Transcript DNA as Drugs

Synthetic DNA as Drugs
Supported by the NSF award 0647129
Simple Methods for Oligonucleotide Purification
Why Can DNA Be Used as Drugs?
The Structure of DNA and RNA
 Four bases in DNA and RNA
 They are A, T (U in RNA), G
and C
 Genetic information is stored
in the sequence of A, T, G, C
in DNA
 DNA double stranded
 RNA double/single stranded
 A pairs to T (U in RNA) only
and G pairs to C only
 DNA-DNA, RNA-RNA, DNARNA
Why Can DNA Be Used as Drugs?
The Base Pairs
DNA-DNA: A-T, G-C
RNA-RNA: A-U, G-C
DNA-RNA: A-U, G-C
Why Can DNA Be Used as Drugs?
The Central Dogma of Molecular Biology
 DNA: double stranded, contains
genetic information
 RNA: single stranded
 Protein: the function molecule of
life, function determined by the
sequence of amino acids
Why Can DNA Be Used as Drugs?
How People Get Disease?
Mono-genetic disorder
 Currently, a total of ~4,000 genetic disorders are known
 Some are single genetic disorder
 Changes (mutations) of the sequence of one gene (DNA)
 Point mutation
 Deletion
 And more
 List of genetic disorders at
http://en.wikipedia.org/wiki/List_of_genetic_disorders
 The mutated genes produce proteins that cannot function
properly, diseases occur
 Examples: Sickle-cell anemia, Cystic fibrosis (1/3900, most
common, difficult breathing, die in 20s-30s, no cure), Color blindness
Why Can DNA Be Used as Drugs?
How People Get Disease?
Poly-genetic disorder
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Mutations occur in many genes
Do not have a clear cut of inheritance
But do “run in families”
The mutated genes produce proteins that cannot
function properly, diseases occur
 Difficult (not impossible) to study and treat because
direct cause is unknown
 Examples: Heart disease, hypertension, diabetes,
obesity, cancers, low IQ
Why Can DNA Be Used as Drugs?
How People Get Disease?
Bacteria and virus infections
• Smallpox
– 300-500 million deaths in 20th century
– Eliminated by vaccination
• HIV/AIDS
– Human immunodeficiency virus/acquire immunodeficiency syndrome
– Still taking many lives each year, in 2000, 2.8 million
• Hepatitis B
– 0.1 million lives taken in 2000
– Liver inflammation, vomiting, and rarely death, but can lead to cancer
• Tuberculosis
– Bacteria, mostly infect lung, but also other parts of body
– In 2004, 1.7 million deaths
Why Can DNA Be Used as Drugs?
How to stop genetic disorder using DNA drugs?
 Design a short DNA sequence that matches the sequence of mRNA
that is transcribed from the mutated gene (which causes diseases)
 The DNA drug binds to the mRNA (A-U, G-C)
 The mRNA cannot be translated to protein
 Because no disease-causing protein, disease is cured
Why Can DNA Be Used as Drugs?
How to stop diseases caused by
microorganisms using DNA drugs?
Select one or more genes that are critical
for the disease-causing bacteria or virus
Design DNAs that can stop the critical
gene expressions
The bacteria or virus dies and diseases
cured
Why Use Synthetic DNA?
Natural DNA will be digested by enzymes,
and also can cause immune response
Synthetic DNA cannot be recognized by
enzymes, so they are stable and may not
cause immune response
So, synthetic DNA can selectively block
gene expression
An Example of DNA Drug
Current Research
At a company
called ISIS
Current Status of Antisense Drugs
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Antisense drugs stop making sense?
18/12/2006 - The once hyped antisense sector of the pharmaceutical industry
has again proved a disappointment after one drug in development was refused
approval and another was scrapped.
Genta,, US, has announced that the lead compound in their antisense programme
failed to win approval from the US Food and Drug Administration (FDA). Genasense
was to be given alongside chemotherapy in the treatment of chronic lymphocytic
leukaemia (CLL).
"We are keenly disappointed by this decision," said Dr Raymond Warrell, Genta's
chief executive.
"We believe that Genasense has amply demonstrated its efficacy and safety in
patients with relapsed and refractory CLL in a carefully designed and executed
randomized clinical trial. "As we decide on next steps with this application, we will
continue working to seek worldwide approval of Genasense for patients who have
advanced cancer."
Genasense inhibits production of Bcl-2, a protein made by cancer cells that prevents
cell death. By reducing the production of Bcl-2, it was hoped that Genasense would
increase the effectiveness of other cancer treatments.
This is not the first time Genta have suffered disappointment with
Current Status of Antisense Drugs
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First Antisense Drug Reaches Review
Applied Genetics News, Sept 1, 1998
The FDA's Ophthalmic Drugs Subcommittee has voted to recommend approval of
trademarked Vitravene (fomivirsen) for the treatment of cCytomegalovirus (CMV)
retinitis in AIDS patients. The product is an antisense drug invented by Isis
Pharmaceuticals (2292 Faraday Ave., Carlsbad, CA 92007; Tel: 619/931-9200, Fax:
619/931-9639) and to be marketed by Ciba Vision Corp., the eye care unit of
Novartis (59 Rte. 10, E. Hanover, NJ).
Vitravene is the first antisense drug to be filed for commercial marketing review. It is
an inhibitor of CMV replication, the virus that causes retinitis. CMV retinitis is a
degenerative opportunistic infection that affects people with AIDS and results in
blindness. The new drug application (NDA) for Vitravene was filed by Isis on April 9,
1998, and is being reviewed under the agency's priority review process. According to
Daniel Kisner, president of Isis, the rising failure rate of protease inhibitor-based
combination AIDS therapy due to resistance, toxicity, and non-compliance signals a
potential resurgence in CMV retinitis and other opportunistic infections.
Vitravene is administered locally into the eye by intravitreal injection. The most
frequently observed ocular side effects were transient--increased intraocular
pressure and generally mild to moderate, reversible intraocular inflammation. The
retinal detachment rate overall in the Phase III program was considerably lower than
the incidence of retinal detachment commonly observed in patients with CMV
retinitis. There was no evidence of any systemic toxicity from the drug.
Current Status of Antisense Drugs
2007
Current Status of Antisense Drugs
Potency---Stability, Affinity
Delivery
Toxicity---Selectivity
Only two drugs are proved
by FDA
Exercise
Design a DNA drug that may cure sickle-cell anemia
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Genetic disease that can cause death (affects 0.4% African Americans, die at ~40s)
Inherited
Hemoglobin is a protein that carries oxygen from lung to cells, it is in red blood cells
in the blood
In sickle-cell Hemoglobin, Glu (negatively charged) is mutated to Val (hydrophobic)
Val binds to hydrophobic pocket of another deoxygenated Hb S, form dimers and
then polymeric fibers
The polymerization cause sickled red blood cells
Sickle cells cannot pass small capillaries of circulation system
Because of insufficient oxygen supply, many organs such as bone and kidney can be
damaged; short breath, paleness, pain, fatigue
The amino acid change in sickle-cell Hemoglobin is resulted from the mutation of the
Hemoglobin gene (following are antisense strands, templates for mRNA synthesis):
– Normal hemoglobin gene, ……CTG ACT CCT GAG (codes Glu) GAG AAG TCT……
– Sickle-cell hemoglobin gene, ……CTG ACT CCT GAG (codes Val) GAG AAG TCT……
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What is the sequence of DNA drug for blocking the expression of the mutated gene?
What might be the problem after blocking the mutated gene?
Exercise
Design a DNA drug that may cure cancer
 Many cancer cells over express Bcl 2 protein, this protein prevents cell
death
 Block the synthesis of the protein will increase the chance of killing
cancer cells by chemotherapy
 The Bcl-2 gene is: ……TGT TCT CCC GGC, TTG, CGC, CAT….
 What should be the sequence of the antisense DNA?
Antisense
Sense strand, non-coding strand
DNA
Antisense strand, coding
strand, template for mRNA
synthesis
mRNA
Sense sequence
DNA drug
Antisense sequence