Biomedical Engineering Reference
In-Depth Information
Treatment of mice with an adeno-associated viral vector expressing
shRNA resulted in fatality by oversaturation of the host's RNAi
machinery [45]. Exportin-5, the enzyme responsible for mediating
the export of both pre-miRNAs and shRNAs out of the nucleus [128],
was determined to be the limiting factor. After this report, several
strategies of shRNA expression were developed to address this
concern. Use of a tissue-specific vector expressing shRNA from the
liver-derived polymerase (Pol) II promoter reduced shRNA levels
and restricted shRNA expression and thereby toxic eff ects, if any, to
liver hepatocytes. Remarkably, Pol II expression of shRNA persisted
for more than 1 year after injection without any evidence of liver
damage [44]. More recently, a tRNA Pol III promoter was used to
express shRNAs as pre-miRs. When generated this way the shRNAs
get processed by alternative mechanisms that do not require the
conventional microprocessor complex and hence do not disrupt
the endogenous miRNA pathway. Thus, anti-HBV shRNA expressed
from a tRNA Lys3 promoter eff ectively inhibited HBV in vivo with
minimal toxicity [32]. Additionally, reducing the dosage by using
Pol II promoters instead of a U6 promoter for expression as well as
shortening the length of the expressed shRNA to a maximum of 19
nucleotides significantly reduces toxicity [45].
A gene therapeutic approach that provides stable shRNA
expression is considered as the ideal option for treating a
chronic infection such as HBV. However, synthetic siRNAs
modified to incorporate 2-fluoro, 2- O -methyl, and 2-deoxysugars,
phosphorothioate linkages, and terminus capping chemistries
displayed extended serum stability of up to 3 days, abrogated
immunostimulation through innate immune recognition pathways
and were at least 1.5 log 10 times more eff ective than unmodified
siRNAs in reducing HBsAg and HBcAg in a HBV vector-based murine
model [91, 92]. This approach minimizes the risk of oversaturating
the exportin-dependent processing pathway. However, the
requirement of three daily doses of 30 mg/kg for several days makes it
prohibitively expensive for HBV therapy. Subsequently the same group
of investigators developed a novel lipid bilayer containing a mixture
of cationic and fusogenic lipids coated with diff usible polyethylene
glycol. These 120 nanometer wide biopolymers labeled SNALPs, for
Stable-Nucleic-Acid-Lipid-Particles, shielded siRNAs from serum
nucleases and allowed cellular endosomal uptake and subsequent
cytoplasmic release of siRNAs [91, 92]. Using the SNALP approach, the
 
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