Biology subjects

Cooney, A.

Publications and source records attributed to Cooney, A..

5 recordsLinked to original sources

bioRxiv2026

Optimized AAV capsids robustly transduce airway epithelial cells

Gene therapies have demonstrated transformative potential for a range of genetic disorders, including immunodeficiencies, hematopoietic conditions, and neuromuscular diseases. However, the application of these approaches to cystic fibrosis (CF) and other airway diseases remains constrained by the challenge of efficient gene delivery to target epithelial cells. Adeno-associated virus (AAV) vectors are widely used for in vivo gene delivery due to their favorable safety profile and capacity for long-term transgene expression in non-dividing cells. Nonetheless, current AAV capsids require high doses to achieve therapeutic efficacy in the airways, raising safety concerns. Here we report the development of novel AAV capsid variants with markedly enhanced transduction efficiency of airway epithelial cells. Using unbiased peptide-modified AAV libraries and round-over-round screening in well-differentiated primary cultures of human airway epithelia (HAE), we identified 20 novel capsids that efficiently transduced cells at doses 10- to 100-fold lower than those required by existing vectors (termed AAV-AE). These variants demonstrated high transgene expression in HAE, primary human basal cells, tracheal explants from nonhuman primates, and murine airways in vivo. These optimized AAV capsids represent a significant advancement in pulmonary gene therapy, offering a versatile platform for the delivery of gene addition and editing reagents to treat CF and other respiratory diseases.

molecular biology↗

bioRxiv2025

Stimuli-driven cyclical content exchange in a composite synthetic cell

Engineering sophisticated behaviours in synthetic cells lacking complex biomolecular machinery remains a central challenge in synthetic biology. Here, we introduce a protein-free approach for dynamic content modulation in liposome-based synthetic cells using an internal gelation strategy. By crosslinking a polymer hydrogel within the lumen of giant vesicles and tethering it to the inner membrane leaflet, we create a composite architecture that enables controlled and reversible membrane permeabilisation via osmotic swelling and shrinking, facilitating externally gated material exchange without reconstituted protein pores or electroporation. Simultaneously, the hydrogel matrix affords control over membrane fluidity and the diffusion of cytoplasmic clients. We deploy the transport-regulation platform to construct a synthetic-cell bioreactor whereby reversible membrane permeabilisation enables content supplementation and fuels a biocatalytic reaction. The composite gel-GUV chassis provides an adaptive, robust and expandable solution for engineering increasingly modular and functional synthetic cellular systems. These findings may echo how primordial cells harnessed environmental fluctuations for content exchange through chemically distinct pathways.

synthetic biology↗

bioRxiv2024

Adolescent Seizure Impacts Oligodendrocyte Development, Neuronal-Glial Circuit Formation, and Myelination

Myelin sheaths, formed by oligodendrocyte cells in the CNS, are vital for rapid conduction of electrical signals down neuronal axons. Oligodendrocyte progenitors differentiate and myelinate axons during development and following demyelinating injury. However, the mechanisms that drive the timing and specificity of developmental myelination are not well understood. It is known that oligodendrocyte progenitors receive synapses from neurons, providing a potential mechanism for neuronal-glial communication. We have previously shown that changing neuronal activity affects the proliferation of oligodendrocyte cells and neuron to OPC connections. We hypothesized that OPC proliferation and differentiation would be affected by pathological neuronal activity during adolescent development, when developmental myelination is occurring, and that this would also impact neuron to OPC connectivity and myelination. We used kainic acid to induce a seizure, then analyzed changes in the rate of OPC proliferation and differentiation five days later in the cerebral cortex, corpus callosum, and hippocampus. We found that OPC proliferation increased, the overall numbers of OPCs increased, and the number of mature oligodendrocytes decreased. We measured changes in the myelination to determine whether seizure activity directly affected myelination rate in adolescent development, and found decreased myelin in the cerebral cortex, corpus callosum, and hippocampus. We used viral monosynaptic circuit tracing to determine whether connections between neurons and OPCs were affected by seizure activity, and found a decrease in neuron to OPC connections in seizure mice compared to controls. Finally, we measured changes in the presence of kir4.1 potassium channels in OPCs, an important regulator of OPC membrane potential as well as an ion channel important for myelination, and found that there was a decrease in the number of potassium channels on OPCs after adolescent seizure. These findings provide insight into the response of the adolescent brain to seizure activity, as well as how seizures affect neuronal glial connections, OPC development and myelin formation, with the goal of understanding how these mechanisms may be important for treatment of demyelination after seizure and in epilepsy.

neuroscience↗

bioRxiv2021

Measles virus exits human airway epithelia via infectious center sloughing

Measles virus (MeV) is the most contagious human virus, but we do not fully understand why. Unlike most respiratory viruses, MeV does not infect the airway epithelium immediately. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for [~]3-5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may slough off while preserving epithelial function. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In sloughed infectious centers, ciliary beating persisted and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, modeling the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center sloughing. 5-ethynyl-2-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the extruded infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeVs strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host. AUTHOR SUMMARYMeasles virus (MeV) is a respiratory pathogen that infects millions worldwide each year. Although sometimes mischaracterized as an innocuous childhood disease, measles remains a leading cause of death for children under five. MeV is the most contagious human virus and requires vaccination rates above 90% to maintain herd immunity. Global decreases in vaccination rates over the past ten years contributed to recent, widespread MeV outbreaks. We uncover here a novel mechanism by which MeV exits the human airways that may explain why it is much more contagious than other viruses. We document that infected cells containing cell-associated virus slough en masse from the airway epithelial sheet. These expelled infectious centers are metabolically active and can transmit infection to primary human monocyte-derived macrophages more efficiently than cell-free virus particles. Thus, cell-associated MeV can transmit host-to-host, a new paradigm for efficient respiratory virus transmission.

microbiology↗

bioRxiv2019

Offspring production of ovarian organoids derived from spermatogonial stem cells by chromatin reorganization

Fate determination of germline stem cells remains poorly understood at the chromatin structure level1,2. Here, we demonstrate successful production of offspring from oocytes transdifferentiated from mouse spermatogonial stem cells (SSCs) with tracking of transplanted SSCs in vivo, single cell whole exome sequencing, and in 3D cell culture reconstitution of the process of oogenesis derived from SSCs. Furthermore, we demonstrate direct induction of germline stem cells (iGSCs) differentiated into functional oocytes by transduction of H19, Stella, and Zfp57 and inactivation of Plzf in SSCs after screening with ovarian organoids. Using high throughput chromosome conformation, we uncovered extensive chromatin reorganization during SSC conversion into iGSCs, which was highly similar to female germline stem cells. We observed that although topologically associating domains were stable during SSC conversion, chromatin interactions changed in a striking manner, altering 35% of inactive and active chromosomal compartments throughout the genome. These findings have important implications in various areas including mammalian gametogenesis, genetic and epigenetic reprogramming, biotechnology, and medicine.

genetics↗