Biotechnology and Genetic Engineering-PBIO 450 ... - Ohio University


14 déc. 2012 (il y a 8 années et 7 mois)

249 vue(s)

Chapter 10

Medical Biotechnology

Gene therapy

New gene therapy approaches

Stem cells and Therapeutic Cloning


Tissue engineering and xenotransplantation

Drug delivery and nanotechnology

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

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.

Vectors/methods used to deliver
genes in Human Gene Therapy



associated viruses

Herpes simplex virus


Naked DNA

Human gene therapy

(# clinical trials 1990

AIDS (19)

Amyotrophic lateral

Cancer (280)

Cardiovasc. dis. (20)

Cystic fibrosis (24)


Gaucher disease (3)

Hemophilia A (2)

Hemophilia B (2)

Hunters disease

Multiple sclerosis

Muscular dystrophy

Rheumatoid arthritis

Severe combined
immunodeficiency (3)

Severe Combined ImmunoDeficiency (SCID)


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

old Ashanthi DeSilva
SCID sufferer
treated with gene therapy
coloring at home
in N Olmstead, OH (March 1993).

Cystic fibrosis transmembrane conductance regulator protein (CFTR)

CFTR involved with chloride
ion transport out of cells; if

builds up inside
cells and draws water inside
resulting in a sticky, sugar
rich extracellular mucus.

Is gene therapy safe?

What do you think?

Jesse Gelsinger story

Jesse Gelsinger

June 18


September 17
) 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

genetic disease

of the
, whose victims are unable to metabolize


a byproduct of

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

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.

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
, but paid the parents an undisclosed amount in settlement.
The Gelsinger case was a severe setback for scientists working in the field.

New Approaches to Gene Therapy

Antisense RNA


RNA interference (RNAi)

mediated RNA trans

Triplex helix oligonucleotide therapy

Inhibition of translation of specific RNA by
antisense nucleic acid molecules


antisense cDNA

poly A addition signal

antisense oligonucleotide



RNA complex

Ribozymes: A. Hammerhead B. Hairpin

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.


Binding of dsRNA
specific nuclease


mRNA is cleaved!

ssRNA complex

Hybridizes to mRNA



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)

Other topics covered in the
chapter, but not in lecture


Tissue engineering


Drug delivery and biosensors