Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 195

0 20 40 60 80 100

Transduced cells (%)

100 80 60 40 20 0

Neuronal
Sub-population

Astrocytes

Neurons

Ubiquitous promoter
(CMV / PGK)

CMV /
PGK

CMV / PGK
+ miR124T

Specific
neuronal
promoter

Pan-neuronal
promoter

Astrocytic
promoter
± miR124T

VSV-G envelope MOK-G envelope

Fig. 5 Schematic diagram of the strategies for targeting specifi c cell types. VSV-G-pseudotyped LVs have a high
neuronal tropism, whereas MOK-G-pseudotyped LVs more frequently target astrocytes. The addition of the
miR124T sequence inhibits transgene expression in neurons, leading to an astrocytic pattern of transgene expres-
sion. Combination with a tissue-specifi c promoter should not change LV tropism but should improve biosafety

factors: the developmental stage considered, the area of the brain
studied, and the subpopulation of cells targeted [ 87 - 90 ]. It is pos-
sible to integrate several miRTs to achieve a synergistic effect ([ 83 ];
see Note 6 ). The miRT sequence may be a natural target of the
endogenous miRNA, which is partly complementary to the miRT
[ 37 ], or a sequence fully homologous to the miR [ 83 ]. miRNA-
mediated gene silencing is based on translational or
posttranscriptional mechanisms [ 91 - 93 ].

3.6 LV Production

A detailed description of LV production is available in a previous
issue of this topic collection and other recent protocols [ 62 , 94 ,
95 ]. In this chapter, we describe the production of LVs pseudo-
typed with the VSV-G and MOK-G envelopes ( see Note 7 ). We
provide information for the scaling up of production and the use
of LVs in preclinical experiments [ 95 - 98 ].
LVs are produced over a period of 4 days (Fig. 6 ). In most
cases, a batch of lentiviral vectors is produced by the transient
cotransfection of 293T cells with various plasmids (Fig. 2 ). Another
way of producing LVs involves the use of a packaging cell line that
stably expresses the packaging, envelope, and rev genes. A single
transfection with the transfer plasmid is suffi cient to produce
recombinant vector. The disadvantage of this approach is that
yields are low and the stable production of VSV-G protein in mam-
malian cells has been shown to be toxic [ 99 , 100 ]. Inducible pack-
aging cell lines have recently been developed for large-scale
production [ 101 - 105 ].