Regenerative Medicine

1
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.

2
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

3
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

4
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

5
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
• RBCs 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)

6
Bone Marrow Transplantation

• Bone marrow is the principal site where blood
  cells are made

7
Bone Marrow Transplantation

• 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

• 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

8
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

9
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

10
Pancreatic b-Islet Transplantation

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

11
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 patients 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

12
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)

13
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.

14
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)

15
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.
16
Adenovirus Entry
17
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.
18
Retrovirus Entry
19
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
• Alzheimers disease
• Leukemia
• Muscular Dystrophy
• Congenital Hypothyroidism
• Several Cancers (ovarian, colorectal, etc.)
• Penetration and Gene Transfer mechanisms are
  similar to the Adenovirus.

20
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.

21
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?