Regenerative Medicine

Regenerative Medicine • The objective of this next set of lectures is to discuss the new field of Regenerative Medicine and how these technologies are applied to provide superior health care. CHEE 340

Need for New Therapies • The clinical need for tissue and organ replacements rapidly increases each year • Estimated cost of these transplantations is about $400 billion annually in the USA alone CHEE 340

Tissue and Organ Transplants • Tissue and organ transplants have been performed for several decades with varying clinical success. • Classifications of transplant materials: • Autogenic (same individual) • no immune response • certain procedures only (e.g. ligament repair) • Allogenic (same species, different individuals) • most common (e.g. organ donation) • can elicit an immunological response • chronic use of immunological suppressants • Xenogenic (different species) • least common (e.g. porcine aortic valves) • greatest potential to elicit an immunological response • must be chemically treated prior to implantation CHEE 340

Regenerative Medicine • Regenerative Medicine is the scientific field that focuses on new approaches to the autologous repair and/or replacement of cells, tissues and/or organs. • Broad research area with several main focuses: • Cellular Therapies • Gene Therapies • Tissue Engineering CHEE 340

Cellular Therapies • Cellular therapies have the promise to become major therapeutic modalities of the next century. • However, cellular therapy is not a new concept: • blood transfusions routinely performed for several decades • RBC’s to anemic patients to restore O2 transport • Examples: • Bone marrow transplantation (currently performed) • Chondrocyte transplantation (in clinical trials) • Pancreatic b-islet transplantation (in clinical trials) CHEE 340

Bone Marrow Transplantation • Bone marrow is the principal site where blood cells are made: CHEE 340

Bone Marrow Transplantation • Allogenic Procedure (e.g. leukemia) • remove BM to hopefully remove disease • donor BM cells harvested and put into circulation • BM stem cells return to marrow cavities and reconstitute marrow function • Autogenic Procedure (e.g. patients receiving chemotherapy) • BM highly susceptible to radiation and chemotherapies • BM cells harvested, cryo-preserved before procedure and returned back to patient • Bone marrow is comprised of 500-1000 billion cells and is the most prolific tissue in the body: • produces ~ 400 billion myeloid cells/day • regenerates every 2-3 days • all of which originate from a small number of stem cells CHEE 340

Autologous Chondrocyte Transplantation • Articular cartilage • dense connective tissue that forms the bearing surfaces of synovial joints • acellular tissue which is has a poor propensity for repair in adults • common to allow for cartilage degeneration to continue until the entire joint can be replaced CHEE 340

Autologous Chondrocyte Transplantation • Procedure (autologous) • biopsy of cartilage and isolation of chondrocytes (non-weight bearing region) • in vitro expansion of chondrocytes (several fold) • lesion cleaned and periosteal flap sutured on top • re-inject chondrocytes under periosteal flap CHEE 340

Pancreatic b-Islet Transplantation • Insulin is required for proper glucose uptake by cells • The Islets of Langerhans (b-cells) of the pancreas produce insulin CHEE 340

Pancreatic b-Islet Transplantation • Diabetic patients have a deficiency to produce the appropriate amount of insulin thus requiring daily insulin injections • Micro-encapsulation of pancreatic b-islet cells is currently under investigation as a suitable long-term therapy • allogenic cells encapsulated in a biomaterial (hydrogel) and transplanted to patient’s pacreas • hydrogel allows the diffusion of semi-permeable small molecules but not the larger molecules of the of the immune system • rejection problem not completely solved since body can “wall-off” the graft with a thick layer of connective tissue CHEE 340

Gene Therapy • Gene Therapy is the technique for correcting defective genes responsible for disease development. • Genes • carried on chromosomes; the basic physical and functional units of heredity • specific sequences of bases that encode how to make proteins • when altered, encoded proteins are unable to carry out their normal functions, genetic disorders can result • Several approaches are currently under investigation: • insertion of the gene into a non-specific location within the genome to replace a non-functional gene (most common) • homologous recombination to swap abnormal gene with a normal gene • selective reverse mutation to return the abnormal gene to its normal function • alteration of gene regulation (degree to which a gene is turned on or off) CHEE 340

Gene Therapy • In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. • A carrier molecule (vector) must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vectors used are viruses which have been genetically altered to carry normal human DNA. • Viruses have evolved a way of encapsulating and delivering their genes to human cells (pathogenic) and scientists have tried to harness this capability and manipulate the viral genome to deliver therapeutic genes. CHEE 340

Gene Therapy Vectors • Some of the different types of viruses currently under investigation for use as gene therapy vectors: • Adenoviruses • Retroviruses • Adeno-Associated Viruses (AAV) CHEE 340

Adenovirus • Adenovirus (non-specific insertion): • A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. The virus that causes the common cold is an adenovirus. • Penetration: • Penetration into the cell by endocytosis. Once inside the cell, the penton of the virus serves to rupture the phagocytic mebrane and release the particle into cytoplasm. • Gene Transfer: • The core migrates to the nucleus where the DNA enters through nuclear pores and becomes incorporated into the genome. CHEE 340

Adenovirus Entry CHEE 340

Retrovirus • Retrovirus (non-specific insertion): • A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus. • Penetration: • Envelope proteins serve as ligands for receptors on cell surface. Viral and cell membranes fuse to release caspid particle into cytoplasm. The reverse transcriptase enzyme (RNA DNA) then synthesizes DNA copies of its RNA. • Gene Transfer: • Transcribed DNA migrates to the nucleus, enters through nuclear pores and becomes incorporated into the genome. CHEE 340

Retrovirus Entry CHEE 340

Adeno-Associated Virus • Adeno-Associated Virus (specific insertion): • A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19. • Chromosome 19 is of particular interest since it: • has almost twice as many genes (1,300 to 1,700) compared to other chromosomes • numerous conditions are related to genes on chromosome 19 (70 known genetic disorders), for example: • Alzheimer’s disease • Leukemia • Muscular Dystrophy • Congenital Hypothyroidism • Several Cancers (ovarian, colorectal, etc.) • Penetration and Gene Transfer mechanisms are similar to the Adenovirus. CHEE 340

Problems with Gene Therapy • Short-Lived Nature • Problems with the stability of therapeutic DNA once in the genome and the rapidly dividing nature of many cells hinder achieving long-term benefits of gene therapy. Patients will have to undergo multiple rounds of gene therapy. • Immune Response • There is a risk of stimulating the immune system when using viral gene delivery vectors, thereby reducing effectiveness. The immune system's enhanced response to repeat invaders makes it difficult for multiple rounds of gene therapy. • Viral Vectors • Viruses present a variety of potential problems to the patient: toxicity, immune and inflammatory responses, gene control and targeting issues. Also, there is the fear that the viral vector, once inside the patient, may recover its ability to cause disease.Multi-Gene Disorders • Conditions arising from mutations in a single gene are the best candidates. However, some common disorders (Alzheimer's, arthritis, diabetes, etc.) are caused by combined effects of variations in many genes making them difficult to treat. CHEE 340

Ethics of Gene Therapy • Some Questions to Consider... • What is normal and what is a disability or disorder, and who decides? • Are disabilities diseases? Do they need to be cured or prevented? • Is somatic gene therapy (which is done in the adult cells of persons known to have the disease) more or less ethical than germline gene therapy (which is done in egg and sperm cells and prevents the trait from being passed on to further generations)? • Preliminary attempts at gene therapy are exorbitantly expensive. Who will have access to these therapies? Who will pay for their use? CHEE 340

Regenerative Medicine