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

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Chapter 11
-
Nucleic Acids as Therapeutic Agents


Nucleic acids


A
ntisense RNA and oligonucleotides


R
ibozymes


A
ptamers
,


I
nterfering RNAs or
RNAi


Gene therapy


Stem cells and therapeutic cloning

Copyright © 2010 ASM Press

American Society for Microbiology

1752 N St. NW, Washington, DC 20036
-
2904

Molecular Biotechnology: Principles and Applications of Recombinant

DNA,

Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 11

Nucleic Acids as Therapeutic Agents

Figure 11.1

Fig. 11.1 Inhibition of translation of specific RNA
by antisense nucleic acid molecules

Promoter


antisense cDNA


poly A addition signal

antisense oligonucleotide

mRNA

-
antisense

RNA complex

Fig. 11.8 Ribozymes: A. Hammerhead B. Hairpin

Figure 11.8

Copyright © 2010 ASM Press

American Society for Microbiology

1752 N St. NW, Washington, DC 20036
-
2904

Molecular Biotechnology: Principles and Applications of Recombinant

DNA,

Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 11

Nucleic Acids as Therapeutic Agents

Figure 11.11

Aptamers
-
nucleic acid
sequences (RNA or DNA) that
bind tightly to proteins, amino
acids or other molecules

Copyright © 2010 ASM Press

American Society for Microbiology

1752 N St. NW, Washington, DC 20036
-
2904

Molecular Biotechnology: Principles and Applications of Recombinant

DNA,

Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 11

Nucleic Acids as Therapeutic Agents

Figure 11.13 Overview of RNA interference (RNAi)

Fig. 11.13 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

RNAi


In 2006, Fire and Mello received a Nobel Prize
for their RNAi work uisng Double Stranded
RNA in C. elegans


see RNA Interference on
YouTube:
http://www.youtube.com/watch?v=UdwygnzI
dVE&feature=related



Discovered in petunia
-

see RNAi Discovered
on YouTube:
http://www.youtube.com/watch?v=H5udFjW
DM3E&feature=related


Copyright © 2010 ASM Press

American Society for Microbiology

1752 N St. NW, Washington, DC 20036
-
2904

Molecular Biotechnology: Principles and Applications of Recombinant

DNA,

Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 11

Nucleic Acids as Therapeutic Agents

Table 11.3

Human Gene Therapy

(disease targets)


AIDS


Amyotrophic lateral
sclerosis


Cancer


Cardiovasc. disease


Cystic fibrosis


Familial
hypercholesterolemia


Gaucher disease


Hemophilia A


Hemophilia B


Hunters disease


Multiple sclerosis


Muscular dystrophy


Rheumatoid arthritis


Severe combined
immunodeficiency

I
n
d
i
c
ati
on
s

G
e
n
e
Th
e
r
ap
y C
l
i
n
i
c
al
Tr
i
al
s


N
u
mb
e
r

%

C
an
c
e
r
d
i
s
e
as
e
s

1186

64.4

C
ar
d
i
ovas
c
u
l
ar
d
i
s
e
as
e
s

155

8.4

G
e
n
e
mar
k
i
n
g

50

2.7

H
e
al
th
y vol
u
n
te
e
r
s

42

2.3

I
n
fe
c
ti
ou
s
d
i
s
e
as
e
s

147

8

I
n
fl
ammator
y d
i
s
e
as
e
s

13

0.7

M
on
oge
n
i
c
d
i
s
e
as
e
s

161

8.7

N
e
u
r
ol
ogi
c
al
d
i
s
e
as
e
s

36

2

O
c
u
l
ar
d
i
s
e
as
e
s

28

1.5

O
th
e
r
s

25

1.4

T
ot
a
l

1843




Human Gene Therapy Clinical Trials

http://www.abedia.com/wiley/indications.php


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.

Two types of gene therapy


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

Vectors used to deliver genes

in Human Gene Therapy


Retroviruses


Adenoviruses


Adeno
-
associated viruses


Herpes simplex virus


Liposomes/Lipofection


Naked DNA/Plasmid DNA


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.


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)