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Approximately 40% of preterm births are preceded by microbial invasion of the intrauterine space; ascent from the vagina being the most common pathway. Within the cervical canal, antimicrobial peptides and proteins (AMPs) are important components of the cervical barrier which help to prevent ascending vaginal infection. We investigated whether expression of the AMP, human β-defensin-3 (HBD3), in the cervical mucosa of pregnant mice could prevent bacterial ascent from the vagina into the uterine cavity. An adeno-associated virus vector containing both the HBD3 gene and GFP transgene (AAV8 HBD3.GFP) or control AAV8 GFP, was administered intravaginally into E13.5 pregnant mice. Ascending infection was induced at E16.5 using bioluminescent Escherichia coli ( E. coli K1 A192PP-lux2). Bioluminescence imaging showed bacterial ascent into the uterine cavity, inflammatory events that led to premature delivery and a reduction in pups born alive, compared with uninfected controls. Interestingly, a significant reduction in uterine bioluminescence in the AAV8 HBD3.GFP-treated mice was observed 24 h post- E. coli infection, compared to AAV8 GFP treated mice, signifying reduced bacterial ascent in AAV8 HBD3.GFP-treated mice. Furthermore, there was a significant increase in the number of living pups in AAV HBD3.GFP-treated mice. We propose that HBD3 may be a potential candidate for augmenting cervical innate immunity to prevent ascending infection-related preterm birth and its associated neonatal consequences., (Copyright © 2020 Suff, Karda, Diaz, Ng, Baruteau, Perocheau, Taylor, Alber, Buckley, Bajaj-Elliott, Waddington and Peebles.)

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.

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.

Preterm birth is a serious global health problem and the leading cause of infant death before 5 years of age. At least 40% of cases are associated with infection. The most common way for pathogens to access the uterine cavity is by ascending from the vagina. Bioluminescent pathogens have revolutionized the understanding of infectious diseases. We hypothesized that bioluminescent Escherichia coli can be used to track and monitor ascending vaginal infections. Two bioluminescent strains were studied: E. coli K12 MG1655-lux, a nonpathogenic laboratory strain, and E. coli K1 A192PP-lux2, a pathogenic strain capable of causing neonatal meningitis and sepsis in neonatal rats. On embryonic day 16, mice received intravaginal E. coli K12, E. coli K1, or phosphate-buffered saline followed by whole-body bioluminescent imaging. In both cases, intravaginal delivery of E. coli K12 or E. coli K1 led to bacterial ascension into the uterine cavity, but only E. coli K1 induced preterm parturition. Intravaginal administration of E. coli K1 significantly reduced the proportion of pups born alive compared with E. coli K12 and phosphate-buffered saline controls. However, in both groups of viable pups born after bacterial inoculation, there was evidence of comparable brain inflammation by postnatal day 6. This study ascribes specific mechanisms by which exposure to intrauterine bacteria leads to premature delivery and neurologic inflammation in neonates., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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 apnea 1 . 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 days 2 . 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.

Argininosuccinate lyase (ASL) belongs to the hepatic urea cycle detoxifying ammonia, and the citrulline-nitric oxide (NO) cycle producing NO. ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia, multiorgan disease and neurocognitive impairment despite treatment aiming to normalise ammonaemia without considering NO imbalance. Here we show that cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress independent of hyperammonaemia. Intravenous injection of AAV8 vector into adult or neonatal ASL-deficient mice demonstrates long-term correction of the hepatic urea cycle and the cerebral citrulline-NO cycle, respectively. Cerebral disease persists if ammonaemia only is normalised but is dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This correlates with behavioural improvement and reduced cortical cell death. Thus, neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Disease amelioration by simultaneous brain and liver gene transfer with one vector, to treat both metabolic pathways, provides new hope for hepatocerebral metabolic diseases.

Major hemoglobinopathies place tremendous strain on global resources. Intrauterine hemopoietic cell transplantation (IUHCT) and gene transfer (IUGT) can potentially reduce perinatal morbidities with greater efficacy than postnatal therapy alone. We performed both procedures in the thalassemic HbbTh3/+ mouse. Intraperitoneal delivery of co-isogenic cells at embryonic days13-14 produced dose-dependent chimerism. High-dose adult bone marrow (BM) cells maintained 0.2-3.1% chimerism over ~24 weeks and treated heterozygotes (HET) demonstrated higher chimerism than wild-type (WT) pups (1.6% vs. 0.7%). Fetalliver (FL) cells produced higher chimerism than BM when transplanted at thesame doses, maintaining 1.8-2.4% chimerism over ~32 weeks. We boosted transplanted mice postnatally with BM cells after busulfan conditioning. Engraftment was maintained at >1% only in chimeras. IUHCT-treated nonchimeras and non-IUHCT mice showed microchimerism or no chimerism. Improved engraftment was observed with a higher initial chimerism, in HET mice and with the addition of fludarabine. Chimeric HET mice expressed 2.2-15.1% engraftment with eventual decline at 24 weeks (vs. <1% in nonchimeras) and demonstrated improved hematological indices and smaller spleens compared with untreated HETmice. Intravenous delivery of GLOBE lentiviral-vector expressing human β-globin (HBB) resulted in a vector concentration of 0.001-0.6 copies/cell. Most hematological indices were higher in treated than untreated HET mice, including hemoglobin and mean corpuscular volume, but were still lower than in WT. Therefore, direct IUGT and IUHCT strategies can be used to achieve hematological improvement but require further dose optimization. IUHCT will be useful combined with postnatal transplantation to further enhance engraftment., (Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Luciferase bioimaging in living animals is increasingly being applied in many fields of biomedical research. Rodent imaging usually involves anaesthetising the animal during data capture, however, the biological consequences of anaesthesia have been largely overlooked. We have evaluated luciferase bioimaging in conscious, unrestrained mice after neonatal intracranial or intravascular administration of lentiviral, luciferase reporter cassettes (biosensors); we present real-time analyses from the first day of life to adulthood. Anaesthetics have been shown to exert both neurotoxic and neuroprotective effects during development and in models of brain injury. Mice subjected to bioimaging after neonatal intracranial or intravascular administration of biosensors, targeting the brain and liver retrospectively showed no significant difference in luciferase expression when conscious or unconscious throughout development. We applied conscious bioimaging to the assessment of NFκB and STAT3 transcription factor activated reporters during the earliest stages of development in living, unrestrained pups. Our data showed unique longitudinal activities for NFκB and STAT3 in the brain of conscious mice. Conscious bioimaging was applied to a neonatal mouse model of cerebral palsy (Hypoxic-Ischaemic Encephalopathy). Imaging of NFκB reporter before and after surgery showed a significant increase in luciferase expression, coinciding with secondary energy failure, in lesioned mice compared to controls.