Biochemical Engineering CEN 551

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Biochemical EngineeringCEN 551

• Instructor Dr. Christine Kelly
• Chapter 15 Medical Applications of Bioprocess
  Engineering

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Schedule

• Thursday, April 1 Dr. Hasenwinkel (hand out
  homework).
• Tuesday, April 6 Finish chapter 15.
• Thursday April 8 Review for exam 3 (Chap. 12, 14
  and 15 homework due).
• Tuesday, April 13 Exam 3 - chapters 12, 14, and
  15 and posters due.
• Poster Presentations Saturday afternoon, April
  17.
• Oral presentations April 15, 20, 22, 27.

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• April 15 Mittal, Sameer, Xu, Anitescu
• April 20 Meka, Chapeaux, Chang, Sayut
• April 22 Pasenello, Prantil, Lu, Menon
• April 27 Price, Reis

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Presentations

• Each student will have to answer written
  questions about each presentation.
• Be sure to include the answers to these questions
  in your presentation.
• WWT, Chromatography and Validation provide me a
  list of questions that you will answer in your
  presentation.

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Questions

• What is the biological product?
•  What is the application for the product?
•  Is the product currently being produced
  commercially?
•  What is the host cell that produces the product?
•  What type of bioreactor is utilized?
•  What types of downstream processes are utilized?
•  What analysis did the author perform on the
  process?

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Outline

• Tissue Engineering
• Gene Therapy
• Bioreactors

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What is Tissue Engineering?

• The application of principles and methods of
  engineering and life sciences toward fundamental
  understanding of structure-function relationships
  in normal and pathological mammalian tissues and
  the development of biological substitutes to
  restore, maintain or improve tissue function
  (Whitaker Foundation Tissue engineering).

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• Developing in vitro tissues based on cells
  derived from donor tissue.
• Used in transplants.
• Commercial examples skin and cartilage.
• Artificial liver outside the body is in trials.
  Uses hollow fiber reactor and pig liver cells.
• Under development liver, pancreas, kidney, fat,
  blood vessel, bone marrow, bone, neurotransmitter
  secreting constructs.

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Skin Engineering
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Introduction

• The term artificial skin was first introduced
  by JF Burke in 1987, and used to designate a
  bilayered dermal- epidermal replacement devised
  by Burke and Yannas.
• Now it can be applied to several on bilayered
  products that have been engineered for permanent
  replacement of lost human dermis and that provide
  either a temporary or potentially permanent
  epidermis.

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The Structure and Function of Skin
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Skin Structure

• Skin has two distinct layers
• epidermis
• keratinocytes
• dermis
• fibroblasts and collagen

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Basic functions of skin

• Thermoregulation.
• Microbial defense (both mechanical barrier and
  immune defense).
• Desiccation barrier.
• Mechanical defense and wound repair.
• Cosmetic appearance, pigmentation, and control of
  contraction.

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Skin Response to Injury

• Epidermal injury (first degree).
• Superficial dermal injury (second degree).
• Epidermal plus near-full to full dermal injury
  (third degree).

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Surgical Management of Skin Loss

• Autograft (Split-thickness skin grafts)
• The best material for wound closure, when
  practical, is the patients own skin (autograft).
  Split-thickness skin grafts (epidermis plus a
  thin layer of dermis) harvested from the
  patients uninjured skin is essential for
  closure.

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• Several disadvantages of autograft
• The donor site is a new wound.
• The donor site is subject to scarring and
  pigmentation changes.
• The dermis taken from the donor site is not
  replaced.
• The donor site is a potential site for microbial
  entry.
• The donor site cannot provide an unlimited supply
  of dermis.
• The limited supply of donor sites on a patient.

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Permanent Dermal Replacement
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A few observations in designing a dermal
replacement

• The thicker the dermal layer of a split-thickness
  skin graft, the less the graft contracts.
• Full-thickness skin grafts contract minimally.
• Full-thickness dermal injuries heal by
  contraction and hypertrophic scarring, producing
  subepithelial scar tissue that is nothing like
  the original dermis.
• Partial-thickness wounds with superficial dermal
  loss heal with less hypertrophic scarring.

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• The two artificial skins that currently exist
  have sought to meet these constraints in two
  different ways
• Integra, devised by Burke Yannas, was designed
  by applying materials science and engineering
  principles to the problem of dermal replacement.
• Bells product, which is being commercially named
  Apligraf was designed by applying the principles
  of tissue culture.

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Artificial Skin as Tissue Regeneration Matrix
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• In order to promptly close the wound, the skin
  substitute had to
• Adhere to the substrate.
• Be durable and sufficiently elastic to tolerate
  some deformation.
• Allow evaporative water loss at the rate typical
  of the stratum corneum.
• Provide a microbial barrier.
• Promote hemostasis.
• Be easy to use.
• Be readily available immediately after injury.
• Elicit a "regeneration-like" response from the
  wound bed without evoking an inflammatory,
  foreign-body, or non-self immunologic reaction.

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Figure 1. Integra, the bilaminate artificial skin
of Burke Yannas, applied to a full-thickness
skin defect.
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Figure 2. Integra 1 week after application to a
full-thickness skin defect.
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Figure 3. Second-stage Integra grafting. At 2
weeks after Integra application, the process of
neodermis formation is complete, the temporary
silicone epidermal analog has been removed.
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Figure 4. Second-stage Integra grafting. A meshed
ultrathin autograft has been applied. The
epidermal cells of the autograft proliferate and
attach to the underlying neodermis, forming a
durable and confluent epithelium.
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Three limitations of Intagra

• First, it has no intrinsic immunologic defenses
  and must be kept free of bacteria.
• Second, the silicone epidermal analog is purely
  prosthetic and must be removed and replaced with
  epidermal autograft.
• A third drawback is that Integra, although it is
  fairly strong and elastic, does not do
  particularly well on those areas such as the
  back, the axilla, and the groin because of shear
  stress.

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Artificial Skin as a Pre-engineered Tissue
Substitute
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In contrast to the materials science and
engineering approach of Burke Yannas, Bell and
colleagues took the approach of reconstituting
dermal injury by applying a preformed tissue. The
resulting product is described as a dermal
equivalent, which, unlike Integra, relies on
living cells in tissue culture to organize the
collagen network.
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The drawback of Apligraf In order to provide
definitive wound closure, an Apligraf-like
product would have to be constructed from a
patients own fibroblasts and keratinocytes. The
production of a patient-specific product (i.e.
with fibroblasts and keratinocytes taken from the
patient) would take several weeks, during which
the wound would have to be covered with a
temporary skin substitute.
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Dermagraft-TC
Deramagraft-TC is a two-layer synthetic material
designed as a temporary skin substitute. The
outer layer is a silicone polymer, and the inner
layer is a nylon mesh.
Scanning electron micrograph of human dermal
fibroblasts grown on a three-dimensional nylon
scaffold (Dermagraft-TC).
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Temporary Dermal Replacement

• Several new products available
• Human cadaveric allograft
• Biobrane
• Dermagraf-TC or Transcyte

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The Future of Artificial Skin

• Materials science and engineering principles
  produced the dermal regeneration template
  Integra.
• Application of tissue culture techniques produced
  Apligraf.

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In the future, a combination of materials science
and tissue culture techniques is likely to
produce a skin substitute that can function as an
autograft for both dermis and epidermis. Although
expensive, the new approach has demonstrated the
feasibility of combining Integra technology with
that of tissue engineering and may be the
forerunner of 21st-century skin replacement.
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Cartilage Engineering
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Introduction

• Most peoples Achilles heel is not their
  achilles heel but their knees. The knee is not
  that simple, it is actually an interwoven system
  of ligaments, cartilage, and muscle.

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Functions of the Components

• Anterior Cruciate Ligament (ACL) responsible
  for stabilizing and preventing excessive
  extension and lateral movements in the joint.
• Posterior Cruciate Ligament (PCL) responsible
  for stabilizing and preventing excessive flexion
  and lateral movements of the joint.
• Medial Collateral Ligament (MCL) provides
  stability against pressure applied to the leg
  that tries to bend the lower leg sideways at the
  knee, away from the other leg.
• Lateral Collateral Ligament (LCL) provides
  stability against pressure applied to the leg
  that tries to bend the lower leg sideways at the
  knee, toward the other leg.
• Patellar Tendon connects the knee cap to the
  tibia.
• Meniscus (Lateral and Medial) rest on the top
  of the tibia and provide a shock absorbing
  effect.
• Articular Cartilage Creates a low friction
  surface for the joint to glide on.

Figure 1 The knee in flexion (bent)
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Why does Articular Cartilage need to be
Engineered?

• Replacement of the articular cartilage is a
  necessity because defects in mature articular
  cartilage do not heal without residues (Reiss,
  Rudert, Schulze, and Wirth 141).
• Meaning that the smooth surface that the joint
  normally glides across becomes rough in that
  area. This roughness leads to swelling, pain,
  and arthritis in the joint.

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History of the Tissue Engineering of Cartilage
Cells

• In the early 1980s the Hospital for Joint
  Diseases in New York started to develop a
  procedure to use the patients own articular
  cartilage cells to use as a transplant into the
  degeneration or defect in the articular
  cartilage. This was do to the poor results
  yielded by methods to repair the articular
  cartilage at that time.
• Starting in 1987 the University of Goteborg and
  Sahlgrenska University Hospital in Goteborg,
  Sweden worked to continue the development of the
  new procedure.
• October of 1994 the Swedish researchers published
  a study in the New England Journal of Medicine.
  The Swedish researchers reported
  "good-to-excellent results" in 14 of 16 patients
  with a cartilage defect on the thigh-bone part of
  the knee treated at least two years earlier. The
  researchers said the vast majority of patients
  treated on the thigh-bone part of the knee had
  developed hyaline-like cartilage, similar to
  normal cartilage, where the defects had been
  (Genzyme The Carticel Treatment Alternative).
• The Harvard Health Letter rated this new
  technique as one of the "Top Ten Medical Advances
  of 1994".

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The Swedish Method
The Swedish Method of Articular Cartilage
Replacement
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The Swedish Method

• The procedure is used on patients who suffer
  from defects in the articular cartilage on the
  bottom of the femur.

Articular cartilage (chondral) defect before
removing damaged articular cartilage.
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• If the defect the same type of defect as shown in
  Figure 2, the an Orthopedic Surgeon will perform
  an arthroscopic surgery, shown in figure 3, to
  collect the sample cartilage cells.
• Arthroscopic surgery is a procedure where the
  surgeon makes three small incisions in the knee
  and works with specialized equipment in a
  relatively noninvasive procedure.

Photograph of an Arthroscopic Surgery
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• In the first incision the arthroscopic scope, a
  device that utilizes fiber optics, is inserted to
  allow the surgeons to see what they are doing.
• In another incision the actual surgical cutting
  tool is inserted.
• In the final incision an irrigating instrument is
  placed to keep the visibility of the area high.
• Figure 4 shows the basic position of each of
  these tools during an arthroscopic surgery.

Arthroscopic Surgery Instrumentation
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• After the cells have been collected they are sent
  to the company Genzyme Tissue Repair in
  Cambridge, Massachusetts.
• At the plant the new cells are grown, by a
  proprietary procedure, for a period of 2-4
  weeks.
• The cells grown are specific for the patient they
  were grown for.
• After enough cells are grown they are shipped
  back to the Orthopedic Surgeon.
• When the cells get back to the Orthopedic Surgeon
  a much more invasive open knee operation is
  performed.

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• In this new surgery first the damaged area of
  the articular cartilage is cut out leaving.

Articular cartilage (surface) defect (circled in
red) after removing damaged articular cartilage.
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• When the defected articular cartilage is gone the
  surgeon will lance off a small amount of
  Periosteum, a tissue that covers the bone, taken
  from the medial tibia.
• The Periosteum is stitched over the hole where
  the defect was.
• The surgeon will then inject the new cells under
  the flap.
• Under the flap the cells do some additional
  growing and eventually connect to the surrounding
  tissues to form the new cartilage.
• After the surgery each patient receives a
  post-operation schedule that is based on
  progressive program of weight-bearing, range of
  motion, and muscle strengthening exercises.

Articular cartilage (surface) defect after
periosteum patch is sewn in place.
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Currently

• Most of the information collect has not been
  updated since 1999 so it is hard to estimate the
  current number of surgeons that have been trained
  in this procedure and just how many patients have
  underwent the operation.
• However, as of March 31, 1998, 2,238 surgeons had
  been trained in the procedure and a total of
  1,271 patients had been treated since Genzyme
  Tissue Repair began marketing the product in
  1995.
• In 1999, the cost of the procedure ranged from
  17,000 to 38,000, with an average cost of
  approximately 26,000 per procedure.
• Genzyme Tissue Repair charged 10,000 per
  procedure for the cells.
• The Orthopedic Surgeons in Sweden who have been
  using this procedure since its conception in 1987
  have recorded anywhere from a 88 to almost a
  100, depending on the type of defect started
  with, improvement in the patients who they
  preformed this procedure on.

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Future

• The research into articular cartilage replacement
  has just about run its course with no major
  breakthroughs in the last five to ten years.
• However, with the number of Orthopedic Surgeons
  being trained in this procedure increasing yearly
  the cost of the procedure should decrease while
  the relative safety will increase.
• As for tissue engineering in general, there are
  still some problems that need to be worked
  through before the engineering of complex organs
  can begin.
• The first issue is the complexity of the organ to
  be engineered. Skin and articular cartilage are
  both geometrically simple organs and thus getting
  the cells to line up in those formations are
  easy. To get the cells to line up properly and
  form a liver for example takes a degree of cell
  control not yet mastered.
• Another issue being faced is the low blood flow
  through the organs. When these organs are being
  grown in the laboratory the blood supply to the
  organs is not yet sufficient enough for the inner
  cells of the thicker organs to survive.

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Conclusions

• While, the Swedish method of cartilage
  replacement is a great innovation in the history
  of mankind, there are still steps in to be taken
  in the cartilage replacement of the knee.
• Also, there are still many organs needed by
  people every year for millions of transplants and
  replacements. Until viable methods to synthesize
  these organs are developed the world of tissue
  engineering is only just beginning.

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References

• 1995 Annual Report of the Whitaker Foundation
  Tissue Engineering. 1995. The Whitaker
  Foundation. 8, April 2002. lthttp//www.whitaker.
  org/95_annual_report/tissue95.htmlgt.
• Burmester, G.R., M. Sittinger, C. Perka, O.
  Schultz, and T. Haupl. Joint Cartilage
  Regeneration by Tissue Engineering. Z Rheumatol
  58.3 (1999) 130-5.
• Genzyme Tissue Repair. 5, June 1999. The Center
  for Orthopedics Sports Medicine. 9, April
  2002. lthttp//www.arthroscopy.com/sp08001.htmlgt.
  
• Kloth, S., W. W. Minuth, and M. Sittinger.
  Tissue Engineering Generation of Differential
  Artificial Tissues for Biomedical Applications.
  Cell Tissue Research 291.1 (1998) 1-11.
• Reiss, G., M. Rudert, M. Schulze, and C. J.
  Wirth. Synthesis of Articular Cartilage-like
  Tissue In Vitro. Arch Orthopedic Trauma Surgery
  117.3 (1998) 141-6.
• Yacobucci, The Gerald N. Yacobucci, M.D.
  Arthroscopic Surgery and Sports Medicine Home
  Page. 1999. YacoSportsMed. 8, April 2002.
  lthttp//members.tripod.com/GeraldY/index.htmlgt.

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Gene Therapy using Viral Vectors
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Gene Therapy

• Transfer of genes into cells for a therapeutic
  effect.
• Patient has faulty gene that does not encode for
  a correctly functioning protein.
• Genes can be delivered ex vivo (outside the body)
  or in vivo (inside the body).
• If ex vivo, the organ is removed, then
  transplanted back in.
• Genes are delivered to the cells with a virus.
• Clinic trials have been problematic.

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• A normal gene may be inserted into a nonspecific
  location within the genome to replace a
  nonfunctional gene. This approach is most common.
• An abnormal gene could be swapped for a normal
  gene through homologous recombination.
• The abnormal gene could be repaired through
  selective reverse mutation, which returns the
  gene to its normal function.
• The regulation (the degree to which a gene is
  turned on or off) of a particular gene could be
  altered.

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http//www.fda.gov/fdac/features/2000/gene.html
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http//www.fda.gov/fdac/features/2000/gene.html
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Viruses used in Gene Therapy

• Retroviruses - 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.
• Adenoviruses - 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.

http//www.ornl.gov/sci/techresources/Human_Genome
/medicine/genetherapy.shtml
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• Adeno-associated viruses - A class of small,
  single-stranded DNA viruses that can insert their
  genetic material at a specific site on chromosome
  19.
• Herpes simplex viruses - A class of
  double-stranded DNA viruses that infect a
  particular cell type, neurons. Herpes simplex
  virus type 1 is a common human pathogen that
  causes cold sores.

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Current Status

• FDA has not yet approved any human gene therapy
  product for sale.
• Current gene therapy is experimental and has not
  proven very successful in clinical trials.
• In 1999, gene therapy suffered a major setback
  with the death of 18-year-old Jesse Gelsinger.
  Jesse was participating in a gene therapy trial
  for ornithine transcarboxylase deficiency (OTCD).
  He died from multiple organ failures 4 days after
  starting the treatment. His death is believed to
  have been triggered by a severe immune response
  to the adenovirus carrier.

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• Another major blow came in January 2003, when the
  FDA placed a temporary halt on all gene therapy
  trials using retroviral vectors in blood stem
  cells. FDA took this action after it learned that
  a second child treated in a French gene therapy
  trial had developed a leukemia-like condition.
  Both this child and another who had developed a
  similar condition in August 2002 had been
  successfully treated by gene therapy for X-linked
  severe combined immunodeficiency disease
  (X-SCID), also known as "bubble baby syndrome."

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Factors that have kept gene therapy from becoming
an effective treatment for genetic disease

• Short-lived nature of gene therapy - Before gene
  therapy can become a permanent cure for any
  condition, the therapeutic DNA introduced into
  target cells must remain functional and the cells
  containing the therapeutic DNA must be long-lived
  and stable. Problems with integrating therapeutic
  DNA into the genome and the rapidly dividing
  nature of many cells prevent gene therapy from
  achieving any long-term benefits. Patients will
  have to undergo multiple rounds of gene therapy.

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• Immune response - Anytime a foreign object is
  introduced into human tissues, the immune system
  is designed to attack the invader. The risk of
  stimulating the immune system in a way that
  reduces gene therapy effectiveness is always a
  potential risk. Furthermore, the immune system's
  enhanced response to invaders it has seen before
  makes it difficult for gene therapy to be
  repeated in patients.

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• Problems with viral vectors - Viruses, while the
  carrier of choice in most gene therapy studies,
  present a variety of potential problems to the
  patient --toxicity, immune and inflammatory
  responses, and gene control and targeting issues.
  In addition, there is always the fear that the
  viral vector, once inside the patient, may
  recover its ability to cause disease.

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• Multigene disorders - Conditions or disorders
  that arise from mutations in a single gene are
  the best candidates for gene therapy.
  Unfortunately, some the most commonly occurring
  disorders, such as heart disease, high blood
  pressure, Alzheimer's disease, arthritis, and
  diabetes, are caused by the combined effects of
  variations in many genes. Multigene or
  multifactorial disorders such as these would be
  especially difficult to treat effectively using
  gene therapy.

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The Gelsinger Case

• OTCD occurs when a baby inherits a broken gene
  that prevents the liver from making an enzyme
  needed to break down ammonia.
• University of Pennsylvania researchers packaged
  it in a replication-defective adenovirus. To
  reach the target cells in the liver, the
  adenovirus was injected directly into the hepatic
  artery that leads to that organ.
• At age 18, Jesse Gelsinger was in good health,
  but was not truly a healthy teenager. He had a
  rare form of OTCD that appeared not to be linked
  to his parents, but the genetic defect arose
  spontaneously in his body after birth.

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• During his youth, he had many episodes of
  hospitalization, including an incident just a
  year before the OTCD trial in which he nearly
  died from a coma induced by liver failure.
• A strict diet that allowed only a few grams of
  protein per day and a pile of pills controlled
  his disease to the point where he appeared to be
  a normally active teenager.
• Gelsinger received the experimental treatment in
  September 1999. Four days later, he was dead.
• It appears that his immune system launched a
  raging attack on the adenovirus carrier.

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• FDA found a series of serious deficiencies in the
  way that the University of Pennsylvania conducted
  the OTCD gene therapy trial,
• Researchers entered Gelsinger into the trial as a
  substitute for another volunteer who dropped out,
  but Gelsinger's high ammonia levels at the time
  of the treatment should have excluded him from
  the study.
• The university failed to immediately report that
  two patients had experienced serious side effects
  from the gene therapy, as required in the study
  design, and the deaths of monkeys given a similar
  treatment were never included in the informed
  consent discussion.

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Models of Viral Infection

• 5 differential equations
• Change in extracellular viruses/cell.
• Change in internalized viruses/cell.
• Difference change surface viruses/cell.
• Change in the endosome viruses/cell.
• Change in the cytoplasmic viruses/cell.

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• Analytical solutions can be found for the 5 virus
  concentrations as a function of time, each other,
  cell concentration, and rate constants (eqns.
  15.8-12).

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Mass Production of Retrovirus

• Two part system cell line and recombinant
  vector (virus).
• Cell line engineered to produce essential viral
  genes that have been deleted from the viral
  genome.
• Virus incapable of causing disease carriers of
  therapeutic genes.
• Retrovirus can only be used with dividing cells
  for integration of therapeutic genes.
• Require high titer of highly active viruses.

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Two Obstacles

• Decay of virus
• Decreasing temperature decreases decay rate more
  than decreases production rate.
• Inhibition by proteoglycans
• Similar molecular weight as virus, so
  concentrated when virus is concentrated.

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Stem Cells

• Differentiated cell has limited reproduction.
• Stem cells are undifferentiated cells capable of
  reproduction to a large number of differentiated
  type cells.

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Hematopoiesis

• Process of generating blood cells (8 major
  types).
• Hematopoitic stem cell ? 2 types of progenitor
  cells capable of replication and a restricted
  range of differentiated progeny.
• Many different growth factors required.
• Different types of bioreactors being evaluated.

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Artificial Liver

• Liver ? metabolism, produces plasma proteins,
  detoxification.
• Liver can repair but requires time.
• An artificial liver can provide regeneration
  time.
• In vitro hollow fiber reactors
• with human or pig liver cells
• have been examined.
• In vitro liver in clinical trials now.