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Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (L...

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models. ispartof: SCIENTIFIC REPORTS vol:10 issue:1 ispartof: location:England status: published

Gene therapy and gene editing technologies are complex and it can be difficult for the public to understand their possible benefits or side effects. However, patient and public support is critical for the successful adoption of any new technology. Given the recent advances in gene therapy and gene editing, their potential clinical benefits, and the significant attention that has been given to the first-known successful attempt at permanent and heritable changes to the human genome, a systematic review was performed to assess beliefs and attitudes toward gene therapy and gene editing for human use, and to highlight the factors that influence acceptability. A systematic search following Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines was undertaken in April 2018 to identify articles examining opinions and attitudes regarding the acceptability of gene therapy and gene editing. Overall, 1,561 records were retrieved from 4 databases (Ovid Medline, PsycINFO, Scopus, and Web of Science). Duplicates were removed, and titles and abstracts independently screened, leaving 86 full-text articles assessed for eligibility. Following full-text review, 33 were included, with 5 articles added after forward/backward searching. An additional three articles were added following an updated search in March 2019 (total

In vivo bioluminescent imaging allows the detection of reporter gene expression in rodents in real time. Here we describe a novel technology whereby we can generate somatotransgenic rodents with the use of a viral vector carrying a luciferase transgene. We are able to achieve long term luciferase expression by a single injection of lentiviral or adeno-associated virus vectors to newborn mice. Further, we describe whole body bioluminescence imaging of conscious mice in a noninvasive manner, thus enforcing the 3R's (replacement, reduction, and refinement) of biomedical animal research.

In vivo bioluminescent imaging allows the detection of reporter gene expression in rodents in real time. Here we describe a novel technology whereby we can generate somatotransgenic rodents with the use of a viral vector carrying a luciferase transgene. We are able to achieve long term luciferase expression by a single injection of lentiviral or adeno-associated virus vectors to newborn mice. Further, we describe whole body bioluminescence imaging of conscious mice in a noninvasive manner, thus enforcing the 3R’s (replacement, reduction, and refinement) of biomedical animal research.

This article focuses on clinical applications of T cells transduced to express recombinant T cell receptor and chimeric antigen receptor constructs directed toward hematological malignancies, and considers newer strategies incorporating gene-editing technologies to address GvHD and host-mediated rejection. Recent data from clinical trials are reviewed, and an overview is provided of current and emerging manufacturing processes; consideration is also given to new developments in the pipeline.

Purpose of Review Alternative approaches to conventional drug-based cancer treatments have seen T cell therapies deployed more widely over the last decade. This is largely due to their ability to target and kill specific cell types based on receptor recognition. Introduction of recombinant T cell receptors (TCRs) using viral vectors and HLA-independent T cell therapies using chimeric antigen receptors (CARs) are discussed. This article reviews the tools used for genome editing, with particular emphasis on the applications of site-specific DNA nuclease mediated editing for T cell therapies. Recent Findings Genetic engineering of T cells using TCRs and CARs with redirected antigen-targeting specificity has resulted in clinical success of several immunotherapies. In conjunction, the application of genome editing technologies has resulted in the generation of HLA-independent universal T cells for allogeneic transplantation, improved T cell sustainability through knockout of the checkpoint inhibitor, programmed cell death protein-1 (PD-1), and has shown efficacy as an antiviral therapy through direct targeting of viral genomic sequences and entry receptors. Summary The combined use of engineered antigen-targeting moieties and innovative genome editing technologies have recently shown success in a small number of clinical trials targeting HIV and hematological malignancies and are now being incorporated into existing strategies for other immunotherapies.

Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The in vitro whole-organ "Bioreactor grown Artificial Liver Model" (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products. ispartof: ISCIENCE vol:23 issue:12 ispartof: location:United States status: published

This is an open access article under the CC BY license.-- et al., The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation., K.E.H. and S.J. were partly funded by St George’s University of London Enterprize Fund. J.M.K.M.D. and S.N.W. are funded by ERC grant Somabio (260862). T.R.M. and S.N.W. are funded by the NC3Rs (NC/L001780/1). J.P.B. is funded by MINECO (SAF2013-41177-R), ISCIII (RD12/0043/0021), EU-SP3-People-MC-ITN (608381), and NIH/NIDA (1R21DA037678-01). V.N.K. is funded by Action Medical Research and The Henry Smith Charity (GN2158), and P.J.K. is funded by Barts and the London Charity (417/2088). T.R.M. and J.B. were each funded by EU Horizon2020; BATCure 666918., Open access funded by European Research Council.

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation.An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days.To further restrict biosensor activity to the CNS, we performed intracerebroventricular injection of each vector. We observed greater restriction of bioluminescence to the head and spine with both vectors. Immunohistochemistry revealed strongest expression in cells of neuronal morphology. LPS administration stimulated a 4-fold increase over baseline bioluminescence.We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner using a model of LPS-induced inflammation. This technology employs the 3R’s of biomedical animal research, complements existing germline-transgenic models and may be applicable to other rodent disease models with the use of different response elements.

The application of luciferase reporter genes to provide quantitative outputs for the activation of promoters is a well-established technique in molecular biology. Luciferase catalyzes an enzymatic reaction, which in the presence of the substrate luciferin produces photons of light relative to its molar concentration. The luciferase transgene can be genetically inserted at the first intron of a target gene to act as a surrogate for the gene's endogenous expression in cells and transgenic mice. Alternatively, promoter sequences can be excised and/or amplified from genomic sources or constructed de novo and cloned upstream of luciferase in an expression cassette transfected into cells. More recently, the development of synthetic promoters where the essential components of an RNA polymerase binding site and transcriptional start site are fused with various upstream regulatory sequences are being applied to drive reporter gene expression. We have developed a high-throughput cloning strategy to develop lentiviral luciferase reporters driven by transcription factor activated synthetic promoters. Lentiviruses integrate their payload cassette into the host cell genome, thereby facilitating the study of gene expression not only in the transduced cells but also within all subsequent daughter cells. In this manuscript we describe the design, vector construction, lentiviral transduction, and luciferase quantitation of transcription factor activated reporters (TFARs) in vitro and in vivo.

Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.

The application of luciferase reporter genes to provide quantitative outputs for the activation of promoters is a well-established technique in molecular biology. Luciferase catalyzes an enzymatic reaction, which in the presence of the substrate luciferin produces photons of light relative to its molar concentration. The luciferase transgene can be genetically inserted at the first intron of a target gene to act as a surrogate for the gene's endogenous expression in cells and transgenic mice. Alternatively, promoter sequences can be excised and/or amplified from genomic sources or constructed de novo and cloned upstream of luciferase in an expression cassette transfected into cells. More recently, the development of synthetic promoters where the essential components of an RNA polymerase binding site and transcriptional start site are fused with various upstream regulatory sequences are being applied to drive reporter gene expression. We have developed a high-throughput cloning strategy to develop lentiviral luciferase reporters driven by transcription factor activated synthetic promoters. Lentiviruses integrate their payload cassette into the host cell genome, thereby facilitating the study of gene expression not only in the transduced cells but also within all subsequent daughter cells. In this manuscript we describe the design, vector construction, lentiviral transduction, and luciferase quantitation of transcription factor activated reporters (TFARs) in vitro and in vivo.

Abnormal microRNA (miRNA) expression profiles have recently been associated with sporadic pituitary adenomas, suggesting that miRNAs can contribute to tumor formation; miRNAs are small noncoding RNAs that inhibit posttranscriptional expression of target mRNAs by binding to target sequences usually located in the 3′-UTR. In this study, we investigated the role played by miR-107, a miRNA associated with different human cancers, in sporadic pituitary adenomas and its interaction with the pituitary tumor suppressor gene aryl hydrocarbon receptor-interacting protein ( AIP). miR-107 expression was evaluated in pituitary adenoma and normal pituitary samples using microRNA screen TLDA (TaqMan Low-Density Array) and RT-qPCR assays. We show that miR-107 expression was significantly upregulated in GH-secreting and nonfunctioning pituitary adenomas. We found that human AIP-3′-UTR is a target of miR-107 since miR-107 inhibited in vitro AIP expression to 53.9 ± 2% of the miRNA control in a luciferase assay and reduced endogenous AIP mRNA expression to 53 ± 22% of the miRNA control in human cells. However, we did not observe a negative correlation between AIP and miR-107 expression in the human tumor samples. Furthermore, we show that miR-107 overexpression inhibited cell proliferation in human neuroblastoma and rat pituitary adenoma cells. In conclusion, miR-107 is overexpressed in pituitary adenomas and may act as a tumor suppressor. We have identified and confirmed AIP as a miR-107 target gene. Expression data in human samples suggest that the expression of AIP and miR-107 could be influenced by a combination of tumorigenic factors as well as compensatory mechanisms stimulated by the tumorigenic process.

The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4−/− mice or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter constructs in somatotransgenic mice, respectively.

Hypoxic Ischemic Encephalopathy (HIE), occurs in 2-3 per 1000 live births. 25% of surviving affected newborns will suffer from severe life-long neurological disability. HIE is associated with reactive astrogliosis and inflammation. During this process the activity of STAT3 and NFkB transcription factors is increased, and glial fibrillary acidic protein (GFAP) expression is upregulated in astrocytes.Light producing transgenic mice, where luciferase expression is controlled by a surrogate promoter or by a minimal promoter downstream of tandem, synthetic, transcription factor binding elements, are used to provide an in vivo readout of disease processes. In this study, we aimed to deliver STAT3 and NFkB activated or GFAP promoter driven luciferase reporter constructs to brains of neonatal mice as a form of assessing the amount of astrogliosis and inflammation in a mouse model of HIE.A rodent GFAP promoter, and STAT3 and NFkB response elements were each cloned into a lentivirus vector upstream of the genes encoding a codon-optimised firefly luciferase and green fluorescent protein. Lentivirus vector pseudotyped with either gp64 or VSV-g viral envelope glycoproteins were injected intra-cranially into CD1 outbred neonatal (P0) mice, and luciferase expression was monitored continually by whole body bioluminescence imaging of conscious mice. Vector containing NFkB response element was also injected into P0 mice, which underwent HIE surgery at P7.The two pseudotypes gp64 and VSV-g exhibited different cellular tropisms within the central nervous system; VSV-g predominately targeting neuronal cells whereas gp64 transducing cells of fibrillary astrocytic morphology. Long-term expression of luciferase was observed. Following induction of HIE, the most severely affected mice lost weight. We observed a significant correlation between the luciferase expression 24 hours after surgery and weight 7 days after surgery (P = 0.0007) in mice which had received the gp64 pseudotyped NFkB biosensor; the most severely affected HIE mice had the lowest luciferase expression and weight. However, the HIE mice which had received the VSV-g pseudotyped NFkB biosensor failed to show this correlation between the luciferase expression and weight (P=0.479). This indicates that NFkB-controlled luciferase expression in astrocytes is predictive of brain injury. We are now investigating the mechanism of this predictive relationship.

Biliary obstruction results in cholestasis characterised by fibrosis, progressive cirrhosis and cholangiocyte hyperplasia leading to liver failure. The underlying molecular mechanisms remain unclear but likely involve deregulation of bi-potent progenitor cells. Transfection of cell lines with vectors containing serial transcription factor (TF) binding sequences upstream of a minimal promoter driving luciferase expression have been widely used to study TF activity in vitro. We have expanded on this technology, both in vitro and in vivo, to enable quantification of bioluminescence output in living cell cultures and animals. We have developed a library of lentiviral vectors expressing either firefly luciferase or a secreted NanoLuc luciferase conditionally activated by a range of TFs. Human HepaRG cells can be cultured as bi-potent progenitors capable of differentiating into cholangiocytes or hepatocytes and act as a valuable tool to understand cholangiocyte hyperplasia. We have evaluated TF activity in living HepaRG cultures subjected to pro-cholestatic agonists by measuring NanoLuc activity in conditioned medium. For in vivo experiments, high-titer VSV-G pseudotyped lentiviral preps containing TF activated reporter cassettes were administered by intravascular injection to P0 neonatal mice. This resulted in liver-restricted transduction and lifelong tolerance of the transgene. After establishing a bioimaging baseline, adult mice injected with either the NF-κB or the Smad2/3 reporter constructs, were subjected to partial bile duct ligation (pBDL) and serial bioimaging thereafter. Responses were observed in NF-kb (p = 0.048) and Smad2/3 (p = 0.009) reporter mice post-BDL. These tools have allowed us to evaluate and compare the temporal activity of candidate TFs during cholestatic insult in living cells and living mice. We envisage that this study could result in the elucidation of novel therapeutic targets.

Germ line light producing transgenic mice, where luciferase expression is controlled by a surrogate promoter or by a minimal promoter downstream of tandem, synthetic, transcription factor binding elements, are used to provide an in vivo readout of disease processes. However, as every cell within the organism contains the luciferase reporter gene, it is therefore not specific to individual organs. We have developed a novel technology for the generation of light emitting somatic transgenic animals using lentiviral vectors. This allows signalling pathways in diseased organs to be monitored specifically, continually and in a non-invasive manner [1]. In this study, we aimed to deliver NFkB driving a luciferase reporter constructs to the nervous system of neonatal mice to generate somatic-transgenic mice using both lentivirus and adeno-associated viral (AAV) vectors. Lentivirus vector pseudotyped with VSV-G viral envelope glycoproteins or AAV8 serotyped vector carrying an NFkB response element was injected intracranially or intravenously to outbred CD1 neonatal (P1) mice and luciferase expression was monitored continually by whole body bioluminescence imaging of conscious mice. The ability to image conscious mice holds advantages when studying neuropathology. After weaning, pathological activation of NFKB was induced by intraperitoneal injection of lipopolysaccharide (LPS). Following intracranial injection, the VSV-G lentivirus NFkB biosensor showed transduction of the brain and spinal cord. Luciferase expression was upregulated 24 hours after administration of LPS. Immunohistochemistry revealed only modest spread throughout the brain. Conversely, intracranial injection of AAV8 NFkB biosensor showed a much wider spread and increased luciferase expression. Finally we administered AAV8 NFkB biosensor intravenously at P1. Whereas previous studies show AAV8 serotype transduces many systemic tissues [2] through this route; we observed luciferase expression predominantly in the brain and spine (see figurefigure). Using a standard Gateway® cloning system we have established a library of more than 25 lentivirus biosensors where some have been tested in vitro and in vivo. We plan on incorporating these response elements into the AAV backbone described above. Therefore, enabling the generation of somatic-transgenic mice which have a wider spread of AAV biosensor. This will complement existing germ line transgenic, light producing technology by maximising the use, and reducing the numbers, of animals used in biomedical research.View Large Image | Download PowerPoint Slide1. Buckley, SMK et al. 2015. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters. Scientific Reports. 2. Inagaki, K et al. 2008. Frequency and spectrum of genomic integration of recombinant AAV serotype 8 vector in neonatal mouse liver. Journal of virology.

Background:Preterm birth is increasing in incidence and current therapies are relatively ineffective. It is responsible for >1million neonatal deaths per annum worldwide and long term complications in survivors. Approximately 50% of PTBs are preceded by microbial invasion of the intrauterine space; current clinical management centres on diagnosis of intrauterine bacterial presence by identifying the resultant inflammatory response. To investigate the relationship between intrauterine bacterial presence and inflammation we developed two separate gene technology approaches:1. Intravaginal bioluminescent bacteria to measure bacterial ascent into the uterus which mimics the ascending vaginal infection seen in preterm birth.2. Lentiviral gene transfer of an NFKB activated luciferase reporter construct to allow bioluminescent imaging of the subsequent systemic NFKB response.Methods:An NFkB response element was cloned into a lentivirus vector upstream of the genes encoding a codon-optimised firefly luciferase. High titred virus was injected intravenously at birth to neonatal female C57BL/6 J-Tyrc-2J mice to achieve luciferase expression predominantly in the liver (to monitor systemic inflammatory response). These mice received Escherichia coli (non-pathogenic K-12, MG1655 with integrated luxABCDE operon) intra-vaginally once reaching adulthood and intraperitoneal lipopolysaccharide (LPS) three weeks later. Luciferase expression was monitored by whole body bioluminescence imaging. Local inflammation was determined using H&E, ICAM-1 (Intracellular adhesion molecule 1) and Ly6g immunohistochemistry and enzyme-linked immunosorbent assays for serum and uterine TNF-α and IL1-β cytokines.Results:Bioluminescent imaging revealed that C57BL/6 J-Tyrc-2J mice were the most susceptible mice breeds for modelling of ascending vaginal infection with E. coli luxABCDE operon. Intraperitoneal LPS induced an NF-KB response in the liver by biosensing (p

Surrogate genetically encoded markers have been utilized in order to analyze gene transfer efficacy, location, and persistence. These marker genes have greatly accelerated the development of gene transfer vectors for the ultimate application of gene therapy using therapeutic genes. They have also been used in many other applications, such as gene marking in order to study developmental cell lineages, to track cell migration, and to study tumor growth and metastasis. This chapter aims to describe the analysis of several commonly used marker genes: green fluorescent protein (GFP), β-galactosidase, firefly luciferase, human factor IX, and alkaline phosphatase. The merits and disadvantages of each are briefly discussed. In addition a few short examples are provided for continual and endpoint analysis in different disease models including hemophilia, cystic fibrosis, ornithine transcarbamylase deficiency and Gaucher disease.

Standard lentiviral vectors (LVs) require sequences from the wild-type virus (most commonly HIV-1) for effective packaging of vector genomes into viral particles. These sequences are preserved in reverse-transcribed proviral DNA, maintaining the packaging signal, major splice sites, the REV-response element (RRE) and CpG islands which amount to approximately 19% of HIV-1 genome in 3rd generation vectors. For gene therapy applications, these regions have been shown to be problematic, or pose potential risks. Vector integrations in proximity to host-cell genes have produced aberrant transcripts through genome-vector splicing. Transcriptional silencing of delivered transgenes can occur through methylation of viral CpG islands and the presence of the packaging signal allows remobilisation of vector genomes in cells expressing LV proteins which could be detrimental in HIV-positive patients.In an attempt to reduce these risks and minimise HIV-1 sequence in the provirus, we have developed the novel LTR-1 vector in which the packaging sequences and RRE are located downstream of the 3’ Long Terminal Repeat (LTR). This location means that the essential cis-elements are contained within the RNA genome for efficient processing and encapsidation, but eliminated from the delivered provirus following reverse transcription. This reduces the amount of HIV-1 genome in the integrated provirus to 4.9%. These vectors can be produced to high titre (>108 TU/ml by eGFP flow cytometry) and the proportion of eGFP positive cells is stable between 3 and 14 days post-transduction. Clonal analysis of plasmid rescue experiments confirm LTR-1 proviral sequences lack HIV packaging and RRE sequences following reverse transcription and sequencing confirms the expected structure. Unlike the 3rd generation vector, LTR-1 cannot be remobilised when transduced cells are transfected with HIV-1 packaging constructs. Investigations into safety improvements produced by the LTR-1 modifications are ongoing.Live bioimaging experiments and immunohistochemistry confirm that LTR-1 also functions effectively in vivo when measured for up to 5 weeks post-injection: intracranial or intravenous injections into neonatal mice result in comparable expression levels to standard 3rd generation vectors in the brain and liver respectively.We suggest that this LTR-1 configuration could have an improved safety profile for the next generation of retroviral gene therapy vectors.

Somatotransgenic bioimaging is a technology for in vivo imaging using lentiviral mediated delivery of a luciferase construct into neonatal mice. This luciferase marker, under the control of transcription factor binding elements or surrogate promoters, can be activated during physiological and pathological processes and can be quantified as photonic readout by whole-body bioluminescence imaging technology. The technique permits lifelong expression with immune tolerance to the transgenic proteins.In this study, we show the utility of somatotransgenic bioimaging using NFkB signaling as a model of inflammation in mouse models of rheumatoid arthritis and contact dermatitis.An NFkB transcription factor response element was inserted upstream of a mouse minimal promoter and the genes encoding firefly luciferase and green fluorescent protein. The cassette was inserted into a lentivirus vector.VSV-G-pseudotyped lentivirus vector was injected subcutaneously into CD1 outbread neonatal (P0) mice either in the footpad or in the abdominal skin to generate paw or skin specific light emitting transgenic mice.Canonical NFkB signaling (through TLR4) was first validated using ultrapure LPS in both the paw and skin of somatic transgenic mice. Following intraperitoneal LPS injection we observed upregulation of luciferase activity in the paw or the skin which returned to background levels within a few days.As a model of inflammatory mediated rheumatoid arthritis, we injected zymosan, which activates NFkB via TLR2 into the mouse paw fat pad. Induction of contact dermatitis was achieved by applying topically oxazolone, which plays an indirect activator role in the NFkB pathway. Luciferase expression was then monitored by whole body bioluminescence.Stimulation with Zymosan of the paw biosensor mice induced a prolonged upregulation of luciferase expression, specifically into the mouse paw which peaked at day 3 post injection and gradually returned to background level after twenty days.Similarly, after topical application of oxazolone onto the skin biosensor mice, we observed a specific, localised and increased luciferase activity which peaked at day 2 and returned to baseline by day 5.We have therefore shown that targeted gene expression can be achieved in two mouse models of inflammation. This also allowed us to quantify NFkB-mediated response to pro-inflammatory stimuli in a disease- and tissue-specific manner. Applied to collagen-induced arthritis, this technique could improve quantitation of disease severity compared to subjective scoring.