Enhancer and promoter chimeras in plasmids designed for intramuscular injection: a comparative in vivo and in vitro study.

Enhancers and promoters from various muscle-specific genes were substituted for or combined with the enhancer/promoter of the human cytomegalovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effort to identify new promoter chimeras with increased expression activity after direct intramuscular injection. The regulatory sequence substitutions or additions varied in content, location, and orientation relative to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo using a standard mouse intramuscular injection assay, and in vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster kidney cells, to test whether cultured cell transfection could substitute for at least some animal experimentation. In vivo, 1 of 19 of the enhancer/promoter chimeras increased expression levels. In vitro, some chimeras showed significant expression augmentation in C2C12 cells, but not in BHK cells. We conclude that because of differences in plasmid expression profiles, these cell culture systems cannot readily substitute for in vivo testing of new plasmid constructs.

Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice.

Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches of the same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Dose-response and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 micrograms of DNA at 7-14 days post-injection. Selected manipulations of the transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no significant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no significant effect on expression. However, linearization of the DNA, injection of the DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And finally, increasing the injection dose by giving multiple injections over a 10-day period increased expression proportionally to the number of injections.

An improved plasmid DNA expression vector for direct injection into skeletal muscle.

In previous work, the direct injection of 50 micrograms of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences. Expression of the modified vectors was then compared to VR1205 using the intramuscular injection assay. In general, modifications to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect. However, modifications to the polyadenylation and transcriptional termination sequences, plasmid backbone elements, and the luciferase gene itself each increased luciferase expression levels. The best-expressing vector, designated VR1255, contained a combination of these incrementally beneficial changes. A single intramuscular injection of 50 micrograms of VR1255 produced 300 ng of luciferase at 7 days postinjection, an expression level 46-fold higher than the VR1205 vector (or 22-fold higher, excluding modifications to the luciferase gene) and 154-fold higher than a commercially available luciferase expression vector. Thus, VR1255 represents an improved plasmid DNA vector that may be useful for gene therapy applications.

Function of neurotrophic factors in the adult and aging brain and their possible use in the treatment of neurodegenerative diseases.

This review summarizes the current knowledge of characterized neurotrophic factors, including nerve growth factor (NGF) which serves as paradigmatic example when studying novel molecules. Special consideration is given to the function of neurotrophic factors in the adult and aging brain. Strategies are discussed for the eventual development of pharmacological applications of these molecules in the treatment of neurodegenerative diseases.

Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons.

Neurons dissociated from the septal area of fetal rat brains were grown in culture. Cholinergic neurons were identified by immunocytochemical visualization of choline acetyltransferase and cytochemical demonstration of acetyl cholinesterase. Choline acetyltransferase immunocytochemistry stained cell bodies and proximal processes while acetylcholinesterase cytochemistry visualized the entire neuron. Choline acetyltransferase-positive neurons could only be identified in cultures grown under conditions that produced the maximal choline acetyltransferase activity, measured biochemically. All of the choline acetyltransferase-positive neurons were double stained for acetylcholinesterase while only 6% of the acetylcholinesterase-positive cells were choline acetyltransferase negative in these cultures. These results indicate that acetylcholinesterase is a reliable marker for cholinergic cells in cultures of dissociated septal neurons. Being the more sensitive method, acetylcholinesterase staining was therefore used to identify cholinergic cells in cultures with choline acetyltransferase levels insufficient for immunocytochemical visualization of this enzyme. Addition of nerve growth factor or antibodies to nerve growth factor to the medium did not affect the number of cholinergic neurons surviving in culture. Furthermore, nerve growth factor and anti-nerve growth factor failed to influence the general morphological appearance and the number of processes of these neurons. However, nerve growth factor elevated the biochemically measured activity of choline acetyltransferase up to two-fold. The nerve growth factor-mediated increase in choline acetyltransferase activity was dose dependent with an ED50 of 10 ng/ml (4 X 10(-10) M). The increase was highly specific for nerve growth factor. It was blocked by anti-nerve growth factor, and epidermal growth factor, insulin and other control proteins failed to exert a similar effect. Nerve growth factor had to be present for at least 3 days in the culture medium to increase choline acetyltransferase activity, suggesting that the increase was due to an elevated choline acetyltransferase synthesis rather than to an activation of the enzyme.

Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions.

Nerve growth factor (NGF) was injected intraventricularly during 4 weeks into adult rats with unilateral partial lesions of the cholinergic septo-hippocampal pathway. On the lesioned side, NGF treatment elevated choline acetyltransferase (ChAT) activity up to 60% above the activity measured on the lesioned side of cytochrome c-treated controls. On the unlesioned side, NGF treatment increased ChAT activity only to an insignificant degree. ChAT activity in the septum of NGF-treated animals was increased by 60% as compared to controls. The NGF-induced increases on the lesioned side and in the septum were not accompanied by elevations in acetylcholinesterase (AChE) activity. Furthermore, histochemical analysis revealed no difference in AChE staining pattern or intensity between NGF-treated and control animals. The lack of effect on AChE strongly suggests that the increases in ChAT activity in hippocampus and septum are due to an elevation of ChAT activity within cholinergic neurons surviving the lesion rather than to a promotion of sprouting of cholinergic fibers.

Implantation of PC12 cells into the corpus striatum of rats with lesions of the dopaminergic nigrostriatal neurons.

PC12 cells, which have the capacity to synthesize and release catecholamines, were implanted into the corpus striatum of adult rats with lesions of the dopaminergic nigrostriatal pathway. PC12 cells survived for 1-2 weeks in the host striatum and degenerated thereafter. The presence of these cells was associated with an attenuation of the apomorphine-induced circling behavior in rats with unilateral nigrostriatal lesions. These findings indicate that cells from a cell line are able, at least temporarily, to survive in the rat brain and to compensate functional deficits caused by lesions of specific neuronal systems. Cell lines might therefore be used for intracerebral grafting instead of neurons derived from fetal brains, if their survival can be prolonged and their proliferation in the host brain prevented.

Effect of thyroid hormone analogs on the activity of choline acetyltransferase in cultures of dissociated septal cells.

Thyroid hormones influence the expression of transmitter-specific enzymes by central cholinergic neurons. Based on the fact that these cholinergic neurons degenerate selectively in human Alzheimer's disease, it was hypothesized that thyroid hormones might be beneficial in its treatment. However, since thyroid hormones influence the function of most peripheral organs, derivatives selective for central cholinergic neurons are necessary. The structural requirements of the receptor mediating the effects of the thyroid hormones on central cholinergic neurons were therefore compared with those of the receptors mediating actions on peripheral organs. Cultures were prepared of dissociated neurons from the septal area of fetal rat brains, and the differentiation of cholinergic neurons was assessed by measuring the activity of choline acetyltransferase (ChAT). Triiodothyronine (T3) was found to stimulate ChAT activity in a dose-dependent manner. The effect of T3 was additive to that of nerve growth factor. The potency of derivatives of T3 in elevating ChAT activity in the cultures was compared with their known anti-goiter activity determined in vivo and their binding affinity to the hepatic nuclear receptor measured in vitro. The findings indicate that the structural requirements of central and peripheral receptors are similar and that it therefore appears unlikely that analogs of thyroid hormones can be developed which selectively affect cholinergic neurons.

Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain.

Nerve growth factor (NGF) receptors were visualized in the basal human forebrain using an immunohistochemical procedure with a monoclonal antibody previously shown to recognize human melanoma cell NGF receptors. The receptors were found to be exclusively located in the medical septal nucleus, the diagonal band of Broca, and the nucleus basalis. This location coincided with that of cell bodies of ascending cholinergic neurons of the basal forebrain. In addition, NGF receptor-positive cells were costained for acetylcholinesterase. These findings indicate that cholinergic neurons of the basal forebrain but none of the other neurons located in this area express receptors for NGF. Results suggest that NGF acts as a trophic factor for cholinergic neurons in the human brain in a similar way as has been established in recent years for the rat brain.

Cyclic AMP promotes the survival of dopaminergic neurons in vitro and protects them from the toxic effects of MPP+.

We have studied how stimulation of protein kinase C and cAMP-dependent protein kinases affect the development of mesencephalic dopaminergic neurons in vitro. IGF-I and bFGF did not activate either second messenger system nor affect the survival of dopaminergic neurons but stimulated dopamine uptake per neuron. Phorbol esters, which stimulate protein kinase C, had no effect on dopamine uptake. Dibutyryl-cAMP caused an increase in dopamine uptake, which was blocked with (Rp)-cAMPS, a specific inhibitor of cAMP-dependent protein kinases. Treating cells with specific phosphodiesterase type IV inhibitors elevated the forskolin-induced increase in dopamine uptake. Furthermore, cAMP, but neither bFGF nor activation dependent astrocyte factor (ADAF), was able to prevent the degeneration of dopaminergic neurons induced by MPP+. These results suggest that increased intracellular cAMP protects dopaminergic neurons in situations of stress and therefore reveal novel possibilities for the treatment of Parkinson's disease.

Adjuvants for plasmid DNA vaccines.

In the late 1980s, Jon Wolff of the University of Wisconsin and Phil Felgner here at Vical were screening cationic lipids for their ability to encapsulate and deliver purified plasmid DNA into mouse tissues. They discovered that direct injection of lipid-DNA complexes into muscle resulted in measurable protein expression. A belated control experiment without lipid led to the serendipitous discovery that "naked" plasmid DNA was taken up and expressed in muscle to a greater extent than DNA-lipid complexes (1). This key observation led to the demonstration that i.m. injection in mice of a standard 50 µg of plasmid DNA encoding a reporter gene becomes readily expressed exclusively in myofiber cells at 180 pg of gene product per muscle (2). More recently, plasmid DNA expression vectors were improved such that an average of 300 ng of gene product could be produced from single intramuscular (i.m.) injections of plasmid DNA, and up to 40 µg of gene product could be produced after multiple injections (3 and J. Hartikka, unpublished observations).

Development and characterization of lyophilized DNA vaccine formulations.

The potential applications of using plasmid DNA for immunization and other gene therapy approaches have been discussed in an increasing number of publications in the past few years. Injection of mouse muscle with naked DNA (plasmid DNA in saline) resulted in significant episomal expression from a number of encoded reporter genes such as firefly luciferase, chloramphenicol acetyltransferase, and ß-galactosidase (1). DNA vaccination has been shown to induce neutralizing antibodies against the gene product, helper T-cell responses of the Th1 phenotype, and cytotoxic T lymphocyte responses (2). Vaccination with plasmid DNA stimulates immunogenicity and provides protection against various infectious diseases in pre-clinical animal models. Examples include hepatitis B in chimpanzees (3), bovine herpes virus in mice (4), influenza A virus in ferrets (5), human immunodeficiency virus in rhesus monkeys (6), Mycobacterium tuberculosis in mice (7,8), malaria in mice (9,10), and genital herpes simplex virus in guinea pigs (11). Recently, DNA vaccines for the protection against influenza (Merck Research Laboratories, Rahway, NJ), malaria (Vical Inc., San Diego, CA), and HIV (Apollon Inc., Philadelphia, PA), have entered phase I human clinical trials. Rapid progress has been made in the areas of adjuvants for DNA vaccines (12), route of immunization (13), industrial scale fermentation and pharmaceutical grade purification (14). One major interest in the commercial development of DNA vaccines, especially for developing countries, is to increase DNA vaccine stability at room temperature, to reduce the requirement for costly cold storage, and to extend product shelf-life.

Comparison of nerve growth factor's effects on development of septum, striatum, and nucleus basalis cholinergic neurons in vitro.

In the central nervous system, nerve growth factor (NGF) affects basal forebrain cholinergic neurons during early development and in the adult mammalian brain. These neurons are located in medial septum, diagonal band of Broca, and nucleus basalis of Meynert. While the effects of NGF on the development of septal cholinergic neurons are well documented, only little is known about the influence of NGF on development of cholinergic neurons in the nucleus basalis. In addition to the basal forebrain cholinergic neurons, there are cholinergic interneurons in the corpus striatum, which form an anatomically and functionally distinct population of cholinergic neurons. These striatal interneurons have been reported to respond to NGF during early development; however, it is not known whether the effects of NGF on their development are similar to those on septal cholinergic neurons. We prepared cultures of dissociated cells from fetal rat septum, striatum, and nucleus basalis and investigated the development of cholinergic neurons localized in these three different areas in the presence or absence of NGF. We now report that, first, cholinergic neurons of striatum and nucleus basalis develop a more extensive fiber network and contain more acetylcholinesterase (AChE) per neuron than do cholinergic neurons of septum. The amount of choline acetyltransferase (ChAT) per cholinergic neuron is approximately the same in all three culture types when grown in the absence of NGF. Second, NGF treatment increases and anti-NGF treatment decreases the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF is able to promote survival of cholinergic neurons of all three areas studied. Third, NGF increases the total length of fibers and the number of branching points of cholinergic neurons in septal cultures but not in cultures of striatum and nucleus basalis. Fourth, NGF treatment increases AChE activity in septal but not in nucleus basalis or striatal cultures, suggesting that AChE activity reflects the extent of the fiber network of cholinergic neurons of all areas. Fifth, NGF treatment produces severalfold elevations in ChAT activity in septal cultures and more modest increases in cultures of nucleus basalis and striatum, suggesting that NGF is able to stimulate ChAT activity also in the absence of a stimulatory effect on survival and fiber growth. Our results demonstrate that, during early development, NGF is able to affect survival and differentiation of all three populations of forebrain cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes.

To characterize the role of NGF in the development of forebrain cholinergic neurons, we established primary cell culture systems to grow these cells under controlled in vitro conditions. Cultures of dissociated cells were prepared from the septal area of fetal (E17) rats, which contained part of the group of basal forebrain cholinergic neurons. Cultures were treated either with NGF (100 ng/ml) or with an antiserum against NGF (1:500 dilution). To assess the influence of non-neuronal cells, 2 types of high-density cultures were prepared: mixed neuronal-glial cultures and pure neuronal cultures. Cholinergic neurons were identified using choline acetyltransferase (ChAT) immunocytochemistry and AChE cytochemistry. Receptors for NGF (NGF-R) were located immunocytochemically using monoclonal antibodies against rat NGF-R. We report that, first, NGF-R are exclusively localized on cholinergic neurons in septal cultures. All neurons labeled with antibodies against NGF-R also contained AChE. Twenty-one percent of all AChE-positive neurons were not stained in NGF-R immunocytochemistry (AChE has earlier been shown to be colocalized with ChAT in septal cultures). Second, NGF treatment increases and anti-NGF treatment reduces the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF promotes survival of septal cholinergic neurons in these cultures. In cultures of high plating density, NGF increased the number of NGF-R and ChAT-positive neurons without affecting the number of AChE-positive neurons in these cultures. These results suggest that exogenous NGF is not required for survival of cholinergic neurons in high-density cultures but stimulates the expression of ChAT and NGF-R. Third, NGF stimulates fiber growth of septal cholinergic neurons, as assessed by computerized image analysis of AChE-positive neurons. Fourth, NGF specifically increases ChAT and AChE activities in septal cultures. These NGF-mediated increases in enzyme activities are more pronounced when neurons are grown together with glial cells. In pure neuronal cultures, NGF increased ChAT and AChE activities by 101 and 16%, and in mixed neuronal-glial cultures by 318 and 87%, respectively. Anti-NGF blocked the effects of NGF but failed to reduce ChAT and AChE activities below control levels in cultures of high plating density. Fifth, astrocytes attenuate the expression of ChAT and AChE by septal neurons in the absence of NGF.(ABSTRACT TRUNCATED AT 400 WORDS)

Gangliosides alter morphology and growth of astrocytes and increase the activity of choline acetyltransferase in cultures of dissociated septal cells.

Administration of gangliosides has been reported to stimulate regeneration of motoneurons and of central dopaminergic and cholinergic neurons. To shed light on the mechanism by which gangliosides mediate the effects on cholinergic neurons, we studied their actions on cultures of cells dissociated from the septal area of fetal rat brains. These cultures contain cholinergic neurons, which, in vivo, give rise to the cholinergic septo-hippocampal pathway. Gangliosides produced prominent changes in the morphological appearance of the cultures. In contrast to control cultures, which contained many process-bearing cells and a confluent layer of flat cells, there were no flat cells in cultures grown in the presence of gangliosides (0.2 to 0.8 mg/ml of medium). Using immunocytochemical visualization of the astrocytic marker glial fibrillary acid protein, it was shown that all astrocytes in cultures grown in the presence of gangliosides exhibited the morphology of process-bearing cells, whereas in control cultures astrocytes represented the majority of the flat cells. Furthermore, gangliosides attenuated astrocytic proliferation. The effects of gangliosides apparently were not mediated by cAMP, since they could be differentiated from actions of forskolin, an activator of adenylate cyclase. Astrocytic growth and morphology were affected by ganglioside mixtures of various sources and composition and also by the pure gangliosides GM1 and GD1a, whereas lipid and carbohydrate components of gangliosides were ineffective. In contrast to the prominent effects on astrocytes, gangliosides failed to significantly alter survival or fiber growth of cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP, but not basic FGF, increases the in vitro survival of mesencephalic dopaminergic neurons and protects them from MPP(+)-induced degeneration.

We studied how stimulation of protein kinase C and cAMP-dependent protein kinases affect the development of mesencephalic dopaminergic neurons in primary cell cultures derived from fetal rats at embryonic day E14. The effects of compounds which activate these second messenger systems were compared to those of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I). In mesencephalic cultures, there was a continuous loss of dopaminergic neurons. Despite this decline in cell number, neurotransmitter uptake per neuron increased with time, indicating that the surviving dopaminergic neurons continued their biochemical differentiation while others degenerated. IGF-I and bFGF did not affect the number of dopaminergic neurons. However, dopamine uptake per neuron was significantly higher in bFGF and IGF-I treated cultures, suggesting that these factors stimulated differentiation. Protein kinase C and cAMP-dependent protein kinases were not involved in mediating the effects of bFGF and IGF-I. Treatment of cultures with phorbol esters did not affect dopamine uptake, whereas elevated levels of intracellular cAMP resulted in an increase in dopamine uptake which was additive to that elicited by bFGF or IGF-I. Further analysis revealed that exposure of mesencephalic cultures to dibutyryl cAMP (dbcAMP) during the first 3 days after plating increased the survival of dopaminergic neurons, whereas prolonged treatment attenuated the development of the dopamine uptake system. Moreover, cyclic AMP, but not bFGF, was able to prevent the degeneration of dopaminergic neurons induced by 1-methyl-4-phenyl-pyridinium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results suggest that increased intracellular levels of cAMP protect dopaminergic neurons in situations of stress like the process of dissociation and plating or the exposure to neurotoxic compounds. Our results reveal novel possibilities for the treatment of Parkinson's disease.

Long-term regulation of serotonergic activity in the rat brain via activation of protein kinase A.

Serotonergic neurons located at the base of the mammalian brain innervate practically every region of the brain and the spinal cord. These neurons exhibit spontaneous electrical discharges in a rhythmical way. Their firing frequency is modulated by serotonin autoreceptors which also regulate intracellular cAMP levels. We have investigated how elevated levels of cAMP alter the development and the functional properties of serotonergic neurons in culture. To study the influence of cAMP on the expression of genes underlying serotonergic activity, a quantitative RT-PCR approach using internal standards was developed. Cultures of embryonic rat brain serotonergic neurons were continuously treated with cAMP analogues. Increased cAMP levels had three effects. First, the neuronal morphology was changed towards that typical for mature serotonergic neurons. Second, the expression of tryptophan hydroxylase, the rate-limiting enzyme in serotonin production, was increased in dibutyryl-cAMP treated cultures. Third, the expression of the inhibitory autoreceptor (5-HT1A) was down-regulated. These results suggest the existence of a mechanism by which the neurons react to synaptic input regulating intracellular cAMP levels. Increased cAMP concentrations affect the development and cause a prolonged activation of serotonergic transmission. Since 5-HT1A receptors inhibit cAMP formation, their down-regulation argues against a negative feedback control in this system, consistent with observations in vivo.

Spatial-temporal patterns of gene expression in mouse skeletal muscle after injection of lacZ plasmid DNA.

Gene therapy for muscular diseases requires the efficient transfection of a large proportion of myofiber cells within a given muscle. In the present experiments, patterns of beta-galactosidase expression were examined in mouse rectus femoris muscles at various time-points after a single injection of lacZ encoded plasmid DNA. beta-Galactosidase expression was detected 3 h after injection and rose to peak levels at 3-14 days, and then stabilized at lower levels. beta-Galactosidase staining was detected in an average of about 6% (up to 15%) of the total 4000 myofiber cells, and in about 70% of those myofibers located in the discrete area containing the greatest proportion of transfected cells. Soon after injection of DNA encoding cytoplasmic or nuclear-targeted beta-galactosidase, expression was noted predominantly in the myotendinous junction areas, after which beta-galactosidase activity progressed toward the central parts of the myofibers. This preferential transgene expression at the myotendinous junction may result from some unique, local property of the myofiber cells and/or from a restricted diffusion or binding of the injected plasmid DNA at tendinous surfaces. A better understanding of the reasons for this pattern of reporter gene expression in muscle may suggest procedures for increasing the number of myofiber cells transfected by direct DNA injections.

Pharmacological activities in thermal proteins: relationships in molecular evolution.

The model of protobiological events that has been presented in these pages has increasing relevance to pharmacological research. The thermal proteins that function as key substances in the proteinoid theory have recently been found to prolong the survival of rat forebrain neurons in culture and to stimulate the growth of neurites. A search for such activity in thermal proteins added to cultures of modern neurons was suggested by the fact that some of the microspheres assembled from proteinoids rich in hydrophobic amino acids themselves generate fibrous outgrowths.

Novel, high expressing and antibiotic-controlled plasmid vectors designed for use in gene therapy.

The promise of effective gene therapy can only be accomplished by high-level expression and regulatable delivery of gene products. To achieve this end, a eukaryotic expression plasmid was modified to make transcription dependent on a tetracycline(Tc)-regulated chimeric transactivator. Mouse muscle injected with this two plasmid cis/trans control system expressed reporter proteins at levels five- to 10-fold greater than the cytomegalovirus immediate-early promoter-controlled parental plasmid. Tetracycline could be useful to either repress or activate transactivator-controlled expression based on the position of the tetO control sequences within the reporter plasmid. Finally, a prototype single plasmid construct was made and shown to express a self-regulating bicistronic transcript containing both the reporter and the transactivator. These Tc-controlled plasmids, termed maximum expression and regulated vectors (MERVs), have the potential to target a variety of gene therapy applications.