V. Treatment of Genetic Disease

Overview Many genetic disorders are characterized by progressive disability or chronic ill health for which there is, at present, no effective treatment. Consequently, one of the most exciting aspects of the developments in biotechnology is the prospect of new treatments mediated through gene transfer, RNA modification, or stem cell therapy.

Conventional Approaches to Treatment of Genetic Disease Most genetic disorders cannot be cured or even ameliorated using conventional methods of treatment. Sometimes this is because the underlying gene and gene product have not been identified. so that there is little, if any, understanding of the basic metabolic or molecular defect. Dietary restriction, as in phenylketonuria (PKU) Hormone replacement, as in congenital adrenal hyperplasia (CAH) Mutations of genes for enzymes mediating the biochemical steps of production of cortisol from cholesterol by the adrenal glands (steroidogenesis)

Protein/Enzyme Replacement If a genetic disorder is found to be the result of a deficiency of or an abnormality in a specific enzyme or protein, treatment could, in theory, involve replacement of the deficient or defective enzyme or protein: Factor VIII concentrate in the treatment of hemophilia A For most of the inborn errors of metabolism in which an enzyme deficiency has been identified, recombinant DNA techniques may be used to biosynthesize the missing or defective gene product; however, injection of the enzyme or protein may not be successful if the metabolic processes involved are carried out within cells and the protein or enzyme is not normally transported into the cell.

Drug Treatment In some genetic disorders, drug therapy is possible: statins can help to lower cholesterol levels in familial hypercholesterolemia In others, avoidance of certain drugs or foods can prevent the manifestation of the disorder Sulfonamides in G6PDH deficiency or Fauvism

Tissue Transplantation Replacement of diseased tissue has been a further option since the advent of tissue typing. Renal transplantation in adult polycystic kidney disease (PKD) Lung transplantation in patients with cystic fibrosis (CF) Islet transplantation for treating type 1 diabetes mellitus

Therapeutic Applications of Recombinant DNA Technology

Gene Therapy UK Gene Therapy Advisory Committee (GTAC): “ The deliberate introduction of genetic material into human somatic cells for therapeutic, prophylactic, or diagnostic purposes “ It includes techniques for delivering synthetic or recombinant nucleic acids into humans; genetically modified biological vectors (such as viruses or plasmids), genetically modified stem cells, oncolytic viruses, nucleic acids associated with delivery vehicles, naked nucleic acids, antisense techniques.

Regulatory Requirements There is universal agreement that germline gene therapy, in which genetic changes could be distributed to both somatic and germ cells, and thereby be transmitted to future generations, is morally and ethically unacceptable. Therefore all programs are focusing only on somatic cell gene therapy, in which the alteration in genetic information is targeted to specific cells, tissues or organs in which the disorder is manifest.

Technical Aspects Gene Characterization Target Cells, Tissue, and Organ Vector System Animal Models

classification Bacterial : Plasmid Viral :Cosmide Artificial: YAC (Yeast artificial chromosome) BAC (Bacterial artificial chromosome)

plasmid Sticky or blunt End

PCR

Gene Transfer Gene transfer can be carried out either : ex vivo : by treatment of cells or tissue from an affected individual in culture, with reintroduction into the affected individual in vivo : if cells cannot be cultured or be replaced in the affected individual.

… Viral: Lenti virus Adeno virus Adeno associated virus Non-viral: Liposome

Target Organs In many instances, gene therapy will need to be, and should be, directed or limited to a particular organ, tissue or body system: Liver Central Nervous System Muscle Bone Marrow

RNA Modification RNA modification therapy targets mRNA, either by suppressing mRNA levels or by correcting/adding function to the mRNA. Antisense Oligonucleotides RNA Interference Targeted Gene Correction (DNA-RNA hybrid containing normal nucleotides)

Diseases Suitable for Treatment Using Gene Therapy Adenosine Deaminase Deficiency Cystic Fibrosis Hemophilia A and B Duchenne Muscular Dystrophy

Stem Cell Therapy Stem cells are defined by their capacity for self-renewal and the ability to differentiate into specialized cells along many lineages.

Classification based on stem cell potency Totipotent / Omnipotent: They can form all the cell types of the adult organism as well as extraembryonic tissues of the embryo Pluripotent: They can form all the cell types of the adult organism. Multipotent: They can form all the differentiated cell types of a given tissue Unipotent: In some cases, a tissue contains only one differentiated lineage and the stem cells that maintain the lineag are described as unipotent.

Classification based on source of extraction Stem cells Embryonic stem cell Embryonic germ stem cell Umbilical cord Somatic stem cell Cancer Stem Cells

Gene Therapy Using Embryonic Stem Cells

Mesenchymal stem cells: Stromal origin MSCs can be isolated from: Placenta Fat tissue Lung Bone marrow Blood Umbilical cord

Induced Pluripotent Stem Cell Therapy Certain kinds of somatic cells seem to have the ability to differentiate into a number of different cell types, given the right conditions.