Bollepalli, L. | bioRxiv Science

bioRxiv2026

Bovine AAV - a promising vector for pulmonary gene therapy

Efficient systemic delivery to the lung remains a major barrier for adeno-associated virus (AAV)-mediated pulmonary gene therapy, particularly when pre-existing immunity limits the use of conventional capsids. Here, we evaluated Bovine AAV, a phylogenetically divergent capsid, as candidate vector for lung-directed gene transfer. In adult C57BL/6J mice, intravenous delivery of Bovine AAV resulted in robust and preferential lung transduction comparable to AAV4, with predominant targeting of alveolar type I pneumocytes and pulmonary endothelial cells. In primary human lung-resident cells, Bovine AAV was particularly effective in microvascular endothelial cells, a target poorly transduced by AAV4 in vitro. Bovine AAV demonstrated scalable production with yield, purification performance, capsid quality, and genome integrity comparable to AAV9. In sera from healthy adults from the United States and Switzerland, Bovine AAV showed intermediate neutralization frequencies, lower than AAV2 and AAV4 but higher than AAV5 and AAV9. Of relevance, Bovine AAV maintained in vivo transduction efficiency in mice previously immunized with a pool of human and non-human primate-derived AAV capsids, including AAV4. Together, these results position Bovine AAV as a promising lung-tropic and immune-distinct vector for pulmonary gene therapy, with particular relevance for applications requiring systemic delivery in the presence of pre-existing immunity to conventional serotypes.

molecular biology↗

bioRxiv2021

Loss of hepatic Lgr4 and Lgr5 promotes nonalcoholic fatty liver disease

Background & AimsThe Rspo-Lgr4/5-Znrf3/Rnf43 module is a master regulator of hepatic Wnt/{beta}-catenin signaling and metabolic zonation, but its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We studied whether liver-specific loss of the Wnt/{beta}-catenin modulators Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4/5 (Lgr4/5) promotes nonalcoholic fatty liver disease (NAFLD). MethodsMice with liver-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were fed with normal diet (ND) or high fat diet (HFD). Livers of these mice were analyzed for lipid and fibrotic content by tissue staining and immunohistochemistry (IHC), and lipoproteins, inflammation and liver enzyme markers were measured in blood. Mechanistic insights into hepatic lipid accumulation were obtained by using ex vivo primary hepatocyte cultures derived from the Lgr4/5dLKO mice. Lipid analysis of mouse livers was performed by mass spectrometry (MS)-based untargeted lipidomic analysis. ResultsWe demonstrated that liver-specific ablation of Lgr4/5-mediated Wnt signaling resulted in hepatic steatosis, impaired bile acid (BA) secretion and predisposition to liver fibrosis. Under HFD conditions, we observed progressive intrahepatic fat accumulation, developing into macro-vesicular steatosis. Serum lipoprotein levels in HFD-fed Lgr4/5dLKO mice were decreased, rather than increased, suggesting that accumulation of fat in the liver was due to impaired lipid secretion by hepatocytes. Our lipidome analysis revealed a severe alteration of several lipid species in livers of Lgr4/5dLKO mice, including triacylglycerol estolides (TG-EST), a storage form of bioactive free fatty acid (FA) esters of hydroxy FAs (FAHFAs). ConclusionsLoss of hepatic Wnt/{beta}-catenin activity by Lgr4/5 deletion led to deregulation of lipoprotein pathways, loss of BA secretion, intrinsic alterations of lipid homeostasis and the onset of NAFLD. Lay summaryThe Wnt/{beta}-catenin pathway plays an important role during development and tissue homeostasis. Loss of Wnt/{beta}-catenin activity in mouse liver leads to loss of liver zonation, but the impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We show that livers of mice developed steatosis upon deletion of the positive pathway regulators Lgr4/5. Livers of knock-out (KO) mice exhibited altered lipid composition due to impaired lipid secretion. Furthermore, livers of these mice developed a nonalcoholic steatohepatitis (NASH)-like phenotype and fibrotic features derived from activated hepatic stellate cells. Our data demonstrate a protective role of Wnt/{beta}-catenin pathway activity towards the development of NAFLD. HighlightsO_LIAbrogation of hepatic Wnt/{beta}-catenin activity and liver zonation upon Lgr4/5 deletion in mice led to hepatic steatosis. C_LIO_LILiver fat accumulation was caused by impaired lipid secretion from hepatocytes. C_LIO_LISteatotic livers contained increased levels of diverse lipid species, including polyunsaturated fatty acids and triglycerol-estolides. C_LIO_LIThese data confirmed that a decrease in Wnt/{beta}-catenin signaling led to the development of nonalcoholic fatty liver disease (NAFLD) in mice. C_LI

physiology↗

bioRxiv2020

DNA-based nanocarriers to enhance the optoacoustic contrast of tumors in vivo

Optoacoustic tomography (OT) enables non-invasive deep tissue imaging of optical contrast at high spatio-temporal resolution. The applications of OT in cancer imaging often rely on the use of molecular imaging contrast agents based on near infrared dyes to enhance contrast at the tumor site. While these agents afford excellent biocompatibility and minimal toxicity, they present limited optoacoustic signal generation capability and rapid renal clearance, which can impede their tumor imaging efficacy. In this work, a synthetic strategy to overcome these limitations utilizing biodegradable DNA-based nanocarrier (DNA-NC) platforms is introduced. DNA-NCs enable the incorporation of near infrared dyes (in this case, IRDye 800CW) at precise positions to enable fluorescence quenching and maximize optoacoustic signal generation. Furthermore, these DNA-NCs show a prolonged blood circulation compared to the native fluorophores, facilitating tumor accumulation by the Enhanced Permeability and Retention (EPR) effect. In vivo imaging of tumor xenografts in mice following intravenous administration of DNA-NCs revealed enhanced OT signals at 24h when compared to free fluorophores, indicating promise for this method to enhance the optoacoustic signal generation capability and tumor uptake of clinically relevant near infrared dyes.

bioengineering↗