gene therapy for hiv infection

For gene therapy of HIV infection to be successful, itwill be necessary to introduce genes that are designed to specifically block or inhibitthe gene expression or function of viral gene

Acquired immunodeficiency syndrome (AIDS) is a rapidly expanding global pandemic Approximately 15 million people worldwide are infected with HIV-1.Despite more than a decade of intense research efforts aimed at understanding theHIV-1 virus and developing an effective therapy for AIDS, HIV-1 infection remains

an incurable and fatal disease However, significant progress has been made in themanagement of HIV-1 replication using traditional drug-based therapies Mostnotable is the advent of the triple-drug regiment, which is composed of three drugsthat inhibit the HIV-1 life cycle at two different stages A protease inhibitor, whichblocks the normal processing of proteins necessary to generate new HIV-1 parti-cles, and AZT and 3TC, which are nucleoside analogs that inhibit replication of theviral genome, are typically the components of the triple-drug cocktail The high rate

of mutation in the viral genome and the generation of drug-resistant strains of

HIV-1 are the major factors that prevent the development of effective drug-based apies The triple-drug regiment has not been sufficiently tested to assess the ability

ther-of the HIV-1 to form drug-resistant mutants The inability ther-of traditional drug-basedtherapies to effectively inhibit the HIV-1 replication has made it necessary todevelop new and innovative therapies for this deadly disease

As part of the normal virus life cycle, the HIV-1 virus integrates into the host

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Copyright © 2001 by Wiley-Liss, Inc ISBNs: 0-471-39188-3 (Hardback); 0-471-22387-5 (Electronic)

cell’s genome and remains there permanently Thus AIDS can be considered as anacquired genetic disorder As previously discussed, gene therapy holds considerablepotential for the treatment of hereditary and acquired genetic disorders Humangene therapy can be defined as the introduction of new genetic material into thecells of an individual with the intention to produce a therapeutic benefit for the patient Therefore, AIDS may be amenable to treatment by gene therapyapproaches to inhibit the replication of HIV-1.

The ultimate goal of gene therapy is to inhibit HIV-1 viral replication and theresulting AIDS pathogenesis For gene therapy of HIV infection to be successful, itwill be necessary to introduce genes that are designed to specifically block or inhibitthe gene expression or function of viral gene products such that the replication ofHIV is blocked or limited.This concept was originally denoted as intracellular immu-nization and is currently being investigated as a therapeutic approach for a widevariety of infectious agents In addition to intracellular interventions, gene therapymay be employed to intervene with the spread of HIV at the extracellular level Inhi-bition of viral spread could be accomplished by sustained expression in vivo of asecreted inhibitory protein or by stimulation of an HIV-specific immune response

GENETIC ORGANIZATION OF HIV

The HIV-1 virus is a member of the family of viruses denoted as retroviruses Theretrovirus classification encompasses a heterogeneous group of viruses containing

a single-stranded, positive-sense ribonucleic acid (RNA) genome and the enzymereverse transcriptase Reverse transcriptase functions by copying the viral genomicRNA into double-stranded deoxyribonucleic and (DNA), which is a critical phase

in the life cycle of retroviruses Retroviruses have historically been subdivided intothree groups primarily based on the pathologic outcome of infection The oncovirussubgroup includes retroviruses that can cause tumor formation in the infected host;however, this group also includes several apparently benign viruses Lentivirusescause slowly progressing, chronic diseases that most often do not contain a tumor-forming component The spumavirus subgroup, although causing marked foamycytopathic effect in vitro, have not yet been clearly associated with any disease.Upon intense investigation into the pathology of HIV infection, it has become clearthat the virus is a member of the lentivirus subgroup Lentiviruses were initially iso-lated in the 1960s when it was found that certain slowly evolving, degenerative dis-eases in sheep were communicable Interestingly, unlike the oncogenic retroviruses,the lentiviruses did not form tumors but were cytopathic (caused cells death).Several members of the lentivirus family have been isolated and described.Members of the lentivirus family include Visna virus, Simian immunodeficiencyvirus, human immunodeficiency virus 1 and 2, caprine arthritis-encephalitis virus,and equine infectious anemia virus

As with all other retroviruses, HIV is an enveloped virus that contains two copies

of single-stranded, positive-sense RNA (Fig 11.1).The genomic organization of HIV

is shown in Figure 11.2 At the ends of the genome are two identical genetic regionssimilar to those found in all retroviruses The genetic elements are called long

terminal repeats (LTRs) The LTRs contain elements that are responsible for the

proper regulation of gene expression during virus replication such as promoters,

enhancers, and elements required for efficient messenger RNA (mRNA)polyadenylation Between the LTRs are the genes that encode all of the viral pro-teins The HIV genome encodes three sets of viral proteins; the structural proteins(Gag, Pol, and Env), the regulatory proteins (Tat, Rev, and Nef), and the matura-tion proteins (Vif, Vpu, and Vpr).

As shown in Figure 11.2, the structural proteins can be subdivided into threegroups: core proteins, enzymes, and envelope proteins These three groups of pro-

teins are encoded by the gag, pol, and env genes, respectively The gag gene refers

to the group antigen and produces the viral core proteins that have antigens reacting with other antigens within large retrovirus groups The Gag proteins are allproduced as a large single polyprotein that is then cleaved into individual proteins

cross-by a virus-encoded protease (p24, p18, and p15) The pol gene products are also

encoded from a single open reading frame as a large polyprotein that is cleaved intothe virus-associated enzymes—protease, reverse transcriptase (RT), ribonuclease,

and integrase The env gene products are surface glycoproteins that are produced

as a polyprotein (gp160), however, they are cleaved by cellular enzymes to producethe two HIV surface glycoproteins (gp120 and gp41)

In addition to the structural elements necessary to assemble the virus particle,the virus genome codes for several nonstructural proteins that play vital roles in theregulation of the viral life cycle The nonstructural proteins produced by the HIVcan be divided into two classes, the regulatory proteins and the maturation proteins.The regulatory proteins include Tat, Rev, and Nef The Tat protein was the first viral

regulatory protein to be described The Tat protein, which is encoded by the tat gene,

is a strong transactivator of viral gene expression In other words, the Tat protein

struc-regulates the function of genes that are not immediately adjacent to its own gene.

The Tat protein binds to the trans-activation response (TAR) element The TAR

element corresponds to an RNA stem-loop structure present within the lated leader sequence of all HIV-1 transcripts, including the RNA genome, and isrequired for HIV-1 Tat function The interaction between Tat and TAR can lead to

untrans-tat

tat tat

rev

rev nef

nef

env

env

vpu vpr

the viral envelope protein (gp 120, gp 41) which is encoded by the env gene and the core proteins (p6, p9, p17, and p24) which are encoded by the gag gene The pol gene generates

the viral-associated reverse transcriptase, integrase, RNase H, and protease enzyme

activities The viral-associated regulatory proteins are encoded by the tat, rev, and nef genes,

respectively The Tat and Rev proteins are powerful regulatory proteins The Tat protein

interacts with the TAR (tat-responsive) element, which leads to a strong transactivation of viral gene expression, while the Rev protein interacts with the RRE (rev response element),

which enhances the nuclear export of unspliced and single-spliced viral mRNA The third

class of viral proteins are the maturation proteins that are encoded by the vif, vpr, and vpu

genes.

a potent transactivation (increasing expression of viral genes by 1000 times their

level of expression in HIV-1 mutants lacking the tat gene) by inducing

transcrip-tional initiation and/or elongation

A second important regulatory protein is Rev, which produced by the rev gene.

The Rev protein is produced early in the replication phase of HIV and interacts

with a 234-nucleotide region of the env open reading frame in mRNA called the Rev response element (RRE) The interaction of the Rev protein with the RRE

markedly enhances nuclear export of single-spliced and unspliced viral mRNAsfrom the nucleus; these RNAs encode the viral structural proteins The production

of Rev protein is an absolute requirement for the replication of the HIV virus, sincemutants of the Rev protein are incapable of inducing synthesis of the viral struc-tural proteins and are, thus, replication defective

The last member of the regulatory protein family is the Nef protein The role of

the Nef protein in HIV-1 replication cycle remains unclear However, the nef gene

product is not required for HIV-1 replication in vitro or SIV in vivo It is clear that

the nef gene plays a role in the down-regulation of CD4 gene expression in infected cells It is also hypothesized that Nef may be involved in the ability of HIV-1 to turn

off its growth and reside dormant in the host cell genome

In addition to the Gag, Pol, and Env, the late gene products encoded by HIVinclude the maturation proteins Vif, Vpu, and Vpr Both the Vif (virion infectivityfactor) and Vpu (viral protein U) proteins play roles in the maturation and pro-duction of infectious HIV virion particles The Vpr (viral protein R) protein hasrecently been described as playing an integral role in causing the cell cycle arrest

of HIV-infected cells Expression of Vpr alone was sufficient to cause arrest of thecell cycle at the G2/M transition phase of the cell cycle Thus, HIV-infected cells areunable to progress normally from the G2phase of the cell cycle through mitosis tocomplete the cell cycle The cell cycle arrest after infection by HIV causes theinfected cell to remain in an activated state and, thus, may maximize virus produc-tion from the infected cell

LIFE CYCLE AND PATHOGENESIS OF HIV-1 INFECTION

As shown in Figure 11.3, the initial stage of infection (the early phase) begins withthe binding of the viral gp120 protein to its cell surface receptor, the CD4 protein.CD4 is present in high concentration on the surface of peripheral blood lympho-cytes (PBL) and at lower concentrations on other cells that can be infected by HIV,including monocytes, macrophages, and dendritic cells However, CD4 is not the solemediator of HIV infection Previous work in murine cell lines expressing humanCD4 are not infected by HIV, which suggested the existence of a human specific co-factor The HIV infection co-factor has recently been identified This co-factor,termed fusin (CXCR4), is absolutely required, in addition to CD4, for the entry ofHIV in to human cells Fusin is an integral membrane glycoprotein and a member

of the chemokine receptor family Several of these co-factor proteins (CXCR4,CCR5, and CCR3) have now been identified on various cell types The binding ofthe HIV gp120/gp41 envelope protein induces conformational changes that allowinteraction with the co-receptor and subsequent fusion of the virus with the hostcell plasma membrane The HIV-1 nucleocapsid is internalized into the cytoplasmwhere the viral-genome is uncoated The RNA genome is reverse transcribed into

a single, negative strand of DNA, by the RT protein encoded by the pol sequences.

The viral-encoded ribonuclease then degrades the viral genomic RNA The RTenzyme then encodes the second (positive strand) of DNA, and this double-stranded viral genome is circularized and transported through the nuclear pore and into the nucleus of the infected cell The newly synthesized viral DNA genome then randomly integrates into the host cell genome by the virally encodedintegrase protein; this integrated form of the virus is denoted as the provirus Theprovirus can replicate immediately or remain latent for extended periods of time and in so doing is passed along to all progeny cells derived from the originalinfected cell

Although the mechanism of proviral activation is unclear, once the provirus isactivated the intermediate stage of viral infection begins Activation induces tran-scription of multiply spliced viral RNAs, which are utilized to produce the Tat andRev proteins that act as powerful regulatory proteins during virus replication Asdiscussed previously, the Tat protein enhances the transcription elongation of viralRNA within the nucleus of the infected cell Whereas, the Rev protein enhancesnuclear export of single-spliced and unspliced viral mRNAs from the nucleus; theseRNAs encode the viral structural proteins

The late phase of HIV-1 infection begins upon the accumulation of significantamounts of structural proteins The late phase consists of assembly of virus parti-cles containing two copies of the viral RNA genome The assembled particles aretransported to the cell membrane where the mature virus particles bud off from theplasma membrane In theory, the life cycle of the HIV-1 virus can be interrupted by

INTEGRATION

VIRION ASSEMBLY

BINDING BUDDING

REVERSE TRANSCRIPTION

nuclear pore

Genomic RNA

Unintegrated Genomic DNA

Core

CD4 gp120

Structural Protein mRNA's

Gag and Env Proteins

Mature

HIV Virions

Tat Rev

Regulatory

mRNA's

FIGURE 11.3 Life cycle and replication of HIV-1.

blocking or inhibiting the function of one or more or the key viral proteins or theircis-acting regulatory elements.

HIV can kill an infected CD4+T lymphocyte in one of two ways As progeny virusparticles are budded off from the cell membrane, the external envelope proteingp120 reacts with CD4 molecules found on the surface of the infected cell to disruptthe integrity of the cell membrane in the areas with high concentrations of CD4.Disruption of the cell membrane kills the infected cell Alternatively, an infectedcell may interact with an uninfected cell through the HIV envelope proteins embed-ded in their cell surface membranes The interaction is again through the CD4 mol-ecules found on the surface of the uninfected cell As the cell fusion occurs, hundreds

of CD4 cells may eventually be involved in the formation of a large syncytium All

of the cells that fused into the syncytium die, and thus the cytopathic effects of HIVcan extend beyond cells directly infected with the virus It is predominantly throughthese two mechanisms that loss of CD4+ lymphocytes occurs in HIV-infectedpatients The outcome of HIV infection in monocyte–macrophage lineage cells isunclear It appears as though the virus is capable of replication, but it does notappear to have any obvious cytopathic effects as in T lymphocytes Similar toinfected T cells, the formation of multinucleated syncytium of macrophage-like cells

is observed in HIV-infected tissues Macrophages that contain replicating virus maynot be destroyed, but evidence suggests that they become dysfunctional

GENETIC APPROACHES TO INHIBIT HIV REPLICATION

Approaches to gene therapy for HIV can be divided into three broad categories:(i) protein approaches such as transdominant negative proteins and single-chainantibodies, (ii) gene therapies based on nucleic acid moieties, including antisenseDNA/RNA, RNA decoys, and catalytic RNA moieties (ribozymes), and (iii)immunotherapeutic approaches using genetic vaccines or pathogen-specific lym-phocytes (Table 11.1) It is further possible that combinations of the aforementionedapproaches may be used simultaneously to inhibit multiple stages of the viral lifecycle or in combination with other approaches, such as hematopoietic stem celltransplantation or vaccination The extent to which gene therapy approaches will

be effective against HIV-1 is the direct result of several key factors: (i) selection ofthe appropriate target cell in which to deliver the gene therapy, (ii) the efficiency

of the gene delivery system on the target cell, (iii) appropriate expression, tion, and stability of the anti-HIV gene product(s), and (iv) the strength of the inhibition of viral replication by the therapeutic entity

regula-TRANSDOMINANT NEGATIVE PROTEINS

Transdominant negative proteins (TNPs) are mutant versions of regulatory or tural proteins that display a dominant negative phenotype that can inhibit replica-tion of HIV By definition, such mutants not only lack intrinsic wild-type activity butalso inhibit the function of their cognate wild-type protein in trans Inhibition mayoccur because the mutant competes for an essential substrate or co-factor that isavailable in limiting amounts, or, for proteins that form multimeric complexes, the

struc-mutant may associate with wild-type monomers to form an inactive mixed mer A potential drawback in the use of transdominant viral proteins is their possi-ble immunogenicity when expressed by the transduced cells The protected cells mayconsequently induce an immune response that might result in their own destruc-tion This may diminish the efficacy of antiviral gene therapy using transdominantproteins HIV-1 regulatory (Tat and Rev) and structural proteins (Env and Gag) arepotential targets for the development of TNPs.

multi-The most thoroughly investigated TNP is a mutant Rev protein denoted RevM10.The Rev protein is rendered a TNP through a series of mutations introduced into

the rev gene (Fig 11.4) The RevM10 still retains the ability to multimerize and bind

to the RRE; but as a result of these mutations, the RevM10 protein can no longerefficiently interact with a cellular co-factor that activates the Rev function Cell linesstably expressing RevM10 are protected from HIV-1 infection in long-term cellculture assays Transduction of RevM10 into T-cell lines or primary PBL delays virusreplication without any detectable negative effects on the cells Recently, it has beendemonstrated that RevM10 inhibits HIV-1 replication in chronically infected T cells

A different TNP Rev protein developed by Morgan et al (1994) inhibited HIV-1

TABLE 11.1 Gene Therapy Strategies to Inhibit HIV Replication

Anti-HIV Strategy Potential Mode of Action

Protein-Based Approaches

Transdominant Negative Proteins

Tat Viral genome transcription/processing

Endogenous Proteins

Soluble CD4 Receptor binding/viral assembly

CD4-KDEL Trapping of Env and Rev in ER

5¢ leader sequence Translation of viral RNA

Multitarget Translation of viral RNA

Antisnese oligonucleotides

RNA decoys Translation of viral RNA

TAR decoy Viral genome transcription/processing RRE decoy Nuclear export of viral mRNA

Immunity Augmentation

DNA Vaccines Induction of cellular and humoral response Env Augments cytotoxic activity to HIV Virus Specific CTL

replication in T-cell lines and PBL when challenged with both laboratory and ical HIV-1 isolates A third type of Rev TNP was generated by deletion of the nucle-olar localization signal sequence This sequence functions as a signal to direct theRev protein to the nucleolar region of the nucleus of an infected cell This TNP Rev

clin-is retained in the cytoplasm and prevented the localization of wild-type Rev to thenucleus by forming inactive oligomers

The HIV-1 regulatory protein Tat was also utilized to generate TNPs A TNP Tatwas mutated in its protein binding domain Upon transduction into T-cell lines, theTNP Tat inhibited HIV-1 replication for up to 30 days The mechanism throughwhich this Tat TNP may function is by sequestration of a cellular factor involved

in Tat-mediated transactivation Interestingly, in this study a retroviral vector wasdeveloped that was capable of expressing both a Tat and Rev TNP The multi-TNPvector was more effective at blocking HIV-1 replication than retroviral vectorsexpressing either TNP Tat or Rev alone This study suggests that the inhibition ofTat and Rev simultaneously may be a more effective HIV-1 gene therapy Recently,

a double transdominant Tat/Rev fusion protein (Trev) was designed in an attempt

to inhibit two essential HIV-1 activities simultaneously Upon transfection or

Rev Rev TNP 1

2 Inhibition of HIV Replication

Genomic RNA

Virion

Assembly

Extra-Nuclear Transport

Structural Protein mRNA's

BUDDING Mature

HIV

virions

FIGURE 11.4 Activity of a transdominant negative Rev protein (1) The normal function

of the Rev protein is to form multimeric complexes (gray circles) which increase the ciency of extranuclear transport of genomic viral RNA(s) and (2) the transdominant nega- tive Rev protein (black circles) forms inactive mixed multimeric complexes with the wild-type Rev protein (gray circles) These inactive Rev complexes interfere with the normal func- tioning of the wild-type Rev complexes and inhibit the extra-nuclear transport of unspliced and singly spliced HIV RNA(s).

effi-transduction of the Trev gene into T cells, they were protected from the cytopathic

effects of HIV-1 Simultaneous inhibition of two HIV-1 functions may have tial advantages over single-function TNPs

poten-TNP moieties based on structural proteins have also been investigated for theiranti-HIV-1 functions The HIV-1 structural proteins (Gag and Env) oligomerize intomultimeric complexes during viral assembly Multimerization makes them ideal can-didates for the generation of TNPs Several Gag TNPs have been investigated andall are capable of inhibiting HIV-1 replication The Gag TNPs function by disrupt-ing distinct stages of the viral life cycle, such as viral assembly, viral budding, uncoat-ing of the viral genome, or initiation of reverse transcription Due to inherently low

levels of transcription of gag genes in the absence of the HIV-1 Rev protein, the

application of Gag TNPs has been limited The low levels of mutant Gag expressedare insufficient to effectively block HIV-1 replication Env TNPs have been gen-erated as well but in initial testing showed only low levels of antiviral activity

Single-Chain Antibodies (Intrabodies)

One of the more novel classes of antimicrobial gene therapies involves the opment of intracellularly expressed single-chain antibodies (also called intra-bodies) The single-chain variable fragment of an antibody is the smallest structuraldomain that retains the complete antigen specificity and binding site capabilities

devel-of the parental antibody Single-chain antibodies are generated by cloning devel-of theheavy- and light-chain genes from a hybridoma that expresses antibody to a spe-cific protein target These genes are used for the intracellular expression of the intra-body, which consists of an immunoglobulin heavy-chain leader sequence that targetsthe intrabody to the endoplasmic reticulum (ER), and rearranged heavy- and light-chain variable regions that are connected by a flexible interchain linker Since thesingle-chain antibody cannot be secreted, it is efficiently retained within the ER,probably through its interaction with the ER-specific BiP protein The BiP proteinbinds incompletely folded immunoglobulins and may facilitate the folding and/oroligomerization of these proteins Intrabodies can directly bind to and prevent genefunction or may sequester proteins in inappropriate cellular compartments so thatthe life cycle of HIV is disrupted

Expression of an intrabody specific for the CD4 binding region of the HIV-1gp120 (Env) markedly reduced the HIV-1 replication by trapping the gp160 in the

ER and preventing its maturation by cleavage into the gp120/gp41 proteins (Fig.11.5) Intrabodies developed to the Rev protein trapped Rev in a cytoplasmic com-partment and blocked HIV-1 expression by inhibiting the export of HIV-1 RNAsfrom the nucleus Additionally, intrabodies containing an SV40 nuclear localizationsignal sequence were developed to Tat The anti-Tat single-chain antibody blockedTat-mediated transactivation of the HIV-1 LTR and rendered T-cell lines resistant

to HIV-1 infection

Endogenous Cellular Proteins as Anti-HIV Agents

Proteins derived from cellular genes have been identified that exhibit specific geneinhibitory activity (Fig 11.5) These activities may act by preventing the binding ofHIV to cells, by binding directly to the regulatory/structural proteins, or indirectly

by inducing or repressing cellular factors that in turn influence viral gene sion One of the most successful in vitro uses of endogenous cellular proteins toinhibit an infectious agent is the use of a soluble version of the HIV receptor CD4(sCD4) The T helper cell antigen CD4 functions as the receptor for the HIVthrough the physical interaction of the HIV envelope glycoprotein gp120 and theCD4 protein Based on these results, investigators have demonstrated that sCD4protein can effectively bind to and inhibit HIV infection in CD4+cells The effect

expres-of this strategy is to compete for binding expres-of HIV to cellular CD4 with high centrations of sCD4 In order for this strategy to be efficacious, a high level of con-tinuous expression of sCD4 will be required Retroviral vectors expressing sCD4have been shown to protect T-cell lines from HIV infection in vitro A significantlimitation to this strategy is the ability to achieve sufficiently high levels of sCD4 toneutralize HIV effectively The use of sCD4 for the gene therapy of HIV infection

con-in a clcon-inical settcon-ing has been disappocon-intcon-ing The con-intravenous con-infusion of nant sCD4 protein in HIV-infected patients failed to show efficacy in phase I clinical trials In contrast to the laboratory strains of the HIV virus, clinical isolates

recombi-HIV Binding

gp 120 CD4 sCD4

gp 120 Expression

gp 120 Expression Lysosome

FIGURE 11.5 Cellular protein-based approaches for the inhibition of HIV-1 replication The intracellular production of a soluble CD4 protein (sCD4) can prevent both the binding

of infectious HIV particles and the production of new virus particles from an infected cells

by saturating all of the available envelope protein The attachment of a endoplasmic lum (ER) retention signal (KDEL) to the CD4 protein brings about the inhibition of virus replication by retaining the gp160 envelope complexes with the endoplasmic reticulum The incorporation of a lysosomal targeting sequence into the CD4 protein leads to the inhibition

reticu-of gp160 expression through targeted degradation in the lysosomes The expression reticu-of chain antibodies (intrabodies) can also lead to the retention of viral proteins in the ER by specific interaction with the BiP protein.

single-have shown a significant increase resistance to the neutralizing characteristics ofsoluble CD4 protein.

A variation on the CD4-based anti-HIV gene therapy approach is the ment of a mutated soluble CD4 molecule that contains a specific ER retention signal(Lys-Asp-Glu-Leu or KDEL) This hybrid molecule blocked secretion of gp120 andcell surface expression of gp120/41, when expressed intracellularly (Fig 11.5) TheCD4-KDEL/gp120 complex was retained within the endoplasmic reticulum, thuspreventing maturation of infectious HIV-1 particles It has also been demonstratedthat the mutations that decrease the affinity of CD4 for gp120 discussed above have little effect on the ability of CD4-KDEL to retain gp120 in the ER A similarapproach is to specifically target soluble CD4/gp160 complexes to the lysosomesthrough the incorporation of lysosome targeting domains onto the soluble CD4 TheCD4-lysosomal domain/gp160 complexes are degraded in the lysosomes of the cellsand production of mature HIV particles is diminished (Fig 11.5)

develop-It has recently been demonstrated that the Rev protein interacts specifically withcellular factors in order to perform its normal function in the infected cell Theeukaryotic initiation factor 5A (eIF-5A) is a cellular transcription factor that inter-acts with Rev by binding to the Rev activation domain The interaction betweenmutants of the eIF-5A and Rev can effectively inhibit HIV-1 replication in vitro.Utilization of the interactions between cellular factors and HIV could provide anadditional approach for the development of HIV genetic therapies Since these areendogenous cellular proteins, they are nonantigenic Therefore, cells engineeredwith these cellular inhibitory genes may not be eliminated by the patient’s immunesystem This is an advantage as compared to the use of genes encoding potentiallyimmunogenic, trans-dominant viral proteins for gene therapy

A novel strategy exploiting the interaction of CD4/fusin with the gp120/gp41protein of the HIV virus has been developed As a consequence of HIV replication,infected cells express the gp120/gp41 envelope protein on their surface in order forthe assembly of new virus particles to occur The expression of gp120/gp41 on thecell surface lead investigators to hypothesize that a virus could be engineered tocontain the HIV receptor and co-receptor in its envelope in place of endogenousviral envelope proteins Generation of these hybrid virus particles would specificallytarget these hybrid virus particles to infected cells where replication of the viruswould kill the cell The vesicular stomatitis virus (VSV) has been engineered in this

manner In the VSV studies, deletion of the VSV gene and substitution with the

genes for CD4 and CXCR4 lead to the formation of recombinant VSV particlesthat specifically infected HIV-infected cells Upon infection, the replication of VSVwas cytopathic in HIV-infected cells

NUCLEIC-ACID-BASED GENE THERAPY APPROACHES

RNA Decoys

This approach disrupts the normal interaction of the HIV regulatory proteins withtheir cis-acting regulatory elements through the overexpression of short RNA mol-ecules (decoys) that compete with viral RNA elements for binding of proteins thatare required for virus replication (Fig 11.6) The TAR (transactivation response)

and RRE (Rev response element) are two such viral regulatory elements found inHIV and are the binding sites for the transactivating proteins Tat and Rev, respec-tively The antiviral activity of the TAR element decoys was examined by retrovi-ral-mediated gene transfer into T-cell lines in vitro Overexpression of the TARdecoys inhibited the transcriptional activation mediated by the Tat protein that, inturn, markedly reduced HIV replication of laboratory HIV isolates The TAR decoyinhibition of virus replication results from the decreased binding of the Tat protein

to the endogenous TAR elements, which in turn inhibits the transcriptional tion necessary for efficient virus replication Expression of a tandem repeat TARdecoy composed of as many as 50 TAR repeats has also been demonstrated to effec-tively inhibit HIV-1 replication in both T-cell lines and primary lymphocytes.Furthermore, the multimerized TAR decoy was shown to efficiently inhibit virusreplication in lymphocytes from late-stage AIDS patients

activa-The enhanced expression of RRE decoys by retroviral vectors resulted in term inhibition of HIV replication in human T-cell lines RRE decoys may function

long-by preventing the binding of REV to the normal RRE sequences, which decreases

Protein Translation

Tar Element

Saturation of Tat Protein

3'

3' 5' 5'

5' 5'

5'

3' 5'

5' 3' 3' 3'

3' Viral mRNA

FIGURE 11.6 Nucleic-acid-based gene therapies for HIV-1 Anti-HIV genes can be expressed in the context of antisense nucleic acids, as antisense oligonucleotides, antisense RNA, or ribozymes All of these antisense approaches promote the destruction of the target sequence by RNAses or block the translation of the mRNA Ribozymes function by specific interaction with a target sequence within the RNA and functionally inactivate it by cleavage

of the phosphodiester backbone Overexpression of short RNA molecules (decoys) that respond to the viral cis-acting elements Transactivation response element (TAR) or the Rev response element (RRE) inhibit the binding of the viral protein to the cognate sequences found on the viral mRNAs.

cor-the levels of singly spliced and unspliced HIV-1 mRNAs that are exported from cor-thenucleus of an infected cell It is clear that the overexpression of RRE decoys hasstrong antiviral activity, but there is some concern as to the long-term effects thatthe expression of the RNA decoys will have on the normal function of the cell Inaddition to viral proteins, both TAR and RRE bind cellular co-factors The overex-pression of the decoys may have negative effects on cell viability or activity throughthe sequestration of proteins required for normal cell function To limit the inter-action between the RRE decoy and cellular proteins, a minimal RRE decoy com-posed of only 13 nucleotides that retained the rev binding domain was tested forantiviral activity This minimal RRE decoy was shown to effectively suppress HIV-

1 replication in vitro

Antisense DNA and RNA

Antisense nucleic acid technology encompasses a broad spectrum of methods alldirected toward the specific silencing of gene expression The silencing of geneexpression is achieved through the introduction into the cell or tissue of an anti-sense RNA or single-stranded DNA moiety (oligodeoxynucleotide), which is com-plementary to a target mRNA (Fig 11.6) In theory, the antisense nucleic acidsutilize Watson–Crick nucleic acid base pairing to block gene expression in asequence-specific fashion One of the most intensely investigated approaches forapplication of antisense RNA is the introduction of DNA oligonucleotides that havebeen chemically modified in an attempt to increase their stability (half-life) within

a cell A variety of synthetic antisense oligonucleotides have been designed thatinhibit the replication of HIV-1 However, their use for the inhibition of HIV-1 hasbeen extremely limited because uptake of free oligonucleotides from the extracel-lular environment in vivo is extremely inefficient, and effective oligonucleotidedelivery systems have not yet been devised Also, the oligonucleotide moieties thatare internalized into the target cells are ultimately degraded by cellular enzymessuch that any inhibitory activity on gene expression is only transient An additionalproblem with the use of DNA oligonucleotides is that the gene inhibition that isobserved is often nonspecific In other words, the inhibition of expression is mostoften not the direct result of the interaction between the oligonucleotide and thetarget sequence, but the interaction with RNA in a broad nonspecific manner.The other approach for antisense nucleic-acid-mediated inhibition of geneexpression is the direct introduction or intracellular production of antisense RNA

in cells or tissues of the organism The direct introduction of RNA transcripts intocells can be accomplished through microinjection of an in vitro transcriptionproduct or as a chemically modified oligonucleotide The direct administration ofantisense RNA transcripts in vivo is not plausible for gene therapy due to the vastnumber of cells that need to receive the therapeutic RNA

An alternative approach to the delivery of antisense RNA for gene therapy isthe use of vector-based systems, which produce the antisense RNA within the cell

or tissue of the organism Most often recombinant viral vector systems, such as viruses, are used because they efficiently target large numbers of cells The use ofretrovirus vector-based systems for the intracellular production of antisense RNAhas an additional advantage That is, the vector integrates into the host cell genomeand, thus, the antisense effects are more prolonged in comparison to oligonu-