Download The use of DNA Microarrys to determine the effect of histone

The use of DNA microarrays to determine the effect of histone
deacetylyase inhibitors on host response to adenovirus gene
therapy vectors
Mike Waters
Davidson College/Virginia Commonwealth University
Mentors: A. Malcolm Campbell, Paul Fawcett
Introduction
A new discovery in the field of gene therapeutics resonates throughout the world
of clinical medicine. Gene therapy regimens are currently under investigation as possible
treatments of cancer9, 8, HIV/AIDS8, 10, cardiovascular diseases4, infectious diseases2, and
neurodegenerative disorders1. Clinical progress however has been slowed due to
problems regarding the vehicles of gene transduction into the patient’s genome. Today,
three major transgene vectors are used in gene therapy studies: retroviral vectors,
adenoviral vectors, and non-viral vectors
(fig 1)8. Of the three, adenoviral vectors
pose the most promise from a
pharmacological standpoint because of their
efficiency of gene transduction, ability to
transduce genes regardless of mitotic cell
status, tissue specificity, and potential
duration of transgene expression6, 7, 8, 9.
However, adenoviral vectors are
problematic pharmacological agents due to
the degree in which they elicit an innate
immune response in their host3, 5, 7, 8. A
robust innate immune response poses 2
major problems for gene therapy: 1) Strong
cytokine activation can result in patient
tissue damage through the stimulation of
NK cells and in extreme cases lead to a
“cytokine storm3, 7.” 2) A robust innate
immune response leads to instability in
Fig 1. Depicts the vectors used in gene therapy
transgene expression 3, 7.
clinical trials in 2003.
Recent literature suggests that a
transient suppression of the innate
Thomas CE. 2003. Nature Reviews Genetics
immune signaling cascade is possible
4:346-358.
through the deacetylation inhibition of the
positive regulatory domain of interferon
regulatory factor 3 (IRF3)7. In mammalian cells the role of deacetylation of immune
response signaling proteins is carried out by a family of histone deacetylase (HDAC)
enzymes. Trichostatin A is an HDAC inhibitor and the agent we will use to attempt to
transiently suppress host innate immune response in order to obtain stable transgene
expression while the host is “infected” with an adenovirus gene therapy vector.
1 hr
6 hr
72 hr
4 weeks
A D V
A D V
A D V
A D V
Igtp
Igtp
Igtp
Stat1
Ifrg15
Parp9
I
MyD88
Ifi203
Ifi47
II
Tlr2
Csf1
Cxcl10
H3f3b
Litaf
Lcn2
Cxcl1
Mt1
III
Mt1
Mt2
Mt2
Vcam1
Mt4
Gstp1
C1qb
C1qa
C1qb
Cfp
IV
V
Methods
The effect of Trichostatin A on the
repression of proteins involved in the innate
immune response will be quantified using DNA
microarray data (fig 2). The mRNA for the
microarrays will be collected ex vivo, from mouse
macrophage cells. The mouse macrophage cells
produce a strong innate immune response that will
make data quantification more apparent. Before the
“infection” with replication deficient adenovirus, a
dose-response experiment will be conducted where
adenovirus concentration vs. transgene expression
will be measured. The reporter transgene expressed
in the dose-response experiment will be GFP in
order to easily detect of the percentage of cells
“infected” with adenovirus.
Possible Results
Before I can begin extracting mRNA from
mouse macrophage cells I first have to familiarize
myself with the subtleties of culturing mammalian
cells. Once I have developed the skills necessary to
work with mammalian cells and obtain mRNA
without contamination I will be able to begin
Fig 2. Microarray depicting the gene
running microarray experiments. The
expression profile of adenovirus
differences in the innate immune response gene
infected mouse liver cells. The top
expression profile of adenovirus infected mouse
two enlarged boxes show an up
macrophages treated with trichostatin A and
regulation of proteins involved in the
that of the control mouse macrophages could
innate immune response at 1 hour and vary from complete repression of innate
6 hours.
immune response to little effect on the innate
immune response signaling cascade, with many
McCaffery & Fawcett. 2008.
possible permutations in between. I am
Molecular Therapy 16(5):931-941
confident that by the end of the summer I will
have produced clean microarray data for
analysis, and have a substantial amount of
mRNA frozen down for analysis at Davidson
College for the academic year.
Hba1-a1
Hba1-a1
Fdx1
Hbb-b1
Hba1-a1
Hba1-a1
References
1. Baekelandt V., DeStrooper B., Nuttin B., Debyser Z. 2000. Gene therapeutic strategies
for neurodegenerative diseases. Current Opinion in Molecular Therapeutics
2:540-554.
2. Bunnell B & Morgan RA. 1998. Gene therapy for infectious deceases. Clinical
Microbiology Reviews 11:42-56.
3. Decker T., Muller Mathias., Stockinger Silvia. 2005. The yin and yang of type I
interferon activity in bacterial infection. Nature Reviews Immunology 5:675-687.
4. Isner, J.M. Myocardial gene therapy. 2002. Nature 415, 234-239.
5. Marshall E. 1999. Clinical Trials- Gene therapy death prompts review of adenovirus
vector. Science 286:2244-2245.
6. McCaffrey AP., Fawcett., Nakai H., McCaffrey L., Ehrhardt A., Pham T., Pandy K.,
Xu H., Feuss S., Storm T., Kay MA. 2008. The host response to adenovirus,
helper-dependent adenovirus, and adeno-associated virus in mouse liver.
Molecular Therapy 16(5):931-941.
7. Nusionzon I & Horvath CM. 2006. Positive and negative regulation of the innate
antiviral response and beta interferon gene expression by deacetylation. Molecular
and Cellular Biology 26(8):3106-3113.
8. Thomas CE., Ehrhardt A., Kay MA. 2003. Progress and problems with the use of viral
vectors for gene therapy. Nature Reviews Genetics 4:346-358.
9. Tseng JF & Mulligan RC. 2002. Gene therapy for pancreatic cancer. Surgical
Oncology Clinics of North America 11(3):537-569.
10. Wolkowicz R., Nolan GP. 2005. Gene therapy progress and prospects: Novel gene
therapy approaches for AIDS. Gene Therapy 12: 467-476.
Works Consulted
Nusinzon I., Horvath CM. 2005. Unexpected roles for deacetylation in interferon- and
cytokine – induced transcription. Journal of Interferon and Cytokine Research
25:745-748.
Akira Shizuo. 2003. Toll-like Receptor Signaling. The Journal of Biological Chemistry
278(40):38015-38108.