Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Rghei, Amira D.; Stevens, Brenna A. Y.; Thomas, Sylvia; Yates, Jacob G. E.; McLeod, Benjamin M; Karimi, Khalil; Susta, Leonardo; Bridle, Byram W.; Wootton, Sarah K.

doi:10.3791/62727

Cited by 10 publications

(7 citation statements)

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“…Vectors pseudotyped with AAV6.2FF were purified by use of a heparin column 59 . AAV vector titres were determined by quantitative polymerase chain reaction (qPCR) analysis as described elsewhere 60 .…”

Section: Methodsmentioning

confidence: 99%

Zoonotic avian influenza viruses evade human BTN3A3 restriction

Pinto

Bakshi

Lytras

et al. 2022

Preprint

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Cross-species transmission of avian influenza A viruses (IAVs) into humans could represent the first step of a future pandemic1. Multiple factors limiting the spillover and adaptation of avian IAVs in humans have been identified, but they are not sufficient to explain which virus lineages are more likely to cross the species barrier1,2. Here, we identified human BTN3A33 (butyrophilin subfamily 3 member A3) as a potent inhibitor of avian but not human IAVs. We determined that BTN3A3 is constitutively expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts at the early stages of virus replication by inhibiting avian IAV vRNA transcription. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F, or rarely 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, several serotypes of avian IAVs that spilled over into humans in recent decades evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure4. Importantly, we identified more than 150 avian IAV lineages with a BTN3A3-resistant genotype. In conclusion, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

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Section: Methodsmentioning

confidence: 99%

Zoonotic avian influenza viruses evade human BTN3A3 restriction

Pinto

Bakshi

Lytras

et al. 2022

Preprint

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“…#LT07-118; Morrisville, NC) with genome and packaging plasmids as previously described [59]. AAV6.2FF vectors were purified using heparin columns [60] and vector titers were determined by quantitative polymerase chain reaction analysis as previously described [61]. [57] and contained a human growth hormone signal peptide (SP) to enable protein secretion.…”

Section: Aav Vector Construction and Virus Productionmentioning

confidence: 99%

AAV-Vectored Expression of the Vascular Normalizing Agents 3TSR and Fc3TSR, and the Anti-Angiogenic Bevacizumab Extends Survival in a Murine Model of End-Stage Epithelial Ovarian Carcinoma

et al. 2022

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Epithelial ovarian cancer is the deadliest gynecological malignancy. The lack of effective treatments highlights the need for novel therapeutic interventions. The aim of this study was to investigate whether sustained adeno-associated virus (AAV) vector-mediated expression of vascular normalizing agents 3TSR and Fc3TSR and the antiangiogenic monoclonal antibody, Bevacizumab, with or without oncolytic virus treatment would improve survival in an orthotopic syngeneic mouse model of epithelial ovarian carcinoma. AAV vectors were administered 40 days post-tumor implantation and combined with oncolytic avian orthoavulavirus-1 (AOaV-1) 20 days later, at the peak of AAV-transgene expression, to ascertain whether survival could be extended. Flow cytometry conducted on blood samples, taken at an acute time point post-AOaV-1 administration (36 h), revealed a significant increase in activated NK cells in the blood of all mice that received AOaV-1. T cell analysis revealed a significant increase in CD8+ tumor specific T cells in the blood of AAV-Bevacizumab+AOaV-1 treated mice compared to control mice 10 days post AOaV-1 administration. Immunohistochemical staining of primary tumors harvested from a subset of mice euthanized 90 days post tumor implantation, when mice typically have large primary tumors, secondary peritoneal lesions, and extensive ascites fluid production, revealed that AAV-3TSR, AAV-Fc3TSR+AOaV-1, or AAV-Bevacizumab+AOaV-1 treated mice had significantly more tumor-infiltrating CD8+ T cells than PBS controls. Despite AAV-mediated transgene expression waning faster in tumor-bearing mice than in non-tumor bearing mice, all three of the AAV therapies significantly extended survival compared to control mice; with AAV-Bevacizumab performing the best in this model. However, combining AAV therapies with a single dose of AOaV-1 did not lead to significant extensions in survival compared to AAV therapies on their own, suggesting that additional doses of AOaV-1 may be required to improve efficacy in this model. These results suggest that vectorizing anti-angiogenic and vascular normalizing agents is a viable therapeutic option that warrants further investigation, including optimizing combination therapies.

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“…Briefly, the plasmid containing the AAV genome was co-transfected into cells along with pDGM6, a packaging plasmid that expresses AAV6 capsid. AAV vector titers were determined by quantitative polymerase chain reaction analysis as previously described [20].…”

Section: Aav Vector Production and Quantificationmentioning

confidence: 99%

Macrophage Depletion via Clodronate Pretreatment Reduces Transgene Expression from AAV Vectors In Vivo

Chow

Bridle

et al. 2021

Viruses

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Adeno-associated virus is a popular gene delivery vehicle for gene therapy studies. A potential roadblock to widespread clinical adoption is the high vector doses required for efficient transduction in vivo, and the potential for subsequent immune responses that may limit prolonged transgene expression. We hypothesized that the depletion of macrophages via systemic delivery of liposome-encapsulated clodronate would improve transgene expression if given prior to systemic AAV vector administration, as has been shown to be the case with adenoviral vectors. Contrary to our expectations, clodronate liposome pretreatment resulted in significantly reduced transgene expression in the liver and heart, but permitted moderate transduction of the white pulp of the spleen. There was a remarkable localization of transgene expression from the red pulp to the center of the white pulp in clodronate-treated mice compared to untreated mice. Similarly, a greater proportion of transgene expression could be observed in the medulla located in the center of the lymph node in mice treated with clodronate-containing liposomes as compared to untreated mice where transgene expression was localized primarily to the cortex. These results underscore the highly significant role that the immune system plays in influencing the distribution and relative numbers of transduced cells in the context of AAV-mediated gene delivery.

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Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Cited by 10 publications

References 0 publications

Zoonotic avian influenza viruses evade human BTN3A3 restriction

Zoonotic avian influenza viruses evade human BTN3A3 restriction

AAV-Vectored Expression of the Vascular Normalizing Agents 3TSR and Fc3TSR, and the Anti-Angiogenic Bevacizumab Extends Survival in a Murine Model of End-Stage Epithelial Ovarian Carcinoma

Macrophage Depletion via Clodronate Pretreatment Reduces Transgene Expression from AAV Vectors In Vivo

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