Lentiviral Vectors as Research Tools in Neurobiology: Design and Production - Viral Vector Approaches in Neurobiology and Brain Diseases - page 3

use of LVs as therapeutic or investigation tool in the Central
Nervous System (CNS) [ 12 , 13 ], whereas other retroviral vectors
are still used for gene therapy, especially of tumors.
From the first generation of LVs, major efforts have been put
to reduce the risk of vector mobilization and recombination and to
improve biosafety. The first turning point was reached with the
development of third-generation conditional packaging system [ 14 ],
were the main transactivator of HIV-1, Tat, were removed and its
requirement offset by a strong constitutive promoter. The following
development of Self-Inactivating (SIN) vectors, certainly contrib-
uted to reach a satisfying level of biosafety; these vectors, in fact,
contain a deletion in the 3′ Untranslated Region that after the
retrotranscription process contributes to the transcriptional inacti-
vation of the upstream Long Terminal Repeat (LTR) [ 15 , 16 ].
Production, concentration, and titration of LVs are now
standard procedures and several state-of-the-art LV-based expres-
sion system are now available [ 17 ].

2 Design of LVs

LVs are very versatile tools, there are a lot of different characteristics
that can be changed and exploited, depending on the intended
use. This section describes the characteristic of the most useful
applications.

The complexity of the genetic material delivered with the vector has
the only limitation of the size of the lentiviral genome. The simplest
configuration has only one gene expressed by a constitutive
promoter. For example, a commonly used and versatile LV is
pCCLsin.cPPT.PGK.eGFP.WPRE [ 18 ]; this construct bear a single
ubiquitous promoter (i.e., human Phosphoglycerate Kinase,
hPGK), which allows the expression of the cDNA (i.e., enhanced
Green Fluorescent Protein, eGFP) in virtually all cell types. It is
also possible to delivery more complex genetic structures, allowing
the expression of multiple genes in the same vector. Three strate-
gies are typically employed: promoter duplication [ 19 ], bidirec-
tional promoters [ 20 ], or the construction of a polycistronic
transcript driven by a unique promoter. In the latter case, the mul-
tiple transcripts are typically linked by an internal ribosome entry site
(IRES) that allows ribosomes to begin transcription at multiple sites
of the mRNA [ 21 ]. This approach is very popular, but often the
expression of the IRES-driven gene is not comparable (weaker) to
the expression of the other gene of the construct. Another approach
that is worth to mention is the introduction of the self-cleaving 2A
peptide [ 22 ]. In this case the two proteins are first translated
together as a fusion polypeptide and only after protein translation
the self-cleaving sequence allows theirs separation.

2.1 Delivery
of Genetic Material