Evidence for Contribution of CD4+CD25+ Regulatory T Cells in Maintaining Immune Tolerance to Human Factor IX following Perinatal Adenovirus Vector Delivery

Following fetal or neonatal gene transfer in mice and other species immune tolerance of the transgenic protein is frequently observed; however the underlying mechanisms remain largely undefined. In this study fetal and neonatal BALB/c mice received adenovirus vector to deliver human factor IX (hFIX) cDNA. The long-term tolerance of hFIX was robust in the face of immune challenge with hFIX protein and adjuvant but was eliminated by simultaneous administration of anti-CD25+ antibody. Naive irradiated BALB/c mice which had received lymphocytes from donors immunised with hFIX developed anti-hFIX antibodies upon immune challenge. Cotransplantation with CD4+CD25+ cells isolated from neonatally tolerized donors decreased the antibody response. In contrast, cotransplantation with CD4+CD25− cells isolated from the same donors increased the antibody response. These data provide evidence that immune tolerance following perinatal gene transfer is maintained by a CD4+CD25+ regulatory population

Bioluminescence monitoring of promoter activity in vitro and in vivo

© 2017, Springer Science+Business Media LLC. 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

In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters.

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

A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice

Many genetic liver diseases present in newborns with repeated, often lethal, metabolic crises. Gene therapy using non-integrating viruses such as AAV is not optimal in this setting because the non-integrating genome is lost as developing hepatocytes proliferate1,2. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR/Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7% – 20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet

Fetal gene therapy for neurodegenerative disease of infants

For inherited genetic diseases, fetal gene therapy offers the potential of prophylaxis against early, irreversible and lethal pathological change. To explore this, we studied neuronopathic Gaucher disease (nGD), caused by mutations in GBA. In adult patients, the milder form presents with hepatomegaly, splenomegaly and occasional lung and bone disease; this is managed, symptomatically, by enzyme replacement therapy. The acute childhood lethal form of nGD is untreatable since enzyme cannot cross the blood–brain barrier. Patients with nGD exhibit signs consistent with hindbrain neurodegeneration, including neck hyperextension, strabismus and, often, fatal apnea1. We selected a mouse model of nGD carrying a loxP-flanked neomycin disruption of Gba plus Cre recombinase regulated by the keratinocyte-specific K14 promoter. Exclusive skin expression of Gba prevents fatal neonatal dehydration. Instead, mice develop fatal neurodegeneration within 15 days2. Using this model, fetal intracranial injection of adeno-associated virus (AAV) vector reconstituted neuronal glucocerebrosidase expression. Mice lived for up to at least 18 weeks, were fertile and fully mobile. Neurodegeneration was abolished and neuroinflammation ameliorated. Neonatal intervention also rescued mice but less effectively. As the next step to clinical translation, we also demonstrated the feasibility of ultrasound-guided global AAV gene transfer to fetal macaque brains