Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 191

3 Methods

3.1 Third-Generation
HIV-1-Based Lentiviral
Vectors

Over the years, the biosafety of LVs has been greatly improved.
The third generation of self-inactivating (SIN) LVs is currently in
use ([ 49 , 50 ]; see Chap. 2 on lentiviral vectors). We use tat-
dependent, SIN LVs split into four plasmids [ 51 ]. This system
contains a deletion in the U3 region of the LTR (Fig. 2 ) to reduce
the risks of recombination and of the formation of replicative LVs
and to decrease interference between the LTR enhancer and the
internal promoter driving transgene expression.

3.2 Pseudotyping
with Various
Envelopes

The fi rst option for modifying the tropism of LVs is to exchange
the envelope glycoprotein (Env), a process known as “pseudotyp-
ing” (Fig. 1 ). By replacing the original Env protein with other viral
glycoproteins, the host range of the vector can be altered and its
transduction effi ciency in specifi c cell types increased. Glycoproteins
homologous to the VSV-G protein have been proposed for CNS
applications. Various G proteins from the rabies (RV-G) and

5'-LTR 3'-LTR

U3 R U5 psi RRE cPPT PGK Transgene WPRE miRT R U5 Transfer plasmid

Δ U3

GAG RRE

Packaging plasmid

CMV poly A+

PRO POL TAT

REV poly A+

RSV REV viral protein plasmid

Envelope plasmid

CMV VSV-G poly A+ or CMV MOK-G poly A+

293T

Fig. 2 Schematic diagram of the third-generation packaging system. Four plasmids encode the genetic materi-
als of the lentiviral vector (1) the transfer plasmid contains the transgene of interest; (2) the packaging plasmid
contains the protein capsid, protease, integrase, and reverse transcriptase; (3) a plasmid expressing the rev
protein; and (4) the envelope plasmid expressing the VSV or MOK glycoprotein. Cotransfected cells produce
lentiviral vectors with a genome consisting exclusively of the sequences present in the transfer plasmid