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Fig. 1 Designing viral vectors for miRNA regulation. Four miRT sites should be placed on the 3
UTR of the
expression cassette, preferably spaced fi ve nucleotides (nt) apart
targeted [ 37 ] ( see Note 9 ). Once the miRNAs have been chosen
for screening, go to the miRBase website ( http://www.mirbase.
org ) and browse for the specifi c specie and miRNA. On the page
for the individual miRNA, scroll to the seed sequence (~22 nucleo-
tides) and the miRT sequence to insert in the vector will be the
reverse complementary DNA of that particular miRNA seed. The
vector should be designed as described in Fig. 1 ( see Note 10 ).
The miRT sequences should be placed in the 3
UTR after the gene
or any postregulatory element present. Four copies of the miRT
should be spaced 4-5 nucleotides apart. Inserting more copies of
the miRT does not bring any additional regulatory benefi t and may
in some cases elicit a sponge effect [ 37 ]. The vector containing the
PD relevant promoter and the miRT sequences may be tested as
above.
3
Anticipated Results
Promoter candidate RNF25, promoter of Ring fi nger protein 25,
was chosen from a microarray study performed on striatal tissue
from parkinsonian patients. The promoter was cloned into a vector-
expressing GFP and validated in rat striatum. The effi ciency and
specifi city of the vector was validated by immunohistochemistry
(Fig. 2a ). The RNF25 promoter showed similar effi ciency (Fig. 2b )
and specifi city (Fig. 2c ) as conventionally used ubiquitous and cell-
specifi c promoters, respectively.
As a proof of principle that promoter targeting an miRNA
detargeting could be combined, we designed astrocytic-specifi c
vectors combining fi brillary acidic promoter (GFAP) and miRNA-
based regulation using miRNA 124. The GFAP promoter can
readily direct expression of lentiviral vectors to astrocytes and is
upregulated during astrogliosis [ 38 ]. Consequently, the GFAP
promoter can further shift tropism to astrocytes that in combina-
tion with mir124T can provide a stringent and autoregulated
astrocytic expression. Delivery of GFAP/mir124T lentiviral vec-
tors expressing GFP to the brain resulted in a robust astrocytic
expression of the vectors (Fig. 3a ). These vectors exhibited an
upregulation in brains lesioned with ibotenic acid that was
comparable to what was observed with the GFAP promoter alone
 
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