Biomedical Engineering Reference
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RNA control, (d) detecting site-specific cleavage products through
5
-RACE, and (e) confirming that knockdown is dose responsive.
While investigating PCKS9 silencing, all of these controls were used.
The levels of PCKS9 mRNA, PCKS9 protein, and serum cholesterol
were also measured as a function of time. 98N
-5 reduced PCKS9
and/or serum cholesterol in all four animal models using modified
siRNA that did not elicit a cytokine response. In wild-type mice,
PCKS9 mRNA was reduced for 28 days after intravenous injections
of 7.5 mg/kg, while PCKS9 protein levels were reduced to the
limit of detection for three days in transgenic mice after injecting
5 mg/kg siRNA. Serum cholesterol levels remained lowered for
30 days in rats. Target mRNA cleavage was confirmed by 5
12
-RACE.
Delivery of off-target siRNA did not reduce target gene expression,
while PCSK9 was silenced in a dose-dependent fashion.
We were curious to examine whether this delivery system
could be used to influence not only host pathology but also the
infectious disease. Malaria is a multi-stage infection that begins
when a mosquito deposits sporozites under the skin of a human
host. Sporozites migrate to hepatocytes, attracted by their highly
sulfated heparan sulfate proteoglycans.
32
After maturing in the
liver, sporozites rupture the hepatocytes, enter the bloodstream,
and infect red blood cells. Malaria continues to devastate parts of
the developing world, particularly in Africa and Asia. Disturbingly, it
was recently reported that mosquitoes may be acquiring resistance
to Artemisinin,
33
the most potent anti-malarial drug produced to
date. If true, it is projected that the 250 million cases and one million
deaths each year caused by malaria will rise dramatically.
To this end, we silenced a hepatic gene that was hypothesized
to influence malaria infection, Heme Oxygenase-1 (HO-1).
33
34
HO-1,
which plays a critical role in the metabolism of heme, is normally
expressed at low levels. Expression increases in response to several
stimuli, including heavy metals, hypoxia, heat shock, and sporozite
infection.
34
HO-1 upregulation is known to inhibit cerebral malaria,
a dangerous stage of the disease that comes after sporozites relocate
from hepatocytes to red blood cells.
35
However, the role of HO-1 in
the liver (pre-erythrocytic stage of malaria) remained unclear. To
study how HO-1 expression influenced susceptibility to sporozite
liver infection, Plasmodium levels were measured in mice expressing
HO-1 (Hmox
+/+
) and in mice genetically modified not to express HO-1
-/-
(Hmox
). In contrast to the anti-malarial role it plays in the
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