Inducible control of gene expression: prospects for gene therapy

512

Inducible control of gene expression: therapy Diane M Harvey* A number of drug-regulated

and C Thomas

the use of small-molecule

mice, recent improvements

systems more amenable

rendering

are likely to make these

of the antiprogestin-regulated

it more sensitive to RU486,

of nonimmunosuppressive dimerization-based

While the in vitro and in

for use in a therapeutic

such as gene therapy. These improvements optimization

through

inducing compounds.

utility of such systems has been demonstrated transgenic

Caskey?

gene expression systems are

available for controlling target gene transcription

context,

include further gene switch,

and the synthesis

rapamycin analogs for use in

strategies

of gene regulation.

Addresses *Merck Research Laboratories, Merck & Co Inc WP26A-3000,

West

Point, PA 19486, USA; e-mail: [email protected] -j-Merck Research Laboratories, Merck & Co Inc PO Box 4, WP26-207, West Point, PA 19486, USA; e-mail: [email protected] Current

Opinion

in Chemical

Biology

1998,

2:512-518

http://biomednet.com/elecref/136759310020051 0 Current Biology Publications ISSN

2

1367-5931

Abbreviations CID

chemical inducer of dimerization

EcR FRAP hGH

ecdysone receptor FKBPl Z-rapamycin-associated human growth hormone

hPR HSV

human progesterone receptor herpes simplex virus

rtTA RXR

reverse tet transactivator retinoid X receptor

tet tTA

tetracycline tetracycline-controlled

prospects for gene

protein

transactivator

Introduction Development of gene therapy vectors that successfully incorporate elements to allow small-molecule control of gene expression will certainly have an impact on the safety and efficacy of future gene therapy protocols, as inclusion of such regulatable elements will afford control over the timing and levels of gene expression within a therapeutic range. Although initial attempts to inducibly regulate gene expression in eukaryotic cells involved the use of endogenous cellular promoters responding to heat shock, heavy metals or hormones, this type of approach is unsuitable for gene therapy because of pleiotropic activity of the inducing agent and high basal transcription in the uninduced state [ 11. Subsequently, inducible systems have been developed that combine functional domains from prokaryotic, eukaryotic and viral proteins to create chimeric transactivators

capable of modulating gene expression in a drug-dependent manner [Z-5]. Such transactivators contain functional element(s) that interact with a small-molecule inducing compound, a DNA-binding domain that does not exhibit cross-reactivity with endogenous cellular sequences, and a transactivation domain. The second component of the regulatable system is an inducible promoter, which most often consists of a minimal promoter linked downstream to repeats of the transactivator recognition sequence. Thus, in the presence of inducer, the chimeric transcription factor should bind specifically to its DNA recognition sequence within the inducible promoter to transactivate expression of the target gene. Ultimately, a number of requirements will need to be fulfilled before regulated systems are included in human gene therapy vectors. First, components of the system should not interfere with endogenous cellular processes. Second, low basal activity and dose-responsive transcriptional inducibility will be important for therapeutic application. Third, the inducer will preferably be an orally bioavailable, physiologically inert compound that is cleared from body tissues within a reasonable length of time. Finally, because of the potential host immune response, the transactivator protein(s) should exhibit low immunogenicity. This review summarizes recent developments in drug-inducible gene expression systems that may be considered for use in gene therapy.

Tetracycline-regulated

systems

A regulatable gene expression system incorporating elements of the tetracycline-resistance operon encoded by the bacterial transposon TN10 was originally described by Gossen and Bujard [6]. In this system, the tetracycline (tet) repressor was fused to a herpes simplex virus (HSV) VP 16 transactivation domain to form a tet-controlled transactivator (tTA). This chimeric protein could transactivate transcription in mammalian cells from a tet-responsive control element consisting of tet operators fused to a minimal promoter. In the presence of tet, tTA is unable to bind operator sequences to activate transcription, making this a tet-repressible system. An inducible system was developed following the successful isolation of tet repressor mutations conferring a reverse phenotype [7]. In contrast to its wild type counterpart, the reverse tet repressor requires tet or a derivative such as doxycycline, to bind tet operator sequences. Correspondingly, the reverse tet transactivator (rtTA) activates gene expression in the presence of either drug, rendering the system more suitable for therapeutic applications (Figure 1).

Inducible

Figure

control

of gene expression:

was apparent over time.

1

PCM”

r [m...............fZ_ rtetR

VP16

f

._

IIIIIIIIC

hCMV,i,

r

Target gene

B

(tet 0)x7 Current Opinion in Chemical Biology

Reverse tet-regulated

1

gene expression. The rtTA consists of the

reverse tetracycline repressor (rtetR) fused to a VP1 6 transactivation domain. In this example, constitutive expression of rtTA is driven by the human cytomegalovirus enhancer (PCMV). In the presence of doxycycline (Dox), rtTA binds and transactivates transcription from the inducible promoter consisting of seven tet operator sites (tet 0) located upstream of the human cytomegalovirus immediate early minimal promoter (hCMV,i,).

Both the original and reverse tet-regulated systems have performed well in vitro, with reported induction levels of gene expression ranging from three to four orders of magnitude above basal levels [6-lo]. Recent experiments describing the behavior of the reverse tet-regulated system in transgenic mice have demonstrated that, although tight regulation of gene expression with attendant transcriptional inducibility can be achieved in some organs, there is a wide range of variation in others. Kistner et nl. [l l] reported a steady state 105-fold induction of reporter activity in pancreatic tissue of transgenic mice receiving doxycycline in drinking water. Varying levels of induction were observed in other organs, however, which was attributed to positional effects on the inducible promoter that are independent of rtTA. Retroviral vectors capable of transducing the reverse tetregulated system have also been described [ 12”, 13’, 141. These vectors permanently integrate their therapeutic transgene(s) into the host genome of infected cells. Doxycycline-regulated control of erythropoietin secretion was maintained for a period of five months in C3H mice receiving implants of primary myoblasts transduced by retroviral vectors carrying rtTA and inducible erythropoietin [lZ**]. C3H is an inbred strain of mice with an intact immune system. The induction profile was limited to about 70-fold because of background transactivation (that is, in the absence of drug). In similar cell implantation experiments, Lindemann et nl. [13*] reported persistence of doxycycline-inducible reporter activity in C3H recipients for 46 days. Basal activity of reporter gene expression under the inducible promoter

for gene therapy

directly

after

Harvey and Caskey

implantation,

but

513

decreased

As inducing agents, tet and its derivatives are orally bioavailable and efficiently penetrate tissues throughout the body [15]. Because tet deposits in bone [16], its slower rate of clearance in viva may affect the reversibility of inducible gene expression. Another concern is potential immunogenicity associated with the prokaryotic and viral components from which rtTA is derived. Interestingly, Bohl et nl. [lZ**] reported no histological evidence of inflammatory infiltrates in muscles of C3H mice

Dox 0

:$ 88

prospects

receiving transduced primary myoblasts expressing rtTA. Furthermore, no circulating antibodies to the tet repressor were detected in the sera of these animals, suggesting that rtTA expression is well tolerated in C3H mice.

Ecdysone-inducible

system

The insect moulting hormone ecdysone stimulates metamorphosis in Drosophila melanogaster. In Drosophila, the functional receptor for this steroid hormone is a heterodimer of the ecdysone receptor (EcR) and the product of the ultraspiracle gene (USP). As a member of the nuclear receptor superfamily, EcR binds ecdysone-response elements in the presence of hormone to transactivate transcription of genes containing these elements. Early systems designed to inducibly regulate gene expression in mammalian cells involved coexpressing the genes for EcR and USP, with an ecdysone-responsive reporter construct. Initially, induction levels were quite low (-three to tenfold) in the presence of ecdysone [17,18]. Increased induction of reporter activity ranging from lOO-150-fold was observed when a chimeric derivative of the EcR gene was coexpressed with USP or with one of its vertebrate counterparts, the retinoid X receptor (RXR) gene-a or y-in the presence of muristerone A, which is a synthetic analog of ecdysone [ 181; moreover, 3000-fold induction of reporter gene activity mediated by chimeric EcR gene expression has been reported in human 293 cells induced with 1 pM muristerone A [19]. More recently, No et al. [ZO] have optimized components of the ecdysone-inducible system to achieve induction levels of over four orders of magnitude above basal levels in stable cell lines induced with 1 pM muristerone A. The optimized system coexpresses human RXR-a and an EcR/VP16 fusion protein (Figure 2). To reduce the possibility of basal transcription from the inducible promoter because of recognition by potentially cross-reactive endogenous nuclear receptors, the DNA-binding specificity of the hybrid receptor was altered to recognize a novel element (E/GRE) consisting of ecdysone and glucocorticoid response element half-sites. No et al. [ZO] established the applicability of this system in viva by demonstrating muristerone-A-dependent reporter activation in transgenic mice expressing the optimized receptor heterodimer.

5 14

Next generation

Figure

2

therapeutics

progesterone, it retains RU486 [26,27].

pCMV

r ........VgEcR .‘,,.

.:.:.:;;;;

In developing an inducible system, a transactivator (GLVP) consisting of a HSV VP16 transactivation domain fused to a yeast GAL4 DNA-binding domain and the truncated hPR LBD was constructed. The inducible promoter was composed of a series of GAL4 recognition sequences (which should not bind endogenous mammalian proteins) upstream of the adenovirus ElB TATA box. Initial experiments with this system involved transient

q

PRS EcR

muristerone

A

-Y

I-+ IIIIIII

A HSPmin

Target

gene

-

(E/GRE)x4 Current Opinion in Chemical Biolqy

Ecdysone-regulated gene expression. The functional receptor heterodimer consists of the human RXRo (hRXRo) and a chimeric protein (VgEcR) consisting of a modified EcR fused to a VP1 6 transactivation domain. Constitutive expression of these proteins is driven by Rous sarcoma virus long terminal repeats (pRSV) and human cytomegalovirus enhancer promoter (pCMV) sequences. In the presence of ecdysone or its synthetic analog muristerone A, EcR binds to hybrid response elements consisting of ecdysone and glucocorticoid response element half-sites (E/GRE) fused to a Drosophila heat shock protein minimal promoter (AHSPmi,) transactivate gene expression.

to

transfection assays and cell implantation in rats and mice in order to determine induction of gene expression in response to RU486. Although overall induction of reporter gene expression was relatively modest (50-fold in transient assays and tenfold in the rat model), transcriptional activation in response to RU486 administration was found to be rapid and sensitive to low concentrations of drug. In vitro, expression of reporter activity could be detected within two hours at RU486 concentrations as low as 0.1 nM, with maximal induction being reached at a concentration of 1 nM. Subsequent experiments testing efficacy of the system in viva utilized transgenic mice expressing the GLVP transactivator and an inducible human growth hormone (hGH) transgene [28”]. In these mice, hGH expression was induced 1500-fold and 3500-fold in a dose-dependent manner following oral administration of RU486 at concentrations

As a means of regulating gene expression within the context of gene therapy, the ecdysone-inducible system presents several advantages and disadvantages. Although No et nl. [ZO] did not directly quantify induction of reporter expression in transgenic mice, basal transcription in the absence of inducer appears to be quite low. In addition, muristerone A is a lipophilic compound that should distribute efficiently in tissues throughout the body; it also appears to be nontoxic, nonteratogenic and has no known effect on mammalian physiology. One disadvantage is the requirement of this system for RXRa expression, which is also an endogenous cellular receptor. Overexpression of RXRa may exert pleiotropic effects by interfering with endogenous signal transduction pathways that respond to retinoid and thyroid hormones, as well as to vitamin D [‘Z-23]. Another disadvantage is the potential immunogenicity associated with EcR and VP16 elements of the chimeric receptor. If recognized as foreign, they could mount an immune response against transduced cells.

Antiprogestin-regulated

the ability to bind the antiprogestin

gene switch

The human progesterone receptor (hPR) is another member of the nuclear receptor superfamily adapted for use in a drug-inducible gene expression system. The original system utilized an hPR with a truncated ligand-binding domain (LBD) [24,25]. Although this mutant form of hPR is no longer able to bind to its natural ligand

of 250 pg/kg

and 500 pg/kg,

respectively.

Further modification of the GLVP increased the transcriptional activation sitivity of the system to inducing Extending the LBD and repositioning

transactivator has potency and sencompound [29**]. the VP16 trans-

activation domain at the carboxyl terminus has produced an optimized transactivator (GLVPc’) capable of activating target gene expression at RU486 concentrations as low as 0.01 nM (Figure 3). As an inducing compound, RU486 is an orally bioavailable, lipophilic hormone with well-characterized pharmacokinetic properties [30]; to be successfully integrated into future gene therapy protocols, however, RU486-mediated induction of gene expression will need to occur at concentrations well below those at which RU486 exhibits anti-progesterone or anti-glucocorticoid activity [31]. Both the original and modified systems apparently achieve this, with maximal induction of gene expression occurring at levels lOO-lOOO-fold below those at which RU486 functions as a progesterone antagonist. Similar to other transactivators described here, functional components derived from yeast and viral proteins may also be immunogenic.

Dimerization-based

regulation

system

The development of regulatable systems utilizing chemical inducers of dimerization (CIDs) stems from earlier studies delineating the mechanism of action of immunosuppressant compounds such as FK506, rapamycin and cyclosporin A (GSA). These drugs inhibit signalling pathways

Inducible

Figure

control

of gene expression:

for gene therapy

Harvey and Caskey

member of the FK506-binding protein family), further nonproductive interactions [37,38].

3

GAL4

HPR-LBD

VP1 6

RU486 0

f

r

nnnn_E’B,i,

Target gene

_

Current Opinion in Chemical Biology

Antiprogestin-regulated gene expression. The optimized transactivator GLVPc, consists of a yeast GAL4 DNA-binding domain fused to human progesterone receptor ligand binding domain and a VP1 6 transactivation domain. In the presence

of RU486, GLVPc, binds to an inducible promoter consisting of four GAL4 DNA-binding sites fused to the adenovirus El B minimal (El B,i,) promoter to transactivate gene expression.

affecting T cell activation and proliferation by mediating the dimerization and inactivation of cellular proteins involved in these processes [32-341. Chemical-binding domains from such cellular proteins, which include the FK506-binding proteins (FKBPs) and the cyclophilin family of proteins, as well as FKBPlZ-rapamycin-associated protein (FRAP), can be used to mediate dimerization of physically distinct DNA-binding and transactivation elements to create an inducible system. [35] reported the synthesis of a hetcontaining protein-binding surfaces from

FK506 and GSA, termed FKCsA. The authors demonstrated the use of FKCsA as a transcriptional regulator in an expression system consisting of physically separate DNA-binding and transactivation domains that dimerized to form a functional transactivator in the presence of FKCsA. With this system, 5000-fold induction of reporter activity from its inducible promoter was observed, with low basal transcription in the uninduced state. Ho et nl. [36] employed FK506, FK1012, as an

a homodimeric derivative inducer of dimerization

to

fusion protein that binds a composite recognition sequence [40] fused to a series of three FKBP12 repeats. These proteins associate in the presence of rapamycin to activate transcription from an inducible promoter containing ZFHDl-binding sites upstream of an human

(GAL4)x4

Belshaw et nl. erodimeric CID

leading

515

Rivera et nl. [39] employed another dimerization-based strategy to develop a humanized system for inducing gene expression at levels up to four orders of magnitude above uninduced (Figure 4). In this system, the transactivation domain is provided by the carboxy-terminal region of the NFKB p65 protein, which is fused to the rapamycinbinding domain of FRAP. The DNA-binding domain, termed ZFHDl, is a composite zinc finger-homeodomain

pCMV

a truncated (HPR-LBD)

prospects

of for

regulation of gene expression. Induction levels were lower than those reported for systems utilizing heterodimeric CIDs, which is probably due to the formation of nonproductive DNA-binding-domain homodimers or transactivation-domain homodimers, as these proteins contain identical drug-binding surfaces. As with the system developed by Belshaw et nl. [35], these chimeric proteins might also interact with endogenous cellular immunosuppressant-binding proteins such as FKBP 12 (a

cytomegalovirus minimal promoter. Upon administering rapamycin to nude mice implanted with cells stably expressing inducible hGH, peak levels of expression were detected within 24 hours, with no detectable expression observed in the absence of inducer [41”]. Overall induction levels were found to be dependent on a number of parameters, including the administered dose of rapamycin. With respect to gene therapy, one advantage of such a regulatable system is that its components are derived from human proteins, thus minimizing the potential for host immune recognition. Another advantage is that the system affords tight control over gene expression in a dose-dependent manner, with apparently low basal activity and high transcriptional inducibility. One disadvantage currently associated with the system is the immunosuppressive nature of rapamycin, making it unsuitable for long-term therapeutic use. Liberles et nl. [42”] have recently reported the synthesis of nonimmunosuppressive rapamycin derivatives with significantly diminished binding affinity for cellular kinase FRAP, which may circumvent problems associated with toxicity of the inducing agent. A mammalian three-hybrid screen was used to identify compensatory mutations in the FKBPlZ-rapamycin binding domain of FRAP that restore binding to the rapamycin derivatives. As these derivatives still bind endogenous FKBP12 [43], nonproductive dimer formation with this ubiquitous cellular protein can still occur.

Conclusions

and future prospects

A number of reports have successfully demonstrated how drug-regulated gene expression systems can be used to study gene function in cell culture and in transgenic mice. For studies in vitro, it is often possible to screen individual cell clones to identify those cells that have the highest levels of inducible gene expression and the lowest basal activity. Similar screening methods can be applied to the generation of transgenic mice, which commonly involves the selection of founder lines that show favorable expression characteristics.

516

Next generation

Figure

4

(a)

therapeutics

hCM”

r

..........

.................... :.:.:.:.:.:.:.:.:.:. :_ yz3’

-e

NLS

ZFHDl

(b)

hCMV

x

NLS

FRB

~65

Rapamycin *

f

3

-(ZFHDl

r Target gene

hCMv,i,

sites)xlz CurrentOpinion in Chemical Biology

Rapamycin-regulated finger-homeodomain

gene expression. (a) The DNA-binding domain (ZFHDl) of the functional transactivator is a composite zinc chimera fused to three FKBPl2 repeats (shown as FKBP). (b) The transactivation domain consists of the carboxyterminal

portion of the NFKB p65 protein (shown as ~65) fused to the FKBPl Z-rapamycin binding (FRB) domain from the cellular protein FRAP. NLS denotes nuclear localization signals. In the presence of rapamycin, the DNA-binding domain and the transactivation domain dimerize via FKBP and FRB to transactivate gene expression from an inducible promoter consisting of 12 ZFHDl minimal promoter (hCMV,i,).

As a tool for gene therapy, performance of regulated systems will probably be influenced by the choice of vector. It is likely that methods of gene delivery involving integration into the host genome will be subject to the same position effects described in transgenic mice. It is unlikely that many gene therapy protocols will allow the selection of clonal cell populations with the appropriate induction profile, thus highlighting the need to maintain tight control of gene expression independent of the integration site. Inclusion of chromosomal insulator sequences into integrating vectors [28”,44,45] may circumvent this problem, allowing position-independent transgene expression regardless of chromosomal location. Similarly, basal expression may be minimized in nonintegrating vectors by flanking sequences with polyadenylation signals to terminate spurious transcription readthrough [46,47]. Although the tet-regulated systems are the most well-characterized with respect to viral vectors, incorporation of other regulated systems into vectors of potential use for gene therapy (such as retroviral and adenoviral vectors) should give a better indication of how well they function in that context. The initiation of primate studies will concerns relating to immunogenicity

of these compounds will disrupt their interaction with endogenous FKBP 12. Likewise, modification of RU486 or other progesterone antagonists may produce derivatives that have decreased anti-progesterone and anti-glucocorticoid activity.

References

and recommended

reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

. ..

of special interest of outstanding interest

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in mammalian

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Wang Y, DeMayo FJ, Tsai SY, O’Malley BW: Ligand-inducible and liver-specific target gene expression in transgenic mice. Nat Biofecbnol 1997, 15:239-243. The RU486-inducible system described in [211 is evaluated in double transgenie mice expressing the GLVP transactivator (a transactivator consisting of a VP16 transactivation domain fused to a GAL4 DNA-binding domain and a truncated human progesterone receptor ligand binding domain) in a liver-specific fashion. Significant dose-dependent induction of human growth hormone secretion was noted when mice received the progesterone antagonist RU486 orally or through intraperitoneal injection. The use of chromosomal insulator sequences to minimize position effects on GLVP transactivator expression is also described. 29. ..

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design of

518

Next generation

therapeutics

Magari SR, Rivera VM, Luliucci JD, Gilman M, Cerasoli F Jr: Pharmacologic control of a humanized gene therapy system implanted into nude mice. J C/in invest 1997, 100:2865-2872. The rapamycininducible system described in [361 is used to demonstrate regulated human growth hormone secretion in a cell implantation model in nude mice. These experiments demonstrate that a number of parameters, such as the activity and pharmacokinetics of rapamycin, as well as responsiveness of the cells to inducing compound and number of cells implanted, influence levels of gene expression upon rapamycin stimulation.

Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBPl2-FRAP interface. Chem Biol 1995, 2:471-481.

41. ..

42. ..

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