Human Secreted Alkaline Phosphatase Research Articles

Novel traceable CRISPR-Cas9 engineered human embryonic stem cell line (E1C3 + hSEAP + 2xKO + pCD47), has potential to evade immune detection in pigs

Here we present the generation of a human embryonic stem cell line with the potential to escape immune rejection upon transplantation to an alternate species, in this case sus scrofa. For in vivo detection the cells were modified by CRISPR-Cas9 to express human secreted alkaline phosphatase. To avoid immune recognition and subsequent rejection by host, genes encoding hB2M and hCIITA were knocked out and the porcine gene for CD47 was introduced. Upon editing and subsequent culture, cells maintained molecular and phenotypic pluripotent charactaristics and a normal karyotype supporting viability and functionality of the engineered cell line.

Successful AAV8 re-administration: Suppression of capsid-specific neutralizing antibodies by a combination treatment of bortezomib and CD20 mAb in a mouse model of Pompe disease.

A major challenge to AAV mediated gene therapy is the presence of anti-AAV capsid neutralizing antibodies (NAbs), which can block viral vector transduction even at very low titers. Here we examined the ability of a combination immunosuppression (IS) treatment with bortezomib and a mouse-specific CD20 mAb to suppress anti-AAV NAbs and enable re-administration of AAV vectors of the same capsid in mice. An AAV8 vector (AAV8-CB-hGAA) that ubiquitously expresses human α-glucosidase was used for initial gene therapy and a second AAV8 vector (AAV8-LSP-hSEAP) that contains a liver-specific promoter to express human secreted alkaline phosphatase was used for AAV re-administration. Plasma samples were used for determination of anti-AAV8 NAb titers. Cells isolated from whole blood, spleen, and bone marrow were analyzed for B-cell depletion by flow cytometry. The efficiency of AAV re-administration was determined by the secretion of hSEAP in blood. In näive mice, an eight-week IS treatment along with AAV8-CB-hGAA injection effectively depleted CD19+ B220+ B cells from blood, spleen, and bone marrow and prevented the formation of anti-AAV8 NAbs. Following administration of AAV8-LSP-hSEAP, increasing levels of hSEAP were detected in blood for up to 6 weeks, indicating successful AAV re-administration. In mice pre-immunized with AAV8-CB-hGAA, comparison of IS treatment for 8, 12, 16, and 20 weeks revealed that the 16-week IS treatment demonstrated the highest plasma hSEAP level following AAV8-LSP-hSEAP re-administration. Our data suggest that this combination treatment is an effective IS approach that will allow retreatment of patients with AAV mediated gene therapy.

Histone deacetylase inhibitors reactivate silenced transgene in vivo.

Gene silencing for plasmid-based vectors and the underlying mechanism are critical factors for development of effective gene therapy. The objective of this study is to explore the role of epigenetic regulation for transgene expression. Two reporter genes, mouse interleukin 10 and human secreted alkaline phosphatase under the control of human cytomegalovirus immediate early promoter for expression, were delivered to mouse liver by hrodydynamics-based procedure and reporter gene expression was monitored. Reporter gene expression reached its peak level one day after gene delivery and declined progressively thereafter, reaching the minimal level in about 3 weeks. Intra-peritoneal injection of vorinostat, valproic acid or sodium butyrate, the known histone deacetylase inhibitors, resulted in a dose-dependent reactivation of reporter gene expression. Repeated administration of histone deacetylase inhibitors blocked gene silencing and maintained reporter gene expression. Mechanistic studies reveal that reactivation of reporter genes is corelated with hyperacetylation of histones H3 and H4, and elevated binding of TATA-box binding protein to the promoter region. These results suggest that epigenetic regulation plays a critical role in controlling transgene expression in vivo and demonstrate that enzymes involved in epigenetic regulation such as histone deacetylase could serve as a target to achieve controlled transgene expression for gene therapy.

Establishment of Novel Reporter Cells Stably Maintaining Transcription Factor-driven Human Secreted Alkaline Phosphatase Expression.

Transcriptional regulation is a very important and pivotal function in myriad biological responses. Thus, methods to determine transcriptional activity are required in not only basic medical research but also in drug discovery. We established novel reporter constructs using human secreted embryonic alkaline phosphatase (SEAP) and Epstein-Barr virus nuclear antigen (EBNA) 1, which can maintain constructs synchronized to host cell replication. We established nuclear factor-kappa B (NFkB) or interferon regulatory factor (IRF) driven SEAP expression constructs and then, introduced them into culture cells. The cells maintain reporter constructs for a long period in the culture and produce SEAP into culture supernatant in response to each specific ligand such as lipopolysaccharide (LPS) and interferon- beta. Measuring SEAP with chemiluminescence makes it possible to get high standard dynamic range applying to high-throughput screening in drug discovery in both 96 and 384 well format. We can also use it to determine transcriptional activity in the cells transfected with expression plasmid or treated with various toll-like receptor (TLR) ligands in a concentration-dependent manner and time-dependent manner. Finally, we demonstrated drug screening using a number of natural products library. We for the first time established the two novel reporter cells and validated their quality and accuracy enough to carry out drug screening.

Production of human secreted alkaline phosphatase in suspension and immobilization cultures of tobacco NT1 cell

The recombinant protein production by a plant cell culture offers several advantages including product safety and ease of the purification of secreted proteins and the capability of plants to correctly fold complex proteins. However, low productivity is generally a limitation of this system, caused by the denaturation and degradation of the secreted proteins due to proteolytic enzyme protease in the medium. In this study, we demonstrated that the transgenic tobacco NT1 cells were immobilized in Ca2+-alginate gel beads coated with a cell-free gel film, which significantly improved the productivity of the recombinant human secreted alkaline phosphatase (SEAP), about 33 times larger than that in the suspension culture. The cells immobilized in the beads were protected from hydrodynamic stress during cultivation and hence did not release the endogenous protease, which caused damage to the secreted SEAP. The immobilization culture without cell leakage could be quite effective for the production of recombinant protein.

Production of human secreted alkaline phosphatase in suspension and immobilization cultures of tobacco NT1 cells

Production of human secreted alkaline phosphatase in suspension and immobilization cultures of tobacco NT1 cells

Synergism of Shrew‐1’s Signal Peptide and Transmembrane Segment Required for Plasma Membrane Localization

Signal peptides (SP) and transmembrane segments (TMS) ensure proper subcellular targeting and localization of proteins. Thus, understanding the molecular variability of this targeting information is essential. In this study, we functionally analyzed the predicted SP and the TMS of adherens junction protein, shrew-1 (Bharti et al. Novel membrane protein shrew-1 targets to cadherin-mediated junctions in polarized epithelial cells. Mol Biol Cell 2004:15:397). We used human secreted alkaline phosphatase (SEAP) as reporter protein. The SP of shrew-1 was able to functionally substitute for SEAP's intrinsic SP and was cleaved, indicating that it acts as a start-transfer signal and not a signal anchor. In turn, the TMS of shrew-1 functions as stop-transfer signal. Notably, clearly detectable plasma membrane localization is only achieved when the fusion protein contains both the SP and the TMS of shrew-1. In combination with the intrinsic SP from SEAP, the shrew-1 TMS is unable to promote stable plasma membrane localization. Hence, it may be assumed that this synergism between an SP and a TMS to mediate plasma membrane localization is essential for structural and/or functional integrity of shrew-1.

Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N‐glycosylation of human secreted alkaline phosphatase

We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins.

Production, Secretion, and Stability of Human Secreted Alkaline Phosphatase in Tobacco NT1 Cell Suspension Cultures

Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.

A genetic redox sensor for mammalian cells

Nutrient and oxygen availability are key metabolic parameters for biopharmaceutical manufacturing. In order to enable mammalian cells to manifest their intracellular nutrient and oxygen levels we engineered a genetic sensor circuitry which converts signals impinging on the cellular redox balance into a robust reporter gene expression readout. Capitalizing on the Streptomyces coelicolor redox control system, consisting of REX modulating ROP-containing promoters in an NADH-dependent manner, we designed a mammalian dual sensor transcription control system by fusing REX to the generic VP16 transactivation domain of Herpes simplex, which reconstitutes an artificial transactivator (REDOX) able to bind and activate chimeric promoters assembled by placing a ROP operator module 5′ of a minimal eukaryotic promoter (P ROP). When nutrient levels were low and resulted in depleted NADH pools REDOX-dependent P ROP-driven expression of secreted (human-secreted alkaline phosphatase; SEAP) or intracellular (Renilla reniformis luciferase; rLUC) reporter genes was high as a consequence of increased REDOX–P ROP affinity. Conversely, at hypoxic conditions leading to high intracellular NADH levels, strongly reduced REDOX–P ROP interaction mediated low-level transgene expression in Chinese hamster ovary (CHO-K1) cells. Other molecules (for example, 2,4-dinitrophenol, cyanide or hydrogen peroxide) which are known to imbalance the intracellular NADH/NAD + poise could also be detected using the REDOX–P ROP sensor circuitry. REDOX's sensor capacity (nutrient and oxygen levels) operated seamlessly in transgenic CHO-K1 cell derivatives adapted for growth in serum-free suspension cultures and enabled precise monitoring of the population's metabolic state. As the first genetic metabolic sensor designed for mammalian cells, REDOX may foster advances in process development and biopharmaceutical manufacturing.

Scalable Generation of High-Titer Recombinant Adeno-Associated Virus Type 5 in Insect Cells

We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required alpha2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of alpha2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene.

330. Effect of Different Inducible Minimal Promoters in the RheoSwitch® Therapeutic System That Give Robust and Long-Term In Vivo Gene Expression

The RheoSwitch|[reg]| Therapeutic System (RTS) is used to regulate gene expression in a ligand-dependent manner, and may have utility in human gene therapies that require the specific regulation of the level of gene expression, or where it is desirable to terminate therapy either when it is no longer required, or where continuation of the therapy would be unsafe. One version of the RTS is comprised of an ecdysone receptor (EcR) fused to a GAL4 DNA binding domain, a chimeric retinoid X receptor fused to a VP16 activation domain (pVP16-Hs-LmRXR), a reporter gene, such as human secreted alkaline phosphatase (hSEAP) placed under the control of a 6xGAL4 response element, and an inducible minimal promoter (p6xGAL4RE- Inducible Promoter- hSEAP). Studies are described that have evaluated the effects of 10 different inducible promoters on ligand- dependent hSEAP transgene expression in vitro in NIH 3T3 cells and in vivo following quadriceps electroporation in C57BL/6 mice. These data can be interpreted to classify the inducible promoters into three performance groups: 1) low expression and undetectable basal expression, 2) intermediate expression with some basal activity in cells, and 3) robust expression in cells and in vivo. One inducible promoter constructed with a consensus TATA box (TATA3) induced only low levels of expression until three Sp1 binding sites (SP1- TATA) were inserted into the promoter, yielding much higher expression. Additionally, a minimal transthyretin (TTR) promoter showed high levels of expression in response to ligand both in cell culture and in vivo. Of these two minimal promoters, Sp1-TATA3 gave higher expression, but higher basal expression was also observed. Additional evaluation of the Sp1-TATA3 and the TTR minimal promoters showed that insulating these promoters with chicken beta globin insulator further increased the expression levels. The expression cassette containing the TTR minimal promoter was chosen to evaluate the long-term expression of the RTS in vivo due to its high ligand-dependent transgene induction relative to basal expression. To assess long-term induction of in vivo expression, hSEAP protein levels and mRNA levels of hSEAP were analyzed in two groups of C57BL/6 mice electroporated (quadriceps muscle) with the RTS. Group one received a single IP dose of ligand 3 days after electroporation. The second group received a single IP dose of ligand on days 3, 185, and 353 after electroporation. Levels of hSEAP protein in serum were assayed over a series of time points and hSEAP was induced at all times. In addition, animals were sacrificed on day 17 and 365, respectively, and muscle tissue was collected for mRNA analysis by RT-QPCR. Levels of mRNA hSEAP were comparable at both 17 and 365 days after RTS electroporation. These studies establish that RTS containing the TTR minimal promoter both induces high levels of gene expression in vivo as well as sustained performance one year after electroporation.

332. Routes of Ligand Administration That Give Efficient and Robust In Vivo Induction with the RheoSwitch® Therapeutic System

The RheoSwitch|[reg]| Therapeutic System (RTS) is used to regulate both the timing and level of gene expression in response to a systemically delivered small molecule ligand, Activator Drug. This inducible gene regulation system may have utility in human gene therapies that require a specifically regulated level of gene expression, where it is desirable to terminate the therapy when it is no longer needed, or where continuation of the therapy would be unsafe. The version of RTS used in these studies was comprised of an ecdysone receptor (EcR) fused to a GAL4 DNA binding domain, a chimeric retinoid X receptor fused to a VP16 activation domain (pVP16-Hs- LmRXR) , a human secreted alkaline phosphatase (hSEAP) reporter gene placed under the control of a 6xGAL4 response element, and a transthyretin minimal promoter (p6xGAL4-TTR-SEAP). The RTS was electroporated into the quadriceps of C57BL/6 mice and Activator Drug was administered by either intraperitoneal (IP) injection, intramuscular (IM) injection, subcutaneous injection, oral gavage, or administration in the rodent chow. Although ligand-dependent gene expression was observed by all five routes of administration, Activator Drug mixed in chow was easiest to administer, could be administered continuously and may mimic administration by capsule. As an example, six groups of mice (n = four/group) were administered various concentrations of Activator Drug in rodent chow 38 days after RTS electroporation. Activator Drug was given at a rate of 300, 150, 100, 50, 25, or 12.5 mg/kg chow for 21 days, corresponding to approximate total daily Activator Drug doses of 45, 23, 15, 8, 4 and 2 mg/kg/day, respectively. In this study, both the rate of ligand-mediated induction and the magnitude of ligand-mediated hSEAP expression were dependent on the concentration of ligand in the chow. Human SEAP reached a ligand- dose dependent plateau in mice receiving 100, 150, or 300 mg/kg chow two weeks after initiation of ligand administration. The threshold for induction was observed with 50 mg Activator Drug/kg chow, which showed a small induction of hSEAP that was slightly above background. In another long-term expression study, four groups of severe combined immunodeficient (SCID) mice (n = four/group) were administered Activator Drug in chow or by single IP injection at various times after electroporation. Activator Drug administration was started at 3, 32, or 76 days after RTS electroporation. In mice receiving Activator Drug by IP injection, gene expression could be cycled on and off by repeated IP administration. Likewise, gene expression could be similarly cycled when mice were put on and taken off chow containing Activator Drug. Gene expression remained |[ldquo]|on|[rdquo]| when mice were chronically maintained on chow containing Activator Drug. These studies establish that ligand-dependent induction of transgene expression can be obtained by various routes of ligand administration, and that gene expression can be sustained for long periods of time by chronic administration of ligand in chow. Further, the results demonstrate that transgene expression under control of RTS can be cycled on and off by repeat exposure to Activator Drug.

Production, Secretion, and Stability of Human Secreted Alkaline Phosphatase in Tobacco NT1 Cell Suspension Cultures

Production, Secretion, and Stability of Human Secreted Alkaline Phosphatase in Tobacco NT1 Cell Suspension Cultures

Mannosamine supplementation extends the N‐acetylglucosaminylation of recombinant human secreted alkaline phosphatase produced in Trichoplusia ni (cabbage looper) insect cell cultures

A major limitation of the insect cell-baculovirus expression vector system is its poor capacity to perform complex glycosylation, since the glycoproteins produced are usually only of the high-mannose and paucimannose types. Nonetheless, recent evidence indicates that, under various conditions, some insect cell lines are capable of producing complex-type glycans. In the present study, we assessed the effects of supplementation with ManN (mannosamine) ManNAc (N-acetylmannosamine) and Cyt (cytidine) on the glycosylation of recombinant human secreted alkaline phosphatase produced by suspension cultures of Trichoplusia ni (cabbage looper) BTI-Tn5B1-4 cells. Addition of ManN in the range 5-20 mM resulted in a 10-fold increase of the terminal GlcNAc (N-acetylglucosamine) associated with the recombinant protein produced after baculovirus infection. Such an increase yielded a maximum of 12.5% hybrid glycans having terminal GlcNAc with respect to total N-linked glycans. In contrast, no changes in the glycan composition associated with recombinant human secreted alkaline phosphatase were observed on supplementation with up to 20 mM ManNAc or up to 1.5 mM Cyt. N-acetyl-beta-glucosaminidase activity in cell extracts was decreased on incubation with 20 mM ManN, but not with 20 mM ManNAc or 1.5 mM Cyt, indicating that the increased proportion of hybrid glycans obtained on the addition of ManN could be a result of N-acetyl-beta-glucosaminidase inhibition.

Tobacco smoke as inducer for gas phase‐controlled transgene expression in mammalian cells and mice

Capitalizing on components evolved to metabolize ethanol in Aspergillus nidulans, we previously designed the first molecular gas-gene expression interface using gaseous acetaldehyde as the major inducer. This fungus-derived acetaldehyde-inducible gene regulation (AIR) system operated perfectly and enabled precise and reversible transgene expression dosing in a variety of mammalian cells. We now validate the use of mainstream cigarette smoke typically containing acetaldehyde at regulation-effective nontoxic concentrations as a noninvasive modality to adjust transgene transcription in mammalian cells and mice. Indeed, tobacco smoke-induced expression fine-tuning of AIR-driven transgenes was successful in mammalian cells. Even mice implanted with cells transgenic for AIR-controlled SEAP (human secreted alkaline phosphatase) production showed serum SEAP levels correlating with inhaled tobacco smoke doses. Tobacco smoke-controlled gene expression may foster clinical opportunities as well as advances in understanding smoke-related pathologies.

Cytochalasin D can improve heterologous protein productivity in adherent Chinese hamster ovary cells

We generated a series of adherent gene-amplified CHO clones expressing human secreted alkaline phosphatase (SEAP) as a model for heterologous protein production. Clones demonstrate a 26- to 52-fold increase in productivity compared to controls after dhfr/methotrexate-mediated gene amplification and clone selection. SEAP is stably expressed in these clones over at least a 6-week period without significant productivity loss. Two-dimensional protein electrophoresis identified 21 proteins that exhibited altered expression in clones of increasing SEAP productivity. Based on MALDI TOF/TOF mass spectrometry of relevant protein spots, changes in translation, energy pathways, chaperones, regulatory proteins, and cytoskeletal proteins were observed, including a 4-fold expression increase in actin capping protein. We hypothesized that an alteration of the actin cytoskeleton using cytochalasin D as a mimic for actin-capping protein could have a beneficial effect on heterologous protein secretion. Treatment with 0.5 mug/mL cytochalasin D increased SEAP productivity 2- to 3-fold compared to an amplified control which resulted in an increase in productivity from 52- to 150-fold compared to a nonamplified parent.

Inducible production of erythropoietin using intramuscular injection of block copolymer/DNA formulation.

We have previously shown that intramuscular injection of plasmid DNA formulated with a non-ionic amphiphile synthetic vector [poly(ethylene oxide)(13)-poly(propylene oxide)(30)-poly(ethylene oxide)(13) block copolymer; PE6400] increases reporter gene expression compared with naked DNA. We have now investigated this simple non-viral formulation for production of secreted proteins from the mouse skeletal muscle. Plasmids encoding either constitutive human secreted alkaline phosphatase or murine erythropoietin inducible via a Tet-on system were formulated with PE6400 and intramuscularly injected into the mouse tibial anterior muscle. PE6400/DNA formulation led to an increased amount of recombinant alkaline phosphatase secreted from skeletal muscle as compared with naked DNA. In the presence of doxycycline, a single injection of 10 microg plasmid encoding inducible murine erythropoietin formulated with PE6400 significantly increased the hematocrit, whereas the same amount of DNA in the absence of PE6400 had no effect. The increase in the hematocrit was stable for 42 days. The tetracycline-inducible promoter permitted pharmacological control of hematocrit level after DNA intramuscular injection. However, 4 months post-injection the hematocrit returned to its pre-injection value, even in the presence of doxycycline. This phenomenon was likely caused by an immune response against the tetracycline-activated transcription factor. Intramuscular injection of plasmid DNA formulated with PE6400 provides an efficient and simple method for secretion and production of non-muscle proteins.

Novel macrolide-adjustable bidirectional expression modules for coordinated expression of two different transgenes in mice.

Precise control of transgene expression is essential for a variety of applications ranging from gene-function analysis, biopharmaceutical manufacturing to next-generation molecular interventions in gene therapy and tissue engineering. The regulation of gene expression is currently a key issue for clinical implementation of gene-therapy-based treatments since desired transgene expression may need to be maintained within a narrow therapeutic window for successful treatment of a particular human disease. We have designed a novel bidirectional expression module that enables adjustable coregulation of two different transgenes in response to clinical doses of macrolide antibiotics. A bidirectional macrolide-responsive promoter consisting of a central operator module (ETR) specific for the macrolide-dependent transactivator (ET1) is flanked by two minimal promoters (P(hCMVmin); P(hsp70min)) which drive expression of two divergently oriented transgenes. Macrolide antibiotics modulate the binding affinity of ET1 to ETR and adjust expression of both transgenes to desired levels. Bidirectional expression configurations enabled excellent macrolide-adjustable coregulation profiles of two secreted reporter genes or one-vector-based autoregulated fine-tuning of a single transgene in various transgenic rodent and human cell lines. Following implantation of microencapsulated CHO-K1 cell derivatives transgenic for macrolide-controlled bidirectional expression of erythropoietin (EPO) and the human secreted alkaline phosphatase (SEAP) intraperitoneally into mice, serum EPO and SEAP levels could be coadjusted to desired levels by administration of different erythromycin doses. Based on their in vivo compatibility, the versatile bidirectional and macrolide-responsive expression modules represent an important advancement on the way to implementing targeted and conditional molecular interventions into a clinical reality.

Streptomyces-derived quorum-sensing systems engineered for adjustable transgene expression in mammalian cells and mice.

Prokaryotic transcriptional regulatory elements have been adopted for controlled expression of cloned genes in mammalian cells and animals, the cornerstone for gene-function correlations, drug discovery, biopharmaceutical manufacturing as well as advanced gene therapy and tissue engineering. Many prokaryotes have evolved specific molecular communication systems known as quorum-sensing to coordinate population-wide responses to physiological and/or physicochemical signals. A generic bacterial quorum-sensing system is based on a diffusible signal molecule that prevents binding of a repressor to corresponding operator sites thus resulting in derepression of a target regulon. In Streptomyces, a family of butyrolactones and their corresponding receptor proteins, serve as quorum-sensing systems that control morphological development and antibiotic biosynthesis. Fusion of the Streptomyces coelicolor quorum-sensing receptor (ScbR) to a eukaryotic transactivation domain (VP16) created a mammalian transactivator (SCA) which binds and adjusts transcription from chimeric promoters containing an SCA-specific operator module (P(SPA)). Expression of erythropoietin or the human secreted alkaline phosphatase (SEAP) by this quorum-sensor-regulated gene expression system (QuoRex) could be fine-tuned by non-toxic butyrolactones in a variety of mammalian cells including human primary and mouse embryonic stem cells. Following intraperitoneal implantation of microencapsulated Chinese hamster ovary cells transgenic for QuoRex-controlled SEAP expression into mice, the serum levels of this model glycoprotein could be adjusted to desired concentrations using different butyrolactone dosing regimes.