Regulation Of Transgene Expression Research Articles

Bioreducible dextran-polyethylenimine conjugates regulate transgene expression distribution in vivo.

The purpose of this work is to design a well-defined cationic dextran for intravenous gene delivery into tumor and examine the effect of the dextran on transfection efficacy in vivo. To this end, disulfide-linked dextran-linear polyethylenimine (Dex-SS-LPEI) conjugates were designed as non-viral vectors for intravenous gene delivery into tumor-bearing Balb/c nude mice. By coupling different molecular weights (2 kDa or 5 kDa) of LPEI disulfide pyridine to thiolated dextran (5 kDa or 10 kDa), Dex-SS-LPEI conjugates were prepared which have dextran as the main chain and disulfide-linked LPEI as the side chain. Dex-SS-LPEI conjugates can condense gene into nanosized polyplexes with moderate surface charge. Besides, the polyplexes of the conjugates have an improved colloidal stability under physiological conditions as compared to that of 22 kDa LPEI lacking dextran and can liberate gene by disulfide cleavage in an intracellular reducing environment. In vitro transfection experiments manifest that Dex-SS-LPEI conjugates mediate efficient gene transfection in different cells. The most efficient transfection in vitro was found for the conjugate with 5 kDa dextran and 5 kDa LPEI. Besides, Dex-SS-LPEI conjugates are practical for systemic gene delivery into tumor-bearing Balb/c nude mice by intravenous injection, affording comparable or higher transgene expression in HepG2 or SKOV-3 tumor than that yielded by 22 kDa LPEI as a positive control. Further, the polyplexes of the conjugate with 10 kDa dextran and 5 kDa LPEI can induce comparable transgene expression in the tumor but lower expression in the lung when compared to those of 22 kDa LPEI. The results of this study indicate that dextran plays a critical role in regulating in vivo gene delivery properties of Dex-SS-LPEI conjugates and transfection efficacy in tumor-xenografted nude mice. Dex-SS-LPEI conjugates have low cytotoxicity in vitro and cause no death of the mice, showing great potential as safe and highly efficient gene delivery vectors towards cancer gene therapy.

Applied RNAi: from fundamental research to therapeutic applications

Applied RNAi: from fundamental research to therapeutic applications

The intron and 5' distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues.

An extended version of an intron-containing soybean polyubiquitin promoter gave very high levels of gene expression using three different validation tools. The intron-containing Glycine max polyubiquitin promoter (Gmubi) is able to regulate expression levels five times higher than the widely used CaMV35S promoter. In this study, eleven Gmubi derivatives were designed and evaluated to determine which regions contributed to the high levels of gene expression, observed with this promoter. Derivative constructs regulating GFP were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. With both expression systems, removal of the intron in the 5'UTR led to reduced levels of gene expression suggesting a role of the intron in promoter activity. Promoter constructs containing an internal intron duplication and upstream translocations of the intron resulted in higher and similar expression levels to Gmubi, respectively, indicating the presence of enhancers within the intron. Evaluation of 5' distal extensions of the Gmubi promoter resulted in significantly higher levels of GFP expression, suggesting the presence of upstream regulatory elements. A twofold increase in promoter strength was obtained when Gmubi was extended 1.5kb upstream to generate GmubiXL (2.4kb total length). In stably transformed soybean plants containing GFP regulated by CaMV35S, Gmubi and GmubiXL, the GmubiXL promoter clearly produced the highest levels of gene expression, with especially high GFP fluorescence in the vascular tissue and root tips. Use of GmubiXL leads to very high levels of gene expression in soybean and represents a native soybean promoter, which may be useful for regulating transgene expression for both basic and applied research.

Conditional and target-specific transgene induction through RNA replacement using an allosteric trans-splicing ribozyme.

Gene therapeutic approaches are needed that can simultaneously induce the well-controlled expression of therapeutic genes and suppress the expression of disease-causing genes for maximization of their efficacy. To address this challenge, we designed an allosteric ribozyme that comprises a Tetrahymena group I-based trans-splicing ribozyme as an active domain for RNA replacement, a small molecule-specific RNA aptamer as a sensor domain, and a communication module as an active transfer domain. The effectiveness of this approach was assessed by constructing various ribozymes in combination with a theophylline-binding aptamer to identify an allosteric ribozyme, which is controlled by theophylline both in vitro and in cells. Moreover, we constructed adenoviral vectors encoding the ribozymes and validated allosteric regulation of trans-gene expression via theophylline-dependent RNA replacement in target RNA-expressing cells. Results demonstrate that an allosteric trans-splicing ribozyme is an applicable RNA-based framework for engineering external ligand-controlled gene expression regulatory systems that exhibit adjustable regulation, design modularity, and target specificity.

Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates.

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.

Effect of peptide content on the regulation of transgene expression by protein kinase Cα-responsive linear polyethylenimine-peptide conjugates

We examined a series of linear polyethylenimine (LPEI)-based nanocarriers that activate transgene expression in response to cancer-specific protein kinase Cα (PKCα). Eight types of LPEI-peptide conjugate differing in peptide content and number were synthesized using click chemistry. The conjugates could form polyplexes with pDNA through electrostatic interaction, but the degree of pDNA condensation, sizes, and surface charges of the resulting polyplexes depended on the pendant-peptide content and number. None of the polyplexes showed significant cytotoxicity toward human hepatoma cells (HepG2). Furthermore, pendant peptide content and number markedly affected transgene activation in response to PKCα. To achieve an all-or-none response to PKCα, we determined the optimum peptide content and number in LPEI-peptide conjugates as ≈6mol% and ≈40peptides/conjugate.

Dual-regulated Lentiviral Vector for Gene Therapy of X-linked Chronic Granulomatosis

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.

MicroRNA Expression Differences in Human Hematopoietic Cell Lineages Enable Regulated Transgene Expression

Blood microRNA (miRNA) levels have been associated with and shown to participate in disease pathophysiology. However, the hematopoietic cell of origin of blood miRNAs and the individual blood cell miRNA profiles are poorly understood. We report the miRNA content of highly purified normal hematopoietic cells from the same individuals. Although T-cells, B-cells and granulocytes had the highest miRNA content per cell, erythrocytes contributed more cellular miRNA to the blood, followed by granulocytes and platelets. miRNA profiling revealed different patterns and different expression levels of miRNA specific for each lineage. miR-30c-5p was determined to be an appropriate reference normalizer for cross-cell qRT-PCR comparisons. miRNA profiling of 5 hematopoietic cell lines revealed differential expression of miR-125a-5p. We demonstrated endogenous levels of miR-125a-5p regulate reporter gene expression in Meg-01 and Jurkat cells by (1) constructs containing binding sites for miR-125a-5p or (2) over-expressing or inhibiting miR-125a-5p. This quantitative analysis of the miRNA profiles of peripheral blood cells identifies the circulating hematopoietic cellular miRNAs, supports the use of miRNA profiles for distinguishing different hematopoietic lineages and suggests that endogenously expressed miRNAs can be exploited to regulate transgene expression in a cell-specific manner.

Transgene expression study of CXCR4 active mutants

Homing and engraftment, a determining factor in hematopoietic stem cell transplantation success is defined as a process through which hematopoietic stem/progenitor cells (HSPCs) lodge recipient bone marrow. SDF-1/CXCR4 axis acts as a principle regulator in homing and engraftment, however, CXCR4 signaling is dependent upon expression of CXCR4 and its ligand SDF-1, which is highly dynamic. Hence, present investigation was aimed to explore the potential of CXCR4 constitutive active mutants (CXCR4-CAMs) in overcoming the limitation of CXCR4 signaling and up-modulate its efficiency in homing and engraftment. Regulated transgene expression study of these mutants revealed their significantly enhanced cell adhesion efficiency to endothelium and extracellular matrix protein. This altogether indicates promising prospects of CXCR4-CAMs in research aimed to improve HSPCs engraftment efficiency.

Temporal and spatial patterning of transgene expression by near-infrared irradiation

We investigated whether near-infrared (NIR) light could be employed for patterning transgene expression in plasmonic cell constructs. Hollow gold nanoparticles with a plasmon surface band absorption peaking at ∼750 nm, a wavelength within the so called “tissue optical window”, were used as fillers in fibrin-based hydrogels. These composites, which efficiently transduce NIR photon energy into heat, were loaded with genetically-modified cells that harbor a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation in the presence of ligand triggered 3-dimensional patterns of transgene expression faithfully matching the illuminated areas of plasmonic cell constructs. This non-invasive technology was proven useful for remotely controlling in vivo the spatiotemporal bioavailability of transgenic vascular endothelial growth factor. The combination of spatial control by means of NIR irradiation along with safe and timed transgene induction presents a high application potential for engineering tissues in regenerative medicine scenarios.

Amino acids regulate transgene expression in MDCK cells.

Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway.

A Simple and Efficient System for Regulating Gene Expression in Human Pluripotent Stem Cells and Derivatives

Regulatable transgene expression in human pluripotent stem cells (hPSCs) and their progenies is often necessary to dissect gene function in a temporal and spatial manner. However, hPSC lines with inducible transgene expression, especially in differentiated progenies, have not been established due to silencing of randomly inserted genes during stem cell expansion and/or differentiation. Here, we report the use of transcription activator-like effector nucleases-mediated targeting to AAVS1 site to generate versatile conditional hPSC lines. Transgene (both green fluorescent protein and a functional gene) expression in hPSCs and their derivatives was not only sustained but also tightly regulated in response to doxycycline both in vitro and in vivo. We modified the donor construct so that any gene of interest can be readily inserted to produce hPSC lines with conditional transgene expression. This technology will substantially improve the way we study human stem cells.

Heritable Epigenetic Variation and its Potential Applications for Crop Improvement

Phenotypic variation within organisms is driven primarily by genetic diversity. However, there is a growing appreciation that epigenetic variation, resulting from a multitude of diverse chemical modifications to the DNA and chromatin, can have profound effects on phenotype. Heritable epigenetic marks persist through meiosis and can be stably transmitted to the next generation, resulting in transgenerational epigenetic inheritance. Importantly, when epigenetic changes occur near coding genes, affecting their transcriptional state, heritable epigenetic variation can result in heritable phenotypic variation. Large-scale interrogation of epigenome inheritance in Arabidopsis has revealed that spontaneous variation in DNA methylation occurs at a rate that is orders of magnitude greater than genetic mutation, indicating the key importance of epigenetic variation during evolution. Thus, there is a potential for epigenetics to play a role in crop improvement, including regulation of transgene expression and creation of novel epialleles. Here, we review cases of naturally occurring and genetically induced epialleles, and discuss how the studies from two epigenetic populations are rapidly increasing our understanding of epigenetic diversity.

Tunable diblock copolypeptide hydrogel depots for local delivery of hydrophobic molecules in healthy and injured central nervous system

Many hydrophobic small molecules are available to regulate gene expression and other cellular functions. Locally restricted application of such molecules in the central nervous system (CNS) would be desirable in many experimental and therapeutic settings, but is limited by a lack of innocuous vehicles able to load and easily deliver hydrophobic cargo. Here, we tested the potential for diblock copolypeptide hydrogels (DCH) to serve as such vehicles. In vitro tests on loading and release were conducted with cholesterol and the anti-cancer agent, temozolomide (TMZ). Loading of hydrophobic cargo modified DCH physical properties such as stiffness and viscosity, but these could readily be tuned to desired ranges by modifying DCH concentration, amino acid composition or chain lengths. Different DCH formulations exhibited different loading capacities and different rates of release. For example, comparison of different DCH with increasing alanine contents showed corresponding increases in both cargo loading capacity and time for cargo release. In vivo tests were conducted with tamoxifen, a small synthetic hydrophobic molecule widely used to regulate transgene expression. Tamoxifen released from DCH depots injected into healthy or injured CNS efficiently activated reporter gene expression in a locally restricted manner in transgenic mice. These findings demonstrate the facile and predictable tunability of DCH to achieve a wide range of loading capacities and release profiles of hydrophobic cargos while retaining CNS compatible physical properties. In addition, the findings show that DCH depots injected into the CNS can efficiently deliver small hydrophobic molecules that regulate gene expression in local cells.

MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation

BackgroundThe in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models.ResultsIn this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression.ConclusionsThis study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo.

378. Tight Regulation of Transgene Expression by Mifepristone-Inducible System after In Vitro Delivery by Lentiviral Vectors to Neural Progenitor Cells

378. Tight Regulation of Transgene Expression by Mifepristone-Inducible System after In Vitro Delivery by Lentiviral Vectors to Neural Progenitor Cells

Regulated expression of CXCR4 constitutive active mutants revealed the up-modulated chemotaxis and up-regulation of genes crucial for CXCR4 mediated homing and engraftment of hematopoietic stem/progenitor cells

SDF-1/CXCR4 axis plays a principle role in the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs), a process that defines cells ability to reach and seed recipient bone marrow niche following their intravenous infusion. However, the proper functioning of CXCR4 downstream signaling depends upon consistent optimal expression of both SDF-1 ligand and its receptor CXCR4, which in turn is variable and regulated by several factors. The constitutive active mutants of CXCR4 (N119A and N119S) being able to induce autonomous downstream signaling, overcome the limitation of ligand-receptor interaction for induction of CXCR4 signaling. Therefore, we intended to explore their potential in Chemotaxis; a key cellular process which crucially regulates cells homing to bone marrow. In present study, Tet-on inducible gene expression vector system was used for doxycycline inducible regulated transgene expression of CXCR4 active mutants in hematopoietic stem progenitor cell line K-562. Both of these mutants revealed significantly enhanced Chemotaxis to SDF-1 gradient as compared to wild type. Furthermore, gene expression profiling of these genetically engineered cells as assessed by microarray analysis revealed the up-regulation of group of genes that are known to play a crucial role in CXCR4 mediated cells homing and engraftment. Hence, this study suggest the potential prospects of CXCR4 active mutants in research and development aimed to improve the efficiency of cells in the mechanism of homing and engraftment process.

Prolonged Gene Expression in Muscle Is Achieved Without Active Immune Tolerance Using MicrorRNA 142.3p-Regulated rAAV Gene Transfer

Gene transfer efficacy is limited by unwanted immunization against transgene products. In some models, immunization may be avoided by regulating transgene expression with mir142.3p target sequences. Yet, it is unclear if such a strategy controls T-cell responses following recombinant adeno-associated viral vector (rAAV)-mediated gene transfer, particularly in muscle. In mice, intramuscular rAAV1 gene delivery of a tagged human sarcoglycan muscle protein is robustly immunogenic and leads to muscle destruction. In this model, the simple insertion of mir142.3p-target sequences in the transgene expression cassette modifies the outcome of gene transfer, providing high and persistent levels of muscle transduction in C57Bl/6 mice. Such regulated vector fails to prime specific CD4 and CD8 T cells; although, transgene tolerance seems to result from ignorance and could be broken by a robust antigenic challenge. While effective in normal mice, the mir142.3p-regulated transgene remains immunogenic in sarcoglycan-deficient dystrophic mice. In these mice, transgene expression is only prolonged but does not persist as effector CD4 and CD8 T-cell responses develop. Thus, using a mir142.3p-regulated transgene can improve rAAV muscle gene transfer results, but the level of efficacy depends on the context of application. In normal muscle, this strategy is sufficient to prevent immunization and functions even more effectively than tissue-specific promoters. In dystrophic models, additional strategies are required to fully control T-cell responses.

Myocardial Gene Transfer: Routes and Devices for Regulation of Transgene Expression by Modulation of Cellular Permeability

Heart diseases are major causes of morbidity and mortality in Western society. Gene therapy approaches are becoming promising therapeutic modalities to improve underlying molecular processes affecting failing cardiomyocytes. Numerous cardiac clinical gene therapy trials have yet to demonstrate strong positive results and advantages over current pharmacotherapy. The success of gene therapy depends largely on the creation of a reliable and efficient delivery method. The establishment of such a system is determined by its ability to overcome the existing biological barriers, including cellular uptake and intracellular trafficking as well as modulation of cellular permeability. In this article, we describe a variety of physical and mechanical methods, based on the transient disruption of the cell membrane, which are applied in nonviral gene transfer. In addition, we focus on the use of different physiological techniques and devices and pharmacological agents to enhance endothelial permeability. Development of these methods will undoubtedly help solve major problems facing gene therapy.

A Double-Switch Vector System Positively Regulates Transgene Expression by Endogenous microRNA Expression (miR-ON Vector)

To better understand and exploit microRNA (miR) regulation, a more precise characterization of miR expression patterns within a tissue or a lineage during development, differentiation, and homeostasis is needed. We previously showed that lentiviral vectors (LV) can be made responsive to miR to stringently control transgene expression as well as to report miR activity "live" and at the single-cell level. Although very useful, this approach reports miR activity by transgene suppression, hampering the direct identification and selection of miR-expressing cells. Here, we describe a strategy to couple transgene expression to the activity of the miR of interest. To this aim, we generated LV encoding two in-series OFF switches: a transcriptional repressor tagged with miR target sequences and a reporter cassette under the control of the repressor. Reporter expression is ON only when the miR is active and represses translation of the transcriptional repressor. We successfully applied this design to different types of repressors, multiple gene encoding vectors and delivered the system either by two separate or a self-contained vector. We demonstrated its performance by live monitoring of two miRs in different stages of human primary hematopoietic stem/progenitor cell differentiation in vivo. Further applications of this approach include imaging of rare miR-expressing cells and positive regulation of a therapeutic or selector gene in target cells identified by the expression of selected miRs.