Biomedical Engineering Reference
In-Depth Information
transgenes controlled by this promoter to be
switched on and off (Lee
et al
. 1981, Scheidereit
et al
.
1983). Fourthly, some retroviruses, such as ampho-
tropic strains of murine leukaemia virus (MLV),
have a broad host range, allowing the transduction
of many cell types. Finally, retroviruses make effici-
ent and convenient vectors for gene transfer because
the genome is small enough for DNA copies to be
manipulated
in vitro
in plasmid cloning vectors, the
vectors can be propagated to high titres (up to 10
8
pfu/ml) and the efficiency of infection
in vitro
can
approach 100%.
The major disadvantage of oncoretroviral vectors
is that they only productively infect dividing cells,
which limits their use for gene-therapy applications
(Miller
et al
. 1990, Roe
et al
. 1993). However, lenti-
viruses, such as human immunodeficiency virus
(HIV), are more complex retroviruses that have
the ability to infect non-dividing cells (Lewis &
Emmerman 1994). These were initially developed
as vectors for the stable transduction of cells display-
ing CD4 (Poznansky
et al
. 1991, Shimada
et al
.
1991, Buchschacher & Panganiban 1992, Parolin
et al
. 1994), but recent advances in lentiviral vector
design provide improved safety and allow the trans-
duction of multiple cell types (Naldini
et al
. 1996;
reviewed by Naldini 1998).
Before discussing the development of retroviral
vectors, it is necessary to briefly describe the genome
structure and the molecular biology of the infection
cycle (for a comprehensive account, see Weiss
et al
.
1985). A typical retroviral genome map is shown
in Fig. 10.8. The infection cycle begins when the
viral envelope interacts with the host cell's plasma
membrane, delivering the particle into the cell. The
capsid contains two copies of the RNA genome, as
well as reverse transcriptase/integrase. Thus, imme-
diately after infection, the RNA genome is reverse-
transcribed to produce a cDNA copy. This is a
complex process involving two template jumps,
with the result that the terminal regions of the RNA
genome are duplicated in the DNA as long terminal
repeats (LTRs). The DNA intermediate then integ-
rates into the genome at an essentially random
site (there may be some preference for actively tran-
scribed regions). The integrated provirus has three
genes (
gag
,
pol
and
env
). The
gag
gene encodes a viral
structural protein,
pol
encodes the reverse transcrip-
tase and integrase and the
env
gene encodes viral
envelope proteins. Viral genomic RNA is synthesized
by transcription from a single promoter located in
the left LTR and ends at a polyadenylation site in the
right LTR. Thus, the full-length genomic RNA is
shorter than the integrated DNA copy and lacks the
duplicated LTR structure. The genomic RNA is
capped and polyadenylated, allowing the
gag
gene to
be translated (the
pol
gene is also translated by read-
through, producing a Gag-Pol fusion protein, which
is later processed into several distinct polypeptides).
Some of the full-length RNA also undergoes splicing,
eliminating the
gag
and
pol
genes and allowing the
downstream
env
gene to be translated. Two copies of
the full-length RNA genome are incorporated into
each capsid, which requires a specific
cis
-acting
packaging site termed
. The reverse transcriptase/
integrase is also packaged.
ψ
Strategies for vector construction
Most retroviral vectors are replication-defective,
because removal of the viral genes provides the
maximum capacity for foreign DNA (about 8 kb).
LTR
LTR
U5
ψ
gag
pol
env
U3 R U5
U3
R
PB(-)
PB(+)
ψ
gag
pol
env
U3 R
polyA
RU5
Fig. 10.8
Generic map of an oncoretrovirus genome. Upper figure shows the structure of an integrated provirus, with long
terminal repeats (LTRs) comprising three regions U3, R and U5, enclosing the three open reading frames
gag
,
pol
and
env
. Lower
figure shows the structure of a packaged RNA genome, which lacks the LTR structure and possesses a poly(A) tail. PB represents
primer binding sites in the viral replication cycle, and
ψ
is the packaging signal. The small circles represent splice sites.
