Explore the vast range of titles available.

During April-July 2022, outbreaks of severe acute hepatitis of unknown etiology (SAHUE) were reported in 35 countries. Five percent of cases required liver transplantation, and 22 patients died. Viral metagenomic studies of clinical samples from SAHUE cases showed a correlation with human adenovirus F type 41 (HAdV-F41) and adeno-associated virus type 2 (AAV2). To explore the association between those DNA viruses and SAHUE in children in Ireland, we quantified HAdV-F41 and AAV2 in samples collected from a wastewater treatment plant serving 40% of Ireland's population. We noted a high correlation between HAdV-F41 and AAV2 circulation in the community and SAHUE clinical cases. Next-generation sequencing of the adenovirus hexon in wastewater demonstrated HAdV-F41 was the predominant HAdV type circulating. Our environmental analysis showed increased HAdV-F41 and AAV2 prevalence in the community during the SAHUE outbreak. Our findings highlight how wastewater sampling could aid in surveillance for respiratory adenovirus species.

Viral vectors have become very popular to overexpress or downregulate proteins of interest in different cell types. They conveniently allow the precise targeting of well-defined tissue areas, which is particularly useful in complex organs like the brain. In theory, each vector should have its own cell specificity that can be obtained by using different strategies (e.g., using a cell-specific promoter). For the moment, there is few vectors that have been developed to alternatively target, using the same capsid, neurons and astrocytes in the central nervous system. There is even fewer examples of adeno-associated viral vectors able to efficiently transduce cells both and . The development of viral vectors allowing the cell-specific downregulation of a protein in cultured cells of the central nervous system as well as within a large brain area would be highly desirable to address several important questions in neurobiology. Here we report that the use of the AAV2/DJ viral vector associated to an hybrid CMV/chicken β-actin promoter (CBA) or to a modified form of the glial fibrillary acidic protein promoter (G1B3) allows a specific transduction of neurons or astrocytes in more than half of the barrel field within the rat somatosensory cortex. Moreover, the use of the miR30E-shRNA technology led to an efficient downregulation of two proteins of interest related to metabolism both and . Our results demonstrate that it is possible to downregulate the expression of different protein isoforms in a cell-specific manner using a common serotype. It is proposed that such an approach could be extended to other cell types and used to target several proteins of interest within the same brain area.

The relationship between LRRK2 mutations and susceptibility to synuclein pathology in Parkinson’s disease (PD) is still unclear. We here investigate whether the mice carrying the D1994S kinase-dead (KD) mutation of LRRK2 show enhanced susceptibility to synucleinopathy. Twelve-month-old LRRK2 KD and WT mice were injected with AAV2/9 carrying human A53T α-synuclein (AAV-h-A53Tα-syn) or AAV2/9-GFP as a control. Three months after injection, α-synuclein pathology and nigrostriatal dopaminergic neuron degeneration were assessed along with motor behaviour. AAV-h-A53Tα-syn-injected LRRK2 KD mice showed a decline in stepping activity in the drag test compared to baseline levels and AAV-GFP-injected controls. This was associated with higher transgene levels and Serine129 α-syn phosphorylation in striatum and substantia nigra measured by immunohistochemistry. Total α-synuclein levels were also elevated in the substantia nigra but not striatum of AAV-h-A53Tα-syn LRRK2 KD mice compared to AAV-h-A53Tα-syn controls. Stereological counting of nigral dopaminergic neurons and densitometric analysis of striatal dopaminergic terminals did not reveal overt nigrostriatal degeneration. We conclude that silencing of kinase activity results in greater α-syn load due to greater viral transduction and/or defective α-syn clearance, possibly related to autophagy-lysosomal pathway impairment, however, with no consequence upon dopaminergic neuron survival in the mouse.

Background Fabry disease (FD) is a progressive multisystemic disease characterized by a lysosomal enzyme deficiency. A lack of α-galactosidase A (α-Gal A) activity results in the progressive systemic accumulation of its substrates, including globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), which results in renal, cardiac, and/or cerebrovascular disease and early death. Enzyme replacement therapy (ERT) is the current standard of care for FD; however, it has important limitations, including a low half-life, limited distribution, and requirement of lifelong biweekly infusions of recombinant enzymes. Methods Herein, we evaluated a gene therapy approach using an episomal adeno-associated viral 2/8 (AAV2/8) vector that encodes the human GLA cDNA driven by a liver-specific expression cassette in a mouse model of FD that lacks α-Gal A activity and progressively accumulates Gb3 and Lyso-Gb3 in plasma and tissues. Results A pharmacology and toxicology study showed that administration of AAV2/8-hGLA vectors (AAV2/8-hGLA) in FD mice without immunosuppression resulted in significantly increased plasma and tissue α-Gal A activity and substantially normalized Gb3 and Lyso-Gb3 content. Conclusions Moreover, the plasma enzymatic activity of α-Gal A continued to be stably expressed for up to 38 weeks and sometimes even longer, indicating that AAV2/8-hGLA is effective in treating FD mice, and that α-Gal A is continuously and highly expressed in the liver, secreted into plasma, and absorbed by various tissues. These findings provide a basis for the clinical development of AAV2/8-hGLA.

Lentivirus and adeno-associated viruses are invaluable tools for biotechnology applications due to their genetic material delivery abilities both in vitro and in vivo. However, their large-scale productions with Good Manufacturing Practices yield low efficiency when adherent and serum dependent HEK293 (Human Embryonic Kidney) cells are used as the host. To increase production efficiency, HEK293 cells are adapted to grow in suspension using commercially available and chemically defined serum-free mediums. Suspended cells can be transiently transfected for viral vector production; however, significant improvements are still needed to increase yield and thereby cost effectiveness. Here, we evaluated four most preferred commercially available mediums that are IVY, FreeStyle293, LV-MAX, and BalanCD HEK293 for the transient transfection feasibility of lentiviral (LV) and adeno-associated virus serotype 2 (AAV2) production in FlorabioHEK293 suspension cells. The highest transfection efficiency was over 90% and obtained by using polyethyleneimine (PEI) 25 K and by media adaptation in IVY without using any transfection enhancer. For the first time the feasibility of HEK293 cells, which were adapted to grow in suspension culture by Florabio and IVY media, were tested for virus production. This study demonstrates the best transfection medium for scalable and optimized production of Lentivirus and Adeno-Associated Virus in suspended HEK293 cell culture.

Background: Adeno-associated viral (AAV) vectors are the leading platform for gene therapy, but common delivery routes show limited spread to distal cortical structures, hence the utility of direct, intrathalamic infusions for broader transgene distribution. In this preliminary study, we recapitulate previous studies targeting the thalamus as a conduit to achieve cortical transgene spread and showcase novel data evaluating biodistribution of a green fluorescent protein (GFP) using cryo-fluorescence tomography (CFT). For the first time in nonhuman primates (NHPs) and coupled with magnetic resonance imaging (MRI)-guidance, we demonstrated the application of CFT as a powerful tool to map out vector distribution in the NHP brain. Methods: Briefly, a single thalamic infusion was performed in African green monkeys using ClearPoint’s navigational platform to deliver an AAV serotype 2 vector containing a GFP payload. Transgene biodistribution was assessed in the left and right hemispheres using CFT and histological analysis, respectively. Results: Infusions were successfully performed with sub-millimetric target accuracy and with minimal error, achieving ~86% thalamic coverage with the largest infusion volume. Histology confirmed the presence of the GFP transgene, with the strongest signal in the cerebral gray/white matter and internal capsule, while CFT allowed for the three-dimensional detection of the transgene starting at the site of infusion and spreading to multiple cortical regions. Conclusions: These findings suggest that by combining MRI-guided technology with CFT imaging, it is feasible to map whole-brain gene biodistribution in NHPs. This proof-of-concept study bridges the gap between cellular microscopy and MRI-guidance to provide a complete picture of disease and treatment with clinical applicability.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been included in the transgene cassette of adeno-associated virus (AAV) in several gene therapy clinical trials, including those for inherited retinal diseases. However, the extent to which WPRE increases transgene expression in the retina is still unclear. To address this question, AAV2 vectors containing a reporter gene with and without WPRE were initially compared in vitro and subsequently in vivo by subretinal delivery in mice. In both instances, the presence of WPRE led to significantly higher levels of transgene expression as measured by fundus fluorescence, western blot, and immunohistochemistry. The two vectors were further compared in human retinal explants derived from patients undergoing clinically indicated retinectomy, where again the presence of WPRE resulted in an enhancement of reporter gene expression. Finally, an analogous approach using a transgene currently employed in a clinical trial for choroideremia delivered similar results both in vitro and in vivo, confirming that the WPRE effect is transgene independent. Our data fully support the inclusion of WPRE in ongoing and future AAV retinal gene therapy trials, where it may allow a therapeutic effect to be achieved at an overall lower dose of vector.

Müller glia are specialized retinal cells with stem cell properties in fish and frogs but not in mammals. Current efforts to develop gene therapies to activate mammalian Müller glia for retinal repair will require safe and effective delivery strategies for recombinant adeno-associated viruses (AAVs), vectors of choice for clinical translation. Intravitreal and subretinal injections are currently used for AAV gene delivery in the eye, but less invasive methods efficiently targeting Müller glia have yet to be developed. : As gene delivery strategies have been more extensively studied in the brain, to validate our vectors, we initially compared the glial tropism of AAV-PHP.eB, an AAV9 that crosses the blood-brain and blood-retinal barriers, for its ability to drive fluorescent protein expression in glial cells in both the brain and retina. We then tested the glial transduction of AAV2/8-GFAP-mCherry, a virus that does not cross blood-brain and blood-retinal barriers, for its effectiveness in transducing Müller glia in murine retinal explants . For assays we used larger rat eyes, performing invasive intravitreal injections, and non-invasive intravenous delivery using focused ultrasound (FUS) (pressure amplitude: 0.360 - 0.84 MPa) and microbubbles (Definity, 0.2 ml/kg). : We showed that AAV-PHP.eB carrying a ubiquitous promoter (CAG) and green fluorescent protein (GFP) reporter, readily crossed the blood-brain and blood-retinal barriers after intravenous delivery in mice. However, murine Müller glia did not express GFP, suggesting that they were not transduced by AAV-PHP.eB. We thus tested an AAV2/8 variant, which was selected based on its safety record in multiple clinical trials, adding a glial fibrillary acidic protein (GFAP) promoter and mCherry (red fluorescent protein) reporter. We confirmed the glial specificity of AAV2/8-GFAP-mCherry, showing effective expression of mCherry in astrocytes after intracranial injection in the mouse brain, and of Müller glia in murine retinal explants. For experiments we switched to rats because of their larger size, injecting AAV2/8-GFAP-mCherry intravitreally, an invasive procedure, demonstrating passage across the inner limiting membrane, leading to Müller glia transduction. We then tested an alternative non-invasive delivery approach targeting a different barrier - the inner blood-retinal-barrier, applying focused ultrasound (FUS) to the retina after intravenous injection of AAV2/8 and microbubbles in rats, using magnetic resonance imaging (MRI) for FUS targeting. FUS permeabilized the rat blood-retinal-barrier and allowed the passage of macromolecules to the retina (Evans blue, IgG, IgM), with minimal extravasation of platelets and red blood cells. Intravenous injection of microbubbles and AAV2/8-GFAP-mCherry followed by FUS resulted in mCherry expression in rat Müller glia. However, systemic delivery of AAV2/8 also had off-target effects, transducing several murine peripheral organs, particularly the liver. : Retinal permeabilisation via FUS in the presence of microbubbles is effective for delivering AAV2/8 across the inner blood-retinal-barrier, targeting Müller glia, which is less invasive than intravitreal injections that bypass the inner limiting membrane. However, implementing FUS in the clinic will require a comprehensive consideration of any off-target tropism of the AAV in peripheral organs, combined ideally, with the development of Müller glia-specific promoters.

The lack of axonal regeneration is the major cause of vision loss after optic nerve injury in adult mammals. Activating the PI3K/AKT/mTOR signaling pathway has been shown to enhance the intrinsic growth capacity of neurons and to facilitate axonal regeneration in the central nervous system after injury. The deletion of the mTOR negative regulator phosphatase and tensin homolog (PTEN) enhances regeneration of adult corticospinal neurons and ganglion cells. In the present study, we used a tyrosine-mutated (Y444F) AAV2 vector to efficiently express a short hairpin RNA (shRNA) for silencing PTEN expression in retinal ganglion cells. We evaluated cell survival and axonal regeneration in a rat model of optic nerve axotomy. The rats received an intravitreal injection of wildtype AAV2 or Y444F mutant AAV2 (both carrying shRNA to PTEN) 4 weeks before optic nerve axotomy. Compared with the wildtype AAV2 vector, the Y444F mutant AAV2 vector enhanced retinal ganglia cell survival and stimulated axonal regeneration to a greater extent 6 weeks after axotomy. Moreover, post-axotomy injection of the Y444F AAV2 vector expressing the shRNA to PTEN rescued ~19% of retinal ganglion cells and induced axons to regenerate near to the optic chiasm. Taken together, our results demonstrate that PTEN knockdown with the Y444F AAV2 vector promotes retinal ganglion cell survival and stimulates long-distance axonal regeneration after optic nerve axotomy. Therefore, the Y444F AAV2 vector might be a promising gene therapy tool for treating optic nerve injury.