer virus based vectors are useful because of natural capability of entering into the cells and can easily integrate the viral genetic information into Host cell genome. -These only infect the cells but lacks to produce viral progeny. *Retro Virus Based Vectors- - Double stranded RNA virus. - Two strands are identical and partially hybridized. - Capacity for the insert is 8 kb. - To use them as a vector following viral genes are removed are- ~ gag (encode core protein), ~ Pol (encode RTase), ~ env (encode envelope protein)

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Retroviral vector binding to host cell surface Endocytosis Uncoating of virus Release of vector viral RNA RTase DNA production and replication Integration of vector into host genome of a dividing cell Expression of Hetrologus Gene USE- In ex-vivo therapy Disadvantage- Random insertion could leads to mutagenesis. - Limited to dividing cells. Advantage- In tumors due to limited dividing cell & highly infectious

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* Adeno Virus Based Vector- - Double Stranded DNA virus -Capacity for insertion is 7.5 Kb -The Adenoviral genes E1A, E2, E4 are deleted for making it to use as vector. Vector Enters into Host Cells Acidic environment Protease in virion gets activated Proteolysis of the virion Viral DNA released into cytoplasm DNA transported to Nucleus

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- USE- In vivo gene therapy - Disadvantage- Can provoke Inflammatory Immune Response. - Advantages - Can used for transfection of Broad spectrum of cells - Can be administered by many route. *Adeno Associated Virus- - Single Stranded DNA Virus - Used for Both in vivo and ex-vivo gene therapy - Disadvantage- Difficulty in producing the virus in large quantity. - Advantages- Cells can easily purified and stored for long period. - Infect a variety of host cells *Others- Vaccinia virus, Herpes simplex virus, Cytomegalo virus, Influenza virus, HIV(Retro Virus) etc are also used.

The First Case :

The First Case The first gene therapy was performed on September 14 th , 1990 Ashanti De Silva was treated for SCID Sever combined immunodeficiency Doctors removed her white blood cells, inserted the missing gene into the WBC, and then put them back into her blood stream. This strengthened her immune system Only worked for a few months . 2000 - The first gene therapy cure was reported when Alain Fischer (Paris) succeeded in totally correcting children with SCID-X1, or “bubble boy” syndrome.

How It Works? :

How It Works? A vector delivers the therapeutic gene into a patient’s target cell The target cells become infected with the viral vector The vector’s genetic material is inserted into the target cell Functional proteins are created from the therapeutic gene causing the cell to return to a normal state

Ex Vivo Versus In Vivo Gene Therapy:

Ex Vivo Versus In Vivo Gene Therapy In vivo gene therapy: - Delivery of new genetic material directly to target cells within the body The challenge lies in ensuring the specificity and in reaching the correct target cells within the body Ex vivo therapy: - Target cells are removed from the body and then genetically modified The cells are then returned to the body after selection and amplification This is a safe method but dependent on the type of cells being targeted

Division Of Gene Therapy:

Division Of Gene Therapy Somatic Gene Therapy Germline Gene Therapy The therapeutic genes are transferred into the somatic cells (non sex-cells), or body, of a patient. The recipient's genome is changed, but the change is not passed along to the next generation. Genetically treated stem cells, when reintroduced into the patient's body are expected to naturally travel through the blood stream to the bone marrow. These cells are targets for gene therapy. Germ cells (sperm or eggs), are modified by the introduction of functional genes, which are integrated into their genomes. The germ cells (sex cells) are changed and the changes is passed along to the next generation. Genetically changes in the reproductive cells (or embryo) before the stage of differentiation, would affect all future offspring of that person. Presently prohibited for application in human beings

Types of Postnatal Gene Therapy:

Types of Postnatal Gene Therapy Gene replacement: -Non-functional or defective gene is replaced by a new functional copy of the gene Can be accomplished by homologous recombination, although efficiency is low. Gene addition: -Introduction of a gene that is able to produce a protein not normally expressed in the cell i.e. Introduction of a so-called “suicide gene” into cancer cells.


TARGET CELLS OF GENE THERAPY THE HEMOPOIETIC CELLS- -Most consistent target for gene therapy. -Has strong potential THE HEPATOCYTES- -Attractive target for gene therapy. -Liver plays a vital role in most metabolic process. MUSCLE CELLS- -Strong potential -Use as an efficient target organ for correlation of intrinsic muscle and non- muscle diseases.

Gene Therapy used to Treat Type I Diabetes:

Gene Therapy used to Treat Type I Diabetes Study by Lee, Kim, Kim, Shin, and Yoon performed in Korea (2000). Their results were published in Nature Type I diabetes is caused by the destruction of insulin-producing pancreatic β cells by an inappropriate autoimmune response. This experiment was performed on mice and rats but the results may result in future implications for humans. Scientists used a recombinant adeno-associated virus (rAAV) to insert a gene that results in the expression of a single-chain insulin analogue (SIA) into streptozotocin-induced diabetic rats and autoimmune diabetic mice.

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First, the gene was cloned under the L-type Pyruvate Kinase (LPK) promoter, which regulates the expression of SIA in response to glucose levels.

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The LPK-SIA gene was then attached to a recombinant adeno-associated virus and integrated into the host chromosomal DNA. After insertion of the rAV -LPK-SIAA , the rats displayed a drop in glucose levels that reached a range of normoglycaemia within one week of treatment. The rates remained in this range for more than eight months.

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In addition to eight months of controlled glucose levels, there were no visible side affects from the gene therapy. While the results did not show permanent remission, the control of glucose levels from the insertion of the SIA gene was promising. This form of gene therapy may provide a cure for type I diabetes for humans in the future (but a lot more research would be required before that can happen).

Gene Therapy as a Treatment for X-SCID:

Gene Therapy as a Treatment for X-SCID X-linked severe combined immunodeficiency (X-SCID) is a disease that affects young children and is usually fatal within their first year of life. Bone marrow transplants are usually the best option for treatment, but with the difficulty in finding a donor who matches the patient, gene therapy has become a new alternative. Clinical trials for treating X-SCID patients have been marked by mixed results.

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One of the patients involved in the Fischer trials has developed leukemia two and a half years after the initial gene therapy treatment ( Gene Therapy). Two of eleven patients involved in a similar study in France have also developed leukemia ( Trends in Biotechnology). The gene therapy treatments have resulted in the over expression of the a gene that may be an oncogene and is located at the site of the retroviral insertion. The site of insertion is the first intron of the LMO-2 gene, which is located on chromosome eleven. LMO-2 is also the site of a translocation that occurs in leukemia. This observation clearly correlates the retroviral insertion as the cause of leukemia in the patients.

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Law of Unintended Consequences? It is still unknown whether the development of leukemia in these clinical studies was the result of a premature treatment (which could be eliminated with further research and development) or if it is a permanent risk. Despite the fact that without treatment, X-SCID is a fatal disease, there is still an ethical question of whether or not it is right to subject a sick child to the possibility of developing another disease through the risks of gene therapy treatments.


Cancer It is basically a disease of cells characterized by the loss of- -Normal cellular growth -Maturation -Multiplication and thus Homeostsis is disturbed. These are immortal cells as they continue to divide infinitely due to the presence of telomerase enzyme.

Gene Therapy For Cancer Treatment:

Gene Therapy For Cancer Treatment Oncogene Inactivation: - Done by using antisense therapy. - Reduces the expression of antigenic proteins responsible for malignancies . Cell- Targeted Suicide: -Herpes Simplex Virus-1 Thymidine Kinase(HSV-1 - TK) is given in combination with Gancyclovir. Causes -Over production of Thymidine Kinase -HSV-1 – TK helps in phosphorylation of Gancyclovir

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Virus Mediated Oncolysis: -Adeno-virus and herpes simplex virus can infect and cause lysis of tumors cells. Augmentation of tumor suppressor gene : - It is done by replacement or repair of the defective tumor suppressor gen es in malignant cells.

Gene Therapy for Cystic Fibrosis:

Gene Therapy for Cystic Fibrosis CF- Fibrosis = Scarring, Cyst = Formation within the pancreas. Also known as MUCOVISCIDOSIS. Caused by Mutation in the gene of Protein named Cystic Fibrosis Transmemberane Regulator(CFTR). CFTR regulates the movement of- -Chloride -Sodium across an epithilium Affects mainly Lungs and also Pancreas, Liver, Intestine. Main symptom--- Difficulty in breathing. CFTR was discovered more than 10 years ago and efforts are done to develop safe and effective vectors for introducing this gene into Respiratory Epithilium.

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After several Phase-1 clinical trial Results that mostly Adenovirus Vectors are used to transduce either nasal or pulmonary epithilium. Disadvantage- -Inefficient amount of transduced cells are transduce. -Transient nature of gene expression last for few days. Later AAV vectors for delivering CFTR are now in clinical trials. Preclinical studies demonstrated- -Long term transgene expression. Long term safety and efficacy study of Adeno associated viral vector are under evaluation. Liposomal vectors are also under evaluation in Cystic Fibrosis. Liposomes may be mildly effective, but their activity does not last.

Successful One Year Gene Therapy Trial For Parkinson's Disease :

Successful One Year Gene Therapy Trial For Parkinson's Disease It is defined as a Parkinsonian syndrome that is Idiopathic, but some Atypical cases have a Genetic origin. The increased risk of PD is due to exposure of certain pesticides and reduced risk in Tobacco smokers. Characterized by -Accumulation of protein called Alpha-Synuclein into inclusions called Lewis bodies in neurons. -Insufficient formation and activity of Dopamine. Neurologix a biotech company announced that they have successfully completed its landmark Phase I trial of gene therapy for Parkinson's Disease.

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This was a 12 patient study with four patients in each of three dose escalating cohorts. All procedures were performed under local anesthesia and all 12 patients were discharged from the hospital within 48 hours of the procedure, and followed for 12 months. Primary outcomes of the study design, safety and tolerability, were successfully met. There were no adverse events reported relating to the treatment. The gene transfer procedure utilized the AAV (adeno-associated virus) vector, a virus that has been used safely in a variety of clinical gene therapy trials, and the vehicle that will be used in all of the company's first generation products, including epilepsy and Huntington's disease. In its Parkinson's disease trial, Neurologix used its gene transfer technology.

Adenosine Deaminase deficiency:

Adenosine Deaminase deficiency First genetic disorder to be clinically treated with gene therapy. Absence of Adenosine Deaminase in children leads to an accumulation of Deoxyadenosine triphosphate (toxic to Lymphocytes). Patient with ADA develops recurrent life threatening infections due to cell-mediated and humoral immune responses.

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Standard therapy is bone marrow trans- plantation along with periodic infusions of PEG-coupled recombinant enzyme(PEG-ADA). In the first clinical trial two patients were infused with periodic blood T lymphocytes that had been tranduced with a retroviral vector containing the human ADA gene. One of these two patients had long term persistence of tranduced T lymphocytes, while the other had a poor response. The responsive patient experienced amelioration of symptoms of the diseases and is living a normal life several years after treatment.

Problems with Gene Therapy:

Problems with Gene Therapy Short Lived Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long time Would have to have multiple rounds of therapy Immune Response new things introduced leads to immune response increased response when a repeat offender enters Viral Vectors patient could have toxic, immune, inflammatory response also may cause disease once inside Multigene Disorders Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to treat because you need to introduce more than one gene May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis

Application of gene therapy:

Application of gene therapy (A)Synthesis Of Therapeutic Protein 1)Coagulation Factor- - Hemophilia A & B causes due to congenital deficiency of the Factor 8 & 9. - Gene responsible for synthesis of Factor 9 with vector is transduce in Skeletal muscle -Vector: Adeno- associated virus 2)Growth Factor (Erythropoietin) : - Due to Chronic Renal Failure. - Standard Treatment- Frequent injection of rEPO. -Disadvantage- High Cost -In Gene Therapy: Gene which is responsible for EPO injection in skeletal muscle --  Transcribe -- mRNA Formation -- Formation of EPO.

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3)Peptide Hormone (Growth Hormone): - AAV vector encoding gene Injected IM in Skeletal Muscle Give long term regulated expression of Human GH (B) In Treatment Of Cancer 1)Breast and Ovarian Cancer by Oncogene Inactivation: - Most common approach is antisense strategies in which Transcription of Oncogene is Inactivated using Adenoviral Gene. 2)Preparation Of DNA Vaccine: - In this approach bacteria plasmid vector carrying a gene coding for an antigenic protein is incubated in SKIN Or Muscle cells.

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Gene + Vector Incubated Transcription and Translation Produces Antigen protein Stimulate the T-helper and T-lymphocytes Produce Antibody 3) Augmentation of Tumor Suppressor Genes (C) In Treatment Of Immunodeficiency Disorders 1) Adenosine Deaminase Deficiency Disorder 2) X- Linked Severe Combined Immunodeficiency (X-SCID)

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(D) In Treatment Of Lung Diseases Cystic Fibrosis (E) In Treatment Of Diabetes (F) Deafness Can Be Cure - Invented by Neurobiologist JOHN BRIGANDE - He Implemented this gene therapy on mouse embryo that were prone to loose hearing as adult mouse . - For this Atoh-1 gene was implanted inside the inner year of the mouse and it converted non-sensory cells to hair cells and thus boosted up the hearing of the mouse

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Thank you…

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