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Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)—a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research .

While the Mediterranean basin is foreseen to be highly affected by climate change (CC) and severe forest fires are expected to be more frequent, international efforts to fight against CC do not consider forest fires’ greenhouse gas (GHG) emissions risk and the possibility of its mitigation. This is partly due to a lack of a methodology for GHG risk spatial assessment and consideration of the high value of carbon stocks in forest ecosystems and their intrinsic risk. To revert this, an innovative GHG emission risk model has been developed and implemented in a pilot forest area. This model considers geospatial variables to build up emission vulnerability based on potential fire severity and resistance of a landscape, value at risk and the hazard of a fire occurrence. The results classify low, moderate and high emission risks in the analysed areas. This identification of hotspots allows the prioritisation of fire prevention measures in a region to maximise the reduction of GHG emissions in the case of a fire event. This constitutes the first step in a holistic and consistent CC mitigation that not only considers anthropic GHG sources but also possible GHG emissions by forest fires that can be actively prevented, managed and reduced.

BackgroundThe 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.AimsWe 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.MethodsWe 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.ConclusionsA 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.

Gene therapies are being developed for rare genetic diseases and offer hope for conditions with few treatment options. Surfactant protein B (SP-B) deficiency is a rare genetic disease of the lung causing severe respiratory distress in neonates and is fatal within the first months of life. Recombinant adeno-associated virus (rAAV) is being investigated for delivery of functional SP-B to the lungs of SP-B deficient neonates, to produce surfactant for secretion onto the lung surface to increase chances of survival. To screen AAV serotypes for efficient targeting of alveolar type II (ATII) cells of the lung parenchyma, rAAV5, rAAV6.2 and rAAV9 expressing reporter transgenes were delivered to murine lungs via intranasal instillation. Serotype 5 mediated the highest transduction levels in the target ATII cells (19.4 %, p< 0.05 versus naive). The in situ hybridisation method RNAscope was adapted to include a DNase digestion step to allow for detection of transgene mRNA expression after rAAV delivery and showed that the hCEFI promoter mediated the highest expression levels in ATII cells (11.1 transgene mRNA copies per cell, p< 0.05 versus rAAV5.SP-B MG and p< 0.0001 versus all other groups). Therefore, rAAV5.hCEFI.SP-B was the rAAV vector selected for use in the murine lung. To validate the vector for human lung delivery, eight AAV capsids were screened in human models of the lung parenchyma: precision cut lung slices (PCLS) generated from lung resections and lung bud organoids (LBO) generated from human embryonic stem cells. The rAAV5 vector did not transduce the PCLS model, or the LBO after microinjection to the lumen; but vectors rAAV6, rAAV6.2 and rAAV6.2FF showed distinct levels of transduction in both models (p< 0.005 versus naive), indicating their suitability for gene delivery to the human lung parenchyma and highlighting the limitations of the murine model for AAV serotype selection in lung. Reporter gene expression using the CMV promoter was more robust than the hCEFI promoter in both the PCLS (hCEFI expression not detected) and LBO model (about 5-fold), but hCEFI mediated distinct levels of expression long-term. To test the capability of rAAV6.2 to mediate therapeutic protein expression in a human model of the SP-B deficient lung, embryonic stem cells were gene edited to incorporate the most common disease-causing 121ins2 mutation in SP-B deficiency. Subsequently, LBOs were generated from this edited cell line by sequential differentiation and delivery of rAAV6.2.CMV.SP-B resulted in expression of mature SP-B protein, highlighting the translational potential of the selected rAAV vector.

Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. Here, we used single-molecule interferometric scattering microscopy to assess the (in)stability of different empty and filled AAV preparations. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with mass photometry can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.Competing Interest StatementAR, MN and MT are employees of Roche Diagnostics GmbH, Penzberg, Germany, a company with interest in employing recombinant AAV vectors for gene delivery purposes. IRSF is an employee of Revvity Gene Delivery, Graefelfing, Germany, a company developing AAV vectors for gene delivery purposes. HMB was an employee of Revvity Gene Delivery and is an employee of Roche Diagnostics GmbH, Penzberg, Germany. The remaining authors declare no competing interests