Dr. Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

Gene Therapy GT Data worldwide Classical & Non Classical Disease Selection for GT Barriers to successful gene therapy GT for Monogenic disorders Examples of GT

GT Clinical Trials (2012)

Clinical Trials by Disease Categories (2012)

Delivery Vectors

Targeting of different organs by viral vectors

Major Types GT Classical Gene Therapy Non Classical Gene Therapy

Classical gene therapy Classical gene therapy is the approach which delivers genes to the appropriate target cells with a goal of attaining optimal expression of the new, introduced gene. Once inside the patient, the expressed genes are intended to: produce a product that the patient lacks, kill the diseased cells directly by producing a toxin, and activate the immune system to help the killing of the diseased cells.

Classical GT Approach This approach involve to deliver gene into targeted cell. Produce a product that a patient lack Kill the disease directly by producing toxin Activate immune system

Non classical Gene Therapy Non classical gene therapy inhibits the expression of genes related to pathogenesis, or corrects a genetic defect and restores normal gene expression.

Non Classical GT Approach This approach involve to inhibit the expression of genes. To correct the genetic defect and to restore the normal gene expression

Why GT ? Many diseases have still not cure & require expensive treatments to prolong the life spam of patients. The idea that therapeutic gene transfer may permanently cure some diseases.

Long term expression Stable and long-term expression of the protein Localized to the injection site possibility of targeting specific cell populations (with aid of cell-type specific promoters or modifying viral coat proteins Continuous supply of the protein of choice

Patient’s Own Cell In gene therapy, the patient’s own cells are used to cure the disease. Hence their will be no rejections (immunologically) by the patient’s body. Brain cells which are hard to target by traditional therapies, can be easily targeted using lentiviral vectors in gene therapy.

Permanent cure The cure is permanent for the patients life time. The genetic manipulation can only done in the somatic cells of the patient and hence there is no risk to the future generations. Even dangerous diseases like AIDS, SCID, Thalassemia can be cured by gene therapy.

Genetic Disorders Thus there is a chance for permanent cure of neurological disorders. Today, gene therapy is a medical necessicity in many types of diseases including cancer and genetic disorders.

A disease suitable for GT Over 4000 diseases results for single gene disorders Many single gene disorders are due to mutations in nucleic acid sequence. In some case a protein is abnormal & is recognized as non-self. This elicit immune response & cause autoimmune disease.

A disease suitable for GT In some cases the mutant protein may have regulatory role, like p53. P53 is involved in 50% cancers P53 play a role is arresting the cell growth/in apoptosis

A disease suitable for GT A disease may be suitable if its life threatening. The effect of the disease must be potentially reversible by the treatment The gene must be fully characterize. The targeted site be feasible. Then its ethically acceptable for GT treatment

Benefits for single gene disorder GT Life saving of the patients Reduce the cost of managing the disease High GT cost per annum could be acceptable

Major Monogenic disorders Familial Hypercholesterolemia. Polycystic kidney disease Huntington’s Chorea Cystic Fibrosis Haemophilia Phenylketonuria Duchene Muscular Dystrophy

Strategy for transfer of gene

Barriers to successful gene therapy Vector development Corrective gene construct Proliferation and maintenance of target cells Efficient transfection and transport of DNA to nucleus for integration into genome Expansion of engineered cells and implantation into patient

Cystic Fibrosis Autosomal Recessive disorder. Abnormal Electrolyte transport in epithelial cells The disease cause respiratory failure Mutation in the gene CFTR (cystic fibrosis transmembrane conductor regulator) have been pinpointed. Patient don’t survive in their mid-thirities.

GT trials for Cystic Fibrosis Several trials have been carried out delivering CFTR gene encoded in adenoviruses. The result of phase-1 study showed 30% improvement over a two week period without any side effect in 2 of 3 patients. The 3rd patient showed inflammatory response to adenovirus vector. Further studies are going on.

Adenosine deaminase deficiency ADA cause sever combined immunodeficiency. Disease cause by ADA enzyme deficiency that result in elevated level of dATP blocking T-lymphocyte differentiation in the thymus.

GT for ADA Current therapies include bone marrow transplantation Or supplement of ADA

Familial Hypercholesterolemia (FH) In this case the arteries become thickened and eventually blocked. The patient can suffer angina and acute myocardial infarction. The disease also treated with by pass or angioplasty. The draw back is it require general anaesthesia & is not an easy process.

GT for FH FH result from LDL receptor This cause high LDL level in the blood while HDL is suppressed. Phase-1 clinical trial is underway. Patient hepatocytes isolated, then transduce ex-vivo with a retrovirus expressing LDL receptors. The modified cell reinfused into inferior mesenteric vein.

GT for FH One patient showed decrease in serum cholesterol level Improvement in LDL:HDL ratio.

Cancer Lungs Cancer Breast Cancer Colorectal cancer Malignant Lymphoma, myeloma Leukemias Ovarian

General Treatments Surgical Radiotherapy Chemotherapy Success rate/Problems: If tumor retained within the treatment area. Inability to differentiate b/w normal & affected cells

GT for Cancer GT provide an alternative route. Problems in cancer GT To target the therapeutic gene to every tumour cell.

Cancer GT Strategies Gene Augmentation Therapy (GAT) Antisense Oligonucleotides Targeted Killing of specific Cells Immunotherapy Targeted Mutation Correction Targeted Inhibition of gene expression

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