This document discusses various topics in medical biotechnology including gene therapy, stem cells, and therapeutic cloning. It describes two types of gene therapy - ex vivo and in vivo gene delivery. It also discusses vectors used in human gene therapy such as retroviruses and adenoviruses. Examples of clinical gene therapy trials are provided for diseases such as cystic fibrosis, hemophilia, and severe combined immunodeficiency. The risks of gene therapy, including the tragic case of Jesse Gelsinger, are examined. New approaches to gene therapy like antisense RNA, ribozymes, and RNA interference are introduced. Finally, stem cell therapy and repairing damaged tissue with stem cells is briefly discussed.

1. Chapter 10 – Medical Biotechnology
•Gene therapy
•New gene therapy approaches
•Stem cells and Therapeutic Cloning
•Vaccines
•Tissue engineering and xenotransplantation
•Drug delivery and nanotechnology

2. Two types of gene therapy (adding a normal
gene to correct a specific gene disorder)
• Ex vivo -cells are removed from the body,
the gene of interest is inserted into them,
the cells are cultured to increase cell
numbers, and they are returned to the
body by infusion or transplantation (time
consuming and expensive)
• In vivo -a gene is introduced directly into
specific cells within the body (quick and
inexpensive), but targeting certain cells
(e.g., bone marrow stem cells) is difficult

3. Consider somatic vs germline gene therapy; the later is currently banned.
Note that gene therapy is limited to somatic cells and disorders that are
caused by a single gene.

5. Vectors/methods used to deliver
genes in Human Gene Therapy
• Retroviruses
• Adenoviruses
• Adeno-associated viruses
• Herpes simplex virus
• Liposomes
• Naked DNA

6. Human gene therapy
(# clinical trials 1990-1999)
• AIDS (19)
• Amyotrophic lateral
sclerosis
• Cancer (280)-p53
• Cardiovasc. dis. (20)
• Cystic fibrosis (24)
• Familial
hypercholesterolemia
• Gaucher disease (3)
• Hemophilia A (2)
• Hemophilia B (2)
• Hunters disease
• Multiple sclerosis
• Muscular dystrophy
• Rheumatoid arthritis
• Severe combined
immunodeficiency (3)

7. Severe Combined ImmunoDeficiency (SCID)
• See http://www.scid.net/about.htm
How is ADA deficiency treated?
There are no real cures for ADA deficiency, but doctors
have tried to restore ADA levels and improve immune
system function with a variety of treatments:
• Bone marrow transplantation from a biological match
(for example, a sibling) to provide healthy immune cells
• Transfusions of red blood cells (containing high levels of
ADA) from a healthy donor
• Enzyme replacement therapy, involving repeated
injections of the ADA enzyme
• Gene therapy - to insert synthetic DNA containing a
normal ADA gene into immune cells
6-yr-old Ashanthi DeSilva-SCID sufferer
treated with gene therapy-coloring at home
in N Olmstead, OH (March 1993).

8. Cystic fibrosis transmembrane conductance regulator protein (CFTR)
CFTR involved with chloride
ion transport out of cells; if
defective Cl- builds up inside
cells and draws water inside
resulting in a sticky, sugar-
rich extracellular mucus.

9. Is gene therapy safe?
• What do you think?
• Jesse Gelsinger story
Jesse Gelsinger (June 18, 1981 - September 17, 1999) was the first person publicly identified as having died in a
clinical trial for gene therapy. He was 18 years old. Gelsinger suffered from ornithine transcarbamylase deficiency,
an X-linked genetic disease of the liver, whose victims are unable to metabolize ammonia - a byproduct of protein
breakdown. The disease is usually fatal at birth, but Gelsinger had not inherited the disease; in his case it was the
result of a genetic mutation and as such was not as severe - some of his cells were normal which enabled him to
survive on a restricted diet and special medications.
Gelsinger joined a clinical trial run by the University of Pennsylvania that aimed to correct the mutation. On Monday,
September 13 1999, Gelsinger was injected with adenoviruses carrying a corrected gene in the hope that it would
manufacture the needed enzyme. He died four days later, apparently having suffered a massive immune response
triggered by the use of the viral vector used to transport the gene into his cells. This led to multiple organ failure
and brain death. Gelsinger died on Friday, September 17th at 2:30 PM.
A Food and Drug Administration (FDA) investigation concluded that the scientists involved in the trial, including the lead
researcher Dr. James M. Wilson (U Penn), broke several rules of conduct:
Inclusion of Gelsinger as a substitute for another volunteer who dropped out, despite having high ammonia levels that
should have led to his exclusion from the trial
Failure by the university to report that two patients had experienced serious side effects from the gene therapy
Failure to mention the deaths of monkeys given a similar treatment in the informed consent documentation.
The University of Pennsylvania later issued a rebuttal [1], but paid the parents an undisclosed amount in settlement.
The Gelsinger case was a severe setback for scientists working in the field.

10. New Approaches to Gene Therapy
• Antisense RNA
• Ribozymes
• RNA interference (RNAi)
• Spliceosome-mediated RNA trans-splicing
• Triplex helix oligonucleotide therapy

11. Inhibition of translation of specific RNA by
antisense nucleic acid molecules
Promoter antisense cDNA poly A addition signal
antisense oligonucleotide
mRNA-antisense
RNA complex

12. Ribozymes: A. Hammerhead B. Hairpin

13. RNA interference (RNAi)
A cellular nuclease binds to the dsRNA cleaving it into ssRNAs of 21-23 nucleotides each.
The nuclease-RNA oligonucleotide complex binds and cleaves specific mRNA.
dsRNA
Binding of dsRNA-specific nuclease
cleavage
mRNA is cleaved!
Nuclease-ssRNA complex
Hybridizes to mRNA
sense
antisense

14. Stem Cells
• Stem cells are the progenitors of many
different cell types, depending upon which
type of stem cell is used (e.g., bone
marrow stem cells, neural stem cells,
embryonic stem cells)
• Stem cell therapy-the goal is to repair
damaged tissue (e.g. Parkinson’s disease,
spinal cord injury)

18. Other topics covered in the
chapter, but not in lecture
• Vaccines
• Tissue engineering
• Xenotransplantation
• Drug delivery and biosensors
• Nanotechnology