Regulatable Gene Expression Systems for Gene Therapy Applications: Progress and Future Challenges

Viral Vectors

Viruses can easily enter cells and deliver their genetic material into the nucleus of target cells; therefore, they are in most cases more efficient than nonviral delivery systems (Fig. 1). Most vectors used for gene delivery are derived from human viral pathogens that have been made nonpathogenic by deleting essential viral genes. They usually have a broad tropism; therefore they can infect and deliver their encoded transgenes to a wide spectrum of cells and/or tissues. The most commonly used viral vectors for gene therapy are adenovirus (Ad), adeno-associated virus (AAV), herpes simplex virus type 1-derived vectors (HSV-1), and retrovirus/lentivirus vectors (Table 1). We will briefly discuss the main groups of viral vectors currently being developed for gene therapy applications. These vectors have also been used to encode regulatable gene expression systems.

Nonviral Vector-Mediated Gene Delivery

In trying to circumvent safety issues inherent to the use of viral vectors, development of numerous nonviral vectors is actively being pursued. The underlying principle of nonviral vector systems is to complex DNA that carries a therapeutic gene with molecules that will facilitate DNA entry into the cells of interest. Complexed DNA binds to the cell membrane, triggering either nonspecific or receptor-mediated endocytosis. Upon entry into the cell these complexes are contained in endosomes. The ability of these complexes to escape from endosomes before lysosomal enzymes destroy them is an essential characteristic of a successful nonviral vector. Once released from the endosomes, these complexes must enter the nucleus to undergo transcription. To be successfully transcribed, complexed DNA must be released from its carrier molecules and stably express RNA. Aside from avoiding the issues of viral safety, nonviral vector DNA remains episomal, allowing long-term and also high levels of gene expression, i.e., in muscle cells for Duchenne muscle dystrophy [70], glial cells and primary neurons [71], fibroblasts for lysosomal storage disorders [72], glial cells for cerebral ischemic diseases [73], and glioblastoma cells [74]. There are several types of nonviral vector systems being explored to find optimal carrier systems. Although uncomplexed DNA has been successfully used to transfect skeletal muscle [75], systemic administration has been unsuccessful due to clearance of DNA by serum nucleases [76,77].

The majority of nonviral vector systems use cationic lipids, polymers, or both as carriers. In these systems negatively charged DNA is strongly attracted to the lipid or polymer, and this interaction causes condensation of the DNA [78,79]. Cationic lipids, which self-assemble into vesicles that adsorb DNA, are known as lipoplexes. The stability of these lipoplexes is dependent on the lipid:DNA ratio [80]. Since lipoplexes are electrostatically, but nonspecifically, attracted to cellular membranes, the specificity of this carrier is low.

Polymers like poly-L-lysine (PLL) and polyethylenimine (PEI) have been used in vitro to introduce DNA more efficiently into cells. The PLL:DNA ratio is essential for successful condensation and efficient intracellular release from endosomes [80]. However, the use of PLL as a carrier agent is limited due to its toxicity. To circumvent this toxicity and prevent rapid clearance from serum, modifications to the basic PLL—DNA complex via conjugation to other substances have been explored. Conjugation to polyethylene glycol (PEG) chains, 2-hydroxypropyl methacryl amide polymer, low-density lipoprotein, and palmitoyl—PEG have all produced more efficient transduction of target cells [81-86]. The targeting specificity of PLL complexes can be increased through the use of specific receptors, antibodies, sugars, peptides, or growth factors [87-95].

PEI polymers are highly branched molecules with an excellent buffering capacity, making efficient the release of DNA from endosomes. As with other polymer systems, the PEI:DNA ratio is an essential consideration for the toxicity and stability of the vector. Conjugation, as with PLL, can improve cell targeting and uptake. Carbohydrates, integrin-binding peptides, transferrin, and antibodies have been successfully used to increase uptake and targeting specifically for PEI [96-100]. While PLL and PEI have been extensively studied, several other cationic polymers are under investigation, including dendrimers [101], Polybrene [102], gelatin [103], tetraminofullerene [104], poly-L-histidine—graft—poly-L-lysine [105], and chitosan [106].

While nonviral vectors are easy to produce and have low immunogenicity, issues of toxicity, specificity, and transfection efficiency must be addressed before wide clinical implementation will be possible.

Cancer

The majority of research into applications for regulatable gene expression vectors is in models of cancer. This is due, in part, to inadequacies of current therapies, including radiotherapy and chemotherapy. One strategy commonly employed is to regulate expression of a cytotoxic gene with a promoter that can be activated by ionizing radiation. The first example of successfully utilizing radiation to induce gene expression in a tumor model was described more than a decade ago. It was found that TNF-α expression, under the control of the Egr-1 promoter, could be increased in response to ionizing X-ray radiation and this was associated with an improved control of tumor growth in comparison with X-ray radiation alone [125]. Since then, other radiation-sensitive promoters, including VEGF, Rec-A, and WAF-1 promoters, have been investigated in preclinical tumor models [126-128]. Induction of gene expression by ionizing radiation offers a number of advantages over other inducible systems for treating cancer. These include the ability to control temporal and spatial gene expression within the ionizing radiation field, reducing damage to adjacent, healthy tissue. In addition, certain genes, including TNF-α and inducible nitric oxide synthase, have synergistic cytotoxic functions when utilized in combination with ionizing radiation [125,129].

The anatomy of growing tumors has been exploited by another commonly employed inducible gene therapy strategy. As a consequence of relatively poor vascularization, the center of most tumors is usually a nutrient-starved and hypoxic environment. Promoters activated by hypoxia or nutrient deficiency have been utilized to drive expression of tumoricidal genes. The glucose-regulated protein 78 promoter was used to express HSV-1 thymidine kinase (TK) and successfully eliminate murine fibrosarcomas [130]. HSV-1-TK was also used successfully to treat hepatocellular carcinoma when expressed by a hypoxic-responsive element/α-fetoprotein promoter [131]. Inducible promoters such as radiation-sensitive or hypoxia-sensitive promoters are highly specific, and ease of use makes them valuable tools for managing certain diseases. Unfortunately, the application of these promoters is limited and they are not suitable for treating the majority of diseases.

Ischemia

Hypoxia-driven promoters are also of great benefit against other diseases, most notably ischemia. Myocardial ischemia can be a chronic illness with repeated bouts of severe hypoxia and can eventually result in myocardial fibrosis and death. By placing hypoxia-responsive elements within the promoter MLC2v, researchers have developed a tissue-specific hypoxia-responsive promoter expressed predominantly in heart tissue that can induce reporter gene expression by up to 400-fold when expressed by adeno-associated viral vectors [132]. Similar vectors expressing VEGF have been demonstrated to increase angiogenesis, improve cardiac function, and reduce myocardial infarcts in mouse model of ischemia [133].

Diabetes

Diabetes is currently treated by daily injections of recombinant insulin to compensate for a deficiency in either production or function of insulin. Although successful for treating the disease, it can be expensive and can also result in a reduction of quality of life due to daily injections and imposed dietary restrictions. Inducible insulin expression by gene therapeutic vectors would circumnavigate many of the disadvantages of conventional insulin therapy. Researchers have investigated the ability to engineer hepatocytes to express and secrete a functional, modified insulin protein under the control of a rapamycin-responsive promoter. Using adenoviral-associated vectors to infect hepatocytes, gene expression was found to be negligible in the absence of rapamycin, but was potently and reversibly expressed in a mouse model of the disease when treated with rapamycin [134]. In a related study, glucose-responsive elements were used to drive insulin expression in hepatocytes in vitro and in vivo. The authors found that fasting blood glucose levels were returned to normal in a rat model of diabetes [135].

Arthritis

Arthritis affects 1% of the population and is primarily a disease of the elderly. It is characterized by chronic and painful inflammation at the joints of individuals. This chronic inflammatory response has been used by researchers attempting to treat symptoms of arthritis with inducible gene therapeutic vectors. Although cytokine-responsive promoters were successfully used to drive the expression of reporter genes in a model of arthritis [136], most current research is now focused on the robust Tet-ON regulatory system for treating this disease [137].

Dynamics of the Tet-OFF and Tet-ON Regulatory Systems

The first studies of Tet-regulatory transcriptional expression systems emerged from experimental studies on the prokaryotic bacterial strain Escherichia coli [138]. Since then, the utilization of Tet-inducible molecular switches to regulate the transcription of genes has become an effective and popular tool to control mammalian gene expression. The Tet-OFF system operates via a coordinated interaction of two important components (Fig. 2A): the Tet repressor protein (TetR) and the tetracycline-response element (TRE). The 23.6-kDa [139,140] protein TetR inhibits the transcription of genes in the tetracycline-resistance operon on the Tn10 transposon. In bacteria, the TetR hinders transcription of these genes by docking to the Tet operator sequences (tetO) in the absence of tetracycline. Fusion of the TetR to a viral protein (VP) domain, VP16, a eukaryotic transactivator derived from herpes simplex virus type 1, converts the TetR from a transcriptional repressor to an activator termed the transactivator (tTA). The tTA is operational under its own promoter and polyadenylation sequences and is autonomous of the TRE-driven transgene encoding cassette. A framework of seven tandem tetO sequences is positioned upstream of the immediate transgene initiation codon sequence and of the minimal cytomegalovirus (CMV) promoter. When the inducer binds to the transactivator, the complex activates the minimal human CMV promoter driving transgene expression. The tetO sequences and the minimal CMV promoter together constitute the TRE. Independent promoters, TRE and CMV or other cell-type-specific promoters, control the transgene and the transactivator. Gene regulation is achieved by the synthesis of transactivators by the tTA-encoded regulatory switch and their further interaction with the tetracycline inducer and TRE. The Tet-OFF system works by switching gene expression on and off in the absence or presence of the inducer, respectively. In the off situation, i.e., upon delivery or presence of the tetracycline derivative antibiotic, the administered inducer binds to the tTA, thereby hindering the capacity of the tTA to become docked to its tetO sequences within the TRE, thus impeding subsequent transcription from the TRE. In the on situation, i.e., upon removal or absence of inducer, tTA binds to its tetO sequences within the TRE, thus activating TRE and inducing gene expression (Fig. 2A).

Randomly induced mutations of the Tet-OFF-based tTA led to the subsequent development of the Tet-ON system [141] (Fig. 2B). Gossen and colleagues induced mutations in the original TetR that is bound to the VP16 domain, which resulted in four amino acid exchanges, yielding a protein that exhibits opposite functions. This mutant, termed rtTA, triggers transcription by activating the TRE only in the presence of the inducer, thereby activating the silent minimal CMV promoter. Transgene expression is switched off in the absence of the inducer. The rtTA reverse transactivator has been utilized successfully by achieving the desired regulation of gene expression in a number of applications, but has some limitations with respect to the precision of gene regulation. Despite the complete withdrawal or absence of the inducer, Tet-ON-based rtTA was found to exhibit some degree of affinity for the Tet operator sequences within the TRE, thus activating the minimal promoter and inducing low levels of gene transcription in the off state. This interaction between the response element and the transactivator, which produces basal activity in the off mode, is the main setback in attaining tight gene regulation using this system.

Since tetracycline-dependent and steroid hormone receptor regulatory systems are the most widely used regulatory systems, studies have attempted to compare both. Adenoviral vectors expressing the chloramphenicol acetyl transferase (CAT) reporter gene under the regulation of either the Tet-ON- or a mifepristone-regulated system were generated and compared. Both systems permit tight control of CAT reporter gene expression in vitro, with induction levels of approximately 1800- (Tet-ON system) and 600-fold (RU486-regulated system) [142]. Though in vitro reporter protein expression with the mifepristone regulatory system was more sensitive to the levels of the inducer, this was not demonstrated in vivo. The ecdysteroid regulatory system was compared with both Tet-ON and Tet-OFF regulatory systems in transient transfection studies. The ecdysteroid muristerone A was reported to boost reporter activity by nearly 1000-fold. In contrast, the Tet-OFF system displayed a 59-fold change in gene expression in the absence of doxycycline and the Tet-ON system showed only a 2.5-fold induction in the presence of doxycycline. The maximum activity of the Tet-ON system was higher than that of all other regulatory systems analyzed but high basal activity of the reporter gene was present even in the absence of doxycycline [121]. Tetracycline is commonly fed to cattle and contaminates fetal bovine serum from sources in the United States and many other countries. The high levels of expression in the absence of doxycycline in the Tet-ON system suggest that medium components were not screened for tetracycline contamination in this study. A number of reports have demonstrated effective silencing of gene expression in the Tet-ON system in the absence of doxycycline [139,143-147]. More recently, novel tetracycline-regulated systems that display high inducibility and very low to negligible basal activity have been developed [148-150].

To improve Tet-ON regulatory systems further, Hillen, Bujard, and colleagues induced random and directed mutagenesis, searching for novel rtTA forms that could enhance tight regulation and minimize background expression in the absence of the inducer [139]. Using Saccharomyces cerevisiae for screening assays, their experiments uncovered five rtTA mutants, from which one mutant transactivator generated virtually negligible basal expression in the absence of the inducer and required a 10-fold lower doxycycline concentration for TRE activation. Quantitative luciferase assay data on the novel mutant transactivator, rtTA2^S-M2, revealed tight regulation of Tet-regulatory switch-dependent β-galactosidase gene expression in transfected HeLa X1/6 cells in the presence and absence of the inducer [139].

Another strategy to reduce unwanted gene expression from the Tet-regulatable system in the off state was also pursued successfully by Bujard and colleagues. A chimeric protein incorporating a modified TetR fused to transcriptional silencing domains of the protein Kid was generated. When this protein (tTS^Kid) was coexpressed with rtTA proteins it was unable to heterodimerize. This was due to modifications on the dimerizing surfaces of the TetR component. In vitro studies of the silencing efficiency demonstrated that aberrant reporter transgene expression was reduced by as much as 1000-fold by tTS^Kid in the absence of Dox. Furthermore, tTS^Kid did not significantly affect maximal expression of the reporter transgene [151].

A multicomponent regulatory switch composed of a tetracycline response promoter coengineered with rtTA2^S-M2 transactivator and a tTS^Kid repressor element, separated by an internal ribosome entry site (IRES), has been shown to be very effective in achieving tight regulation of transgene expression in mice and nonhuman primates [148,149]. Lamartina and co-workers engineered and examined ex vivo a bicistronic vector containing a single regulatory cassette, encompassing the rtTA2^S-M2 and tTS^Kid elements. In the absence of doxycycline, the rtTA2^S-M2/tTS^Kid-encoded Tet-regulatory switch produced negligible basal activity. In the presence of doxycycline, there was a 1000-fold inducibility of serum alkaline phosphatase gene expression and elevated sensitivity to doxycycline [148]. Such novel transcriptional regulatory switches are advantageous for tight regulation of transgene expression to facilitate further their application for gene therapies [150].

Effects of Different Ratios of TRE to Transactivators on Regulation, Kinetics, and Levels of Transgene Expression Using the Tet-Regulatable System

The VP16 transactivator domain is an important unit of the regulatory switch that interacts with a diverse pool of transcriptional regulatory proteins, exhibiting tight regulation of a gene of interest [152]. The use of different versions of the transactivator elements revealed important data on the ratios of the TRE promoter and tTA transactivator elements required to produce the desired transgene induction levels, while simultaneously minimizing basal expression of the transgene in the uninduced state. Studies on the rtTA and tTA transactivator ratios versus levels of TRE appear to produce varying results as to what proportion of each element is required for optimal induction levels and negligible basal expression [153,154].

While some authors report that a 1:1 tTA:TRE ratio is required for optimal induction and negligible background expression [155], others report the need for excess tTA vs TRE [153,156,157]. This highlights the dependency of the system on the promoters used for driving the expression of the transactivator, cell lines, or in vivo target organ; the delivery system used; and the sensitivity of the method used to evaluate levels of transgene expression. It is therefore important to set up the optimal conditions for each application or target organ.

Characteristics of an Ideal Tet-ON Regulatory System for Clinical Gene Therapy Applications

It has become apparent that tight regulation of therapeutic genes is of critical importance in clinical gene therapy. Tet-ON-based regulatable gene-expression systems are preferred for use in clinical gene therapeutics principally because of the constant administration of antibiotics needed to silence gene expression with the Tet-OFF system. The Tet-OFF switch system would allow for transgene expression only in the absence of antibiotics, and a continuous administration of tetracycline would be required to switch off the system. The constant high levels of antibiotics in the general circulation could lead to other complications such as patients’ increased tolerance to the tetracycline derivative, cytotoxicity, or adverse side effects. Therefore, novel Tet-ON transactivator systems are more attractive in a clinical setting as they do not require constant administration of the inducer and can be switched on only when needed. Nevertheless, choosing the optimal Tet-dependent regulatory system will rely on the pathogenicity and etiology of the disease.

The optimal Tet-ON gene regulatory system should exhibit (a) no basal activity of the tTA/TRE unit in the absence of the tetracycline inducer, (b) good regulation and induction kinetics, (c) quick response to the administration or removal of the inducer, (d) stringent transgene regulation, and (e) negligible cytotoxic or inflammatory responses associated with the regulatory elements within the switch system. For a regulatory system to be used in clinical gene therapy, stringent gene regulation must be achieved such that transgene induction and protein synthesis do not occur in the absence of the inducer. When dealing with therapeutic transgenes, the basal expression of the transgene in the absence of a tetracycline-derived inducer, known as “leakiness,” has been the major obstacle in achieving stringent regulation kinetics. Newly emerging transactivators, like the rtTA2^S-M2, provide superior control of gene expression in vivo, allowing tightly regulated gene expression [148,149].

The regulatory switch engineered with a strong promoter, i.e., mouse CMV [158], to drive transactivator gene expression, will exhibit higher levels of transgene expression upon induction with a given dose of the inducer [150]. The higher levels of therapeutic transgene expression attained in the on state will allow the use of lower titers of the vector to express and regulate the therapeutic gene during treatment (Fig. 3). This will minimize the chance of induced toxicity and inflammation due to high vector doses [158,159]. High sensitivity of the transactivator to the tetracycline inducer is a desirable feature in this kind of system since enhanced sensitivity means that lower antibiotic concentrations will be required to trigger transgene expression. The quantity, concentration, and frequency of antibiotic administration needed to attain the desirable levels of therapeutic product are crucial factors that require careful evaluation when considering antibiotic-based gene control systems for clinical use (Table 2). The well-characterized tetracycline derivative doxycycline is a broadly employed antibiotic in medical treatment largely due to its nontoxic effects. A recent study by Chtarto et al. [146] revealed an additional tetracycline derivative that was shown to be effective and less cytotoxic than doxycycline in vivo. Minocycline, an antibiotic that is considered to possess antiapoptotic and anti-inflammatory properties, exhibits reduced cytotoxicity and more rapid elimination from the body than doxycycline. Although improved doxycycline-based Tet-ON regulatory systems show promising results, additional studies are needed to advance the effectiveness and minimize the side effects of antibiotics used in conjunction with the Tet-ON system.

Regulating Transgene Expression from First-Generation Adenoviral Vectors

A number of studies utilizing first-generation adenoviral vectors with Tet-regulatory systems targeted at specific cell types produced successful transgene regulation within the brain in vivo [156,160-162]. Smith-Arica and co-workers [156] have established effective doxycycline-dependent transcriptional regulation and cell-type-specific transcriptional targeting, producing successful regulatable transgenesis in cell lines, primary brain cultures, and localized regions within the rat brain. A first-generation adenoviral vector encoding the glial fibrillary acidic protein promoter driving the expression of β-galactosidase under the control of the Tet-OFF regulatory switch produced localized doxycycline-dependent β-galactosidase expression within glial cells in vitro and in vivo in a dose-dependent manner [156]. The neuron-specific enolase (NSE) promoter was used to induce expression of β-galactosidase in the absence of doxycycline within neurons. Although the NSE promoter did not exhibit neuronal-restricted transgene expression in vitro, it attained successful neuronal specificity in vivo [156]. These results indicate that the choice of a specific promoter designed for expressing a therapeutic or cytotoxic gene does not always result in the expected localized transgene expression when tested in vitro; therefore it is imperative to test these systems in vivo. Another study that employed the prolactin promoter, specific to anterior pituitary lactotrophic cells, encoded within a first-generation adenoviral vector driving the Tet-OFF regulatory cassette elicited inducible β-galactosidase expression within lactotrophs, both in vitro and in vivo [160].

Using the Tet-OFF system, successful regulation of tyrosine hydroxylase (TH) expression elicited effective regression of estrogen-induced pituitary prolactinomas in vivo [163]. TH is the rate-limiting enzyme in the dopamine biosynthetic pathway. Dopamine, in addition to its principal role in keeping the motor circuitry of the basal ganglia intact, is recognized as having inhibitory effects on adenohypophysial prolactin-producing cells of the pituitary gland. An adenoviral vector engineered with a TH transgene cassette and a Tet-OFF regulatory switch produced regulated TH expression in anterior pituitary (AP) cell lines [163]. Also, rats with estrogen-induced hyperprolactonemia and pituitary lactotroph hyperplasia showed regression of pituitary mass and normalization of serum prolactin levels when injected with the Ad engineered vector into the AP gland, in the absence of doxycycline, resulting in normal levels of circulating prolactin [163]. A first-generation Ad was used in mammary cancer cell lines for the expression of human H2 preprorelaxin (hH2), a precursor of the protein relaxin whose role is critical in tumor cell migration, attachment, and invasion. These in vitro experiments demonstrated that the addition of inducer led to hH2 expression, tumor cell migration, and proliferation [164]; this represents a new tool for the study of mammalian tumor physiology. Tietge and co-workers [168] generated a RAd encoding the human group IIA secretary phospholipase A2 (sPLA2), a protein that has been shown to have the ability to degrade plasma membrane phospholipids [165,166] and possibly to play a role in removal of metabolically compromised and injured cells [167]. Unregulated expression of sPLA2 might be toxic to 293 cells, thereby preventing RAd generation and scale up. When a RAd encoding a group IIA secretary phospholipase A2 under the Tet-OFF regulatable system (Adtet-OFF.hsPLA (2)) was utilized, the authors describe sPLA2 expression in vitro but they could not detect plasmatic sPLA2 protein in vivo. Providing additional tTA protein either by stable transfection or by co-infection with a tTA-expressing Ad resulted in increased levels of sPLA2 and β-galactosidase expression. Plasmatic sPLA2 was detectable in a tTA dose-dependent pattern that lasted for 7 days, and β-gal expression was observed in the lung and liver exhibiting a tTA dose-dependent pattern [168]. This system represents an interesting tool for the generation of RAds encoding potentially toxic genes, allowing vector multiplication in the absence of transgene expression. Gene delivery using viral vectors can result in long-term sustained transgene expression in the subretinal space, the area that separates the photoreceptor outer segments from the retinal pigment epithelium (RPE), resulting in vector transduction of RPE [169,170]. Due to the unique immune-privileged status of this site, systemic immune responses are minimized [171], making it possible to deliver genes to the ocular cells and control them much like any other conventional drug, administered as needed for therapeutic purposes. Dejneka et al. [147] described that when a RAd encoding the hGH under the Tet-ON system was administered to the subretinal space in mice, 100% of RPE cells were transduced within 48 h of administration. The hGH produced in the retina was secreted into the bloodstream in a doxycycline dose-dependent manner [147]. The delivery route and the gene regulation described in this paper offer a unique way to evaluate gene function in the eye and represent a novel method for introducing therapeutic proteins into the retina. One example of a Tet-OFF-based regulatory system using a bidirectional tetracycline-responsive promoter driving both the transgene and the regulatory switch cassettes is the regulation of single-chain interleukin 12 (IL-12) gene expression using first-generation adenoviral vectors [172,173]. Block and co-workers [172] described the adenoviral-mediated expression of murine IL-12 under Tet regulation, encoded within the same Ad vector. IL-12 is chosen for cancer gene therapy because it can stimulate antitumoral immune responses and it exerts proliferation of T lymphocytes and NK cells. Thus, the cytolytic activity of these cells is enhanced by IL-12-mediated production of interferon-γ. Furthermore, IL-12-secreting tumor cells were shown to induce a strong antitumoral response in different models [173,174]. The authors demonstrate that IL-12 expression was suppressed up to 6000-fold in the presence of doxycycline, resulting in a considerable decrease in interferon-γ secretion by activated splenocytes and, consequently, a marked reduction in immune response [175]. Considering that the systemic administration of recombinant human IL-12 has been associated with severe side effects like hemorrhagic colitis, leukopenia, and elevated liver enzymes; the development of a RAd providing regulatable single-chain IL-12 expression for the transduction of tumor cells would significantly contribute to the safety of these innovative approaches in cytokine gene therapy. For applications in neurological diseases, direct injections into the striatum and the substantia nigra of RAds encoding the neurotropic factor glial-derived neurotropic factor (GDNF) have been shown to induce growth and protection of viable dopaminergic neurons [176-181]. These results using Ad-vector-mediated GDNF gene transfer may substantially slow dopaminergic neuronal cell loss in patients with Parkinson disease [180]. Kojima and co-workers [182] described the Tet-regulated expression of human GDNF preventing dopamine depletion in brain striatum, in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced injury to nigrostriatal neurons [182]. They found human GDNF to be expressed at high levels in vitro in primary cultures of rat nigral neurons. By injecting 2 × 10⁶ PFUs into MPTP-treated mice in vivo, they found a decreased loss of dopamine content in the injected side of the striatum (to 16%), whereas administration of the control virus expressing β-gal did not have this effect (9%). Although the administration of a higher dose was supposed to provide a more beneficial effect, the delivery of 5 × 10⁷ PFUs showed a lower protection against MPTP-induced dopaminergic cell loss, probably due to vector-induced neurotoxicity [183].

Regulating Transgene Expression from High-Capacity Adenoviral Vectors

The first in vivo gene transfer and expression study employing a combined Tet-ON transactivator and repressor system using HC-Ad vectors produced tight regulation kinetics with maximal induction in the on state, while minimizing leakiness of gene expression in the off state [143]. Engineered with the Tet-ON transactivator rtTA2^S-M2 and the transcriptional repressor tTS^Kid, doxycycline-dependent secreted alkaline phosphatase reporter gene expression was tightly regulated upon intramuscular injection of the engineered HC-Ad vector in C57/B6 and Balb/C nude mice [143]. In this study, the IRES addition to ensure translation of the tTS^Kid silencer downstream of the CMV promoter-driven transactivator produced an almost 100,000-fold increase in transgene expression in stable HeLa cell lines. Tight regulation of reporter gene expression was achieved using HC-Ad vector constructs with head-to-head configuration of the promoters driving the transgene and regulatable switch cassettes [143]. These results are also consistent with another report of adeno-associated viral vector-based transcriptional gene regulation using the Tet-ON system. In this study, the two promoters driving the expression of the transgene and transactivator were maximally spaced from each other in a head-to-head configuration, producing tight regulation kinetics of transgene expression while sustaining minimal levels of background activity in the uninduced state [184]. When analyzed together, these data suggest that the distance between the individual promoters and the orientation of the transgene- and transactivator-encoding cassettes could play an important role in achieving good induction kinetics. Also, the promoters positioned closer together might induce possible cross talk between them, thus increasing the likelihood of transactivator activation, thereby inducing transgene expression in the absence of the inducer. The first studies of intrastriatal injections of the Tet regulatory switch encoded within first-generation vectors, i.e., RAd hGDNF, demonstrated tight regulation of the transgene expression in both neuronal and glial cells [182]. Our own results using HC-Ad indicate that these vectors engineered with the rtTA2^S-M2/IRES/tTS^Kid regulatory switch produced strong β-galactosidase expression within the striatum in the induced state and negligible basal activity in the uninduced state (Fig. 4) [150,185]. Immunohistochemistry and fluorescence confocal microscopy analysis revealed colocalization of transduced β-galactosidase expression within neurons and astrocytes in the striatum (Fig. 5) [150]. The strong murine CMV promoter driving the transactivator produced high transgene expression levels with negligible basal activity in the uninduced state compared to the Ad vectors encoding β-galactosidase driven by the constitutive mCMV or hCMV promoter [8,186].

Thus utilizing these HC-Ad vectors, engineered to encode specific neurotropic factors under the control of a regulatory switch containing the rtTA2^S-M2 transactivator, in conjunction with transcriptional silencers, tight regulation of neurotropic factor gene expression as would be needed for gene therapy of Parkinson disease should be achievable. Another useful application of tetracycline-controlled transactivators emerged from studies conducted on the molecular basis of long-term potentiation (LTP) to understand further the mechanisms of learning and memory. Synaptic plasticity, a key player in the LTP phenomenon, has been thought to be under the control of protein kinases and protein phosphatases that are dependent on the enzyme calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) [187]. Using the original Tet-ON rtTA system, Hedou and Mansuy [188] demonstrated that protein activity of phosphatase CN, a protein opposing CaMKIIα, is important for determining synaptic strength and subsequently LTP. The application of upgraded tetracycline-dependent molecular switches may be utilized to examine further and establish the role of these important kinases in the LTP pathway.

Since HC-Ad are much safer than their predecessor first-generation adenoviral vectors, regulatable switches engineered within these viral vectors will be critical to acquiring safe levels and regulation of transgene expression in vivo. The current issues that require further research are to examine the extent of HC-Ad vector-induced immune responses, establishing viral vector stability and spread once injected into the targeted tissue or organ, and to determine safe viral vector titers needed for effective reversion of an affected phenotype in a clinical setting (Table 2).

Regulating Transgene Expression from Retroviral Vectors

Engineered retroviral vectors produced successful transgene regulation using the Tet-OFF-based tetracycline-dependent system. One of the first Tet-regulatory systems to be used in a retroviral vector involved a modified Tet-OFF/tTA that produced successful and stable transgene expression of the VSV-G toxin gene in mammalian cell lines in the absence of the doxycycline inducer [189]. In this study, a modified version of the tetracycline-controlled inducible system was produced by the addition of a ligand-binding domain from the estrogen receptor (ER) to the carboxy terminus of the tTA, yielding tTA-ER. A single retroviral vector, encoding the VSV-G gene regulated by the tTA-ER element, exhibited a 30-fold induction in the absence of the inducer, 17β-estradiol, in transfected mammalian cell lines, producing stable expression of the tTA-ER. Moreover, the addition of the ER ligand-binding domain to the tTA was shown to reduce tTA toxicity in the induced state in the absence of 17β-estradiol [189]. In another study using the Tet-OFF tetracycline-responsive expression system, a single retroviral vector containing TRE and the HSV-1-TK cytotoxic gene under the control of the tetO synthetic promoter produced high levels of TK gene expression in transduced murine NIH 3T3 cells in the absence of the inducer tetracycline [190]. They observed a 417-fold increase in HSV-1-TK enzyme activity in NIH 3T3 cells compared to enzyme activity in control cells. These in vitro studies demonstrate good efficiency in attaining high levels of HSV-1-TK expression to improve gene therapy strategies for brain tumors. Studies by Kenny and co-workers [191] established successful Tet-OFF-based regulation from retroviral vectors and demonstrated the effectiveness of the TRE promoter in achieving stringent regulation of gene expression [191]. Upon evaluation of different promoters to drive tTA expression, such as CMV, elongation factor 1α, and phosphoglycerate kinase-1, in combination with the TRE they observed that only the CMV promoter in combination with the TRE promoter produced successful regulatable β-galactosidase expression when controlled by the Tet-OFF regulatory switch in HC11 mouse mammary epithelial cell lines [191].

Although stable and efficient regulatable transgenesis can be attainable with retroviral vectors, their restricted cloning size of 8 kb constrains their wide use in gene therapy. Hofmann et al. [192] describe a single autoregulatory cassette, containing tandem Tet operator sequences and the cytomegalovirus minimal promoter driving the expression of a bicistronic mRNA, leading to transcription of the gene of interest (β-gal) and the internal ribosome entry site-controlled transactivator (Tetrepressor—VP16 fusion protein). This system allowed reversible induction of transgene expression in response to Tet; in the absence of Tet there was a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of Tet, gene expression was prevented [192]. The use of this retroviral vector resulted in rapid gene delivery of inducible genes into cell types, i.e., primary mouse myoblasts, in which direct transfection has low efficiency [192].

Regulating Transgene Expression from Lentiviral Vectors

Lentiviral vectors have been demonstrated to transduce successfully and drive gene expression in a number of mammalian cells and animal models [42,193] and have been engineered using the Tet-ON- and Tet-OFF-based tetracycline-dependent systems to achieve successful regulation of gene expression in vitro into 293 cells [194], ex vivo in cells of the central nervous system [195], and in vivo in a rat model for Huntington disease [196]. Kafri et al. [194] demonstrated that after administration of a green fluorescent protein (GFP)-expressing lentiviral vector to rats, in a variety of tissues including differentiated neurons, myotubes, hepatocytes, and hematopoietic stem cells, under the regulation of the Tet system, GFP expression was tightly regulated in vivo following addition or withdrawal of Dox in the drinking water. They described a 500-fold increase in cellular GFP levels upon withdrawal of the inducer from the culture medium in transfected mammalian cell lines. Successful regulatable transgenesis was also achieved in vivo following transduction of rat brain; doxycycline-regulated GFP expression was observed in terminally differentiated neurons and was regulated by adding or withdrawing doxycycline from the rats’ drinking water [194]. In addition to single-gene-engineered lentiviral vectors, doxycycline-mediated regulation was also successfully attained from bicistronic lentiviral vectors, as demonstrated by Reiser and co-workers [144]. A dual HIV-1-based vector system, one vector encompassing the rtTA-based Tet-ON construct and the other encoding the TRE and an enhanced (E) GFP reporter gene, generated effective regulatable EGFP expression in primary human skin fibroblasts (HSFs) in the presence of doxycycline [144]. Fluorescence microscopy of HSFs transduced with the NL-rtTA/TRE-EGFP bicistronic vector demonstrated a 1.7-fold difference in fluorescence intensities in the presence and absence of doxycycline. CD81 is a transmembrane protein that is upregulated in the mesolimbic dopaminergic pathway after acute administration of high doses of cocaine [197]. Its expression contributes to some of the behavioral changes associated with cocaine sensitization [198]. A lentiviral vector encoding CD81 under the control of the Tet-ON system was stereotaxically delivered into the ventral tegmental area (VTA) of two groups of rats that were given water alone (CD81 expression) or water with doxycycline solution (down-regulates CD81 expression). A 3.2-fold augmented locomotor activity was observed in those animals expressing CD81 in the VTA vs animals placed on doxycycline, which downregulated CD81 expression. Additionally, when the administration of the virus was to the nucleus accumbens, it resulted in higher effects, yielding a 4.2-fold increase in locomotor activity [198].

Regulating Transgene Expression from Adeno-associated Vectors

One of the first successful applications of achieving regulatable gene expression within the rAAV vectors was the efficient Tet-ON-based regulation of the green fluorescent protein. Two rAAV vectors, one engineered with a GFP-encoding cassette and the other with the silencer-flanked rtTA Tet-ON transactivator tTS^Kid-rtTA, were constructed [199]. Subretinal injection of these engineered rAAV vector particles into rats produced tightly regulated GFP expression. In vitro studies performed by the same group also demonstrated that a higher ratio of silencer-encoded AAV vector to transactivator-encoded AAV vector produced higher levels of gene expression in the on state compared with an equal ratio of the two vectors. The data also showed that an 8:1 ratio of the silencer-encoded vector to the transactivator-encoded vector produced an almost 12-fold increase in gene expression. However, compared to an equal ratio of the two vectors, basal activity in the absence of the inducer was not further suppressed when using the 8:1 ratio [199]. Studies by Rendahl et al. [145] also reported Tet-ON-based stringent gene regulation in vivo by utilizing two rAAV vectors, one with a regulatory switch-encoding vector and the other with an erythropoietin (Epo) transgene-encoding vector. The tTS^Kid repressor and rtTA-engineered rAAV vector produced both tight and prolonged levels of Epo expression upon intramuscular injection of the engineered vector in mice in the presence of doxycycline [145]. Periodic administration of the inducer over a 32-week period produced constant Epo expression, achieving successful long-term transgene regulation [145]. It has been described that leptin, the product of the Ob gene, acts on satiety centers in the hypothalamus to both decrease food intake and increase energy expenditure. Virus-mediated leptin gene delivery has been shown to cause a rapid and complete disappearance of white adipose tissue in genetically normal animals [200]. In a study by Wilsey et al. [201] the use of two rAAV vector systems yielded concise Tet-ON-dependent regulation of the expression of leptin in young rats injected with the viral vector into the hypothalamus, producing weight gain in the induced state [201]. The transgene-engineered rAAV vector contained the inducible TetR promoter, driving a bicistronic construct for the expression of Ob and GFP with the addition in the construct of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream of the transgene to augment expression. In the presence of doxycycline, the silencer-encoded rtTA induced Ob gene expression, producing an 8-fold increase in hypothalamic leptin [201].

In addition to successful tetracycline-inducible transgene expression using dual AAV vectors, the utilization of a single AAV vector encoding an autoregulatory cassette that encompasses a bidirectional tetracycline-responsive promoter using the Tet-dependent regulatory system was also effective [146]. Tet-OFF systems with bidirectional tetracycline-responsive promoter-encoded cassettes designed into AAV vectors produced successful transgene expression in the uninduced state [202,203]. Folliot et al. [204] used a single rAAV vector engineered with the Tet-OFF regulatory switch devoid of the silencer, which elicited efficient and prolonged transgene regulation of GFP reporter gene expression. Intravitreal injections of a single rAAV vector coengineered with a destabilized GFP gene and Tet-OFF tTA regulatory cassette produced only 5% residual expression of GFP 48 h after doxycycline administration, sustaining GFP transduction shutdown for as long as 1 week in ganglion cells.

In an another study by Chtarto and co-workers [146], an autoregulatory rAAV vector was engineered with the Tet-ON rtTA and the EGFP reporter gene, both driven by a bidirectional Tet-responsive promoter and the complete cassette flanked with bidirectional SV40 polyadenylation sites at both ITRs. The autoregulatory cassette produced around 50-fold induction of EGFP expression in the presence of doxycycline in human tumor cell lines, and in vivo data revealed an 80-fold increase in the induced state [146]. Slow regulation kinetics was observed after doxycycline administration but rapid termination of the gene expression was observed in the uninduced state. However, the use of new Tet-ON-based transactivators encoded with silencers and post-transcriptional elements will be difficult using rAAV vectors due to the constrained cloning space within the core rAAV vector. One feasible approach of regulating transgene expression using rAAV vectors is through a bidirectional autoregulatory cassette, in which more cloning capacity is available for including silencer elements and other short transgenes. In addition to the type of Tet-ON transactivator employed to attain optimal gene regulation, the configuration of the transgene and regulatory switch cassettes was found to be an important factor in achieving maximum gene expression and virtually negligible expression in the on and off state, respectively. In a recent report by Chenuaud et al. [184], an optimal design of a single recombinant AAV using two independent promoters was established with the use of the Tet-ON transactivator rtTA2^S-M2. Promoters driving the transactivator and the erythropoietin DNA reporter gene-encoding cassettes situated distant from each other, but oriented in the same direction, produced maximal induction and minimal background activity in the off state when injected intramuscularly into mice [184].

Recombinant AAV vector-based studies suggest advantages to using these vectors to regulate gene expression, but there are also possible drawbacks with respect to their application in human gene therapy. Due to the predetermined cloning capacity of 5 kb of the AAV vectors, most of the studies described to date utilize two vector systems to deliver both the transgene and the regulatory cassettes, limiting the choice of transgenes to clone. Second, in the clinical perspective, the administration of a higher ratio of silencer- and transactivator-encoding rAAV vector to therapeutic gene-encoding rAAV vector to achieve higher transgene expression levels can result in delivery of a combined higher vector dose, thus creating a greater potential for adverse immune responses. In a Tet-OFF rAAV vector, gene expression has been quantified by very precise techniques [205]; Tet-regulated transgene expression has been studied in rat retinal ganglion cells [204], the regulated expression of erythropoietin from a AAV vector has been proven to improve anemia in a mouse model of β-thalassemia [206], and Tet-inducible IL-10 expression has been applied to the experimental arthritis model [207]. The interaction between the Tet-ON/OFF regulatory systems and the mammalian immune system has yet to be fully characterized. The degree of immune response to a viral vector can be a consequence of one or more factors that include the method and route of vector delivery at the target site [208], cell-type-specific gene expression [209], and immunogenicity of the vector capsid and the transactivator elements making up the regulatory switch [210].

Regulating Transgene Expression from Herpes Simplex Vectors

A number of studies generated successful and efficient regulatable transgenesis from HSV-1-derived amplicon vectors engineered with the Tet-OFF tetracycline-responsive system. In one study, localized and high levels of β-galactosidase reporter gene expression were observed in the dendrate gyrus of the mouse brain with over 10-fold suppression in gene expression and controlled cytotoxicity in the presence of doxycycline [211]. In another study, effective inducible reporter gene expression was effectively regulated in vitro and in vivo with a Tet-OFF system-based HSV-based amplicon vector [212]. Hippocampal cultures infected with the engineered amplicon vectors produced over 50-fold repression of firefly luciferase reporter gene expression in the presence of tetracycline, with maximal gene expression levels achieved within 10–12 h after the withdrawal of tetracycline [212]. Adding tetracycline to cultures without prior tetracycline exposure resulted in quick silencing of gene expression, by which luciferase reporter gene activity was reduced to less than 8% within 24 h. Maximum gene expression levels from the amplicon vectors occurred within 2–3 days postinfection and gradually declined subsequently [212]. Combinations of emerging amplicon vectors and superior Tet-ON- and Tet-OFF-based tetracycline-regulatable system technologies have been shown to generate better and effective HSV-1-derived ampliconmediated therapeutic gene transfer. One example of successful implementation of adapted Tet-regulatable systems can be shown from work by Schmeisser and co-workers [213]. In an attempt to find suitable tetracycline-inducible HSV vector-based systems that exhibit enhanced induction characteristics and flexibility, several combinations of TRE regulatory/promoter elements were constructed to regulate β-galactosidase reporter gene expression in cells [213]. These promoters contained a TRE linked to a minimal CMV immediate early promoter, a minimal HSV ICP0 promoter, or a truncated HSV ICP0 promoter. The ICP0 promoter constructs expressed the highest and most sustained levels of β-gal. Vectors were also constructed that coexpressed an inducible Tet transactivator in addition to the inducible β-gal marker gene. This resulted in tetracycline-inducible gene expression that was not restricted to specific cell lines, and this vector was capable of inducible expression in irreversibly differentiated NT2 cells (NT neurons) for several days [213]. In another study, Herrlinger et al. demonstrated tetracycline-mediated regulation of the luciferase reporter gene under the control of either the Tet-OFF or the Tet-ON system from HSV-1 mutants in infected Vero cells [214]. The study revealed the role and importance of selective transactivating factors encoded by immediate early genes and their effect on the TRE complex and transgene regulation. The presence of specific transactivating factor proteins and viral proteins has been shown to induce the tetO/minimal TRE complex in the uninduced state, resulting in their interference in generating effective tetracycline-regulated transgene expression [214]. Transient transfection into Vero cells of a mutant HSV-1 expressing ICP4 and VP16 transactivating factors failed to minimize or eliminate basal levels of transgene expression in the uninduced state with the Tet-OFF and Tet-ON systems. In contrast, mutant HSV-1 vectors devoid of ICP4 and VP16 abolished this basal activity in the uninduced state, suggesting the likely role of specific viral proteins in generating an effective tetracycline-based regulatory system [214]. This study demonstrates that in the HSV-1 genome, especially the transactivating immediate early gene products (ICP4, ICP27, and ICP0) and the VP16 tegument protein can activate the tetO/minimal CMV promoter and thereby interfere with the integrity of tetracycline-regulated transgene expression [214].

Regulatable Expression Systems as Applied to Clinical Trials: Importance and Key Challenges for Their Implementation in Human Patients

The goal of gene therapy is to provide treatment for diseases that currently have limited or no therapeutic options. Below, we will review the importance and challenges of the future implementation of regulatable gene expression systems in gene therapy clinical trials. To date, phase I, II, and III clinical trials have utilized retroviral or adenoviral vectors in treating malignant glioma; meanwhile, the first trial utilizing an adeno-associated virus for Parkinson disease treatment has recently been approved [215]. Several gene therapeutic strategies have been used to treat glioblastoma. In two such trials, although some degree of immune response was elicited, a combination of herpes simplex-thymidine kinase/ganciclovir after surgical resection of recurrent glioblastoma was observed to be relatively safe and showed statistically significant efficacy [216,217]. In this scenario, the use of an inducible gene expression system would allow shutting down of expression of HSV-1 TK once tumor regression occurs as assessed by MRI or other imaging systems. Another gene therapy trial for glioblastoma involved intratumoral injection of an adenoviral vector carrying wild-type p53 [218]. A replication-disabled Semliki Forest viral vector carrying the human interleukin 12 gene, which has been shown to be safe and effective in treating breast and prostate cancer in prior animal studies [219], was also used in glioblastoma treatment. Again it would be clinically advantageous to be able to turn therapeutic gene expression off once the therapy is no longer needed. Clinical trials that use viral vectors to treat other neurological diseases such as Parkinson disease are also in progress. One such trial is being proposed for the use of subthalamic, glutamic acid decarboxylase gene transfer via an adeno-associated viral vector [220]. If successfully transduced, the transgene will increase subthalamic nucleus levels of GABA, an inhibitory neurotransmitter involved in the pathological pathway. Although none of the vectors in current use contain regulatable systems within their viral vector constructs, utilization of such regulatable systems will provide greater safety, thereby increasing the potential for clinical use of viral and nonviral vectors for in vivo gene delivery.

Systems in which the transgene must be repressed by the constant presence of a substance such as antibiotics may pose potential clinical problems, such as side effects, making them undesirable. However, the use of regulatable systems in which high-level, specific, and transient expression of a therapeutic gene can be induced, i.e., the Tet-ON regulatable gene expression system, may prove to be a therapeutic powerful tool. Instances in which regulatable switches would have a powerful impact might include turning on a transgene used to eliminate a tumor and subsequently turning it off to prevent aberrant destruction of other tissues or inflammatory reactions. Also when expressing a factor to stimulate growth of specific cells, i.e., GDNF for Parkinson disease, it would be crucial to be able to turn gene expression off or be able to regulate levels of expression tightly to prevent adverse side effects due to abnormal cell growth, aberrant connections between cells, or abnormal neurotransmitter release.

In solid tumors, radiation-inducible promoters have proven very successful in preclinical models of the disease. One such vector, called TNFerade, has been tested in phase I clinical trials on patients with solid tumors and soft-tissue sarcoma’s [221,222]. This replication-deficient adenoviral vector contains the TNF-α gene and expression is regulated by the Egr1 promoter. Although patient numbers were small in these phase I clinical trials, objective responses were high even in patients with refractory tumors. This therapy utilizes the ability to focus ionizing radiation at the tumor to limit damage to normal tissues and side effects were minor in the majority of individuals. Consequently, this novel therapy appears to warrant further investigation in larger, randomized trials.

Myocardial ischemia is also a system in which inducible gene therapy vectors have proven very successful in preclinical models of the disease. Although no clinical trials are yet under way, the vector system currently in development utilizes a hypoxia-sensitive promoter to regulate gene expression [223] and affords us a glimpse of an exciting future in gene therapy in which inducible vectors may lie dormant for months or even years before therapeutic gene expression is switched on in response to danger signals such as hypoxia. Clinical trials in humans with diabetes are unfortunately farther into the future and many technical challenges must still be overcome, including correct processing of the insulin transgene and stable expression of an inducible system that must be switched on and off many thousands of times. However, early research in liver cells has validated the use of gene therapy to replace insulin levels and perhaps an approach unifying stem cell research and gene therapy can go some way to surmounting these obstacles in the future.

Combining regulatable switches with other genetic elements, i.e., the WPRE and regions of the HSV-1-TK gene, known to increase mRNA stability and levels of transgene expression will further enhance their usefulness by promoting long-term and widespread distribution of transgene expression [224]. Alternatively, the development of strategies to promote predetermined therapeutic gene insertion within the host genome will also enhance the utility and safety of gene transfer vectors. Utilization of regulatable switches driven by strong promoters will allow high levels of transgene expression, thus requiring less viral vector dose to be utilized. This in turn will minimize possible toxicity and inflammation, making these viral vectors safer for clinical use. Development of tools that allow for greater control of gene expression will reduce untoward side effects, creating wider and safer clinical settings in which gene therapies could be implemented.