A promising future to disease treatment HUMAN GENE THERAPY A promising future to disease treatment BY, Wasim Sajjad Muuhammad Huzaifa

INTRODUCTION Gene therapy is the introduction of genes into existing cells to prevent or cure a wide range of diseases. It is a technique for correcting defective genes responsible for disease development. The first approved gene therapy experiment occurred on September 14, 1990 in US, when Ashanti DeSilva was treated for ADA-SCID. Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery. Gene therapy is a novel approach to treat, cure, or ultimately prevent disease by changing the expression of a person’s genes.  Replacing mutated genes Fixing mutated genes Making diseased cells more evident to the immune system

Somatic CELL gene therapy TYPES OF GENE THERAPY Somatic CELL gene therapy Germ line gene therapy Therapeutic genes transferred into the somatic cells. Eg. Introduction of genes into bone marrow cells, blood cells, skin cells etc. Will not be inherited later generations. At present all researches directed to correct genetic defects in somatic cells. Therapeutic genes transferred into the germ cells. Eg. Genes introduced into eggs and sperms. It is heritable and passed on to later generations. For safety, ethical and technical reasons, it is not being attempted at present. Somatic cells are non-reproductive. Somatic cell therapy is viewed as a more conservative, safer approach because it affects only the targeted cells in the patient somatic cell therapy are short-lived. Because the cells of most tissues ultimately die and are replaced by new cells, repeated treatments over the course of the individual's life span are required to maintain the therapeutic effect. Gene therapy using germ line cells results in permanent changes that are passed down to subsequent generations. If done early in embryologic development, such as during preimplantation diagnosis and in vitro fertilization, the gene transfer could also occur in all cells of the developing embryo. 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 possibility of eliminating some diseases from a particular family, and ultimately from the population, forever.

APPROACHES IN GENE THERAPY In vivo gene therapy:- direct delivery of genes into the cells of a particular tissue in the body Ex vivo gene therapy:- transfer of genes to cultured cells and reinsertion.

EX VIVO GENE THERAPY Isolate cells with genetic defect from a patient Grow the cells in culture Introduce the therapeutic genes . Select genetically corrected cells and grow. Transplant the modified cells to the patient.

EXAMPLE OF EX VIVO GENE THERAPY 1st gene therapy – to correct deficiency of enzyme, Adenosine deaminase (ADA). Performed on a 4yr old girl Ashanthi DeSilva. Was suffering from SCID- Severe Combined Immunodeficiency. Caused due to defect in gene coding for ADA. Deoxy adenosine accumulate and destroys T lymphocytes. Disrupts immunity , suffer from infectious diseases and die at young age. People with SCID have a defect in their immune system that leaves them vulnerable to potentially deadly infections. Mostly used

IN VIVO GENE THERAPY Direct delivery of therapeutic gene into target cell into patients body. Carried out by viral or non viral vector systems. It can be the only possible option in patients where individual cells cannot be cultured in vitro in sufficient numbers (e.g. brain cells). In vivo gene transfer is necessary when cultured cells cannot be re-implanted in patients effectively. . In-vivo gene therapy is less commonly used than ex-vivo gene therapy. In-vivo gene therapy holds more risks than its ex-vivo counterpart, such as a possible immune reaction from the organism. Vectors used in in-vivo gene therapy include viruses, bacterial plasmids, nanoparticles, and more. 

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 due to a gene defect. In the absence of CFTR chloride ions concentrate within the cells and it draws water from surrounding. This leads to the accumulation of sticky mucous in respiratory tract and lungs. Treated by in vivo replacement of defective gene by adenovirus vector .

VECTORS IN GENE THERAPY To transfer the desired gene into a target cell, a carrier is required. Such vehicles of gene delivery are known as vectors. 2 main classes Viral vectors Non viral vectors

VIRAL VECTORS 1) RETROVIRUS VECTOR SYSTEM The recombinant retroviruses  have the ability to integrate into the host genome in a stable fashion. Can carry a DNA of size – less than 3.4kb Replication defective virus particles Target cell - dividing

VIRAL VECTORS ADENO VIRUS VECTOR SYSTEM Adeno virus with a DNA genome – good vectors. Target- non dividing human cell. Eg. Common cold adenovirus.

VIRAL VECTORS 3) ADENO ASSOCIATED VIRUS VECTOR It is a human virus that can integrate into chromosome 19. 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. They infect and persist in nervous cells.

NON VIRAL VECTOR SYSTEM 1. PURE DNA CONSTRUCT Direct introduction of pure DNA construct into target tissue . Efficiency of DNA uptake by cells and expression rather low. Consequently, large quantities of DNA have to be injected periodically. 2. LIPOPLEXES Lipid DNA complexes; DNA construct surrounded by artificial lipid layer. Most of it gets degraded by lysosomes.

NON VIRAL VECTORS 4) HUMAN ARTIFICIAL CHROMOSOME DNA MOLECULAR CONJUGATES Commonly used synthetic conjugate 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 lysosomal breakdown of DNA. 4) HUMAN ARTIFICIAL CHROMOSOME Can carry a large DNA ie, with one or more therapeutic genes with regulatory elements.

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 Microinjection  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.

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 chemicals disturbs the cell membrane, widens the pore size and allows cDNA to pass through the cell. LIPOFECTION 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.

OTHER TYPES OF GENE THERAPY GENE AUGMENTATION THERAPY Most common form of gene therapy Foreign gene replaces missing or defective gene. Eg. Replacement of defective p53 gene by a normal one in liver cancer. GENE INHIBITION THERAPY Done to block the overproduction of some proteins. 2 types – Antigene and antisense therapy. Antigene – blocks transcription using antigene oligonucleotide Antisense – blocks transalation using antisense oligonucleotide.

Gene Therapy for Genetic Disorders Severe Combined Immune Deficiency (ADA-SCID)  ADA-SCID is also known as the bubble boy disease. Affected children are born without an effective immune system Mutation on Chromosome 20 is often to blame. 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 in all treated patients without noticeable side effects, who now live normal lives without the need for further treatment. 

Gene Therapy for Genetic Disorders Chronic Granulomatous Disorder (CGD)  CGD is a genetic disease in the immune system. Leads to the patients' inability to fight off bacterial and fungal infections that can be fatal. Using similar technologies as in the ADA-SCID trial, investigators in Germany treated two patients with this disease Patient’s immune systems have since been able to provide them with full protection against microbial infections for at least two years.

Gene Therapy for Genetic Disorders Hemophilia  Patients born with Hemophilia are not able to induce blood clots Suffer from external and internal bleeding that can be life threatening. In a clinical trial 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 however, was transient because the genetically corrected liver cells were recognized as foreign and rejected by the healthy immune system in the patients.

Gene Therapy for Diseases Cancer  Multiple gene therapy strategies have been developed to treat a wide variety of cancers, including suicide gene therapy, oncolytic virotherapy, anti-angiogenesis and therapeutic gene vaccines. Two-thirds of all gene therapy trials are for cancer and many of these are entering the advanced stage Including a Phase III trial of Ad.p53 for head and neck cancer and two different Phase III gene vaccine trials for prostate cancer and pancreas cancer.

ADVANTAGES Gene therapy has the potential to eliminate and prevent hereditary diseases such as cystic fibrosis, ADA- SCID etc. It is a possible cure for heart disease, AIDS and cancer. It gives someone born with a genetic disease a chance to life. It can be used to eradicate diseases from the future generations. Preventing cancer cells from developing new blood vessels, or angiogenesis

DISADVATAGES 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.

DISADVATAGES The working gene might be slotted into the wrong spot. The working gene might produce too much of the missing enzyme or protein, causing other health problems. The patient may have to undergo several therapy treatments.

ETHICAL ISSUES Who will have access to therapy? Is it interfering with God’s plan? Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence etc.? Is it alright to use the therapy in the prenatal stage of development in babies?

CONCLUSION Theoretically, gene therapy is the permanent solution for genetic diseases. But it has several complexities. At its current stage, it is not accessible to most people due to its huge cost. A breakthrough may come anytime and a day may come when almost every disease will have a gene therapy Gene therapy have the potential to revolutionize the practice of medicine.