Gene Therapy (2005) 12, 467–476
& 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00
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REVIEW
Gene therapy progress and prospects: Novel gene
therapy approaches for AIDS
R Wolkowicz1,2 and GP Nolan1,2
1
Department of Microbiology and Immunology, School of Medicine, Stanford University, Stanford, CA, USA; and
Baxter Laboratory of Genetic Pharmacology, School of Medicine, Stanford University, Stanford, CA, USA
2
Acquired immunodeficiency syndrome (AIDS), caused by
human immunodeficiency virus (HIV), kills millions worldwide
every year. Vaccines against HIV still seem a distant
promise. Pharmaceutical treatments exist, but these are
not always effective, and there is increasing prevalence of
viral strains with multidrug resistance. Highly active antiretroviral therapy (HAART) consists of inhibitors of viral
enzymes (reverse transcriptase (RT) and protease). Gene
therapy, first introduced as intracellular immunization, may
offer hopes for new treatments to be used alone, or in
conjunction with, conventional small molecule drugs. Gene
therapy approaches against HIV-1, including suicide genes,
RNA-based technology, dominant negative viral proteins,
intracellular antibodies, intrakines, and peptides, are the
subject of this review.
Gene Therapy (2005) 12, 467–476. doi:10.1038/sj.gt.3302488
Published online 10 February 2005
Keywords: HIV; vectors; RNA interference; ribozyme
In brief
Progress
Vectors based on human immunodeficiency virus
(HIV), simian immunodeficiency virus (SIV), and
feline immunodeficiency virus (FIV) have been
developed for delivery of transgenes to T cells and
hematopoietic cells.
Dominant-negative effectors against HIV-1 expressed
as peptides with antiviral effects, truncated or
mutated host or viral proteins that inhibit viral
machinery have been identified.
Intrabodies against viral or viral-related host proteins
can be delivered to successfully decrease viral
replication.
Transcription factors designed to specifically bind the
HIV-1 promoter and decrease viral transcription have
been assessed.
Combinations of RNA-based strategies comprising
ribozymes, RNA decoys, and short interfering RNA
(siRNA) to efficiently block viral replication are being
evaluated.
Cells haven been engineered to express gp41derived peptides on their surface to block HIV-1
entry.
Introduction
Human immunodeficiency virus (HIV)-1 continues to be
a global pandemic of enormous consequence to humanity. At the heart of the virus lies its replication inside cells
Correspondence: Professor G Nolan, Department of Microbiology and
Immunology, Stanford University, School of Medicine, 269 Campus Dr.,
CCSR 3205, Baxter Lab, Stanford, USA
Published online 10 February 2005
Prospects
Novel vectors based on viral chimeras and/or
specific receptor-pseudotypes will be developed.
Peptide libraries, expressed within or on the surface of
the cell, will be useful as tools for the discovery of
small molecules that interfere with the HIV-1 life cycle.
Specific targeting of infected HIV-1 cells, based on
gp120 expression, can be developed for killing of
target cells or gene delivery.
The multivesicular body (MVB) machinery and Gag
protein might be targeted to inhibit HIV-1 budding.
The species-specific human restriction factor Trim5a
(Trim5ahu) can be engineered to increase affinity for
HIV-1 capsid; binding to engineered factors will
result in efficient destruction of the viral particle.
Blocking the antiviral APOBEC3G deaminase/Vif
(the virion infectivity factor) interaction will inhibit
viral replication.
Factors that render a cell nonpermissive to infection,
such as Murr1, will be characterized and used as
targets in gene therapy regimen.
Novel secreted factors will account for CD8+ T cells
antiviral activity and be expressed in different cell
types to inhibit infectivity.
and the comparatively brief moments, it spends in the
intracellular spaces. Against this backdrop we consider a
therapeutic approach still unperfected for HIV-1 disease.
Gene therapy has shown many potential targets.
The toxicity associated with anti-HIV-1 drugs, together
with the appearance of newly resistant strains to current
drugs, drives the continued search for novel strategies to
fight HIV-1. One aim of gene therapy is to provide a cell
an intrinsic advantage that inhibits replication of the
Gene therapy approaches for AIDS
R Wolkowicz and GP Nolan
468
virus. Perhaps the most exciting information that has
become available in recent years has been a further
understanding of host processes that HIV-1 exploits to
further its own replication. Ongoing research on HIV-1 is
continuously uncovering host genes and their protein
products that appear to play a role in aspects of the viral
life cycle. In most of these cases, HIV-1 proteins depend
upon interactions with host proteins. We strongly believe
that such novel targets for gene therapy present a unique
set of opportunities for further progress against HIV-1.
While these targets themselves might not present a valid
consideration for gene therapy, gene therapy approaches
in animal models, or even in compassionate care
experiments in human, might validate them for further
consideration when new pharmaceutical approaches
become available to target.
Infection with HIV-1 (Figure 1) starts with the
recognition of, and binding to, the CD4 receptor and
coreceptor (mainly CCR5 or CXCR4) allowing fusion
with the cell membrane. Entry then proceeds with the
uncoating of the viral particle, releasing the main enzyme
reverse transcriptase (RT), the product of the pol gene. RT
generates proviral DNA from the viral genome, which is
then transported into the nucleus as part of the
preintegration complex. In the nucleus, integrase is
responsible for the insertion of the proviral DNA into
the host chromosome. The provirus can then be silent for
long periods of time until the transcriptional machinery
of the host cell activates transcription of viral genes
leading to viral replication. mRNA copies are trans-
ported into the cytosol where translation of polypeptides
and protease proceeds. The genomic RNA and structural
proteins form a complex, while protease cleaves the
polypeptides into their final active form. The viral
particle can then undergo assembly and be released
from the cell by budding or lysis. As will be assessed in
the course of this review, multiple steps in the life cycle
are potential targets for intervention. Proof-of-concept
experiments will be reviewed that have validated each of
these and their relative merit will be discussed.
We will assume for this review that gene transfer of
suitable long-term progenitor cells, such as stem cells,
will be the basis of a gene therapy approach. That,
coupled to appropriate cell-type specific promoters will
provide appropriate expression in relevant cells of
antiviral or protective genes.
Vectors based on HIV-1, SIV, and FIV have
been developed for delivery of transgenes to
T cells
Although ironic, perhaps the best means of delivery of
genes to target cells for gene therapy of HIV-1 depends
upon the use of vectors derived from HIV-1 or related
retroviruses. Retroviral and lentiviral viruses have been
the basis for development of improved vectors for gene
transfer for the last 20 years.1 HIV-1 and Moloney murine
leukemia virus (MLV)-based vectors have been pseudotyped with different envelopes including MLV-A, MLV-
Figure 1 The figure depicts the life cycle of HIV. The different steps are shown and the yellow boxes indicate those steps that are targeted by classical
approaches of therapy, including RT inhibitors (RTI) and protease inhibitors (PI). Red boxes indicate steps that have been validated in initial studies by the
gene therapy approaches addressed in this Review. For the specifics of the cellular components and viral proteins targeted for gene therapy, the reader should
refer to the body of the text. Some of the targets or factors described are shown in green circles.
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R Wolkowicz and GP Nolan
10A1, GaLV, RD114, and VSV-G, showing similar
transduction efficiency of preactivated T lymphocytes
(CD4(+) and CD8(+)). These vectors show differential
efficacy in transducing resting T lymphocytes.2 Lentiviral
vectors have been used successfully for stable transfer of
short interfering RNA (siRNA), as demonstrated by
reduction of luciferase levels in human cells,3 and
discussed below. HIV-1 based vectors have also been
shown to have activity against HIV-1 itself.4–6 Importantly, HIV-1 vectors especially have shown excellent
utility in infecting hematopoietic stem cells.7–9
Despite the appeal of certain features of HIV-1-based
vectors, non-HIV-1-based lentiviral vectors that are able
to infect nondividing cells might have a distinct
‘psychological’ advantage over HIV-1-based vectors for
human gene therapy. Patients are understandably wary
of being infected with HIV-based vector even when such
vectors are completely safe, and there is no comprehensible possibility of them causing an acquired immunodeficiency syndrome (AIDS)-like syndrome. One such
example of a non-HIV-1-based lentiviral vector is
derived from the a-pathogenic molecular virus clone
SIVagm3mc of the simian immunodeficiency virus (SIV)
infecting African green monkeys. This vector, pseudotyped with the vesicular stomatitis virus G-protein (VSVG), transduced both proliferating and growth-arrested
mammalian cell lines, including human lines. The SIVbased vector, pseudotyped with its own envelope
(homologous SIV(SIV)) was able to selectively transduce
human CD4(+)/CCR5(+) cells.10 Nonprimate retroviral
vectors, like the feline immunodeficiency virus (FIV)based vectors have also been developed.11–13
Importantly, although the virus used in delivery may
vary (HIV, SIV, or FIV), the main objective of such
approaches is the same. All these viruses are capable of
delivery of genetic materials directly into the genome of
the cell. This not only allows for the cell to which the
gene has been delivered to express the protective gene
therapy but also any daughter cells that derive from that
cell should also carry the protective vectors. Since a
hallmark of HIV-1 is to infect and debilitate the immune
system by destroying cells that are expanding to fight
infections, this is of course a key consideration in any
gene therapy approach. For the purposes of this review,
we will assume that gene therapy will be carried out by
infection of appropriate target cells (such as hematopoietic stem cells) using vectors that integrate into the
target DNA. Other targets could include neural cells that
are occasionally believed to be infected with HIV-1, and
lentiviral-based vectors have been used to transduce
neuronal cells efficiently. Although the vector for
delivery is critical, and the target cell type is as
important, a major consideration must be the viral–host
processes to be inhibited.
Dominant-negative effectors against HIV-1
inhibit viral replication
Dominant-negative effects against HIV-1 have been
aimed at interfering with a variety of different processes
in the viral life cycle. For instance, studies have been
aimed at interfering with the expression of the host cell
membrane protein CCR5 (chemokine receptor 5), since
CCR5 serves as an important coreceptor for certain
subtypes of HIV-1 in the body. Much of the expectation
of success in such an approach comes from the fact that
individuals homozygous for the 32-bp deletion mutant
D32-CCR5 are more resistant to macrophage-tropic (Mtropic) HIV-1 infection than individuals who are wild
type for the receptor. In a recent study, the D32-CCR5
construct was fused to the amino-acid motif KDEL
(KDEL can drive retention of fused proteins in the
endoplasmic reticulum). This construct significantly
decreased CCR5 membrane expression by a transdominant negative manner,14,15 partially inhibiting HIV-1 Mtropic infection in T cells expressing this construct.
Another fusion construct based on an intracellular
chemokine or intrakine approach relies on the same
D32-CCR5 fused to RANTES, showing significant interference with R5 strain HIV-1 infection and replication in
human peripheral blood lymphocytes (PBL). Thus, this
suggests that downregulation of the first phase of the
viral entry process can successfully prevent viral entry.
Post entry of the cell, the virus offers a number of
enticing targets for interfering with its replication. For
instance, the multimerization of Vif (the virion infectivity
factor) has been shown to be essential in the life cycle of
the virus.16 Vif is absolutely necessary for productive
HIV-1 infection of PBL and macrophages. It overcomes
the antiviral effects of host factors such as the C-to-U
deaminase enzyme APOBEC3G (see below) and facilitates thereby efficient infection of nonpermissive cells.
This protein appears to interact with the HIV-1 Gag
polyprotein P55Gag; this seems to drive the efficiency of
infectivity of progeny virions by modulating assembly,
budding, and/or maturation. Disrupting Vif assembly is
an attractive target for gene therapy and several
published studies16,17 are aimed at finding peptides that
in a transdominant negative manner disrupt its oligomerization. In one study, a 12-mer containing the aminoacid sequence PXP motif, proline-X-proline, identified
through phage display, inhibited Vif oligomerization in
vitro. Other peptides, derived from the Vif sequence, had
a similar ability to inhibit Vif oligomerization. In
particular, a 15-mer containing the amino-acid sequence
motif PPLP was found to be significant for inhibiting Vif
multimerization. More importantly, these peptides
strongly inhibited HIV-1 replication in nonpermissive
H9-cells,17 showing their broad utility.
Another example based on oligomerization involves
the HIV-1 Vpr protein. Vpr is capable of inducing G2 cell
cycle blockade.18,19 A mutation at residue 73 in Vpr
results in a virus with both lower transcriptional activity
and an inability of Vpr to block the cell cycle at G2. This
effect is due to heterodimerization between wild-type
and mutant proteins, thereby inactivating the complex.20
Thus, delivery of debilitated viral proteins can interfere
with formation of ‘normal’ viral complexes and could be
high on the list of therapies to be considered.
As the viral preintegration complex approaches its
‘safe harbor’ in the DNA of the cell, an interesting
transdominant negative target is the integrase machinery
itself. HIV-1 assembles host proteins to facilitate its
integration into the genomic DNA. One host protein,
termed Integrase interactor 1 (INI1), has been shown to
interact with HIV-1 integrase (IN). A fragment of INI1
spanning the minimal integrase-interaction domain
(amino acids 183–294) strongly inhibited virion production and replication. The ectopic expression of this
469
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R Wolkowicz and GP Nolan
470
fragment inhibited HIV-1 replication in a transdominant
manner by binding to integrase within the context of
Gag-Pol. This interaction possibly interferes with the
proper multimerization of Gag and Gag-Pol.21
As viral proteins are expressed post integration, there
are numerous places at which virus assembly offers a
choice for disabling virus functions. One promising
juncture could be to inhibit the processing of precursor
proteins of HIV-1 (akin to protease inhibition, but by a
different process). In such experiments, a1-antitrypsin
(AT) was delivered into PBL via a simian virus (SV-40)
vector. Processing of both gp160 and p55 were reduced.22
HIV-1 replication may be blocked or decreased by
interfering with cellular serine proteases or viral aspartyl
proteases.23 Possibly by targeting multiple aspects of
viral postprocessing one might attain a synergistic effect
with more standard orally delivered protease inhibitors.
Since the virus co-opts protein processing machinery
from the host, these targets must be carefully chosen to
minimize side effects on the host cell itself. It is likely,
however, that some targets can be found in which the
virus takes advantage of a cellular process that is not
directly essential to the biology of the cell, but is critical
to the virus. Such efficacy-toxicity issues are a standard
consideration in pharmacology with small molecules, so
this is not a new concept, just a different application of
well-understood caveats.
Intrabodies against viral or viral-related host
proteins can be delivered to successfully
decrease viral replication
The effectiveness of intracellular expression of recombinant antibodies, or intrabodies, in intracellular immunization against HIV-1 is shown in an increasing number of
studies. Targeting of Vif may have important repercussions on viral assembly and reverse transcription, as Vif
is needed for efficient infectivity, viral replication, and
completion of proviral DNA synthesis. Another way to
interfere with Vif function is to create and express a Vifspecific single-chain antibody (scFv) in the cytoplasm of
T cells.24 In this study, the intrabody was capable of
binding Vif and cells expressing the a-Vif intrabody were
more resistant to infection by several strains of HIV-1
than were cells without the intrabody. Moreover, the
viral particles produced by intrabody-expressing cells
were not able to complete reverse transcription in
subsequently infected cells. Finally, peripheral blood
mononuclear cells (PBMC) transduced with HIV-1-based
vectors carrying the a-Vif intrabody were resistant to
laboratory-adapted and primary HIV strains.24
In another example, retroviral plasmid-based delivery
was used to express intrabodies to the a-HIV-1 Gag p17
protein in the cytoplasm of primary human T cells and in
Jurkat cells (a human CD4(+) T cell line).25 When these
cells were challenged with several strains of HIV-1,
replication was strongly inhibited, although the degree
of resistance varied and was not efficient in cells that
sustained high viral replication. The inhibition was at
post-entry and reverse transcription levels, as only p24
and not proviral DNA differed between the scFvtransduced human primary T cells and control. Several
scFv specifically binding to RT have been found26 that
inhibit RT activity in vitro. Naturally, their blocking
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activity has yet to be confirmed when expressed as
intrabodies. Clearly, more genes expressing antiviral
epitopes antibody motifs or other intrabodies have yet
to be exploited, but should prove an interesting avenue
for further study. The biggest drawback of the technique
is that the virus would obviously attempt to elaborate
mutations in the epitopes recognized by the intrabody to
create ‘resistance’.
One approach around this hurdle is that intrabodies
might be efficient for AIDS gene therapy when targeted
not against viral proteins themselves, but against
proteins interacting with them. And in this manner, the
assumption is that the virus could not readily evolve a
manner to dislodge the blocking antibody from its
proposed suitor. Potential targets for such an approach
are the receptors for the virus, such as the chemokine
coreceptor CXCR4, as shown in some studies. An scFv
derived from the a-CXCR4 monoclonal antibody 12G5
was delivered into HeLa cells expressing the CD4 main
receptor. These cells were able to specifically inhibit
replication of T-tropic HIV-1 strains.27 Another recent
example employs intrabodies to target the transcription
activation machinery of the virus. ScFv were isolated
from a phage display assay for their affinity to the
human cyclinT1 subunit (hCyclinT1), one of the components of the positive transcription elongation factor
complex (P-TEFb). Tat is known to interact with
hCyclinT1 and activate elongation of RNA polymerase
II at the HIV-1 long terminal repeat (LTR). In this study,
several scFv were able to inhibit HIV-1 replication when
expressed as intrabodies in non-Hodgkin’s T-cell lymphoma-derived Supt1 cells.28 The question obviously
arises whether such targets are reasonably considered in
the context of normal primary cells. Appropriate studies
in normal hematopoietic cells in the context of a normal
immune system in higher mammals would be warranted
prior to any human studies.
Transcription factors to specifically bind the
HIV-1 promoter and decrease viral
transcription have been assessed
In an elegant set of experiments, intracellular immunity
was induced by transcriptional repression. Those experiments take advantage of the zinc-finger DNA-binding
domain for the engineering of a transcription repressor
of the HIV-1 LTR. The Kruppel-associated box (KRAB)
repressor domain from KOX1 has been fused to zincfingers. One of such hybrid proteins is ZFP HIVB-KOX,
shown to bind a region overlapping two Sp1 sites within
the 50 LTR. The protein repressed a Tat-activated 50 LTR
cellular HIV-reporter assay. HIVBA’-KOX, another protein overlapping the three Sp1 sites present in the
promoter efficiently inhibited both basal and Tat-activated transcription in unstimulated and mitogen-stimulated T cells and more importantly, strongly inhibited
HXB2 replication.29 Zinc-finger-based transcription factors were also engineered to bind the HIV-1 promoter at
sites both accessible in chromatin structure and highly
conserved among the various HIV-1 subgroups.30 Transient transfection assays showed that KRAB-HLTR3 was
able to achieve a 100-fold repression of the HIV-1
promoter. The replication of several HIV-1 strains was
reduced in T-cell lines and primary human PBMC when
Gene therapy approaches for AIDS
R Wolkowicz and GP Nolan
KRAB-HLTR3 was expressed. Repression lasted at least
18 days with no significant cytotoxicity. Stable T-cell lines
expressing KRAB-HLTR3 did not show obvious signs of
cytotoxicity.30 This factor and others could become
attractive candidates for intracellular immunization
against HIV-1.
Other factors occurring naturally have been shown to
efficiently inhibit viral transcription. Such a factor is the
RelA-associated inhibitor (RAI) recently identified as a
novel inhibitor of nuclear factor kappa B (NF-kB)
interacting with the p65 (Rel A) subunit. RAI has been
shown to inhibit the DNA binding of Sp1 and consequently the basal HIV-1 promoter activity.31 Once again,
toxic side effects on target cells have to be considered
here as there could be effects on other host genes that are
deleterious to cell function. Nevertheless, because RAI is
not constitutively expressed in susceptible HIV-1 cells,
such as CD4 T lymphocytes, macrophages, and microglial cells, its ectopic expression may render them
specifically resistant to viral replication.31
Combinations of RNA-based strategies
comprising ribozymes, RNA decoys, and
siRNA to efficiently block viral replication
are being evaluated
RNA-based gene therapy against HIV-1 includes mainly
ribozymes, RNA decoys, and siRNAs (Michienzi et al32
and references therein). Catalytic RNAs also referred to
as ribozymes, aimed at disrupting transcription regulation, can be envisioned as an efficient approach for gene
therapy. Ribozymes have been engineered to ensure the
proper colocalization of ribozyme and target and the
high affinity of binding. Catalytic antisense RNA hybrid
molecules are composed of ribozymes and stem-loops
domains. Such a molecule, composed of the antisense
stem-loop of the HIV-1 TAR sequence and a hairpin
ribozyme, cleaved TAR-containing RNAs.33 The same
approach was undertaken with hammerhead ribozymes.34 Artificial RNA substrates containing the TARRNA stem-loop were cleaved more efficiently by the
hybrid ribozyme-stem-loop antisense domain molecule
than the parental hammerhead ribozyme alone. The
enhancement is due to the interaction of both complementary stem-loop motifs as deletion of the TAR
sequence abolishes the effect. Similar results were
obtained with hammerhead- and hairpin-based catalytic
antisense RNAs targeted against the HIV-1 LTR, demonstrating that the TAR domain can be used as ribozymetargeting to TAR-containing RNAs.34
In another study, the envelope-coding region of HIV-1
RNA was targeted at several conserved sites in nine
related HIV B-subtype with a multimeric hammerhead
ribozyme referred as to Rz1-9. Rz1-9 was shown to
inhibit viral replication.35 Another multimeric ribozyme,
Rz10-14, targeted five highly conserved regions in all
HIV-1 variants. Rz1-14, a ribozyme that combines Rz1-9
and Rz10-14, was expressed in a human CD4+ T
lymphoid cell line. The cells were highly refractory to
HIV-1 infection for at least 60 days. The combined
version of the multimeric ribozyme was more effective
than either Rz1-9 or Rz10-14 alone against all major
subtypes of HIV-1.35 While ribozymes are a promising
tool for gene therapy against HIV-1 they may suffer from
the same vulnerability as siRNA, that is, the outgrown of
HIV-1-resistant mutations as well as limitations in the
specificity of the siRNA to a given individual target (and
not bystander effects).
The sequence-specific mRNA degradation triggered
by double-stranded RNA (dsRNA) in animal and plant
cells, referred as to RNA interference (RNAi), has
become a powerful tool for gene silencing. Short
duplexes or siRNAs complementary to a particular
mRNA elicit this degradation in human cells. This
approach has been applied against early and late steps
of HIV-1 replication in human cell lines and primary
lymphocytes, targeting both viral and host genes needed
for the HIV-1 life cycle. siRNAs against several regions of
the HIV-1 genome have been delivered into human cells
both as synthetic siRNA duplexes and via plasmidderived expression. HIV-1 RNA was specifically degraded to some extent, impacting HIV-1 infection.36
Although the siRNA must be optimized for more
effective degradation of the HIV target, this study proved
the value of RNAi for targeting genomic HIV-1 RNA
within the nucleoprotein reverse transcription complex.
U6-driven siRNAs targeting HIV significantly inhibited
HIV-1 gene expression when cotransfected with provirus.37 Other studies specifically targeted Rev38 or Tat
and Rev.39 Although clearly efficient for short-lived
inhibition, RNAi might be less efficient in the long run,
mainly due to point mutations at HIV-1 target sites,
leading to resistance. This indeed occurs as shown in one
study targeting the Tat protein with short hairpin RNA
(shRNA), where appearance of closely related yet
significantly different viral genomes, or quasi-species,
was accompanied with point mutation in the Tat
region.40 In a recent study siRNA targeting the Nef gene
did confer resistance to HIV-1 replication; nevertheless,
HIV-1 variants appear that are immune to interference
owing to nucleotide substitutions or deletions within the
Nef gene.41 RNAi should therefore be used in combination with other strategies in order to prevent the
appearance of resistant viruses.41
Other studies have targeted the cellular genes involved in the viral life cycle. In this case, the question is
whether silencing of host genes will have detrimental
effects on the cells. A recent example addressed this issue
with P-TEFb (composed of hCycT1 and CDK9) involved
in the transcriptional regulation of many genes other
than HIV-1 (through Tat). Both hCycT1 and CDK9 were
targeted for gene silencing in HeLa cells. Surprisingly,
silencing of P-TEFb was not lethal and inhibited Tat
transactivation and HIV-1 replication.42 In another
siRNA experiment, lysyl-tRNA synthetase (LysRS)
was chosen as a target.43,44 The enzyme is packaged
into the virion, apparently via its interaction with Gag,
and carries with it the two lysine tRNAs or tRNALys
Lys
Lys
isoacceptors tRNALys
is used as a
1,2 and tRNA3 . tRNA3
primer for reverse transcription.43 Interestingly, although
the overall cellular pool of LysRS was reduced, little or
no cellular toxicity was observed. Viruses produced from
cells transfected with siRNA showed a reduction in
packaging of tRNALys, in annealing to viral RNA, and in
viral infectivity.44 This experiment is particularly interesting as it shows that some antiviral candidates,
although cellular in nature, may be excellent targets
since a reduction in activity might be more detrimental to
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R Wolkowicz and GP Nolan
472
the virus than the cell itself. Other studies include those
aimed at targeting the CXCR4 coreceptor, decreasing
receptor expression on the cell surface and consequently
lowering infectivity by HIV-145 or the V3 loop and CD4
binding domains of Env.46 Some examples target both
viral and host genes, for example, Tat and CCR5 (the
main coreceptors in macrophage lines).47
A combination of ribozyme and RNA decoy technologies also seems promising. In one experiment, an antiCCR5 ribozyme (CCR5RZ) and nucleolar localizing TAR
RNA decoy were transduced into cultured and primary
cells via lentiviral vectors. A single vector backbone
harboring both CCR5RZ and U16TAR decoy transmitted
a high degree of resistance against HIV-1 in both primary
T cells and CD34(+)-derived monocytes.48 This strategy
has been extended to include siRNA.49 Ribozymes
against CCR5, Tat, rev, and env-coding mRNAs together
with RNA decoy targeting rev and siRNA directed
against a sequence common to rev and tat mRNAs were
transduced into CD34 hematopoietic progenitor cells via
retroviral or lentiviral vectors. The cells were differentiated into macrophages in vitro and T cells in vivo in a
murine thymopoiesis model. Both transgene-containing
macrophages and T cells were phenotypically normal
and, importantly, were highly resistant to HIV-1 infection, demonstrating the utility of RNA-based anti-HIV
constructs for gene therapy.49
Cells have been engineered to express
gp41-derived peptides on their surface
to block HIV-1 entry
The entry process is mediated by both gp120 and gp41
envelope glycoproteins, anchored to the viral surface as
trimers through the gp41 transmembrane domain. Upon
binding of gp120 to CD4 and coreceptor, the envelope
undergoes multiple conformational changes. This results
in the gp41 hydrophobic N-terminal fusion peptide
insertion into the membrane of the infected cell (Eckert
et al50 and references therein). The T20 peptide that
overlaps the C-terminal heptad repeat (HR) of gp41 is an
efficient fusion-inhibitor and is now in clinical trials.51,52
T20 blocks entry by inhibiting the conformational switch
that leads to the six-helix bundle formation comprised of
three sets of N- and C-terminal HRs. The T20 peptide,
when expressed on the surface of PM-1 cells as an
anchored-membrane construct, effectively inhibited HIV1 entry.53 A retroviral vector, the peptide itself, and a
scaffold for its presentation were optimized for maximal
expression and localization on the cell surface. The
membrane-anchored C-peptide binds to the free version
of gp41 N peptides, suggesting that the membraneanchored C peptides exert their biological effect by
binding gp41.54 Although the appearance of resistant
variants might be inevitable,55 entry inhibitors as those
that interfere with fusion carry great hope for anti-HIV
therapy.
Prospects
Gene therapy success relies on two prerequisites: firstly,
the choice of the right genetic information for transfer
and, secondly, efficient transfer of that information into
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the target cell. Obviously novel approaches for efficient
delivery are required. This will include creation of
vectors such as viral chimeras and novel pseudotyped
vectors based on envelopes that bind to specific receptors
expressed on subpopulations of HIV-1-targeted cells.
Ways to increase transfer efficiency into hematopoietic
progenitors and resting T cells must be developed. Early
tries at anti-HIV-1 therapy in neurons has failed due to
difficulty in gene transfer efficiency,23 but this might be
overcome in later attempts. Nonretroviral vectors such as
Simian foamy virus (SFV-1)-based vectors have been
used to transduce several chemically arrested cell lines
and terminally differentiated human neurons,56 and may
become a vector of choice.
As we learn more about the life cycle of HIV-1, more
protein targets will arise. One fast developing field
centers on multivesicular bodies (MVB) or the late
endosome, a vesicle biogenesis machinery crucial for
virion release. Tsg101, a component of the E vacuolar
protein sorting (Vps) machinery57,58 is necessary for
efficient budding. The p6 product of Gag interacts with
Tsg101 through its ‘late’ domain. Overexpression of the
N terminus of Tsg101 strongly inhibited virus production
by affecting budding.59 Expression of the mutant Vps4
ATPase was shown to block HIV-1 formation60 and
disrupted the interaction between AIP1 and CHMP
proteins (ESCRT-III),61,62 all parts of the MVB machinery.
It is possible to envision small molecules or peptides
targeted against the p6-Tsg101 interaction or similar
pathways. The receptors involved in targeting Gag to the
plasma membrane or to the MVB have not yet been
discovered,63 but might become novel targets. Strategies
will include expression of peptides that interfere with the
MVB machinery specifically needed for viral release or
expression of dominant mutants involved in viral
assembly for budding.
HIV-1-infected cells express gp120 on their surface.
One can envision genetic intervention with HIV-1infected cells, while noninfected cells are avoided. This
could be achieved by pseudotyping a vector with a
protein that harbors a peptide with high affinity to
gp120. Once this is accomplished, knocking out or
introducing a killer enzyme should be straightforward.
In the fast developing field of RNA interference, an
increasing number of genes could be targeted for
destruction. Lack of effectiveness of siRNA molecules
due to high mutational rates can be overcome if
degenerative sublibraries of specific target siRNA molecules are used or if targets are highly conserved. RNAbased technologies can be combined with DNA enzymes
(Dz’s) as another nucleic acid-based approach. Dz’s
against TAR have shown some protection against HIV-1
in T lymphocytes and human PBMCs.64 Nucleic acidbased technologies, including aptamers (Joshi et al65 and
references therein) will certainly join forces for AIDS
therapy.
One particular area of interest to the authors is the
development of intracellularly selected peptide libraries.66,67 In such an approach, retrovirally expressed
peptides are screened inside cells for those that inhibit
certain aspects of viral replication. Once peptides are
genetically selected, they are used to ‘discover’ the host
protein or viral process with which they interact and
interfere. As such, the peptides are both a means to a
gene therapy end as well as a means to discover host cell
Gene therapy approaches for AIDS
R Wolkowicz and GP Nolan
processes on which the virus depends. These host
proteins could then be the subject of other antiviral
therapies. In our group, we have already used such an
approach to derive dozens of peptides that interfere with
host cell processes in a variety of fascinating manners.
One class of these peptides, interestingly, seems to
converge on the inhibition of the COP9 signalosome
complex of cells. Different peptides have been independently identified that appear to bind to different
members of this multiprotein complex and result in
healthy cells, but inhibited HIV-1 replication. Interestingly, some of these peptides interfere with HIV-1
replication, but not MMULV replication. These peptides
may be delivered using gene therapy, or may be used as
the basis for development of small molecule drugs.
Clinical trials with peptides derived from gp41, like
T20,51,52 are underway. Other peptidic compounds such
as the 18-mer T22, the 14-mer T134, and the 9-mers
ALX40-4C and CGP 64222 have been identified as
CXCR4 antagonists, and as such show anti-T-tropicspecific HIV-1 strain activity.68 Peptide T, targeting the
CCR5 receptor, similarly blocks M-tropic-specific
strains.69 Other studies involved the Vif and protease
proteins of HIV-1 that have been shown to interact with
each other.70 A peptide derived from the N-terminus of
protease seems to block that interaction and consequently inhibit HIV-1 replication in restrictive cells.71 It
yet has to be seen if these peptides and similar will have
antagonistic effects when expressed inside the cell.
Obviously, there is room not only for improvement of
the inhibitory characteristics of this latter peptide but
also for the discovery of other peptides with different
targets as well.
Animals already seem to have developed innate
inhibitory activities against viral pathogens. For instance,
Trim5a from rhesus monkeys (Trim5arh) has been
identified as a species-specific restriction factor against
HIV-1 in rhesus monkeys. The capsid protein is believed
to be the target of the restriction, as Trim5arh seems to
interact with capsid and ubiquitinate it for destruction.72,73 Human Trim5a (Trim5ahu) could be engineered
to increase its affinity for the HIV-1 capsid, or rhesus
Trim5a could be expressed in human cells. Other
interesting restriction factors include the retroviral innate
resistance C-to-U deaminase APOBEC3G involved in the
late block of HIV-1 infection.74–77 Vif bears two motifs,
one that binds APOBEC3G and the second that targets it
for proteosomal degradation.78 Vif blocks APOBEC3G
incorporation into nascent virions, but once incorporated
does not seem to have any effect on it.79 Clearly, any
molecule that will induce APOBEC3G or block vif
interaction would be an attractive target for gene therapy.
The human cellular prion protein (PrPc) has been
shown to have nucleic acid binding and chaperone
properties similar to those of nucleo-capsid of HIV-1.80
Expression of PrPc in 293T cells impaired HIV-1 production at the post-transcriptional level, especially processing of Env and Vpr.81 By reducing Env incorporation
into the virion, PrPc decreased infectivity efficiency of the
produced virion. However, using prion-like molecules in
a therapy of any sort would come under considerable
scrutiny, given the notoriety of the molecule.
The search for factors that convert an infectionpermissive cell into a nonpermissive cell is clearly far
from over. In a recent study, Murr1, a protein involved in
copper regulation, was implicated in infectivity. HIV-1
efficiently infects quiescent and proliferating CD4+
lymphocytes but does not infect resting T cells. This is
due at least in part to Murr1 inhibition of basal and
cytokine-stimulated NF-kB activity as shown by the
increase in NF-kB activity and decrease in IkBa concentrations upon Murr1 knockdown.82 By altering the
expression of Murr1, one could alter the signaling
threshold for effective NF-kB activity83 and hopefully
inhibit HIV-1 infection. A recent report involves the heatshock protein 70 (Hsp70) as a possible novel innate
immunity factor against HIV-1. Hsp70 seems to block
both G2 and apoptosis in a Vpr-dependent manner,84
inhibiting the nuclear localization of Vpr. Interestingly, in
Vpr-deficient HIV-1-infected macrophages, Hsp70 seems
to have an opposite effect on nuclear import and
replication.85
Finally, an important field of study includes a search
for immunoregulatory secreted factors that might suppress viral replication without killing the host cell. Such
protein or proteins are widely referred to as CD8
antiviral factor or CAF (Devico et al86 and references
therein) and their search is still underway. The secretion
of RANTES, MIP-1a and MIP-1b alone cannot account
for the CD8+ T cells’ antiviral activity. a-Defensins
1, 2, and 3,87 important in antiviral post reverse
transcription activity, were inappropriately believed to
be produced by CD8+ T cells88 and therefore cannot
account for CAF. In addition, they have been shown not
to be responsible for the Stat1-mediated inhibition of the
viral promoter transcriptional activation.89 Protease
activity can also play a role as protease inhibitors such
as leupeptin restore HIV-1 infectivity in CD8+ T cells.90
Other factors, such as ribonucleases may also have
antiviral activity.91 Overall, anti-X4 viruses CAF has yet
to be accounted for. The importance of these factors
and others to be discovered is clear and it is straightforward to envision how through gene therapy one
could endogenously express them in naturally nonproducing cells.
473
Conclusions
The fight against HIV-1 has hardly begun, and is an
evolving dilemma. Classical vaccination approaches
appear far off, although studies to boost immune
function through vaccination might eventually offer
some small hope. However, perhaps the best option still
remains to deliver inhibitory genetic blockage to the cells
the virus must infect. Of course the solution, as it is in
many cases of complex biological phenomena, may rely
on a combination of targeted proteins and processes.
Inhibitors delivered might be siRNA molecules, dominant negative viral proteins, intracellular antibodies,
intrakines, or peptides, or a combination of the above.
Inhibitors in the form of small molecules, although less
specific and needed in high concentration, might be part
of the combination of treatment against HIV-1. We favor
the delivery of small peptides (either genetically selected
or derived) to interfere dominantly with HIV-1-host cell
functions. We believe that such peptides could be
delivered as propeptide complexes of multiple peptides
that are enzymatically cleaved within cells and then
allowed to inhibit their disparate targets.
Gene Therapy
Gene therapy approaches for AIDS
R Wolkowicz and GP Nolan
474
Finally, many of the therapies envisioned here and
being considered in laboratories around the world are
quite expensive. Given the recent issues regarding the
cost and access of treating HIV-1 in the developing
world, and threats by countries hard-hit,92–96 are some of
the costly approaches listed above ever going to become
standard treatment? How does one make some of the
treatments noted herein, if they work, affordable? We
believe that the therapies outlined, while not economically viable in the long run, could provide proof-ofprinciple stand-in until pharmaceutical sophistication
eventually reaches the specificity of the gene therapy
approach. The notion of institutionalizing a network for
HIV-1 gene therapy similar to the AIDS Clinical Trial
Groups might be a way to determine the best clinical
approaches to be undertaken,97 and point to such proofof-principle targets. Whatever the approaches eventually
applied in the clinic, it is becoming obvious that they will
rely on the combination of different therapeutic strategies to combat a virus that has taught us as much about
the foibles and abilities of our own immune system as
any pathogen ever encountered. It will be with some
satisfaction, and considerable resolve, that we lay HIV-1
to rest among the great plagues of the past. It is safe to
say that gene therapy, to date, has already played a major
role in defining the path of that final outcome.
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