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

whooshdisguisingBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

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Chapter 10


Medical Biotechnology


Gene therapy


New gene therapy approaches


Stem cells and Therapeutic Cloning


Vaccines


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


Retroviruses


Adenoviruses


Adeno
-
associated viruses


Herpes simplex virus


Liposomes


Naked DNA


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)

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).

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.

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.


New Approaches to Gene Therapy


Antisense RNA


Ribozymes


RNA interference (RNAi)


Spliceosome
-
mediated RNA trans
-
splicing


Triplex helix oligonucleotide therapy


Inhibition of translation of specific RNA by
antisense nucleic acid molecules

Promoter


antisense cDNA


poly A addition signal

antisense oligonucleotide

mRNA

-
antisense

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.


dsRNA

Binding of dsRNA
-
specific nuclease

cleavage

mRNA is cleaved!

Nuclease
-
ssRNA complex

Hybridizes to mRNA

sense

antisense

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


Vaccines


Tissue engineering


Xenotransplantation


Drug delivery and biosensors


Nanotechnology