Gene Therapy for Parkinson’s Disease - Viral Vector Approaches in Neurobiology and Brain Diseases - page 176

resulting in gene silencing. On the other hand, if the viral vector
transduces a cell that lacks that specifi c miRNA, the mRNA will be
translated and the gene will be expressed. Therefore, the presence
of miRNA target (miRT) in the messenger RNA will detarget gene
expression in cells expressing that specifi c miRNA. This approach
has successfully directed gene expression of viral vectors in a num-
ber of systems including the central nervous system.
As a proof of principle, Colin and colleagues showed that it
was possible to redirect expression of lentiviral vectors in the brain,
more specifi cally from neurons to astrocytes by addition of the tar-
get for miRNA 124a (mir124T) [ 30 ]. This miRNA is highly
expressed in mature neurons; therefore, delivery of transduction of
lentiviral vectors containing copies of mir124T should result in
degradation of vector messenger RNA in neurons [ 31 , 32 ]. Indeed,
addition of mir124T to lentiviral vectors shifted the tropism of the
vectors from a neuronal to an astrocytic tropism. The simplicity
and versatility of miRNA regulated targeting expression shows that
it is a powerful to modulate viral tropism in the brain [ 31 ]. In addi-
tion, several miRTs for different miRNA can be combined to fur-
ther direct vector tropism in vivo and thus detarget several cell
types simultaneously [ 29 ].

2 Methods

2.1 Selection of
Promoter Candidates
and Promoter Parts

Analysis of microarray data from patients with PD has shown that
many genes are highly expressed in parkinsonian brain compared
to healthy brain [ 33 ]. A list of genes that are highly expressed in
PD can be constructed using this type of data and potential pro-
moter candidates, relevant for PD, can be selected from this list.
A literature study should then be performed on the genes of all
potential promoter candidates. This step is essential to exclude any
candidates associated with for example blood, an unwanted CNS
cell type or, if the promoter is to be used in cell-specifi c vectors, a
wide variety of cell types. The literature study may also help to
identify any known regulatory elements in the promoter of the
gene that should be included or excluded in the promoter con-
struct. The promoters of some genes, such as the promoter of the
microtubule-associated protein 1A (MAP1a), contain elements
that have been reported to confer an exclusive expression in one
cell type [ 34 ]. An appropriate promoter construct including, or
excluding, as many of these elements as possible can then be cho-
sen for all of the selected promoter candidates ( see Notes 1 and 2 ).

It is important to evaluate the effi ciency and specifi city of the cho-
sen promoter candidates to ensure the vector has suitable charac-
teristics for the planned PD gene therapy paradigm. These

2.2 Evaluation
of Promoters In Vivo