Expression In Transgenic Mice Research Articles

Glial reactivity in a mouse model of beta-amyloid deposition assessed by PET imaging of P2X7 receptor and TSPO using [11C]SMW139 and [18F]F-DPA

BackgroundP2X7 receptor has emerged as a potentially superior PET imaging marker to TSPO, the gold standard for imaging glial reactivity. [11C]SMW139 is the most recently developed radiotracer to image P2X7 receptor. The aim of this study was to image reactive glia in the APP/PS1-21 transgenic (TG) mouse model of Aβ deposition longitudinally using [11C]SMW139 targeting P2X7 receptor and to compare tracer uptake to that of [18F]F-DPA targeting TSPO at the final imaging time point. TG and wild type (WT) mice underwent longitudinal in vivo PET imaging using [11C]SMW139 at 5, 8, 11, and 14 months, followed by [18F]F-DPA PET scan only at 14 months. In vivo imaging results were verified by ex vivo brain autoradiography, immunohistochemical staining, and analysis of [11C]SMW139 unmetabolized fraction in TG and WT mice.ResultsLongitudinal change in [11C]SMW139 standardized uptake values (SUVs) showed no statistically significant increase in the neocortex and hippocampus of TG or WT mice, which was consistent with findings from ex vivo brain autoradiography. Significantly higher [18F]F-DPA SUVs were observed in brain regions of TG compared to WT mice. Quantified P2X7-positive staining in the cortex and thalamus of TG mice showed a minor increase in receptor expression with ageing, while TSPO-positive staining in the same regions showed a more robust increase in expression in TG mice as they aged. [11C]SMW139 was rapidly metabolized in mice, with 33% of unmetabolized fraction in plasma and 29% in brain homogenates 30 min after injection.Conclusions[11C]SMW139, which has a lower affinity for the rodent P2X7 receptor than the human version of the receptor, was unable to image the low expression of P2X7 receptor in the APP/PS1-21 mouse model. Additionally, the rapid metabolism of [11C]SMW139 in mice and the presence of several brain-penetrating radiometabolites significantly impacted the analysis of in vivo PET signal of the tracer. Finally, [18F]F-DPA targeting TSPO was more suitable for imaging reactive glia and neuroinflammatory processes in the APP/PS1-21 mouse model, based on the findings presented in this study and previous studies with this mouse model.

Brain-Wide Transgene Expression in Mice by Systemic Injection of Genetically Engineered Exosomes: CAP-Exosomes.

The bottleneck in drug discovery for central nervous system diseases is the absence of effective systemic drug delivery technology for delivering therapeutic drugs into the brain. Despite the advances in the technology used in drug discovery, such as Adeno-Associated Virus (AAV) vectors, the development of drugs for central nervous system diseases remains challenging. In this manuscript, we describe, for the first time, the development of a workflow to generate a novel brain-targeted drug delivery system that involves the generation of genetically engineered exosomes by first selecting various functional AAV capsid-specific peptides (collectively called CAPs) known to be involved in brain-targeted high-expression gene delivery, and then expressing the CAPs in frame with lysosome-associated membrane glycoprotein (Lamp2b) followed by expressing CAP-Lamp2b fusion protein on the surface of mesenchymal stem cell-derived exosomes, thus generating CAP-exosomes. Intravenous injection of green fluorescent protein (GFP) gene-loaded CAP-exosomes in mice transferred the GFP gene throughout the CNS as measured by monitoring brain sections for GFP expression with confocal microscopy. GFP gene transfer efficiency was at least 20-fold greater than that of the control Lamp2b-exosomes, and GFP gene transduction to mouse liver was low.

Serum biomarkers indicate sex differences in response to pathology inhibition in aged mice with tau pathology

AbstractBackgroundThe failure of some clinical trials using drugs that remove brain amyloid to prevent progression of dementia suggest that neurodegenerative disease may not progress in a linear fashion and threshold levels of one pathology may initiate alterations that are unable to be reversed upon removal of the initiating pathological process. We used the inducible Tg4510 mouse tauopathy model to determine whether neurodegeneration could be halted following removal of transgene expression in mice aged to 18 monthsMethodPathology was induced in mice from 8 weeks until 18 months of age (n = 20 equal male/female; both tau transgenic and non‐transgenic) by removal of doxycycline (dox) diet. Dox diet was returned to half the animals from 18‐23 months to inhibit pathology. Levels of serum Nfl were measured using SIMOA assay throughout disease progression and p‐tau181 along with other serum biomarkers were measured at endpoint. Immunohistochemical assays were performed to quantitate numbers of neurons (NeuN) and tau load (PHF‐tau) in hippocampus.ResultSerum Nfl increased over aging in all mice and was significantly higher in tau transgenics relative to non‐transgenics at 18 months (p<0.05). Between 18 and 23 months, the rate of serum Nfl increase was significantly(p<0.05) reduced in male transgenic mice on dox compared to female transgenic mice, so that by 23 months levels were not different from non‐transgenic mice. Similarly, the numbers of neurons in the hippocampus of male mice on dox at 23 months was significantly higher than their female counterparts (p<0.05). When looking at levels of serum p‐tau181 at 23 months, there was significantly (p<0.05) higher levels in male mice on dox than female miceConclusionThese data suggest that aged female mice have ongoing neurodegeneration following removal of pathology induction that is similar to mice with continued pathology induction. The differences in serum p‐tau‐181 may indicate that female mice fail to clear pathological tau from the brain. Further studies will examine the relationship between histopathology and blood biomarkers and determine whether female mice on dox have a higher tau pathology load than their male counterparts.

Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability

During the COVID-19 pandemic, Pfizer-BioNTech and Moderna successfully developed nucleoside-modified mRNA lipid nanoparticle (LNP) vaccines. SARS-CoV-2 spike protein expressed by those vaccines are identical in amino acid sequence, but several key components are distinct. Here, we compared the effect of ionizable lipids, untranslated regions (UTRs), and nucleotide composition of the two vaccines, focusing on mRNA delivery, antibody generation, and long-term stability. We found that the ionizable lipid, SM-102, in Moderna’s vaccine performs better than ALC-0315 in Pfizer-BioNTech’s vaccine for intramuscular delivery of mRNA and antibody production in mice and long-term stability at 4 °C. Moreover, Pfizer-BioNTech’s 5′ UTR and Moderna’s 3′ UTR outperform their counterparts in their contribution to transgene expression in mice. We further found that varying N1-methylpseudouridine content at the wobble position of mRNA has little effect on vaccine efficacy. These findings may contribute to the further improvement of nucleoside-modified mRNA-LNP vaccines and therapeutics.

A Ubiquitous Chromatin Opening Element and DNA Demethylation Facilitate Doxycycline-Controlled Expression during Differentiation and in Transgenic Mice.

Synthetic expression cassettes provide the ability to control transgene expression in experimental animal models through external triggers, enabling the study of gene function and the modulation of endogenous regulatory networks in vivo. The performance of synthetic expression cassettes in transgenic animals critically depends on the regulatory properties of the respective chromosomal integration sites, which are affected by the remodeling of the chromatin structure during development. The epigenetic status may affect the transcriptional activity of the synthetic cassettes and even lead to transcriptional silencing, depending on the chromosomal sites and the tissue. In this study, we investigated the influence of the ubiquitous chromosome opening element (UCOE) HNRPA2B1-CBX3 and its subfragments A2UCOE and CBX3 on doxycycline-controlled expression modules within the chromosomal Rosa26 locus. While HNRPA2B1-CBX3 and A2UCOE reduced the expression of the synthetic cassettes in mouse embryonic stem cells, CBX3 stabilized the expression and facilitated doxycycline-controlled expression after in vitro differentiation. In transgenic mice, the CBX3 element protected the cassettes from overt silencing although the expression was moderate and only partially controlled by doxycycline. We demonstrate that CBX3-flanked synthetic cassettes can be activated by decitabine-mediated blockade of DNA methylation or by specific recruitment of the catalytic demethylation domain of the ten-eleven translocation protein TET1 to the synthetic promoter. This suggests that CBX3 renders the synthetic cassettes permissive for subsequent epigenetic activation, thereby supporting doxycycline-controlled expression. Together, this study reveals a strategy for overcoming epigenetic constraints of synthetic expression cassettes, facilitating externally controlled transgene expression in mice.

Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants

BackgroundLipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments.ResultsSo far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction.ConclusionsThe expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery.Graphical

Methylation silencing and reactivation of exogenous genes in lentivirus-mediated transgenic mice.

Taking advantage of their ability to integrate their genomes into the host genome, lentiviruses have been used to rapidly produce transgenic mice in biomedical research. In most cases, transgenes delivered by lentiviral vectors have resisted silencing mediated by epigenetic modifications in mice. However, some studies revealed that methylation caused decreased transgene expression in mice. Therefore, there is conflicting evidence regarding the methylation-induced silencing of transgenes delivered by lentiviral transduction in mice. In this study, we present evidence that the human TTR transgene was silenced by DNA methylation in the liver of a transgenic mouse model generated by lentiviral transduction. The density of methylation on the transgene was increased during reproduction, and the expression of the transgene was completely silenced in mice of the F2 generation. Interestingly, 5-azacytidine (5-AzaC), a methyltransferase inhibitor, potently reactivated the silenced genes in neonatal mice whose hepatocytes were actively proliferating and led to stable transgene expression during development. However, 5-AzaC did not rescue liver transgene expression when administered to adult mice. Moreover, 5-AzaC at the given dose had low developmental toxicity in the newborn mice. In summary, we demonstrate the methylation-induced silencing of an exogenous gene in the liver of a mouse model generated by lentiviral transduction and show that the silenced transgene can be safely and efficiently reactivated by 5-AzaC treatment, providing an alternative way to obtain progeny with stable transgene expression in the case of the methylation of exogenous genes in transgenic mice generated by lentiviral transduction.

A Small-Molecule-Responsive Riboswitch Enables Conditional Induction of Viral Vector-Mediated Gene Expression in Mice.

Adeno-associated viral (AAV) vector-mediated gene therapy holds great potential for future medical applications. However, to facilitate safer and broader applicability and to enable patient-centric care, therapeutic protein expression should be controllable, ideally by an orally administered drug. The use of protein-based systems is considered rather undesirable, due to potential immunogenicity and the limited coding space of AAV. Ligand-dependent riboswitches, in contrast, are small and characterized by an attractive mode-of-action based on mRNA-self-cleavage, independent of coexpressed foreign protein. While a promising approach, switches available to date have only shown moderate potency in animals. In particular, ON-switches that induce transgene expression upon ligand administration so far have achieved rather disappointing results. Here we present the utilization of the previously described tetracycline-dependent ribozyme K19 for controlling AAV-mediated transgene expression in mice. Using this tool switch, we provide first proof for the feasibility of clinically desired key features, including multiorgan functionality, potent regulation (up to 15-fold induction), reversibility, and the possibility to fine-tune and repeatedly induce expression. The systematic assessment of ligand and reporter protein plasma levels further enabled the characterization of pharmacokinetic-pharmacodynamic relationships. Thus, our results strongly support future efforts to develop engineered riboswitches for applications in clinical gene therapy.

Analysis of a limb-specific regulatory element in the promoter of the link protein gene

Link protein is encoded by the Hapln1 gene and is a prototypical protein found in the cartilage matrix. It acts as an important component of the endochondral skeleton during early development. To study its transcriptional regulation, promoter fragments derived from the link protein gene were coupled to the β-galactosidase reporter and used to study in vivo transgene expression in mice. In day 15.5 mouse embryos, a link promoter fragment spanning −1020 to +40 nucleotides demonstrated highly specific β-galactosidase staining of skeletal structures, including the appendicular and axial cartilaginous tissues. Two shorter promoter fragments, spanning −690 to +40 and −315 to +40 nucleotides, demonstrated limb- and genitalia-specific expression resembling that of homeodomain-regulated tissues. Bioinformatic analysis revealed a highly conserved, Hox-like binding site (HLBS) at approximately −220 bp of the promoter, shared by both constructs, which contained the Hox-core consensus sequence TAATTA. Electromobility shift assays demonstrated binding of Hox-B4 recombinant protein to the HLBS, which was eliminated with nucleotide substitutions within the core-binding element. Co-transfection analysis of the HLBS demonstrated a 22-fold transcriptional activation by HoxA9 expression, which was ablated with a substitution within the core HLBS element. Together these findings establish promoter regions within the link protein gene that are important for in vivo expression and identify the potential role of homeodomain-containing proteins in controlling cartilage and limb gene expression.

No enhancing effects of plasmid-specific histone acetyltransferase recruitment system on transgene expression in vivo

Altered levels of histone acetylation are associated with changes in chromosomal gene expression. Thus, the specific acetylation of histones bound to plasmid DNA might increase transgene expression. Previously, the expression of the histone acetyltransferase domain of CREB-binding protein fused to the sequence-dependent DNA binding domain of GAL4 (GAL4-HAT) successfully improved reporter gene expression in cultured cells [J. Biosci. Bioengng. 123, 277–280 (2017)]. In this study, the same approach was applied for transgene expression in mice. The activator and reporter plasmid DNAs bearing the genes for GAL4-HAT and Gaussia princeps luciferase, respectively, were co-administered into the mouse liver by hydrodynamics-based tail vein injection, and the Gaussia luciferase activity in serum was measured for two weeks. Unexpectedly, the co-injection of the GAL4-HAT and luciferase plasmid DNAs seemed to decrease, rather than increase, luciferase expression. Moreover, the co-injection apparently reduced the amount of luciferase DNA in the liver. These results indicated that this system is ineffective in vivo and suggested the exclusion of hepatic cells expressing GAL4-HAT.

AP-1 and the injury response of the GFAP gene.

Increased GFAP gene expression is a common feature of CNS injury, resulting in its use as a reporter to investigate mechanisms producing gliosis. AP-1 transcription factors are among those proposed to participate in mediating the reactive response. Prior studies found a consensus AP-1 binding site in the GFAP promoter to be essential for activity of reporter constructs transfected into cultured cells, but to have little to no effect on basal transgene expression in mice. Since cultured astrocytes display some properties of reactive astrocytes, these findings suggested that AP-1 transcription factors are critical for the upregulation of GFAP in injury, but not for its resting level of expression. We have examined this possibility by comparing the injury response in mice of lacZ transgenes driven by human GFAP promoters that contain the wild-type AP-1 binding site to those in which the site is mutated. An intact AP-1 site was found critical for a GFAP promoter response to the three different injury models used: physical trauma produced by cryoinjury, seizures produced by kainic acid, and chronic gliosis produced in an Alexander disease model. An unexpected additional finding was that the responses of the lacZ transgenes driven by the wild-type promoters were substantially less than that of the endogenous mouse GFAP gene. This suggests that the GFAP gene has previously unrecognized injury-responsive elements that reside further upstream of the transcription start site than the 2.2kb present in the GFAP promoter segments used here.

Edaravone, a cytoprotective drug, enhances transgene expression mediated by lipoplexes in HepG2 cells and mice

A requirement of gene therapy is efficient nucleic acid delivery. However, the application of cationic liposomes to gene therapy is restricted by their inefficient transfection capacity, which may be caused by cytotoxicity. This cytotoxicity is highly dependent on cationic lipid-induced reactive oxygen species (ROS). Here, to provide cellular protection, we used edaravone, an efficacious anti-oxidative drug, to scavenge ROS during transfection using cationic liposome/plasmid DNA complexes (lipoplexes). Both free edaravone and edaravone-loaded liposomes (EDLPs) enhanced transgene expression in the human hepatoma cell line, HepG2, while EDLPs decreased the effective dose of edaravone. The cellular protective effect of edaravone was found to decrease the cytotoxicity of cationic liposomes. Edaravone was also effective in the commercial product, Lipofectamine® 3000, which may expand the application of edaravone to promote transfection efficiency. Compared with free edaravone, EDLPs also showed superior transgene expression in mice. Our findings will promote the development of efficient and safe gene therapy.

Spray-Dried Nanoparticle-in-Microparticle Delivery Systems (NiMDS) for Gene Delivery, Comprising Polyethylenimine (PEI)-Based Nanoparticles in a Poly(Vinyl Alcohol) Matrix.

Nucleic acid-based therapies rely on efficient formulations for nucleic acid protection and delivery. As nonviral strategies, polymeric and lipid-based nanoparticles have been introduced; however, biological efficacy and biocompatibility as well as poor storage properties due to colloidal instability and their unavailability as ready-to-use systems are still major issues. Polyethylenimine is the most widely explored and promising candidate for gene delivery. Polyethylenimine-based polyplexes and their combination with liposomes, lipopolyplexes, are efficient for DNA or siRNA delivery in vitro and in vivo. In this study, a highly potent spray-dried nanoparticle-in-microparticle delivery system is presented for the encapsulation of polyethylenimine-based polyplexes and lipopolyplexes into poly(vinyl alcohol) microparticles, without requiring additional stabilizing agents. This easy-to-handle gene delivery device allows prolonged nanoparticle storage and protection at ambient temperature. Biological analyses reveal further advantages regarding profoundly reduced cytotoxicity and enhanced transfection efficacies of polyethylenimine-based nanoparticles from the nanoparticle-in-microparticle delivery system over their freshly prepared counterparts, as determined in various cell lines. Importantly, this nanoparticle-in-microparticle delivery system is demonstrated as ready-to-use dry powder to be an efficient device for the inhalative delivery of polyethylenimine-based lipopolyplexes in vivo, as shown by transgene expression in mice after only one administration.

Combining fibroblast isolation with lentiviral gene transfer to validate transgene expression in mice following pronucleus injection.

The binary tetracycline-based expression system in transgenic mice relies on the expression of the tetracycline transactivator (tTA or rtTA) in a particular cell type together with a transcription unit encoding the gene of interest under a tetracycline or doxycycline-responsive promoter. Transgenic mice containing this transcription unit are produced via pronucleus injection. As the chromosomal integration site of the injected DNA influences transgene expression, several founder lines have to be crossed with (r)tTA-expressing mice to find a line showing low background and high transgene expression following doxycycline stimulation. Here, we describe a method to analyze primary fibroblasts derived from the founder lines to quickly test transgene expression and inducibility. Fibroblasts isolated from a small piece of mouse ear were infected with a recombinant lentivirus expressing rtTA. Transgene expression was verified by both RT-PCR and western blot, following stimulation with doxycycline. Transgene expression could easily be detected on the RNA and protein levels in primary fibroblasts derived from transgenic founder lines. An enzymatic function of the transgene was not required for the identification of transgene expression. Thus, the method allows a quick and easy discrimination of transgenic founder lines according to transgene expression and inducibility.

Cosmetics-triggered percutaneous remote control of transgene expression in mice.

Synthetic biology has significantly advanced the rational design of trigger-inducible gene switches that program cellular behavior in a reliable and predictable manner. Capitalizing on genetic componentry, including the repressor PmeR and its cognate operator OPmeR, that has evolved in Pseudomonas syringae pathovar tomato DC3000 to sense and resist plant-defence metabolites of the paraben class, we have designed a set of inducible and repressible mammalian transcription-control devices that could dose-dependently fine-tune transgene expression in mammalian cells and mice in response to paraben derivatives. With an over 60-years track record as licensed preservatives in the cosmetics industry, paraben derivatives have become a commonplace ingredient of most skin-care products including shower gels, cleansing toners and hand creams. As parabens can rapidly reach the bloodstream of mice following topical application, we used this feature to percutaneously program transgene expression of subcutaneous designer cell implants using off-the-shelf commercial paraben-containing skin-care cosmetics. The combination of non-invasive, transdermal and orthogonal trigger-inducible remote control of transgene expression may provide novel opportunities for dynamic interventions in future gene and cell-based therapies.

147. Transcription Factor Binding Site in Plasmid Regulates Persistence of Transgene Expression in Mouse Liver

Persistent expression of transgene is most desirable for treatment of genetic diseases. It is primarily controlled by genetic elements in the promoter region where transcription factors bind. The objective of this study is to assess the role of the transcription factor binding sites (TFBS) present in the most commonly used CMV and albumin promoters containing the promoter core sequence and several TFBS for ATF1, NFκB, SRF, AP1, Sp1, NF1α, CEBPA, HNF4α, and HNF1 in determining persistency of transgene expression in mice. Secreted embryonic alkaline phosphatase (SEAP) and interleukin 10 (IL-10) were used as a reporter to monitor transgene expression. Variousdeletion mutation plasmids at the promoter region were made to examine the function of each of the TFBS in regulating reporter gene expression. Similarly, a series of new plasmids containing only one TFBS inserted at the 5’ end of the TATA-box region was made to assess its unique activity in affecting transgene expression. Our results showed that TFBSs are essential for effective transgene expression in mouse liver after a hydrodynamic tail vein gene delivery. Plasmid with a TATA-box with all TFBS deleted resulted in minimal level of gene product, 700-fold lower than the original plasmids. Reporter gene expression appears transient reaching the background level in less than 2 weeks. The more TFBS the plasmid has, the higher level of gene product was seen. Plasmid containing a single TFBS either NFκB, ATF1, CEBPA, HNF4α, or HNF1 exhibits persistent transgene expression for more than 2 months, at which animals injected with plasmids containing NFkB, ATF1, CEBPA, HNF4α, or HNF1 exhibited gene product level 60, 130, 1020, 2250, and 2076 folds higher than that of animals injected with plasmids without TFBS, respectively. Mechanism study employing real time PCR, Western Blotting, and ChIP reveals involvement of histone acetylation and the binding of acetylated histones to plasmid DNA sequences in TFBS-mediated enhancement in persistency of reporter gene expression. TFBSs for HNF4α appear most potent in recruiting H4AC and TBP proteins to the plasmids. Our results suggest that TFBSs for NFkB, ATF1, CEBPA, HNF4α, and HNF1 are important elements to include into the promoter for achieving longer-term gene expression. In addition, histone acetylation on plasmid is a probable pathway that TFBS regulate the duration of transgene expression.

574. Sustaining Helper-Dependent Adenoviral Vector-Mediated Human CFTR Expression in Mouse Airways Through Transient Immunosuppression

Cystic Fibrosis (CF) is a common life-threatening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. Gene therapy for CF lung disease has been tested extensively. However, sustained expression of the CFTR gene is a major challenge to gene delivery with either viral or no viral vectors. One strategy to improve the persistent CFTR expression is to modulate host immune responses by transient immunosuppression.We showed previously that cyclophosphamide significantly enhanced human CFTR expression in mice received HD-Ad vector readministration, through inhibition of host immune reactions. In this study, we examined cyclophosphamide, dexmethoson, and combination of cyclosporine, methylprednsolone and azathioprine, for their effects on long term expression of the human CFTR delivered with helper-dependent adenoviral (HD-Ad) vectors to mouse airways. Four group mice were nasally delivered with an HD-Ad-K18-CFTR vector at 1.5×10E10 particles per mouse. Three groups were treated with immunesuppresants and one group without any treatment was used as a control. The immunosuppressants were administrated twice, at 6 hours before and 48 hours after nasal delivery of the HD-Ad-CFTR vector. Expression of the CFTR gene was assessed 4 months after vector administration. We found that cyclophosphamide has significantly improved the sustained expression of the CFTR gene (2.5 times higher) compared to the no treatment group as determined by real-time qPCR. It alsogreatly reduced the levels of anti-adenoviral and neutralizing antibodies in both BALF and serum. However, cyclophosphamide did not show major effects on the innate immune reaction to HD-Ad vectors, such as NK cell infiltration and activation, 4 days after vector delivery. Dexmethesone also greatly reduced neutralizing antibodies production, but not affected transgene expression. A combination of cyclosporine, methylprednsolone and azathioprine which is used to suppress immune reactions in lung transplantation, also did not affect CFTR gene expression, nor immune reactions following HD-Ad-CFTR vector delivery. These data demonstrate that transient administration of cyclophosphamide can significantly improve sustained HD-Ad-mediated transgene expression in mice and it is more effective than dexmethsone or the combination of cyclosporine, methylprednsolone and azathioprine.

Molecular mechanism regulating myosin and cardiac functions by ELC

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgMhVLC-1) or E56G-mutated hVLC-1 (hVLC-1E56G; TgME56G). hVLC-1 or hVLC-1E56G expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgMhVLC-1 (1.67pN/nm and 2.3μm/s, respectively) were significantly higher than myosin with hVLC-1E56G prepared from TgME56G (1.25pN/nm and 1.7μm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41pN/nm and 1.5μm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgMhVLC-1 (80.0mmHg) were significantly higher than hearts from TgME56G (66.2mmHg) or C57/BL6 (59.3±3.9mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1>hVLC-1E56G≈mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations.

Reversible transgene expression in mice: now you see it, now you don’t (1121.1)

Transgenic mice can suffer from the “Position effect” whereby the transgene integration site, per se, influences the observed phenotype. Multiple ‘Founder’ animals must therefore be studied to determine the actual phenotype of each transgene. We describe a method whereby a transgene can be switched off and on, allowing reliable validation of phenotype.We modified the endothelial specific Tie2 promoter with LacO sequences derived from the E. coli LacO/LacI system and generated transgenic mice expressing defective Connexin(Cx)40 molecules, with EGFP as reporter. Transgene expression was down‐regulated by breeding with mice expressing the inhibitor protein LacI and up‐regulated by Isopropyl β‐D‐1‐thiogalacto‐pyranoside (IPTG) in the drinking water. EGFP was quantified using immunohistochemistry and Western blotting. Blood pressure was measured by tail cuff plethysmography and telemetry.EGFP expression was exclusively endothelial and correlated with transgene copy number. Defective Cx40 expression increased blood pressure. Breeding with LacI mice significantly reduced EGFP expression and blood pressure by 70‐100%. EGFP expression could be reinstated by IPTG in some, but not all, founder lines in a time dependent manner.We conclude that the LacO/LacI system allows temporal transgene regulation and can reliably control for artifactual phenotypes arising from the “Position effect”.

Long-Term Elimination of Hepatitis C Virus from Human Hepatocyte Chimeric Mice After Interferon-γ Gene Transfer

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, liver failure, and hepatocellular carcinoma. Although the combination therapy employing pegylated interferon (IFN)-α and ribavirin is effective, this treatment is effective in only approximately 50% patients with genotype 1 HCV infection. IFN-γ is a potent anti-HCV agent that exhibits its antiviral action through a receptor distinct from that for IFN-α. Therefore, IFN-γ application might provide an alternative approach to IFN-α-based therapies. However, recombinant IFN-γ protein exhibits a poor pharmacokinetic property, that is, a very short half-life. It is our hypothesis that sustained IFN-γ serum concentrations produced by gene transfer could effectively eliminate HCV in vivo. We examined the in vivo antiviral activity in human hepatocyte chimeric mice infected with genotype 1b HCV at high HCV RNA titers (10(5)-10(7) copies/ml). The human IFN-γ-expressing plasmid vector pCpG-huIFNγ exhibited prolonged transgene expression in mice compared with the plasmid vector pCMV-huIFNγ. Moreover, the gene transfer of pCpG-huIFNγ eliminated HCV from the liver of the chimeric mice for a sustained period. On the contrary, administration of pCMV-huIFNγ could not eliminate HCV. In conclusion, we found that a single pCpG-huIFNγ injection resulted in long-term elimination of HCV RNA in chimeric mice, providing, for the first time, direct evidence that chronic infection with high titer HCV in vivo can be treated by sustained IFN-γ treatment.