production-adeno-associated-virus-vectors-cell-stacks-for-preclinical

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

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. ...

University of Guelph

An integral step in the development of solid tumors is the recruitment of blood vessels to fuel tumor growth. Antiangiogenic therapies can inhibit this process and control solid tumor growth. Thrombospondin-1 is an antiangiogenic protein possessing three type I repeats (3TSR) near the center of the protein and a CD47-binding peptide (CD47) in its C-terminus. Previously, we showed that treatment with recombinant 3TSR induces tumor regression, normalizes tumor vasculature, and improves uptake of chemotherapy drugs in an orthotopic, syngeneic mouse model of advanced stage epithelial ovarian cancer (EOC). While effective, this intervention required daily intraperitoneal injections. To circumvent this, here we employ adeno-associated virus (AAV) gene therapy vectors to express 3TSR alone or in combination with the CD47-binding peptide of TSP-1 and evaluate the impact on tumor development and survival in a mouse model of EOC. A single intraperitoneal injection of 1 × 10 vg of AAV expressing 3TSR, CD47-binding peptide, or 3TSR + CD47 effectively suppressed primary tumor growth; however, only AAV-3TSR was able to inhibit development of secondary lesions at 90-days post-tumor implantation and significantly improve survival. Taken together, AAV-mediated expression of 3TSR appears safe and effective at inhibiting tumor development and represents a novel, less invasive approach for treating ovarian carcinoma.

AAV-mediated expression of 3TSR inhibits tumor and metastatic lesion development and extends survival in a murine model of epithelial ovarian carcinoma.

Filoviruses are among the deadliest infectious agents known to man, causing severe hemorrhagic fever, with up to 90% fatality rates. The 2014 Ebola outbreak in West Africa resulted in over 28,000 infections, demonstrating the large-scale human health and economic impact generated by filoviruses.  is responsible for the greatest number of deaths to date and consequently there is now an approved vaccine, Ervebo, while other filovirus species have similar epidemic potential and remain without effective vaccines. Recent clinical success of REGN-EB3 and mAb-114 monoclonal antibody (mAb)-based therapies supports further investigation of this treatment approach for other filoviruses. While efficacious, protection from passive mAb therapies is short-lived, requiring repeat dosing to maintain therapeutic concentrations. An alternative strategy is vectored immunoprophylaxis (VIP), which utilizes an adeno-associated virus (AAV) vector to generate sustained expression of selected mAbs directly in vivo. This approach takes advantage of validated mAb development and enables vectorization of the top candidates to provide long-term immunity. In this review, we summarize the history of filovirus outbreaks, mAb-based therapeutics, and highlight promising AAV vectorized approaches to providing immunity against filoviruses where vaccines are not yet available.

AAV Vectored Immunoprophylaxis for Filovirus Infections.

Clostridium difficile is the leading cause of antibiotic-associated nosocomial diarrhea in the developed world. When the host-associated colon microbiome is disrupted by the ingestion of antibiotics, C. difficile spores can germinate, resulting in infection. C. difficile secretes enterotoxin A (TcdA) and cytotoxin B (TcdB) that are responsible for disease pathology. Treatment options are limited as the bacterium demonstrates resistance to many antibiotics, and even with antibacterial therapies, recurrences of C. difficile are common. Actotoxumab and bezlotoxumab are human monoclonal antibodies that bind and neutralize TcdA and TcdB, respectively. In 2016, the US food and drug administration (FDA) approved bezlotoxumab for use in the prevention of C. difficile infection recurrence. To ensure the long-term expression of antibodies, gene therapy can be used. Here, adeno-associated virus (AAV)6.2FF, a novel triple mutant of AAV6, was engineered to express either actotoxumab or bezlotoxumab in mice and hamsters. Both antibodies expressed at greater than 90 μg/mL in the serum and were detected at mucosal surfaces in both models. Hundred percent of mice given AAV6.2FF-actoxumab survived a lethal dose of TcdA. This proof of concept study demonstrates that AAV-mediated expression of C. difficile toxin antibodies is a viable approach for the prevention of recurrent C. difficile infections.

AAV-mediated delivery of actoxumab and bezlotoxumab results in serum and mucosal antibody concentrations that provide protection from C. difficile toxin challenge.

The avian paramyxovirus, Newcastle disease virus (NDV), is a promising oncolytic agent that has been shown to be safe and effective in a variety of pre-clinical cancer models and human clinical trials. NDV preferentially replicates in tumor cells due to signaling defects in apoptotic and antiviral pathways acquired during the transformation process and is a potent immunostimulatory agent. However, when used as a monotherapy NDV lacks the ability to consistently generate durable remissions. Here we investigate the use of viral sensitizer-mediated combination therapy to enhance the anti-neoplastic efficacy of NDV. Intratumoral injection of vanadyl sulfate, a pan-inhibitor of protein tyrosine phosphatases, in combination with NDV significantly increased the number and activation status of natural killer (NK) cells in the tumor microenvironment, concomitant with increased expression of interferon-β, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1, leading to rapid tumor regression and long-term cures in mice bearing syngeneic B16-F10 melanomas. The anti-tumor efficacy of this combination therapy was abrogated when NK cells were depleted and when interferon-β expression was transiently suppressed. Tumor-specific CD8 T cell responses were not detected, nor were mice whose tumors regressed protected from re-challenge. This suggested efficacy of the combination therapy predominantly relied on the innate immune system. Importantly, efficacy was not limited to melanoma; it was also demonstrated in a murine prostate cancer model. Taken together, these results suggest that combining NDV with vanadyl sulfate potentiates an innate immune response that can potentiate rapid clearance of tumors, with type I interferon signaling and NK cells being important mechanisms of action.

Combining vanadyl sulfate with Newcastle disease virus potentiates rapid innate immune-mediated regression with curative potential in murine cancer models.

More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector () expressing human ACE-2 (). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 (). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 10 TCID SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions.

Amira D. Rghei

Department of Pathobiology

University of Guelph

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