01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

designed by immunization with portions or the entirety of
CCR-5 and/or gp120 to generate appropriate antibodies
(D’Souza and Harden, 1996). Inactivation of the CCR-5
co-receptor to mimic the natural resistance of the CCR-5defective
individuals, in cultured lymphocytes, rendered
them viable and resistant to macrophage-tropic HIV-1
infection (Yang et at, 1997).
B. Therapeutic strategies against HIV
A number of therapy strategies emerged soon after
identification of HIV as the etiologic agent of AIDS.
Virtually every stage in the viral life cycle and every viral
gene product is a potential target. Albeit major efforts for
the combat of HIV have focused on the development of
antiviral drugs and preventive vaccines, a number of
studies have been aimed at eliminating HIV with gene
therapy. The intracellular immunization approach
(Baltimore, 1988) has prompted the advent of molecular
tactics for inhibiting replication and infection of HIV
(Trono et al, 1989; Malim et al, 1989). Four main targets
have been defined in HIV therapeutics: (i) viral RNAs
using ribozymes and antisense RNAs; (ii) viral proteins
using RNA decoys, trans-dominant viral proteins,
intracellular single-chain antibodies, and soluble CD4; (iii)
infected cells aimed at eliminating those with transfer and
expression of suicide genes; and (iv) the immune system
by in vivo immunization (see Corbeau et al, 1996). Such
gene therapeutic approaches can be combined with potent
antiretroviral drugs especially the potent reverse
transcriptase and protease inhibitors (Junker et al, 1997).
The elucidation of the chemokine receptor mechanisms
for entry of HIV-1 into T cells and macrophages provides
new tactics for intervention at the level of interaction of
gp120 with CCR-5. Inactivation of the CCR-5 gene might
be achieved (i) with triplex oligonucleotide technologies
once critical transcription factor binding sites in the
regulatory region of the gene have been determined; (ii)
with saturation of the blood of infected individuals with
ligands, selected from peptide libraries, that block the
extracellular domains of the CCR-5 receptor and preclude
interaction with gp120; (iii) with antagonists of RANTES
(see above) that lack chemotactic activity but can block
HIV infections (Arenzana-Seisdedos et al, 1996).
Specificity for the ablation of HIV Tat-expressing cells
has been achieved through the use of the promoter element
from the long terminal repeat (LTR) of HIV (Venkatesh et
al, 1990; Caruso et al, 1992).
C. Gene therapy against HIV in cell
culture
Strategies for HIV gene therapy include the
inactivation of the CCR-5 coreceptor which is
accomplished by targeting a modified CC-chemokine to
the endoplasmic reticulum to block the surface expression
Gene Therapy and Molecular Biology Vol 1, page 95
95
of newly synthesized CCR-5 (Yang et al, 1997). A
different gene therapy strategy proposed is targeting Tat,
an early regulatory protein that is critical for viral gene
expression and replication and which transactivates the
LTR of HIV-1 via its binding to the transactivation
response element (TAR); Tat also superactivates the HIV-
1 promoter via activation of NF-κB in a pathway
involving protein kinase C and TNF-α; combinations of
the NF-κB inhibitors, pentoxifylline and Go-6976, with a
stably expressed anti-Tat single-chain intracellular
antibody suppressed HIV-1 replication and LTR-driven
gene expression (Mhashilkar et al, 1997). Production of
recombinant retrovirus containing the HSV-tk gene
coupled to the HIV-2 promoter and Tat responsive region
(TAR) has been used on human and mouse cells in culture
for the specific elimination of HIV Tat-expressing cells;
since the HIV-2 promoter can sustain a considerable level
of basal expression in the absence of its activator, Tat, a
number of modifications were made to the HIV-2
promoter in order to minimize toxicity to non-infected
cells.
Retroviruses export unspliced, intron-containing RNA
to the cytoplasm of infected cells despite the fact that
intron-containing cellular RNAs cannot be exported; this
export pathway is a critical step in the HIV-1 life-cycle. In
HIV-1 this is accomplished through an interaction
between the viral regulatory Rev protein and the Rev
response element (RRE) RNA. In the absence of Rev,
these intron-containing HIV-1 RNAs are retained in the
nucleus. The nuclear export sequence (NES)
LQLPPLERLTL has been identified on Rev that is
responsible for its export to the cytoplasm (see Boulikas,
1998, this volume for more details). Targeting of Rev has
provided a framework for novel interventions to reduce
virus production in the infected host. Because disruption
of either Rev or the RRE will completely inhibit HIV-1
replication, an anti-HIV-1 intracellular immunization
strategy was developed based on RRE region-specific
hammerhead ribozymes and on the intracellular expression
of an anti-HIV-1 Rev single chain variable fragment (Sfv),
which specifically targets the Rev activation domain. This
combination resulted in a potent inhibition of HIV-1
replication in cell culture that holds promise as a future
therapeutic regimen (Duan et al, 1997). To disable Rev
function, primary T cells or macrophages were transduced
with a recombinant AAV carrying an anti-Rev singlechain
immunoglobulin (SFv) gene or an RRE decoy gene
or with combinations of the two genes to disrupt the
interaction between Rev and the RRE; when the
transfected cells were then challenged by either clinical or
laboratory HIV-1 isolates, this genetic antiviral strategy
effectively inhibited infection (Inouye et al, 1997).
A synergic effect of anti-Tat and anti-Rev molecules
was found when the RRE sequence was cloned 3' to a tat
transdominant negative mutant (tat22/37) gene; for this
strategy Jurkat cells were transduced with the recombinant