Transcript Nucleic Acid Therapeutics

Chapter 11-Nucleic Acids as Therapeutic Agents
•Nucleic acids
•Antisense RNA and oligonucleotides
•Ribozymes
•Aptamers,
•Interfering RNAs or RNAi
•Gene therapy
•Stem cells and therapeutic cloning
Chapter 11
Nucleic Acids as Therapeutic Agents
Figure 11.1
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Fig. 11.1 Inhibition of translation of specific RNA
by antisense nucleic acid molecules
Promoter
antisense cDNA
antisense oligonucleotide
poly A addition signal
mRNA-antisense
RNA complex
Fig. 11.8 Ribozymes: A. Hammerhead B. Hairpin
Figure 11.8
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
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 11
Nucleic Acids as Therapeutic Agents
Figure 11.13 Overview of RNA interference (RNAi)
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Fig. 11.13 RNA interference (RNAi)
dsRNA
sense
antisense
Binding of dsRNA-specific nuclease
Nuclease-ssRNA complex
Hybridizes to mRNA
cleavage
mRNA is cleaved!
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.
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
Chapter 11
Nucleic Acids as Therapeutic Agents
Table 11.3
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
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
Human Gene Therapy Clinical Trials
Indications
Gene Therapy Clinical Trials
Number
%
Cancer diseases
1186
64.4
Cardiovascular diseases
155
8.4
Gene marking
50
2.7
Healthy volunteers
42
2.3
Infectious diseases
147
8
Inflammatory diseases
13
0.7
Monogenic diseases
161
8.7
Neurological diseases
36
2
Ocular diseases
28
1.5
Others
25
1.4
Total
1843
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, sugarrich 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)