We have shown that a lentiviral vector (rSIV.F/HN) pseudotyped with the F and HN proteins from Sendai virus generates high levels of intracellular proteins after lung transduction. Here, we evaluate the use of rSIV.F/HN for production of secreted proteins. We assessed whether rSIV.F/HN transduction of the lung generates therapeutically relevant levels of secreted proteins in the lung and systemic circulation using human α1-anti-trypsin (hAAT) and factor VIII (hFVIII) as exemplars. Sedated mice were transduced with rSIV.F/HN carrying either the secreted reporter gene Gaussia luciferase or the hAAT or hFVIII cDNAs by nasal sniffing. rSIV.F/HN-hAAT transduction lead to therapeutically relevant hAAT levels (70 μg/ml) in epithelial lining fluid, with stable expression persisting for at least 19 months from a single application. Secreted proteins produced in the lung were released into the circulation and stable expression was detectable in blood. The levels of hFVIII in murine blood approached therapeutically relevant targets. rSIV.F/HN was also able to produce secreted hAAT and hFVIII in transduced human primary airway cells. rSIV.F/HN transduction of the murine lungs leads to long-lasting and therapeutically relevant levels of secreted proteins in the lung and systemic circulation. These data broaden the use of this vector platform for a large range of disease indications.
Background The COVID-19 pandemic continues to be a worldwide threat and effective antiviral drugs and vaccines are being developed in a joint global effort. However, some elderly and immune-compromised populations are unable to raise an effective immune response against traditional vaccines. Aims We hypothesised that passive immunity engineered by the in vivo expression of anti-SARS-CoV-2 monoclonal antibodies (mAbs), an approach termed vectored-immunoprophylaxis (VIP), could offer sustained protection against COVID-19 in all populations irrespective of their immune status or age. Methods We developed three key reagents to evaluate VIP for SARS-CoV-2: (i) we engineered standard laboratory mice to express human ACE2 via rAAV9 in vivo gene transfer, to allow in vivo assessment of SARS-CoV-2 infection, (ii) to simplify in vivo challenge studies, we generated SARS-CoV-2 Spike protein pseudotyped lentiviral vectors as a simple mimic of authentic SARS-CoV-2 that could be used under standard laboratory containment conditions and (iii) we developed in vivo gene transfer vectors to express anti-SARS-CoV-2 mAbs. Conclusions A single intranasal dose of rAAV9 or rSIV.F/HN vectors expressing anti-SARS-CoV-2 mAbs significantly reduced SARS-CoV-2 mimic infection in the lower respiratory tract of hACE2-expressing mice. If translated, the VIP approach could potentially offer a highly effective, long-term protection against COVID-19 for highly vulnerable populations; especially immune-deficient/senescent individuals, who fail to respond to conventional SARS-CoV-2 vaccines. The in vivo expression of multiple anti-SARS-CoV-2 mAbs could enhance protection and prevent rapid mutational escape.
Tamoxifen is an antiestrogen frequently used in the treatment of breast cancer and is currently being assessed as a prophylactic for those at high risk of developing tumors. We have found that tamoxifen and its derivatives are high-affinity blockers of specific chloride channels. This blockade appears to be independent of the interaction of tamoxifen with the estrogen receptor and therefore reflects an alternative cellular target. One of the clinical side effects of tamoxifen is impaired vision and cataract. Chloride channels in the lens of the eye were shown to be essential for maintaining normal lens hydration and transmittance. These channels were blocked by tamoxifen and, in organ culture, tamoxifen led to lens opacity associated with cataracts at clinically relevant concentrations. These data suggest a molecular mechanism by which tamoxifen can cause cataract formation and have implications for the clinical use of tamoxifen and related antiestrogens.
The UK CF GTC has been working for several years to determine the clinical benefit of CFTR gene therapy. Our premise was that for such a therapy to achieve clinical benefit, repeated administration would be required, and that therefore a non-viral approach was needed. We demonstrated in laboratory and preclinical models that GL67A (Genzyme Corp) was the optimal gene transfer agent, and designed a plasmid, pGM169, completely depleted of pro-inflammatory CpG motifs and driven by the novel hCEFI promoter, designed for prolonged expression. In a longitudinal observational study (the Run-in) we measured the variability of multiple outcome measures, both conventional and novel. These data have allowed us to perform power calculations and a) choose our primary outcome (FEV1), b) secondary efficacy outcomes (lung clearance index, various parameters on CT scan, Quality of life questionnaire [CFQ-R], exercise capacity and activity, and selected sputum and serum inflammatory markers), and c) safety measures (clinical findings, exacerbation rate, gas transfer, sputum culture, serum inflammatory markers, renal and hepatic markers). We have recently completed a single-dose safety and dose ranging study. In this trial, 130 patients, aged 12 years and above are being randomised in a 1:1 fashion to active treatment or placebo and will receive the nebulised agent at monthly intervals for 12 doses. The group size was determined on the basis of a 6% relative improvement in FEV1. An adaptive design will be used for additional safety; the first 20 patients will receive 3 doses ahead of the rest of the cohort. Patients will be invited to participate in either one or two substudies, being conducted to explore mechanisms; a) nasal administration followed by nasal potential difference (PD) and brushings for mRNA expression and b) pre and post-treatment bronchoscopies for lower airway PD, gene expression and histology. The double-blinded nature of the trial means that final outcome data will only be available upon completion of the study. The trial was initiated in April 2012; here we will update on recruitment, projected time-lines and progress. Funded by the MRC and NIHR through the EME programme.
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