Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 192

Mokola (MOK-G) lyssaviruses have been proposed for this func-
tion [ 28 , 31 , 37 , 52 - 57 ]. Following the injection of RV-G-
pseudotyped LV (EIAV, RabERA strain) into rat brain, 80 % of the
cells transduced were neurons and 20 % were glial cells [ 58 ].
Retrograde transport of the viral particles was observed in project-
ing areas [ 58 ] ( see Note 1 ). However, -Gal staining was weaker

with the RV-G vector than with the VSV-G vector, a limitation of
lyssavirus pseudotyping that has also been reported for the MOK-G
envelope (viral vector production and transduction effi ciency lower
than for the wild-type envelope, by a factor of 2-6; [ 28 , 37 ]).
Following the intrastriatal injection of MOK-G (MOKZIM strain)-
pseudotyped LV (HIV-1) into rats, 71 % of the transduced cells
were neurons [ 28 ]. However, the proportions of neurons trans-
duced may differ slightly between brain areas and species. In mouse
striatum infected with a MOK-G-pseudotyped LV, the percentage
of

β

-positive cells (glial cells) was 68 %,
whereas only 9 % of cells were positive following transduction with
VSV-G-LV [ 37 ]. Species differences (rats vs. mice), the strain of
MOK-G used and variability of the lentiviral vector (EIAV vs.
HIV-1) may account for these discrepancies.
As an alternative LV pseudotyping strategy, the use of enve-
lopes from neurotropic viruses has been proposed. In evaluations
of the tropism of LVs pseudotyped with glycoproteins from Ross
River Virus (RRV, an alphavirus), which replicates in neurons and
glial cells [ 54 , 59 ], 56 % of

β -galactosidase S100 β

-galactosidase-positive cells were
astrocytes and 27 % were oligodendrocytes [ 59 ]. Finally, the lym-
phocytic choriomeningitis virus (LCMV, an arenavirus), which is
taken up by cells after binding to

β

-dystroglycan, shows a strong
preference for astrocytes in the neonatal rat brain [ 60 , 61 ], despite
the presence of

α

α -dystroglycan on both neurons and astrocytes.

The lentiviral transfer plasmid is a complex DNA molecule
containing the various elements required for transgene expression
(Fig. 3 ). We use tat-dependent SIN transfer vectors [ 62 ] containing
a central polypurine tract (cPPT; [ 63 ]). The expression cassette
consists of a promoter (ubiquitous or specifi c to a subpopulation of
cells), a transgene ( see Note 2 ) and, in most cases, the woodchuck
posttranscriptional regulatory element (WPRE), which increases
transgene expression [ 64 ]. Finally, an miR-target (miRT) can be
inserted after the WPRE sequence to restrict transgene expression.

3.3 Design
of Lentiviral Transfer
Plasmids Containing
the Transgene
Expression Cassette

One strategy for overcoming the lack of specifi city of LVs is based
on the use of tissue-specifi c promoters to modulate the transcription
of transgenes. However, cellular promoters often have weak tran-
scriptional activity, limiting their potential use in LVs. Furthermore,
only a small number of promoters have been characterized. However,
this may change in the future, as large consortia are currently mak-
ing use of transcriptome databases for astrocytes, neurons, and

3.4 Tissue-Specifi c
Promoters