Vector Targeting for Therapeutic Gene Delivery (Curiel/Therapeutic Gene Delivery) || Promoter Optimization and Artificial Promoters for Transcriptional Targeting in Gene Therapy

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22PROMOTER OPTIMIZATIONAND ARTIFICIAL PROMOTERSFOR TRANSCRIPTIONALTARGETING IN GENETHERAPY

DIRK M. NETTELBECK, PH.D., DAVID T. CURIEL, M.D., AND ROLFMULLER, PH.D.

INTRODUCTION

Selectivity of intervention is essential to most therapeutic strategies. In genetherapy, optimization of gene transfer vectors, including vector targeting, iscrucial for the development of efficient clinical protocols (Anderson, 1998;Nabel, 1999). Considering the diversity of diseases, it is evident that any sin-gle gene delivery system cannot be universally applicable. Individually opti-mized vectors are therefore required. Transcriptional targeting facilitates spa-tially controlled, inducible, or physiologically regulated therapy by utilizingregulatory DNA sequences—promoters, enhancers, and/ or silencers—to drivetargeted expression of the therapeutic gene. Furthermore, a specific therapeuticstrategy for treatment of malignancy, viral oncolysis, is based on the replace-ment of viral regulatory elements by selective promoters to control the expres-sion of essential viral genes with the goal of obtaining tumor-specific condi-

Vector Targeting for Therapeutic Gene Delivery, Edited by David T. Curiel and Joanne T. DouglasISBN 0-471-43479-5 Copyright 2002 Wiley-Liss, Inc.

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tionally replicating viruses. Of note, transcriptional targeting is the only fea-sible strategy for certain applications, such as radiation-induced gene ther-apy, which link genetic and conventional therapeutic approaches. Furthermore,selective or inducible promoters can be combined with transductional target-ing to form a multilevel targeting approach. This combination may result inincreased selectivity (1) by superimposing two levels of selectivity for the sametarget or (2) by combining different targeting modalities, for example tissuetargeting with proliferation-dependent gene expression (dual specificity).

DESIGNER PROMOTERS

Key to the realization of the full potential of transcriptional targeting is theavailability of highly specific and effective promoters for a wide spectrum ofdiseases. In addition, the promoter of choice must be functional in the heterol-ogous context of a given gene therapy vector (vector compatibility). Thus, sizerestrictions and potential interference between vector sequences and promoteractivity are of potential concern. Few promoters that fulfill these requirementshave been described to date. Furthermore, for some applications, suitable nat-ural promoters might not exist. Therefore, recent research efforts have focusedon the development of “designer promoters” for a specific disease or vectorcontext. In this regard, the following criteria have to be considered: (1) an ade-quate level of promoter activity in the target cell (ON status), comparable tothe activity of strong, but not selective promoters used in standard nontargetedgene therapy studies (like the cytomegalovirus [CMV] promoter/ enhancer), (2)negligible promoter activity in nontarget tissues (OFF status), that is minimal“leakiness,” and (3) size limitations imposed by the vector of choice.

In general, the necessary activity and tightness of a candidate promoterdepend on the therapeutic gene of choice, that is, on the potency of its encodedproduct on target versus nontarget cells. The goal is to achieve the highest pos-sible therapeutic index for the specific combination of selective promoter andtherapeutic gene. Technical strategies to achieve this aim include (1) the opti-mization of native promoters, (2) the construction of synthetic promoters, and(3) the engineering of vector backbone DNA sequences in order to retain theexpression profile of incorporated promoters.

Fortunately, progress in elucidating basic questions of molecular cell biol-ogy provides gene therapists with the necessary tools for the developmentof targeted vectors. For example, the determination of viral cell binding andcell entry mechanisms facilitates the development of transductionally targetedvectors. Likewise, manipulation of promoter activity and size as well as thedesign of synthetic promoters are based on the characterization of moleculesand mechanisms of transcriptional control. Specifically, the modular structureof both cis-acting (promoters, enhancers, and silencers) as well as trans-act-ing (transcription factors) regulators represents the basis for promoter opti-mization and design. The number, diversity, orientation, and placement of reg-

PROMOTER OPTIMIZATION: DELETION AND MULTIMERIZATION 483

ulatory elements within a candidate promoter are critical parameters deter-mining its performance. Their precise combination must be determined bydetailed structure–function analyses. Similarly, recombinant transcription fac-tors can be engineered by fusing natural or artificial domains that mediateDNA-binding, transactivation, or ligand-dependent regulation, thus derivingdesigner molecules able to regulate gene expression in the desired fashion.

The strategies for promoter optimization and development of artificial pro-moters described in this chapter represent a scenario for the translation ofprogress in basic biological research into therapeutic approaches of modernmolecular medicine.

PROMOTER OPTIMIZATION: DELETION AND MULTIMERIZATION

Cell-specific or otherwise selective promoters are frequently inefficient activa-tors of transcription or are comprised of extended DNA sequences too largefor incorporation into gene transfer vectors. Naturally occurring or disease-associated activating point mutations within promoters have been exploited toincrease transgene expression. However, activating mutations have been foundonly in a few cases, such as the a-fetoprotein (AFP) and multiple drug resis-tence (mdr) promoters (Ishikawa et al., 1999; Stein, Walther, and Shoemaker,1996), and the levels of activation were minimal. Clearly, a more potent strat-egy is to optimize promoters by recombinant DNA technology. The simplestapproach for expression optimization and simultaneous size reduction is toeliminate promoter sequences that do not obviously contribute to its transcrip-tional strength and/ or selectivity. In addition, the multimerization of importantregulatory elements can be advantageous. Both strategies have been success-fully applied to several promoters as illustrated in the following paragraphs.

An exhaustive promoter analysis and optimization study for the regulatoryregion of the carcinoembryonic antigen (CEA) gene, which is transcriptionallyupregulated in adenocarcinomas, has been performed by Richards, Austin, andHuber, 1995. An upstream enhancer element and a tetramer of a tumor-spe-cific promoter element fused to the core promoter generated a highly active andselective promoter. Simultaneously, the promoter size was reduced consider-ably. The fusion of distant enhancer elements to a core promoter is a strategysuccessfully applied to several transcriptional regulatory regions, such as thehepatocellular carcinoma-specific AFP gene (Wills et al., 1995; Mawatari et al.,1998), the prostate specific genes for prostate-specific antigen (PSA) (Pang etal., 1997; Latham et al., 2000) and human kallikrein 2 (hK2) (Xie et al., 2001),and the melanocyte-specific human and murine tyrosinase genes (Siders, Hal-loran, and Fenton, 1996, 1998; Park et al., 1999).

For both tyrosinase constructs an increase in activity and a size compati-ble with most gene therapy application have been achieved by combining anenhancer dimer with the core promoters (Siders, Halloran, and Fenton, 1996).For the human construct, the size was reduced from 2.7 kb to 650 bp, with a


five-fold increase in activity. Melanocyte-specific cytotoxicity was shown forthese promoter constructs in combination with the herpes simplex virus thymi-dine kinase (HSV-tk)/ gancyclovir (GCV) and purine nucleoside phosphorylase(PNP)/ 6-methyl purine phosphorylase (6-MPDR) prodrug activation systemsin both adenoviral and liposomal vectors (Siders et al., 1998; Park et al., 1999).

Of note, optimized albumin, AFP, PSA, and hK2 promoters have beensuccessfully applied for the transcriptional targeting of herpes simplex virus(HSV) or adenovirus (Ad) replication to tumor cells (Miyatake et al., 1997,1999; Rodriguez et al., 1997; Yu, Sakamoto, and Henderson, 1999; Hallenbecket al., 1999). In this scenario, expression of the essential immediate early viralgenes ICP4 (HSV) or E1 (Ad) is driven by a tumor/ tissue-specific promoter.

Transcriptional regulatory sequences located within the transcribed sequencehave also been utilized for transcriptional targeting. For example, in theendothelium-specific and angiogenesis associated flk-1 gene, a tissue-specificenhancer was located within the first intron. Accordingly, transcriptional target-ing to tumor endothelium was achieved with a construct containing the flk-1promoter and a 3′-located intron (Kappel et al., 1999; Heidenreich, Kappel,and Breier, 2000).

CHIMERIC PROMOTERS

Synthetic promoters with optimized activity and/ or specificity can also be engi-neered by linking transcriptional control elements derived from different genes,yielding chimeric promoters. Different strategies have been pursued in this con-text: (1) combining defined elements of different genes with the same speci-ficity, (2) combining specific cellular promoters with viral enhancers to increasetranscriptional activity, (3) combining transcriptional control elements medi-ating different types of specificity, (4) utilizing disease-specific transcriptionfactor binding elements fused to heterologous core promoters, and (5) utilizingspecific repressor elements in combination with ubiquitously active promoters.

Combining Defined Elements of Different Genes of the Same(Tissue–) Specificity

Chimeric promoters for enhanced liver- or hepatocellular carcinoma (HCC)-specific gene expression have been derived by fusing a tetramer of the apolipo-protein E enhancer to the human alpha-antitrypsin (hAAT) promoter (Okuyamaet al., 1996), or by linking the AFP enhancer to the albumin promoter (Su etal., 1996, 1997). For the latter construct, the IC50 value for HSV-tk/ GCV cellkilling in vitro was inversely correlated to the level of AFP (but not albumin)expression of HCC cells.

A particularly sophisticated approach for generating chimeric promoters isthe random combination of (tissue-) specific elements followed by the isolationof the most active and specific construct. Li and co-workers assembled 5 to

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20 muscle-specificity-mediating elements in random order in such a way thatthey were exposed on the same side of the DNA double helix (Li et al., 1999).This synthetic enhancer library was cloned upstream of a minimal alpha–actinpromoter driving the luciferase reporter gene and subsequently analyzed forgene expression. The most promising constructs showed specific activity thatwas stronger than the CMV promoter/ enhancer in differentiating muscle cellsin vitro and after intramuscular injection in mice.

Activity Enhancement by Strong Viral Enhancers

This strategy has been pursued by cloning the CMV promoter/ enhancerupstream of a 600 bp PSA promoter, resulting in a prostate-specific increasein promoter activity (Pang et al., 1995). However, this strategy is not univer-sally applicable, as it depends on the presence of a tissue-specific repressorelement to ablate the activity of the viral enhancer in nontarget tissues. Thus,linking the SV40 enhancer to the vascular smooth muscle cell alpha actin pro-moter (Keogh et al., 1999) or to the endothelium-specific von Willebrand factor(vWF) promoter (Nettelbeck, Jerome, and Muller, 1998) resulted in activation,but loss of specificity.

In a different approach, fusion of muscle-specific elements to the CMVpromoter/ enhancer or partial replacement of the CMV promoter/ enhancer withthese elements, resulted in chimeric promoters with enhanced activity both inmuscle cells and after intramuscular injection in vivo (Barnhart et al., 1998).However, these constructs showed considerable activity in nontarget cells.Interestingly, this study did not show a clear correlation between promoteractivity in vivo and in vitro, thus underscoring the importance of in vivo pro-moter analysis in the screening process.

Combining Transcriptional Control Elements Mediating DifferentTypes of Specificity

This strategy has been investigated in two studies, establishing combinedtissue- or tumor-specific and inducible transcriptional control. In the first study,the human beta-interferon promoter was linked to the B-cell specific IgHCenhancer, resulting in B-cell-specific, virus-inducible gene expression (Engel-hardt et al., 1990). The second study combined a 300 bp AFP promoter withthe VEGF gene-derived hypoxia-responsive element for the targeting of livertumors. This construct resulted in hypoxia-induced, HCC-specific and, relativeto the AFP promoter alone, increased reporter gene expression and therapeuticgene mediated toxicity (Ido et al., 2001).

Utilizing Disease-Specific Transcription Factor Binding Elements inCombination with Heterologous Core Promoters.

To utilize disease-specific transcription factors for transcriptional targeting ingene therapy, the corresponding DNA elements, or multiple copies thereof, are


Figure 22.1 Outline of a synthetic promoter activated by disease-specific transcrip-tion factors. A synthetic promoter induced by disease-specific transcription factors con-sists of multiple copies of the corresponding transcription factor binding site and aminimal core promoter. Binding of the transcription factors to their binding sites intarget cells activates transcription from the core promoter, which shows no activityper se. Tumor specific transcription factors can result from genomic translocations thatgenerate fusion molecules with novel properties. The Epstein-Barr virus nuclear anti-gen 1 (EBNA-1) is an example for a tumor-associated viral transcription factor. Othertranscription factors are induced by tumor-associated hypoxia or conventional ther-apeutic regimens like irradiation. Synthetic transcription factor responsive promotershave been utilized to drive expression of therapeutic genes (gene therapy) or to controlexpression of essential viral genes to trigger selective viral replication (virotherapy).

cloned within a synthetic construct upstream of a core promoter (Fig. 22.1).The core promoter is the locale for binding of the transcription initiation com-plex, but this is insufficient for appreciable transcriptional activity. Efficientgene expression from the core promoter depends on the additional binding oftranscriptional activators to their cognate elements. Like the core promoter, theupstream elements are normally not capable of mediating efficient transcriptionon their own.

An example of this strategy is the exploitation of the tumor-specificchimeric transcription factor PAX3-FKHR, a fusion protein of the pairedbox gene 3 (PAX3) DNA-binding domain and the forkhead in rhabdomyo-sarcoma (FKHR) transactivation domain that results from a genomic rear-rangement found in alveolar rhabdomyosarcoma (ARMS). Since expressionof the parental transcription factor PAX3 is restricted to prenatal development,the tumor-specific chimeric transcription factor can be applied to target geneexpression to alveolar rhabdomyosarcoma. For this purpose, an artificial pro-moter consisting of repeated PAX3 binding sites in front of a heterologousminimal adenoviral E1B promoter was engineered and shown to be strictlyPAX3-FKHR-dependent (Massuda et al., 1997). This sophisticated approachmight find broader application because translocations that generate chimerictranscription factors are frequently found in human cancers.

In a similar approach, viral transcription factors have been utilized for thetranscriptional targeting of transgenes to virus-associated tumors. Specifically,

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multimerized binding sites for the Epstein-Barr Virus (EBV) encoded transcrip-tion factors EBNA-1 or -2, cloned upstream of a minimal core promoter, havebeen applied to the targeting of Burkitt’s lymphoma and other EBV-associ-ated tumors (Judde et al., 1996; Gutierrez et al., 1996; Franken et al., 1996).Because viruses have been associated with several human malignancies, thisapproach may be applicable to a variety of cancers. An interesting aspect ofboth approaches described previously is the repressor activity of the PAX3 andEBNA-1 binding elements in the absence of the corresponding transcriptionfactors, which leads to tight transcriptional control and remarkable inducibil-ity.

In a similar scenario, artificial promoters responsive to physiologically acti-vated transcription factors have been engineered. For the targeting of hypoxictumors, hypoxia-responsive elements (HREs) from different genes have beencloned upstream of the minimal SV40 promoter and analyzed for inductionby oxygen deprivation. A trimer of the phosphoglycerate kinase gene HREwas shown to mediate a >100-fold activation in the presence of 0.1% oxygen,resulting in stronger gene expression than the CMV promoter/ enhancer (Boastet al., 1999; Binley et al., 1999). Another study demonstrated that the identityof the core promoter is critical in such a setting. Hence, a pentamer of theVEGF-derived HRE mediated hypoxia-dependent gene expression in combi-nation with the CMV or adenovirus E1B minimal promoters, but not with theelongation factor 1 a (EF-1 a) core promoter (Shibata, Giaccia, and Brown,2000). Clearly, an advantage of these artificial hypoxia-dependent promotersis their applicability to a broad range of solid tumors. In addition, increasedspecificity was achieved by combining the HRE repeats with a tumor-specificcore promoter (see foregoing text).

A further scenario is the utilization of multimerized binding sites fortherapy-induced transcription factors. The goal of this concept is to combinegene therapy with conventional therapies, for example, the temporally and/ orspatially restricted expression of proteins, which sensitize cells to the inducingconventional therapeutic regimen. In this regard, the heat shock factor 1 (HSF-1) transcription factor was utilized for hyperthermia-induced gene expressionby fusing multiple HSF-1 binding sites, termed heat shock elements (HSE),upstream of the heat shock protein 70b promoter. This synthetic promoterresulted in increased heat-inductivity of reporter gene expression compared tothe hsp70b promoter alone (Brade et al., 2000). Another therapy-inducible syn-thetic promoter consists of multiple radiation responsive elements upstream ofthe minimal CMV promoter. Radiation inducibilty of this synthetic promoterhas been shown to be superior to the wild-type radiation-responsive egr-1 pro-moter (Marples et al., 2000).

A different concept is the utilization of disease-specific transcription factorsto target viral oncolysis. Recently, an adenovirus with colon cancer restrictedreplication competence was generated by replacing the promoters of the essen-tial viral genes E1B and E2 with multimerized binding sites for the transcrip-tion factor Tcf4 (Brunori et al., 2001). This transcription factor is constitu-


tively activated by mutations in the beta-catenin and adenomatous polyposicoli (APC) genes, a hallmark of colon cancer.

Utilizing Specific Repressor Elements in Combination withUbiquitously Active Enhancers or Promoters

Repressor elements that mediate repression selectively in nontarget tissues rep-resent a potentially interesting option to achieve tissue-specific gene expres-sion. Their utilization for gene therapy purposes is feasible, if combined withubiquitously active transcriptional control elements. Millecamps and co-work-ers achieved targeted gene expression in the context of adenoviral vectorswith a promoter construct harboring multiple copies of the neuron-restrictivesilencer factor (NRSF) binding sites upstream of the ubiquitous phosphoglyc-erate kinase promoter (Millecamps et al., 1999). Since NRSF is expressed onlyin nonneuronal cells, this artificial promoter is specifically active in neurons.

RECOMBINANT TRANSCRIPTIONAL ACTIVATORS: INDUCIBILITY,AMPLIFICATION, AND DUAL SPECIFICITY

Transcriptional control is mediated by cis-acting regulatory elements and trans-acting transcription factors that bind to these elements. Artificial transcrip-tional control systems can therefore also be based on engineered designertranscription factors or recombinant transcriptional activators (RTAs, Fig.22.2A). Transcription factors are composed of multiple functionally indepen-dent domains (modules), including a DNA-binding domain (DBD) that directsthe protein to specific binding sites, and a transactivation domain (TAD) thatcontrols the transcription rate through interactions with other transcription fac-tors and the transcription machinery. RTAs can be engineered by combining

Figure 22.2 Enhancing promoter activity by use of recombinant transcriptional acti-vators (RTAs). (A) RTAs are synthetic fusion proteins containing a potent transactiva-tion domain (for example of the HSV-VP16 protein) fused to a DNA-binding domain(for example of the yeast Gal4 or the bacterial LexA protein). In addition, a ligand-binding domain (for example of a steroid hormone receptor) can be incorporated totrigger ligand-dependent RTA activity. The RTA is encoded by a corresponding fusiongene. Binding of the RTA to its binding site results in activation of gene expression. Foramplification of specific promoter activity (B) The RTA is expressed from the (tissue-)specific but weak promoter. The RTA binds to synthetic binding sites upstream of asecond promoter and activates transcription and gene expression via its strong transac-tivation domain. A further level of specificity can be achieved if the second promoteris also specific and thus activated in target cells only (C). (B See Segawa et al., 1998;C see Nettelbeck, Jerome and Muller, 1998).

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modules that exhibit the desired characteristics. Frequently, a DBD of non-mammalian origin—which does not bind to mammalian sequences, thus pre-venting adverse effects on endogenous gene expression—is combined with aTAD of choice (for example a strong activator). In addition, ligand-bindingdomains (LBDs), derived, for example, from steroid hormone receptors, canbe incorporated into the RTA, resulting in ligand-controlled activity of the RTA


and RTA-dependent promoters. Artificial promoters regulated by RTAs havealso been shown to be functional in vivo (Oligino et al., 1996; Fang et al.,1998). They have been developed for different purposes, including pharma-cologically regulated gene expression, amplification of promoter activity, andcombining tissue-specific and cell cycle regulated gene expression.

Pharmacologically Regulated Gene Expression

Several RTA-driven artificial promoter systems have been developed to imple-ment inducible gene expression (for references see Harvey and Caskey, 1998).A common feature of these systems is that the gene of interest is expressedunder control of a synthetic RTA responsive promoter. The RTA itself isexpressed constitutively, whereas DNA-binding of the RTA, and thus transgeneexpression, is drug dependent. One drug-inducible promoter system, termedTet-OFF, utilizes the bacterial tet repressor fused to the HSV VP16 transactiva-tion domain (tTA). This RTA binds to the tet operator DNA element in absenceof tetracycline or its analogue doxocycline. A reverse phenotype mutant of thetet repressor fused to the VP16 TAD (rtTA) binds to the tet operator in pres-ence of doxocycline, thus establishing the Tet-ON system. A further variationof the tet system is the fusion of the tetR to the transcription repression domainof the transcription factor KRAB. In this approach, gene expression dependson dissociation of the RTA from the tet operator.

A further group of pharmacologically controlled synthetic promoters is rep-resented by the hormone-inducible promoter systems. Here, the RTA consistsof the yeast Gal4 DBD fused to a hormone receptor and the VP16 TAD orthe KRAB repression domain. DNA binding of the RTA, and consequentlygene expression or repression, are induced, for instance, by the progesteroneanalogue RU486. This component, but not endogenous progesterone, binds toa truncated progesterone receptor module of the RTA.

The rapamycin inducible or dimerizer system is triggered by an RTA con-sisting of two subunits, a DBD and a TAD fusion protein. These fusion pro-teins dimerize in the presence of rapamycin, thus reconstituting a functionalRTA capable of inducing expression of the gene of interest via a correspondingDNA element.

Of note, spatial targeting of inducible transcriptional control is feasible byexpressing the drug responsive RTA from an appropriate tissue-specific pro-moter (Smith-Arica et al., 2000).

Amplification of Promoter Activity

A further application of RTAs is the amplification of the activity of specificbut weak promoters in order to obtain gene expression appropriate for ther-apeutic purposes. In this scenario, the weak promoter drives expression of astrong RTA (harboring, for example, the powerful HSV VP16 transactivation


domain). In a second construct, an RTA-responsive promoter drives expres-sion of the gene of interest (Fig. 22.2B and C). This strategy is more generallyapplicable than the deletion/ multimerization approach for enhancing promoteractivity, since the latter depends on the presence of defined promoter elements.However, as even low-level expression of the powerful RTA in nontarget cellswould result in loss of specificity, the “tightness” of the applied tissue-specificpromoter is critical for the development of an RTA-driven promoter amplifi-cation system. Segawa et al. used the PSA promoter to drive expression ofa Gal4-VP16 fusion protein. This RTA then activates transgene expressionthrough Gal4 binding sites placed upstream of a minimal tk promoter, resultingin enhanced transgene expression (Fig. 22.2B; Segawa et al., 1998).

In a different system, the specific promoter drives expression of both thegene encoding the RTA, here a LexA-DBD/ VP16-TAD fusion protein, andthe transgene. Incorporation of multimeric LexA binding sites upstream of thespecific but weak promoter results in specific amplification of gene expression.With this approach both the endothelium-specific vWF and the gastrointestinal-specific sucrase isomaltase promoters were enhanced >100-fold in target cells.No increase in transgene expression over the activity of promoterless constructswas observed in control cells, resulting in high levels (>1000-fold) of speci-ficity (Nettelbeck, Jerome, and Muller, 1998). The tightness of this approachwas achieved by two mechanisms conveying specificity: (1) specific expres-sion of the RTA and (2) restriction of RTA-mediated transactivation to targetcells by the specific promoter (Fig. 22.2C).

Dual-Specific Transcriptional Control Combining Tissue-Specific andCell Cycle-Regulated Gene Expression

Unrestrained proliferation is a hallmark of cancer. Thus, anticancer therapeuticsfrequently target proliferating cells. Limitations of this strategy are imposed by(1) the resistance of tumor cells not proliferating at the time of therapy and (2)a dose-limiting toxicity due to adverse effects on healthy proliferative tissues,such as the bone marrow and the epithelium of digestive organs. In gene ther-apy, transcriptional targeting strategies can potentially circumvent these draw-backs by constructing proliferation-dependent vectors rather than effectors byutilizing cell cycle regulated promoters (Nettelbeck et al., 1998). To target non-proliferating tumor cells, effector systems with a local bystander effect on non-proliferating neighboring cells can be utilized. Furthermore, the dual-specificitystrategy combining tissue-specific and cell cycle-regulated transcription controlcan abrogate toxicity to proliferating nontarget tissue.

Two generally applicable dual-specificity designer promoter systems havebeen described. The first approach (Fig. 22.3A) is based on a heterodimericRTA, which drives expression of the therapeutic gene. Dual-specificity resultsfrom tissue-specific expression of the first RTA subunit, here by the optimizedtyrosinase promoter described previously, and proliferation-specific expression

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of the second subunit, here by the cyclin A promoter (Jerome and Muller, 1998,2001). The second dual specificity system (Fig. 22.3B) is based on the mech-anism of cell cycle dependent transcriptional repression of the cyclin A pro-moter. Here, the RTA, a fusion protein of the Gal4-DBD and the TAD of thetranscription factor NF-Y, is expressed under control of the tissue-specific opti-mized tyrosinase promoter. In the target tissue, the RTA subsequently binds tomultimerized Gal4 binding sites replacing the upstream regulatory sequence ofthe cyclin A promoter. Cell cycle regulated gene expression is mediated by atranscriptional repressor, which binds to the cyclin A core promoter specificallyin non-proliferating cells. This repressor specifically inhibits transactivation bythe NF-Y transactivation domain. For this system, a cell cycle specificity simi-lar to that of the wild-type cyclin A promoter, but restricted to melanoma cells,has been achieved (Nettelbeck, Jerome, and Muller, 1999).

The described RTA-dependent promoter systems involve the expression ofnonhuman proteins, which are potentially immunogenic in patients. For clini-cal gene therapy it may be advantageous to avoid an immune response to thetransduced cells. To this end, it has been reported that the viral VP16 domaincan be replaced without loss of activity with the TAD of human p65, a sub-unit of the transcription factor NFkB (Rivera et al., 1996). On the other hand,RTAs with a DBD of a human transcription factor could bind to their cognateDNA sites in the genome of transduced cells potentially triggering detrimentalinterferences with endogenous gene expression. Recently, human zink-fingerderived DBDs with altered DNA sequence specificities have been engineeredby exchanging only a few amino acids. These DBDs combine both lowerimmunogenicity than viral DBDs with high specificity for the transgenic targetsequence—an 18 bp sequence of choice—relative to genomic sites (Xu et al.,2001).

Figure 22.3 Dual specificity promoter systems: combining tissue specificity and cellcycle regulation by utilization of RTAs. (A) A DNA-binding subunit expressed from atissue-specific promoter and a transactivating subunit expressed from a cell cycle-reg-ulated promoter interact to form a heterodimeric RTA via fused dimerization domains.Expression of the functional, heterodimeric RTA is therefore restricted to proliferatingcells of a certain tissue type. The RTA binds to Gal4 binding sites (bs) of a syn-thetic reporter/ effector construct and activates gene expression from the downstreamcore promoter (Jerome and Muller, 1998, 2001) (B) An RTA that consists of the Gal4DNA-binding domain fused to the NF-YA transactivation domain is expressed froma tissue-specific promoter and binds constitutively to Gal4 binding sites of a syntheticreporter/ effector construct. The transcriptional repressor CDF-1 binds to the down-stream CDE/ CHR element specifically in the G0/ G1 phases of the cell cycle andinhibits transactivation by NF-YA. As a consequence, expression of the transgene isrestricted to proliferating cells of a specific type (from Nettelbeck, Jerome and Muller1999).


THE CRE/ LOX SYSTEM

The bacteriophage P1-derived Cre/ lox system consists of the Cre recombinaseand a pair of loxP DNA elements. The Cre recombinase mediates site-spe-cific excisional deletion of a DNA sequence that is flanked by a pair of loxPsequences resulting in a residual loxP site left in the original DNA sequenceand the excised DNA circularized at the loxP site (Fig. 22.4). For transcrip-tional targeting in gene therapy, the Cre/ lox system is utilized for these con-ceptual strategies (see Fig. 22.4): (1) as an ON switch for the amplificationand prolongation of promoter activity (gene activation strategy) or (2) as anOFF switch to selectively delete a target gene (gene inactivation strategy).

The Cre/ lox Gene Activation Strategy

The Cre/ lox ON switch (Fig. 22.4A) has been described in several recent genetherapy studies. In this scenario, the reporter or therapeutic gene of interestseparated from a strong (and constitutive) promoter by a transcriptional stopcassette, that is a marker gene or a polyA transcription termination sequence,flanked by a pair of loxP sites. In a second construct, the specific promoter ofchoice drives expression of the Cre recombinase. Activity of the specific pro-moter in target cells results in expression of Cre and subsequent excision of thestop cassette in the reporter/ therapeutic construct leading to strong and consti-tutive expression of the gene of interest. By means of this strategy, thyroglobu-lin or CEA promoter driven HSV-tk/ GCV therapies of thyroid carcinoma andadenocarcinoma, respectively, have been improved significantly without loss ofspecificity (Nagayama et al., 1999; Kijima et al., 1999). In a different context,the Cre/ lox ON system switches the transient activity of a radiation-activatedpromoter into enhanced and persistent expression of the gene of interest (Scottet al., 2000).

Figure 22.4 Switching genes ON and OFF with the Cre/ lox system. (A) In theCre/ lox gene activation strategy a transcription stop cassette flanked by loxP sites isdeleted by Cre recombinase selectively in target cells where the specific promoter thatdrives Cre expression is active. This strategy results in strong and prolonged expres-sion of the gene of interest. In cells not targeted by the specific promoter, therapeuticgene expression is prevented by the transcription termination signal, the stop cassette,which is located between constitutive promoter and gene of interest. (B) In contrast,in the Cre/ lox gene inactivation strategy the gene of interest is excised by Cre recom-binase. This strategy has been recently applied for the development of conditionallyreplicative adenoviruses. Here, Cre expression is controlled by a p53 responsive pro-moter resulting in excision of the essential E1A gene from the virus genome in healthycells (p53 wild-type), but not in p53 mutant tumor cells. Thus, the virus specificallyreplicates in tumor cells that lack functional p53 (Nagayama et al., 2001).

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The Cre/ lox Gene Inactivation Strategy

The Cre/ lox gene inactivation strategy (Fig. 22.4B) has been applied to thetranscriptional targeting of lytic adenovirus replication to p53 mutant tumorcells. p53, a transcriptional activator, is mutated, deleted, or inactivated inroughly 50% of human tumors. Flanking the essential viral E1A gene by apair of loxP sites and regulating Cre expression with a p53-responsive pro-


moter results in deletion of the viral gene and loss of viral replication capacityin nonmalignant, p53 wild-type cells. In contrast, in tumor cells without func-tional p53, the virus stays intact and retains its replicative potency, resultingin oncolysis (Nagayama et al., 2001).

NONPROMOTER ELEMENTS FOR OPTIMIZED TRANSCRIPTIONALTARGETING AND PROMOTER FIDELITY IN VIRAL VECTORGENOMES

Noncoding DNA Sequences Other Than Promoters, Enhancers, andSilencers

Noncoding DNA sequences other than promoters, enhancers, and silencers haveshown benefit for gene therapeutic purposes. For instance, gene expression canbe increased by DNA elements mediating posttranscriptional events, as exempli-fied by the incorporation of an intron into the nontranslated region of the thera-peutic gene. Increased gene expression results from splicing of the intron-con-taining pre-mRNA, as splicing can promote mRNA export and translation. Nev-ertheless, the activity of such elements depends on the candidate gene (Scham-bach et al., 2000) and has to be reevaluated in the context of a given promoter.

Virus-derived internal ribosome entry site (IRES) sequences are other ele-ments of interest for transcriptional targeting in gene therapy. These sequencesmediate cap-independent internal translation initiation. By this means, a sin-gle specific promoter can drive expression of two transgenes via a bicistronicIRES containing mRNA (Harries et al., 2000). However, the activity of IRESsequences depends on the fused cDNA and in some instances it can be advan-tageous to apply two promoters rather than an IRES sequence (Nettelbeck,Jerome, and Muller, 1998).

Noncoding sequences not triggering transcriptional control can also mediate(or improve) specific gene expression. This possibility was revealed for anelement located in the 3′ nontranslated sequence of hypoxia-dependent genesthat regulates RNA stability in a hypoxia-dependent manner (Damert et al.,1997; Boast et al., 1999; Shibata, Giaccia, and Brown, 2000).

Promoter Fidelity in the Context of the Vector of Choice

Crucial for the application of transcriptional targeting in gene therapy is thefidelity of promoters in the context of the vector of choice (vector compatibilityof a candidate promoter). Viral vectors contain sequences with transcriptionalregulatory activity, which can be detrimental to the fidelity of a candidate-specific promoter, as reported for adenoviral and retroviral vectors (Vile etal., 1994, 1995; Ring et al., 1996; Shi, Wang, and Worton, 1997). Whereassome promoters might not be influenced by viral sequences, others lose theirspecificity in the vector context.

CONCLUSIONS 497

A potentially detrimental adenoviral regulatory element is the E1Aenhancer, which cannot be deleted, as it overlaps with the packaging signal. Itis located in proximity to the conventional site of transgene incorporation. Inthis context, promoter interference has been avoided by two strategies: engi-neering of the adenovirus vector backbone or incorporation of insulator ele-ments. The first strategy has been realized by the creation of transgene incorpo-ration sites less prone to promoter interference in the virus genome (Rubinchiket al., 2001). Insulator elements are defined as DNA sequences that do notinfluence promoter activity per se, but block promoter activation by enhancerswhen placed between them. Insulator activity in the context of adenoviral vec-tors has been shown for a 1.2 kb element derived from the chicken beta-globinlocus (Steinwaerder and Lieber, 2000) and for the bovine growth hormonepolyadenylation signal (Vassaux, Hurst, and Lemoine, 1999).

In retroviral vectors, enhancer elements within the long terminal repeat(LTR) represent potential interfering sequences. Whereas some promotershave been shown to lose specificity in an internal position within the retro-viral genome, other reports demonstrate specific transcriptional control byreplacing the viral LTR enhancer with these or other promoters, for exam-ple the muscle creatine kinase enhancer (Ferrari et al., 1995), the tyrosinasepromoter/ enhancer (Vile et al., 1995; Diaz et al., 1998), the human prepro-endothelin promoter (Jager, Zhao, and Porter, 1999), or a synthetic hypoxia-responsive promoter (Boast et al., 1999). Transcriptional targeting in retroviralgene transfer faces a further level of promoter deregulation resulting from thechromosomal integration of the vector genome and interference by neighbor-ing chromosomal elements. In this regard, flanking the transgene by matrixor scaffold attachment regions (MARs or SARs) or by a locus control region(LCR) can result in position-independent transgene activity after genomic inte-gration (McKnight et al., 1992; Kalos and Fournier, 1995; Dang, Auten, andPlavec, 2000; Kowolik, Hu, and Yee, 2001).

CONCLUSIONS

There is no doubt that vector optimization and vector specificity are essentialfor translating gene therapy concepts into successful clinical regimens. To thisend, transcriptional targeting is a powerful strategy for mediating pharmaco-logically, physiologically, or spatially regulated gene therapy. Furthermore, theconditionality of viral replication required by the recent therapeutic approachof viral oncolysis can be achieved by transcriptional targeting. Preclinical stud-ies have proven the feasibility of transcriptional targeting for mediating effec-tive and selective gene therapy and viral oncolysis, resulting in significantlyreduced toxic side effects. Clinical studies applying transcriptional targetingare currently underway.

Based on the rapid progress in research on transcriptional regulation in recentyears, a plethora of novel strategies for the optimization of promoter activity


and for the development of artificial promoters has been successfully estab-lished. These strategies resulted in reduced promoter leakiness and in increasedpromoter activity and specificity in different vector systems. The utilization ofthis transcriptional targeting science, in combination with novel technologies todetermine disease signatures, might promote the derivation of a new generationof targeted vectors and their application in molecular medicine.

ACKNOWLEDGMENTS

We are grateful to Joel Glasgow for critical reading of the manuscript. Researchin the authors’ laboratories was supported by grants from the National Can-cer Institute R01 CA74242, R01 CA86881, N01 CO-97110, R01 CA68245,P50 CA89019, R01 HL50255, and from the Juvenile Diabetes Foundation 1-2000-23 to DTC; and from the Deutsche Forschungsgemeinschaft and the Dr.Mildred Scheel Stiftung to RM. DMN is supported by a postdoctoral fellow-ship from the Deutsche Forschungsgemeinschaft.

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Vector Targeting for Therapeutic Gene Delivery (Curiel/Therapeutic Gene Delivery) || Promoter Optimization and Artificial Promoters for Transcriptional Targeting in Gene Therapy