Adenoviral vectors infect B lymphocytes in vivo.

B cells are the antibody-producing arm of the adaptive immune system and play a critical role in controlling pathogens. Several groups have now demonstrated the feasibility of using engineered B cells as a therapy, including infectious disease control and gene therapy of serum deficiencies. These studies have largely utilized ex vivo modification of the cells. Direct in vivo engineering would be of utility to the field, particularly in infectious disease control where the infrastructure needs of ex vivo cell modification would make a broad vaccination campaign highly challenging. In this study we demonstrate that engineered adenoviral vectors are capable of efficiently transducing murine and human primary B cells both ex vivo and in vivo. We found that unmodified human adenovirus C5 was capable of infecting B cells in vivo, likely due to interactions between the virus penton base protein and integrins. We further describe vector modification with B cell-specific gene promoters and successfully restrict transgene expression to B cells, resulting in a strong reduction in gene expression from the liver, the main site of human adenovirus C5 infection in vivo.

In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector.

Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Adenoviral vector vaccine platforms in the SARS-CoV-2 pandemic.

Adenoviral vectors have been explored as vaccine agents for a range of infectious diseases, and their ability to induce a potent and balanced immune response made them logical candidates to apply to the COVID-19 pandemic. The unique molecular characteristics of these vectors enabled the rapid development of vaccines with advanced designs capable of overcoming the biological challenges faced by early adenoviral vector systems. These successes and the urgency of the COVID-19 situation have resulted in a flurry of candidate adenoviral vector vaccines for COVID-19 from both academia and industry. These vaccines represent some of the lead candidates currently supported by Operation Warp Speed and other government agencies for rapid translational development. This review details adenoviral vector COVID-19 vaccines currently in human clinical trials and provides an overview of the new technologies employed in their design. As these vaccines have formed a cornerstone of the COVID-19 global vaccination campaign, this review provides a full consideration of the impact and development of this emerging platform.

Advances in Alpha-1 Antitrypsin Gene Therapy.

AAT (alpha-1 antitrypsin) deficiency (AATD), characterized by low levels of circulating serine protease inhibitor AAT, results in emphysematous destruction of the lung. Inherited serum deficiency disorders, such as hemophilia and AATD, have been considered ideal candidates for gene therapy. Although viral vector-meditated transduction of the liver has demonstrated utility in hemophilia, similar success has not been achieved for AATD. The challenge for AAT gene therapy is achieving protective levels of AAT locally in the lung and mitigating potential liver toxicities linked to systemically administered viral vectors. Current strategies with ongoing clinical trials involve different routes of adeno-associated virus administrations, such as intramuscular and intrapleural injections, to provide consistent therapeutic levels from nonhepatic organ sites. Nevertheless, exploration of alternative methods of nonhepatic sourcing of AAT has been of great interest in the field. In this regard, pulmonary endothelium-targeted adenovirus vector could be a key technical mandate to achieve local augmentation of AAT within the lower respiratory tract, with the potential benefit of circumventing liver toxicities. In addition, incorporation of the CRISPR/Cas9 (CRISPR-associated protein 9) nuclease system into gene-delivery technologies has provided adjunctive technologies that could fully realize a one-time treatment for sustained, lifelong expression of AAT in patients with AATD. This review will focus on the adeno-associated virus- and adenoviral vector-mediated gene therapy strategies for the pulmonary manifestations of AATD and show that endeavoring to use genome-editing techniques will advance the current strategy to one fully compatible with direct human translation.

Beta-cell excitability and excitability-driven diabetes in adult Zebrafish islets.

Islet ß-cell membrane excitability is a well-established regulator of mammalian insulin secretion, and defects in ß-cell excitability are linked to multiple forms of diabetes. Evolutionary conservation of islet excitability in lower organisms is largely unexplored. Here we show that adult zebrafish islet calcium levels rise in response to elevated extracellular [glucose], with similar concentration-response relationship to mammalian ß-cells. However, zebrafish islet calcium transients are nor well coupled, with a shallower glucose-dependence of cytoplasmic calcium concentration. We have also generated transgenic zebrafish that conditionally express gain-of-function mutations in ATP-sensitive K+ channels (KATP -GOF) in ß-cells. Following induction, these fish become profoundly diabetic, paralleling features of mammalian diabetes resulting from equivalent mutations. KATP -GOF fish become severely hyperglycemic, with slowed growth, and their islets lose glucose-induced calcium responses. These results indicate that, although lacking tight cell-cell coupling of intracellular Ca2+ , adult zebrafish islets recapitulate similar excitability-driven ß-cell glucose responsiveness to those in mammals, and exhibit profound susceptibility to diabetes as a result of inexcitability. While illustrating evolutionary conservation of islet excitability in lower vertebrates, these results also provide important validation of zebrafish as a suitable animal model in which to identify modulators of islet excitability and diabetes.

In vivo monitoring of intracellular Ca2+ dynamics in the pancreatic ß-cells of zebrafish embryos.

Assessing the response of pancreatic islet cells to glucose stimulation is important for understanding ß-cell function. Zebrafish are a promising model for studies of metabolism in general, including stimulus-secretion coupling in the pancreas. We used transgenic zebrafish embryos expressing a genetically-encoded Ca2+ sensor in pancreatic ß-cells to monitor a key step in glucose induced insulin secretion; the elevations of intracellular [Ca2+]i. In vivo and ex vivo analyses of [Ca2+]i demonstrate that ß-cell responsiveness to glucose is well established in late embryogenesis and that embryonic ß-cells also respond to free fatty acid and amino acid challenges. In vivo imaging of whole embryos further shows that indirect glucose administration, for example by yolk injection, results in a slow and asynchronous induction of ß-cell [Ca2+]i responses, while intravenous glucose injections cause immediate and islet-wide synchronized [Ca2+]i fluctuations. Finally, we demonstrate that embryos with disrupted mutation of the CaV1.2 channel gene cacna1c are hyperglycemic and that this phenotype is associated with glucose-independent [Ca2+]i fluctuation in ß-cells. The data reveal a novel central role of cacna1c in ß-cell specific stimulus-secretion coupling in zebrafish and demonstrate that the novel approach we propose - to monitor the [Ca2+]i dynamics in embryonic ß-cells in vivo - will help to expand the understanding of ß-cell physiological functions in healthy and diseased states.

Expression pattern of the human ABC transporters in pluripotent embryonic stem cells and in their derivatives.

BACKGROUND: ATP-binding cassette (ABC) transporters have key roles in various physiological functions as well as providing chemical defense and stress tolerance in human tissues. In this study, we have examined the expression pattern of all ABC proteins in pluripotent human embryonic stem cells (hESCs) and in their differentiated progenies. We paid special attention to the cellular expression and localization of multidrug transporter ABC proteins. METHODS: Stem cell differentiation was carried out without chemical induction or cell sorting, and specialized cell types were separated mechanically. Cellular features regarding pluripotency and tissue identity, as well as ABC transporter expression were studied by flow cytomtery, immuno-microscopy, and qPCR-based low-density arrays. RESULTS: Pluripotent hESCs and differentiated cell types (cardiomyocytes, neuronal cells, and mesenchymal stem cells) were distinguished by morphology, immunostaining markers, and selected mRNA expression patterns. We found that the mRNA expression levels of the 48 human ABC proteins also clearly distinguished the pluripotent and the respective differentiated cell types. When multidrug and lipid transporter ABC protein expression was examined by using well characterized specific antibodies by flow cytometry and confocal microscopy, the protein expression data corresponded well to the mRNA expression results. Moreover, the cellular localization of these important human ABC transporter proteins could be established in the pluripotent and differentiated hESC derived samples. CONCLUSIONS: These studies provide valuable information regarding ABC protein expression in human stem cells and their differentiated offspring. The results may also help to obtain further information concerning the specialized cellular functions of selected ABC transporters.