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1
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Molina E, Tejero M, Duzenli OF, Kuoch H, Caine C, Krotova K, Paulaitis M, Aslanidi G. Insights in AAV-mediated antigen-specific immunity and a strategy for AAV vaccine dose reduction through AAV-extracellular vesicle association. Mol Ther Methods Clin Dev 2024; 32:101358. [PMID: 39559560 PMCID: PMC11570487 DOI: 10.1016/j.omtm.2024.101358] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Accepted: 10/16/2024] [Indexed: 11/20/2024]
Abstract
We previously showed therapeutic advantages of using a capsid-modified and encoded antigen-optimized AAV-based cancer vaccine to initiate strong antigen-specific immune responses and increase survival in a syngeneic mouse model of melanoma. In this study, we further explore AAV vaccine dose reduction and possible mechanisms of the immune response. Immunization with extracellular vesicle (EV)-associated AAV6-S663V encoded ovalbumin (OVA) or tyrosinase-related protein 1 (TRP-1) induced significantly higher levels of antigen-specific CD8+ T cells compared with standard AAV in mice. Importantly, a higher number of specific CD8+ T cells was achieved with EV-AAV several logs lower than optAAV-based doses. EV-optAAV-OVA was used in a dose 100 times lower, and EV-optTRP-1 10 times lower than optOVA and optTRP-1 correspondingly. Our data suggest that significant dose reduction for optimized AAV-based vaccines is possible without sacrificing efficiency. In addition, we studied the role of conventional type 1 dendritic cells (cDC1) in optimized AAV-based immunization using a C57BL/6-Irf8em1Kmm (Irf8 + 32-/-) mouse model lacking cDC1. Interestingly, we found that cDC1 are not essential for conveying effector T cell responses to AAV-encoded tumor antigens.
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Affiliation(s)
- Ester Molina
- The Hormel Institute, University of Minnesota, Austin, MN 55912, USA
| | - Marcos Tejero
- The Hormel Institute, University of Minnesota, Austin, MN 55912, USA
| | | | - Hisae Kuoch
- The Hormel Institute, University of Minnesota, Austin, MN 55912, USA
| | - Colin Caine
- The Hormel Institute, University of Minnesota, Austin, MN 55912, USA
| | - Karina Krotova
- The Hormel Institute, University of Minnesota, Austin, MN 55912, USA
| | - Michael Paulaitis
- Department of Ophthalmology, Center for Nanomedicine at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
| | - George Aslanidi
- The Hormel Institute, University of Minnesota, Austin, MN 55912, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 5455, USA
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2
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Mnyandu N, Jacobs R, Arbuthnot P, Maepa MB. Recent Advances in Designing Adeno-Associated Virus-Based Vaccines Against Viral Infections. Pharmaceutics 2024; 16:1360. [PMID: 39598484 PMCID: PMC11597783 DOI: 10.3390/pharmaceutics16111360] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2024] [Revised: 10/21/2024] [Accepted: 10/23/2024] [Indexed: 11/29/2024] Open
Abstract
Over 80% of the world's deadliest pandemics are caused by viral infections, and vaccination remains the most effective way to prevent these infections from spreading. Since the discovery of the first vaccine over two centuries ago, several vaccine design technologies have been developed. Next-generation vaccines, based on mRNA and viral vector technologies, have recently emerged as alternatives to traditional vaccines. Adenoviral vector-based vaccines against coronavirus disease 2019 have demonstrated a more sustained antibody response as compared to mRNA vaccines. However, this has not been without complications, with a few cases of severe adverse events identified in vaccinated individuals, and the underlying mechanism is the subject of intense investigation. Adeno-associated viral vectors induce a weaker cellular immune response compared to adenoviral vectors, and it is mainly for this reason that there has been a diminished interest in exploring them as a vaccine platform until recently. This review will discuss recent developments and the potential of adeno-associated viral vectors as anti-viral vaccines.
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Affiliation(s)
| | | | | | - Mohube Betty Maepa
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (N.M.); (R.J.); (P.A.)
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3
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Chauhan M, Daugherty AL, Khadir FE, Duzenli OF, Hoffman A, Tinklenberg JA, Kang PB, Aslanidi G, Pacak CA. AAV-DJ is superior to AAV9 for targeting brain and spinal cord, and de-targeting liver across multiple delivery routes in mice. J Transl Med 2024; 22:824. [PMID: 39237935 PMCID: PMC11375878 DOI: 10.1186/s12967-024-05599-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2024] [Accepted: 08/12/2024] [Indexed: 09/07/2024] Open
Abstract
Highly efficient adeno associated viruses (AAVs) targeting the central nervous system (CNS) are needed to deliver safe and effective therapies for inherited neurological disorders. The goal of this study was to compare the organ-specific transduction efficiencies of two AAV capsids across three different delivery routes. We compared AAV9-CBA-fLucYFP to AAV-DJ-CBA-fLucYFP using the following delivery routes in mice: intracerebroventricular (ICV) 1 × 1012 vg/kg, intrathecal (IT) 1 × 1012 vg/kg, and intravenous (IV) 1 × 1013 vg/kg body weight. Our evaluations revealed that following ICV and IT administrations, AAV-DJ demonstrated significantly increased vector genome (vg) uptake throughout the CNS as compared to AAV9. Through the IV route, AAV9 demonstrated significantly increased vg uptake in the CNS. However, significantly fewer vgs were detected in the off-target organs (kidney and liver) following administration of AAV-DJ using the IT and IV delivery routes as compared to AAV9. Distributions of vgs correlate well with transgene transcript levels, luciferase enzyme activities, and immunofluorescence detection of YFP. Overall, between the two vectors, AAV-DJ resulted in better targeting and expression in CNS tissues paired with de-targeting and reduced expression in liver and kidneys. Our findings support further examination of AAV-DJ as a gene therapy capsid for the treatment of neurological disorders.
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Affiliation(s)
- Monika Chauhan
- Department of Neurology, Greg Marzolf Jr. Muscular Dystrophy Center, University of Minnesota Medical School, 420 Delaware Street SE, MMC 295, Minneapolis, Minnesota, MN, 55455, USA
| | - Audrey L Daugherty
- Department of Neurology, Greg Marzolf Jr. Muscular Dystrophy Center, University of Minnesota Medical School, 420 Delaware Street SE, MMC 295, Minneapolis, Minnesota, MN, 55455, USA
| | - Fatemeh Ellie Khadir
- Department of Neurology, Greg Marzolf Jr. Muscular Dystrophy Center, University of Minnesota Medical School, 420 Delaware Street SE, MMC 295, Minneapolis, Minnesota, MN, 55455, USA
| | - Ozgun F Duzenli
- The Hormel Institute, University of Minnesota, Austin, MN, USA
| | | | - Jennifer A Tinklenberg
- Department of Neurology, Greg Marzolf Jr. Muscular Dystrophy Center, University of Minnesota Medical School, 420 Delaware Street SE, MMC 295, Minneapolis, Minnesota, MN, 55455, USA
| | - Peter B Kang
- Department of Neurology, Greg Marzolf Jr. Muscular Dystrophy Center, University of Minnesota Medical School, 420 Delaware Street SE, MMC 295, Minneapolis, Minnesota, MN, 55455, USA
| | - George Aslanidi
- The Hormel Institute, University of Minnesota, Austin, MN, USA
| | - Christina A Pacak
- Department of Neurology, Greg Marzolf Jr. Muscular Dystrophy Center, University of Minnesota Medical School, 420 Delaware Street SE, MMC 295, Minneapolis, Minnesota, MN, 55455, USA.
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4
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Araujo AE, Bentler M, Perez Garmendia X, Kaleem A, Fabian C, Morgan M, Hacker UT, Büning H. Adeno-Associated Virus Vectors-a Target of Cellular and Humoral Immunity-are Expanding Their Reach Toward Hematopoietic Stem Cell Modification and Immunotherapies. Hum Gene Ther 2024; 35:586-603. [PMID: 39193633 DOI: 10.1089/hum.2024.114] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/29/2024] Open
Abstract
All current market-approved gene therapy medical products for in vivo gene therapy of monogenic diseases rely on adeno-associated virus (AAV) vectors. Advances in gene editing technologies and vector engineering have expanded the spectrum of target cells and, thus, diseases that can be addressed. Consequently, AAV vectors are now being explored to modify cells of the hematopoietic system, including hematopoietic stem and progenitor cells (HSPCs), to develop novel strategies to treat monogenic diseases, but also to generate cell- and vaccine-based immunotherapies. However, the cell types that represent important new targets for the AAV vector system are centrally involved in immune responses against the vector and its transgene product as discussed briefly in the first part of this review. In the second part, studies exploring AAV vectors for genetic engineering of HSPCs, T and B lymphocytes, and beyond are presented.
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Affiliation(s)
- Angela E Araujo
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Martin Bentler
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | | | - Asma Kaleem
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Claire Fabian
- Laboratory for Vector based immunotherapy, Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany
- Medical Department II, University Cancer Center Leipzig (UCCL), Leipzig University Medical Center, Cancer Center Central Germany, Leipzig, Germany
| | - Michael Morgan
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Ulrich T Hacker
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
- Laboratory for Vector based immunotherapy, Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany
- Medical Department II, University Cancer Center Leipzig (UCCL), Leipzig University Medical Center, Cancer Center Central Germany, Leipzig, Germany
| | - Hildegard Büning
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
- German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Braunschweig, Germany
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5
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Stamataki M, Rissiek B, Magnus T, Körbelin J. Microglia targeting by adeno-associated viral vectors. Front Immunol 2024; 15:1425892. [PMID: 39035004 PMCID: PMC11257843 DOI: 10.3389/fimmu.2024.1425892] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2024] [Accepted: 06/21/2024] [Indexed: 07/23/2024] Open
Abstract
Microglia play a crucial role in maintaining homeostasis of the central nervous system and they are actively involved in shaping the brain's inflammatory response to stress. Among the multitude of involved molecules, purinergic receptors and enzymes are of special importance due to their ability to regulate microglia activation. By investigating the mechanisms underlying microglial responses and dysregulation, researchers can develop more precise interventions to modulate microglial behavior and alleviate neuroinflammatory processes. Studying gene function selectively in microglia, however, remains technically challenging. This review article provides an overview of adeno-associated virus (AAV)-based microglia targeting approaches, discussing potential prospects for refining these approaches to improve both specificity and effectiveness and encouraging future investigations aimed at connecting the potential of AAV-mediated microglial targeting for therapeutic benefit in neurological disorders.
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Affiliation(s)
- Maria Stamataki
- ENDomics Lab, Department of Oncology, Hematology & Bone Marrow Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Björn Rissiek
- Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Tim Magnus
- Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Jakob Körbelin
- ENDomics Lab, Department of Oncology, Hematology & Bone Marrow Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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6
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Ball JB, Frank MG, Green-Fulgham SM, Watkins LR. Use of adeno-associated viruses for transgenic modulation of microglia structure and function: A review of technical considerations and challenges. Brain Behav Immun 2024; 118:368-379. [PMID: 38471576 PMCID: PMC11103248 DOI: 10.1016/j.bbi.2024.03.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/02/2023] [Revised: 02/08/2024] [Accepted: 03/03/2024] [Indexed: 03/14/2024] Open
Abstract
Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches.
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Affiliation(s)
- Jayson B Ball
- Department of Psychology and Neuroscience, and the Center for Neuroscience, University of Colorado, Boulder, CO 80309, USA.
| | - Matthew G Frank
- Department of Psychology and Neuroscience, and the Center for Neuroscience, University of Colorado, Boulder, CO 80309, USA
| | - Suzanne M Green-Fulgham
- Department of Psychology and Neuroscience, and the Center for Neuroscience, University of Colorado, Boulder, CO 80309, USA
| | - Linda R Watkins
- Department of Psychology and Neuroscience, and the Center for Neuroscience, University of Colorado, Boulder, CO 80309, USA
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7
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Hadi M, Qutaiba B Allela O, Jabari M, Jasoor AM, Naderloo O, Yasamineh S, Gholizadeh O, Kalantari L. Recent advances in various adeno-associated viruses (AAVs) as gene therapy agents in hepatocellular carcinoma. Virol J 2024; 21:17. [PMID: 38216938 PMCID: PMC10785434 DOI: 10.1186/s12985-024-02286-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2023] [Accepted: 01/02/2024] [Indexed: 01/14/2024] Open
Abstract
Primary liver cancer, which is scientifically referred to as hepatocellular carcinoma (HCC), is a significant concern in the field of global health. It has been demonstrated that conventional chemotherapy, chemo-hormonal therapy, and conformal radiotherapy are ineffective against HCC. New therapeutic approaches are thus urgently required. Identifying single or multiple mutations in genes associated with invasion, metastasis, apoptosis, and growth regulation has resulted in a more comprehensive comprehension of the molecular genetic underpinnings of malignant transformation, tumor advancement, and host interaction. This enhanced comprehension has notably propelled the development of novel therapeutic agents. Therefore, gene therapy (GT) holds great promise for addressing the urgent need for innovative treatments in HCC. However, the complexity of HCC demands precise and effective therapeutic approaches. The adeno-associated virus (AAV) distinctive life cycle and ability to persistently infect dividing and nondividing cells have rendered it an alluring vector. Another appealing characteristic of the wild-type virus is its evident absence of pathogenicity. As a result, AAV, a vector that lacks an envelope and can be modified to transport DNA to specific cells, has garnered considerable interest in the scientific community, particularly in experimental therapeutic strategies that are still in the clinical stage. AAV vectors emerge as promising tools for HCC therapy due to their non-immunogenic nature, efficient cell entry, and prolonged gene expression. While AAV-mediated GT demonstrates promise across diverse diseases, the current absence of ongoing clinical trials targeting HCC underscores untapped potential in this context. Furthermore, gene transfer through hepatic AAV vectors is frequently facilitated by GT research, which has been propelled by several congenital anomalies affecting the liver. Notwithstanding the enthusiasm associated with this notion, recent discoveries that expose the integration of the AAV vector genome at double-strand breaks give rise to apprehensions regarding their enduring safety and effectiveness. This review explores the potential of AAV vectors as versatile tools for targeted GT in HCC. In summation, we encapsulate the multifaceted exploration of AAV vectors in HCC GT, underlining their transformative potential within the landscape of oncology and human health.
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Affiliation(s)
- Meead Hadi
- Department of Microbiology, Faculty of Basic Science, Central Tehran Branch, Islamic Azad University, Tehran, Iran
| | | | - Mansoureh Jabari
- Medical Campus, Xi'an Jiaotong University, Xi'an, Shaanxi Province, China
| | - Asna Mahyazadeh Jasoor
- Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran
| | - Omid Naderloo
- Department of Laboratory Sciences, Faculty of Medicine, Islamic Azad University of Gorgan Breanch, Gorgan, Iran
| | | | | | - Leila Kalantari
- School of Medicine, Kashan University of Medical Sciences, Kashan, Iran.
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8
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Dogbey DM, Torres VES, Fajemisin E, Mpondo L, Ngwenya T, Akinrinmade OA, Perriman AW, Barth S. Technological advances in the use of viral and non-viral vectors for delivering genetic and non-genetic cargos for cancer therapy. Drug Deliv Transl Res 2023; 13:2719-2738. [PMID: 37301780 PMCID: PMC10257536 DOI: 10.1007/s13346-023-01362-3] [Citation(s) in RCA: 28] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/29/2023] [Indexed: 06/12/2023]
Abstract
The burden of cancer is increasing globally. Several challenges facing its mainstream treatment approaches have formed the basis for the development of targeted delivery systems to carry and distribute anti-cancer payloads to their defined targets. This site-specific delivery of drug molecules and gene payloads to selectively target druggable biomarkers aimed at inducing cell death while sparing normal cells is the principal goal for cancer therapy. An important advantage of a delivery vector either viral or non-viral is the cumulative ability to penetrate the haphazardly arranged and immunosuppressive tumour microenvironment of solid tumours and or withstand antibody-mediated immune response. Biotechnological approaches incorporating rational protein engineering for the development of targeted delivery systems which may serve as vehicles for packaging and distribution of anti-cancer agents to selectively target and kill cancer cells are highly desired. Over the years, these chemically and genetically modified delivery systems have aimed at distribution and selective accumulation of drug molecules at receptor sites resulting in constant maintenance of high drug bioavailability for effective anti-tumour activity. In this review, we highlighted the state-of-the art viral and non-viral drug and gene delivery systems and those under developments focusing on cancer therapy.
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Affiliation(s)
- Dennis Makafui Dogbey
- South African Research Chair in Cancer Biotechnology, Division of Chemical and Systems Biology, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa
| | | | - Emmanuel Fajemisin
- South African Research Chair in Cancer Biotechnology, Division of Chemical and Systems Biology, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa
| | - Liyabona Mpondo
- South African Research Chair in Cancer Biotechnology, Division of Chemical and Systems Biology, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa
| | - Takunda Ngwenya
- South African Research Chair in Cancer Biotechnology, Division of Chemical and Systems Biology, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa
| | - Olusiji Alex Akinrinmade
- South African Research Chair in Cancer Biotechnology, Division of Chemical and Systems Biology, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa
| | - Adam W Perriman
- School of Cellular and Molecular Medicine, University of Bristol, BS8 1TD, Bristol, UK
| | - Stefan Barth
- South African Research Chair in Cancer Biotechnology, Division of Chemical and Systems Biology, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa.
- Medical Biotechnology and Immunotherapy Research Unit, Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
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9
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Tejero M, Duzenli OF, Caine C, Kuoch H, Aslanidi G. Bioengineered Hybrid Rep 2/6 Gene Improves Encapsulation of a Single-Stranded Expression Cassette into AAV6 Vectors. Genes (Basel) 2023; 14:1866. [PMID: 37895215 PMCID: PMC10606878 DOI: 10.3390/genes14101866] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2023] [Revised: 09/18/2023] [Accepted: 09/21/2023] [Indexed: 10/29/2023] Open
Abstract
The production of clinical-grade recombinant adeno-associated viral (AAV) vectors for gene therapy trials remains a major hurdle in the further advancement of the gene therapy field. During the past decades, AAV research has been predominantly focused on the development of new capsid modifications, vector-associated immunogenicity, and the scale-up vector production. However, limited studies have examined the possibility to manipulate non-structural components of AAV such as the Rep genes. Historically, naturally isolated, or recombinant library-derived AAV capsids have been produced using the AAV serotype 2 Rep gene to package ITR2-flanked vector genomes. In the current study, we mutated four variable amino acids in the conservative part of the binding domain in AAV serotype 6 Rep to generate a Rep2/6 hybrid gene. This newly generated Rep2/6 hybrid had improved packaging ability over wild-type Rep6. AAV vectors produced with Rep2/6 exhibited similar in vivo activity as standard AAV6 vectors. Furthermore, we show that this Rep2/6 hybrid also improves full/empty capsid ratios, suggesting that Rep bioengineering can be used to improve the ratio of fully encapsulated AAV vectors during upstream manufacturing processes.
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Affiliation(s)
- Marcos Tejero
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
| | - Ozgun F. Duzenli
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
| | - Colin Caine
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
| | - Hisae Kuoch
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
| | - George Aslanidi
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
- Institute Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
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10
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Kwak G, Gololobova O, Sharma N, Caine C, Mazur M, Mulka K, West NE, Solomon GM, Cutting GR, Witwer KW, Rowe SM, Paulaitis M, Aslanidi G, Suk JS. Extracellular vesicles enhance pulmonary transduction of stably associated adeno-associated virus following intratracheal administration. J Extracell Vesicles 2023; 12:e12324. [PMID: 37272896 PMCID: PMC10241173 DOI: 10.1002/jev2.12324] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2022] [Accepted: 03/29/2023] [Indexed: 06/06/2023] Open
Abstract
Adeno-associated virus (AAV) vector has shown multiple clinical breakthroughs, but its clinical implementation in inhaled gene therapy remains elusive due to difficulty in transducing lung airway cells. We demonstrate here AAV serotype 6 (AAV6) associated with extracellular vesicles (EVs) and secreted from vector-producing HEK-293 cells during vector preparation (EVAAV6) as a safe and highly efficacious gene delivery platform for inhaled gene therapy applications. Specifically, we discovered that EVAAV6 provided markedly enhanced reporter transgene expression in mucus-covered air-liquid interface (ALI) cultures of primary human bronchial and nasal epithelial cells as well as in mouse lung airways compared to standard preparations of AAV6 alone. Of note, AAV6 has been previously shown to outperform other clinically tested AAV serotypes, including those approved by the FDA for treating non-lung diseases, in transducing ALI cultures of primary human airway cells. We provide compelling experimental evidence that the superior performance of EVAAV6 is attributed to the ability of EV to facilitate mucus penetration and cellular entry/transduction of AAV6. The tight and stable linkage between AAV6 and EVs appears essential to exploit the benefits of EVs given that a physical mixture of individually prepared EVs and AAV6 failed to mediate EV-AAV6 interactions or to enhance gene transfer efficacy.
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Affiliation(s)
- Gijung Kwak
- Center for Nanomedicine at Wilmer Eye InstituteJohns Hopkins University School of MedicineBaltimoreMarylandUSA
- Department of OphthalmologyJohns Hopkins University School of MedicineBaltimoreMarylandUSA
| | - Olesia Gololobova
- Department of Molecular and Comparative PathobiologyJohns Hopkins University School of MedicineBaltimoreMarylandUSA
| | - Neeraj Sharma
- Department of Genetic MedicineJohns Hopkins University School of MedicineBaltimoreMarylandUSA
| | - Colin Caine
- Hormel InstituteUniversity of MinnesotaAustinMinnesotaUSA
| | - Marina Mazur
- Gregory Fleming James Cystic Fibrosis Research CenterHeersink School of MedicineUniversity of Alabama at BirminghamBirminghamAlabamaUSA
| | - Kathleen Mulka
- Department of Molecular and Comparative PathobiologyJohns Hopkins University School of MedicineBaltimoreMarylandUSA
| | - Natalie E. West
- Department of Pulmonary and Critical Care MedicineJohns Hopkins University School of MedicineBaltimoreMarylandUSA
| | - George M. Solomon
- Gregory Fleming James Cystic Fibrosis Research CenterHeersink School of MedicineUniversity of Alabama at BirminghamBirminghamAlabamaUSA
| | - Garry R. Cutting
- Department of Genetic MedicineJohns Hopkins University School of MedicineBaltimoreMarylandUSA
| | - Kenneth W. Witwer
- Department of Molecular and Comparative PathobiologyJohns Hopkins University School of MedicineBaltimoreMarylandUSA
| | - Steven M. Rowe
- Gregory Fleming James Cystic Fibrosis Research CenterHeersink School of MedicineUniversity of Alabama at BirminghamBirminghamAlabamaUSA
| | - Michael Paulaitis
- Center for Nanomedicine at Wilmer Eye InstituteJohns Hopkins University School of MedicineBaltimoreMarylandUSA
| | - George Aslanidi
- Hormel InstituteUniversity of MinnesotaAustinMinnesotaUSA
- Masonic Cancer CenterUniversity of MinnesotaMinneapolisMinnesotaUSA
- Institute for Molecular VirologyUniversity of MinnesotaMinneapolisUSAMinnesota
| | - Jung Soo Suk
- Center for Nanomedicine at Wilmer Eye InstituteJohns Hopkins University School of MedicineBaltimoreMarylandUSA
- Department of OphthalmologyJohns Hopkins University School of MedicineBaltimoreMarylandUSA
- Department of Chemical and Biomolecular EngineeringJohns Hopkins UniversityBaltimoreMarylandUSA
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11
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Wu F, Luo S, Zhang Y, Ou Y, Wang H, Guo Z, He C, Bai S, He P, Jiang M, Chen X, Du G, Sun X. Single-shot AAV-vectored vaccine against SARS-CoV-2 with fast and long-lasting immunity. Acta Pharm Sin B 2023; 13:2219-2233. [PMID: 35846427 PMCID: PMC9273293 DOI: 10.1016/j.apsb.2022.07.004] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2022] [Revised: 06/28/2022] [Accepted: 06/30/2022] [Indexed: 12/05/2022] Open
Abstract
Due to the insufficient long-term protection and significant efficacy reduction to new variants of current COVID-19 vaccines, the epidemic prevention and control are still challenging. Here, we employ a capsid and antigen structure engineering (CASE) strategy to manufacture an adeno-associated viral serotype 6-based vaccine (S663V-RBD), which expresses trimeric receptor binding domain (RBD) of spike protein fused with a biological adjuvant RS09. Impressively, the engineered S663V-RBD could rapidly induce a satisfactory RBD-specific IgG titer within 2 weeks and maintain the titer for more than 4 months. Compared to the licensed BBIBP-CorV (Sinopharm, China), a single-dose S663V-RBD induced more endurable and robust immune responses in mice and elicited superior neutralizing antibodies against three typical SARS-CoV-2 pseudoviruses including wild type, C.37 (Lambda) and B.1.617.2 (Delta). More interestingly, the intramuscular injection of S663V-RBD could overcome pre-existing immunity against the capsid. Given its effectiveness, the CASE-based S663V-RBD may provide a new solution for the current and next pandemic.
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Affiliation(s)
| | | | - Yongshun Zhang
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Yangsen Ou
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Hairui Wang
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Zhaofei Guo
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Chunting He
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Shuting Bai
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Penghui He
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Min Jiang
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Xiaoyan Chen
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Guangsheng Du
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Xun Sun
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
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12
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Tumor antigen-loaded AAV vaccine drives protective immunity in a melanoma animal model. Mol Ther Methods Clin Dev 2023; 28:301-311. [PMID: 36851984 PMCID: PMC9957711 DOI: 10.1016/j.omtm.2023.01.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2022] [Accepted: 01/29/2023] [Indexed: 02/04/2023]
Abstract
We previously described therapeutic opportunities provided by capsid- and expression cassette-optimized adeno-associated virus serotype 6 (AAV6) vectors to suppress tumor growth in both solid and metastatic mouse models by using artificial ovalbumin (OVA) immunogen. In the current study, we further elucidated the mechanism of function of a novel AAV-based vaccine loaded with the melanoma tumor-associated antigens premelanosome protein gp100, tyrosinase (Tyr), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (TRP2). We showed that the AAV6-based vaccine creates cellular and humoral antigen-specific responses, while antigen expression at the site of vaccine injection was temporal, and the clearance of antigen coincided with T cell infiltration. Our data revealed the superior protective immune response of optimized AAV6-TRP1 compared with other self-antigens in a disease-free mouse model. We further assessed the ability of AAV6-TRP1 to protect animals from metastatic spread in the lungs and to extend animal survival by inhibiting solid tumor growth. Flow cytometry-based analysis indicated significant infiltration of CD8+ T cells and natural killer (NK) cells in the tumor site, as well as changes in the polarization of intratumoral macrophages. Altogether, our data strongly support the use of optimized AAV vectors for cancer vaccine development.
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13
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Hacker UT. On the way to developing AAV-based vaccines as novel tools for cancer immunotherapy. Mol Ther Methods Clin Dev 2023; 28:394-395. [PMID: 36874246 PMCID: PMC9982205 DOI: 10.1016/j.omtm.2023.02.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/06/2023]
Affiliation(s)
- Ulrich T Hacker
- Department of Medicine II, University Cancer Center Leipzig (UCCL), Leipzig University Medical Center, Leipzig, Germany
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14
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Kuoch H, Krotova K, Graham ML, Brantly ML, Aslanidi G. Multiplexing AAV Serotype-Specific Neutralizing Antibodies in Preclinical Animal Models and Humans. Biomedicines 2023; 11:biomedicines11020523. [PMID: 36831059 PMCID: PMC9953293 DOI: 10.3390/biomedicines11020523] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2022] [Revised: 01/29/2023] [Accepted: 02/07/2023] [Indexed: 02/16/2023] Open
Abstract
The accurate assessment of AAV-specific pre-existing humoral immunity due to natural viral infection is critical for the efficient use of clinical gene therapy. The method described in the present study applies equivalent infection conditions to each AAV serotype (AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVAnc80L65). In the current study, we validated the assay by assessing AAV-neutralizing antibody titers in a limited cohort of random human donors and well-established preclinical large animal models, including dogs and non-human primates (NHPs). We achieved a rapid and accurate evaluation of neutralizing titers for each individual subject that can be used for clinical enrollment based on specific AAV serotypes and individualized selection of the most suitable AAV serotype for each specific patient.
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Affiliation(s)
- Hisae Kuoch
- The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA
| | - Karina Krotova
- The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA
| | - Melanie L. Graham
- Department of Surgery, Medical School, University of Minnesota, Minneapolis, MN 55108, USA
| | - Mark L. Brantly
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Medical School, University of Florida, Gainesville, FL 32610, USA
| | - George Aslanidi
- The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA
- Correspondence: ; Tel.: +1-507-437-9622; Fax: +1-507-437-9606
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15
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Marino M, Holt MG. AAV Vector-Mediated Antibody Delivery (A-MAD) in the Central Nervous System. Front Neurol 2022; 13:870799. [PMID: 35493843 PMCID: PMC9039256 DOI: 10.3389/fneur.2022.870799] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Accepted: 02/21/2022] [Indexed: 11/13/2022] Open
Abstract
In the last four decades, monoclonal antibodies and their derivatives have emerged as a powerful class of therapeutics, largely due to their exquisite targeting specificity. Several clinical areas, most notably oncology and autoimmune disorders, have seen the successful introduction of monoclonal-based therapeutics. However, their adoption for treatment of Central Nervous System diseases has been comparatively slow, largely due to issues of efficient delivery resulting from limited permeability of the Blood Brain Barrier. Nevertheless, CNS diseases are becoming increasingly prevalent as societies age, accounting for ~6.5 million fatalities worldwide per year. Therefore, harnessing the full therapeutic potential of monoclonal antibodies (and their derivatives) in this clinical area has become a priority. Adeno-associated virus-based vectors (AAVs) are a potential solution to this problem. Preclinical studies have shown that AAV vector-mediated antibody delivery provides protection against a broad range of peripheral diseases, such as the human immunodeficiency virus (HIV), influenza and malaria. The parallel identification and optimization of AAV vector platforms which cross the Blood Brain Barrier with high efficiency, widely transducing the Central Nervous System and allowing high levels of local transgene production, has now opened a number of interesting scenarios for the development of AAV vector-mediated antibody delivery strategies to target Central Nervous System proteinopathies.
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Affiliation(s)
- Marika Marino
- Laboratory of Glia Biology, VIB-KU Leuven, Center for Brain & Disease Research, Leuven, Belgium
- Department of Neurosciences, KU Leuven, Leuven, Belgium
| | - Matthew G. Holt
- Laboratory of Glia Biology, VIB-KU Leuven, Center for Brain & Disease Research, Leuven, Belgium
- Department of Neurosciences, KU Leuven, Leuven, Belgium
- Leuven Brain Institute, Leuven, Belgium
- Synapse Biology Group, Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, Porto, Portugal
- *Correspondence: Matthew G. Holt
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16
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Maes ME, Wögenstein GM, Colombo G, Casado-Polanco R, Siegert S. Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2021; 23:210-224. [PMID: 34703843 PMCID: PMC8516996 DOI: 10.1016/j.omtm.2021.09.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Accepted: 09/07/2021] [Indexed: 12/13/2022]
Abstract
Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1CreERT2 mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.
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Affiliation(s)
- Margaret E Maes
- Institute of Science and Technology (IST) Austria, 3400 Klosterneuburg, Austria
| | | | - Gloria Colombo
- Institute of Science and Technology (IST) Austria, 3400 Klosterneuburg, Austria
| | | | - Sandra Siegert
- Institute of Science and Technology (IST) Austria, 3400 Klosterneuburg, Austria
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17
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Colon-Cortes Y, Hasan MA, Aslanidi G. Intra-tracheal delivery of AAV6 vectors results in sustained transduction in murine lungs without genomic integration. Gene 2021; 763S:100037. [PMID: 32904225 PMCID: PMC7452375 DOI: 10.1016/j.gene.2020.100037] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Revised: 05/20/2020] [Accepted: 07/30/2020] [Indexed: 11/25/2022]
Abstract
Despite the progress made in AAV-based gene therapy targeting different organ systems, lung-targeted gene therapy using AAV vectors has not been effective, mostly due to the poor transduction and un-sustained gene expression in airway epithelium. Furthermore, concerns over possible harmful insertional mutagenesis seen in other cell types, particularly hepatocytes, raised a question about AAV safety. In this study, we evaluate the long-term persistence of this vector in mouse lungs and any possible harmful integration of these vectors into the host genome. AAV6 vectors expressing reporter gene (firefly luciferase) were delivered to the lungs of C57BL/6 mice through intra-tracheal intubation. Despite the large variation among individual animals, most animals had high and sustained luciferase activity with a peak from 2 to 3 weeks post-transduction before a significant decline between 15 and 19 weeks post-transduction. More importantly, even after its decline, most animals maintained detectable luciferase expression for 150 days or more, which was confirmed by post-necropsy qPCR analysis of luciferase gene expression. At the termination point of experiments, an average of one copy of AAV expression cassette per mouse genome was detected. We also found that partial overlaps between the AAV6 expression cassette and the mouse genome were distributed broadly with no apparent systematic preference in any mouse chromosomal map location. In summary, our data suggest that AAV6 mediated long-term gene expression in the lungs with no evidence of genomic integration, and thus, any insertional mutagenesis.
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Affiliation(s)
| | | | - George Aslanidi
- The Hormel Institute, University of Minnesota, Austin, MN 55912, United States of America.
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18
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Bower JJ, Song L, Bastola P, Hirsch ML. Harnessing the Natural Biology of Adeno-Associated Virus to Enhance the Efficacy of Cancer Gene Therapy. Viruses 2021; 13:v13071205. [PMID: 34201599 PMCID: PMC8309980 DOI: 10.3390/v13071205] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2021] [Revised: 06/08/2021] [Accepted: 06/10/2021] [Indexed: 12/25/2022] Open
Abstract
Adeno-associated virus (AAV) was first characterized as small “defective” contaminant particles in a simian adenovirus preparation in 1965. Since then, a recombinant platform of AAV (rAAV) has become one of the leading candidates for gene therapy applications resulting in two FDA-approved treatments for rare monogenic diseases and many more currently in various phases of the pharmaceutical development pipeline. Herein, we summarize rAAV approaches for the treatment of diverse types of cancers and highlight the natural anti-oncogenic effects of wild-type AAV (wtAAV), including interactions with the cellular host machinery, that are of relevance to enhance current treatment strategies for cancer.
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Affiliation(s)
- Jacquelyn J. Bower
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA
- Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; (L.S.); (P.B.)
- Correspondence: (J.J.B.); (M.L.H.)
| | - Liujiang Song
- Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; (L.S.); (P.B.)
- Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Prabhakar Bastola
- Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; (L.S.); (P.B.)
- Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Matthew L. Hirsch
- Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; (L.S.); (P.B.)
- Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
- Correspondence: (J.J.B.); (M.L.H.)
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19
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O'Carroll SJ, Cook WH, Young D. AAV Targeting of Glial Cell Types in the Central and Peripheral Nervous System and Relevance to Human Gene Therapy. Front Mol Neurosci 2021; 13:618020. [PMID: 33505247 PMCID: PMC7829478 DOI: 10.3389/fnmol.2020.618020] [Citation(s) in RCA: 50] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Accepted: 12/11/2020] [Indexed: 12/12/2022] Open
Abstract
Different glial cell types are found throughout the central (CNS) and peripheral nervous system (PNS), where they have important functions. These cell types are also involved in nervous system pathology, playing roles in neurodegenerative disease and following trauma in the brain and spinal cord (astrocytes, microglia, oligodendrocytes), nerve degeneration and development of pain in peripheral nerves (Schwann cells, satellite cells), retinal diseases (Müller glia) and gut dysbiosis (enteric glia). These cell type have all been proposed as potential targets for treating these conditions. One approach to target these cell types is the use of gene therapy to modify gene expression. Adeno-associated virus (AAV) vectors have been shown to be safe and effective in targeting cells in the nervous system and have been used in a number of clinical trials. To date, a number of studies have tested the use of different AAV serotypes and cell-specific promoters to increase glial cell tropism and expression. However, true glial-cell specific targeting for a particular glial cell type remains elusive. This review provides an overview of research into developing glial specific gene therapy and discusses some of the issues that still need to be addressed to make glial cell gene therapy a clinical reality.
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Affiliation(s)
- Simon J O'Carroll
- Spinal Cord Injury Research Group, Department of Anatomy and Medical Imaging, School of Medical Sciences, University of Auckland, Auckland, New Zealand
| | - William H Cook
- Molecular Neurotherapeutics Group, Department of Pharmacology and Clinical Pharmacology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
| | - Deborah Young
- Molecular Neurotherapeutics Group, Department of Pharmacology and Clinical Pharmacology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
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20
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Krotova K, Aslanidi G. Modifiers of Adeno-Associated Virus-Mediated Gene Expression in Implication for Serotype-Universal Neutralizing Antibody Assay. Hum Gene Ther 2020; 31:1124-1131. [PMID: 32495655 PMCID: PMC7588322 DOI: 10.1089/hum.2020.074] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Adeno-associated virus (AAV)-based gene therapy is undergoing major expansion into clinical practice, with two treatments currently being granted Food and Drug Administration (FDA) approval. However, the presence of pre-existing neutralizing antibodies (NAB) is one of the significant hurdles for the clinical application of AAV vectors that significantly limits the patient population, which benefits from the treatment. A reliable diagnostic to evaluate the patient's seropositivity is required to ensure the effectiveness of the AAV-mediated therapeutic. Here, we describe a simple method for the determination of AAV NAB activity based on our finding that Compound C makes HEK293 cell highly permissive for infection by 10 commonly used AAV serotypes.
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Affiliation(s)
- Karina Krotova
- Hormel Institute, University of Minnesota, Austin, Minnesota, USA
| | - George Aslanidi
- Hormel Institute, University of Minnesota, Austin, Minnesota, USA
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21
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Zhang L, Rossi A, Lange L, Meumann N, Koitzsch U, Christie K, Nesbit MA, Moore CBT, Hacker UT, Morgan M, Hoffmann D, Zengel J, Carette JE, Schambach A, Salvetti A, Odenthal M, Büning H. Capsid Engineering Overcomes Barriers Toward Adeno-Associated Virus Vector-Mediated Transduction of Endothelial Cells. Hum Gene Ther 2020; 30:1284-1296. [PMID: 31407607 DOI: 10.1089/hum.2019.027] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-VEC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.
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Affiliation(s)
- L Zhang
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.,Institute of Pathology, University Hospital of Cologne, Cologne, Germany
| | - A Rossi
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,International Center for Research in Infectiology (CIRI), INSERM U1111, CNRS UMR5308, Lyon, France
| | - L Lange
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - N Meumann
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.,Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - U Koitzsch
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.,Institute of Pathology, University Hospital of Cologne, Cologne, Germany
| | - K Christie
- Biomedical Sciences Research Institute, Ulster University, Ulster, Northern Ireland
| | - M A Nesbit
- Biomedical Sciences Research Institute, Ulster University, Ulster, Northern Ireland
| | - C B T Moore
- Biomedical Sciences Research Institute, Ulster University, Ulster, Northern Ireland.,Avellino Labs USA, Menlo Park, California
| | - U T Hacker
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,1st Medical Department, University Cancer Center Leipzig, University Leipzig Medical Center, Leipzig, Germany
| | - M Morgan
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - D Hoffmann
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - J Zengel
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California
| | - J E Carette
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California
| | - A Schambach
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.,Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts
| | - A Salvetti
- International Center for Research in Infectiology (CIRI), INSERM U1111, CNRS UMR5308, Lyon, France
| | - M Odenthal
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.,Institute of Pathology, University Hospital of Cologne, Cologne, Germany
| | - H Büning
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.,Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.,REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.,German Center for Infection Research (DZIF), Partner Sites Bonn-Cologne and Hannover-Braunschweig, Braunschweig, Germany
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22
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Krotova K, Day A, Aslanidi G. An Engineered AAV6-Based Vaccine Induces High Cytolytic Anti-Tumor Activity by Directly Targeting DCs and Improves Ag Presentation. Mol Ther Oncolytics 2019; 15:166-177. [PMID: 31720373 PMCID: PMC6838889 DOI: 10.1016/j.omto.2019.10.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2019] [Accepted: 10/01/2019] [Indexed: 12/30/2022] Open
Abstract
We have previously shown that an AAV6-based vaccine generates high levels of antigen-specific CD8+ T cells. Further modifications described here led to significantly increased levels of antigen-specific CD8+ and CD4+ T cells, enhanced formation of memory cells, and superior antigen-specific killing capacity in a murine model. By tracking reporter-gene-positive dendritic cells, we showed that they were directly targeted with modified AAV6 in vivo. Our vaccine's anti-cancer potential was evaluated with the antigen ovalbumin against a B16F10 melanoma cell line stably expressing ovalbumin. The vaccination showed superior protection in a murine model of metastatic melanoma. The vaccination significantly delayed solid tumor growth but did not completely prevent tumor development. We show that tumors in immunized mice escaped vaccine-induced killing by losing ovalbumin expression. The vaccine induced massive tumor infiltration with NK and CD8+ T cells with upregulated PD-1 expression. Thus, a vaccination of a combination of anti-PD-1 antibodies demonstrated significant improvement in the treatment efficacy. To summarize, we showed that a bioengineered AAV6-based vaccine elicits strong and long-lasting cellular and humoral responses against an encoded antigen. To increase AAV vaccine efficiency and mitigate tumor escape through antigen loss, we intended to target several antigens in combination with treatments targeting the tumor microenvironment.
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Affiliation(s)
- Karina Krotova
- The Hormel Institute, University of Minnesota, Austin, MN, USA
| | - Andrew Day
- The Hormel Institute, University of Minnesota, Austin, MN, USA
| | - George Aslanidi
- The Hormel Institute, University of Minnesota, Austin, MN, USA
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23
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Maes ME, Colombo G, Schulz R, Siegert S. Targeting microglia with lentivirus and AAV: Recent advances and remaining challenges. Neurosci Lett 2019; 707:134310. [PMID: 31158432 PMCID: PMC6734419 DOI: 10.1016/j.neulet.2019.134310] [Citation(s) in RCA: 96] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2018] [Accepted: 05/30/2019] [Indexed: 02/06/2023]
Abstract
Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In neuroscience, recombinant viruses such as lentiviruses and adeno-associated viruses (AAVs) have been successfully used to target various cell types in the brain, although effective transduction of microglia is rare. In this review, we provide a short background of lentiviruses and AAVs, and strategies for designing recombinant viral vectors. Then, we will summarize recent literature on successful microglial transductions in vitro and in vivo, and discuss the current challenges. Finally, we provide guidelines for reporting the efficiency and specificity of viral targeting in microglia, which will enable the microglial research community to assess and improve methodologies for future studies.
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Affiliation(s)
- Margaret E Maes
- Institute of Science and Technology (IST) Austria, Am Campus 1, 3400 Klosterneuburg, Austria
| | - Gloria Colombo
- Institute of Science and Technology (IST) Austria, Am Campus 1, 3400 Klosterneuburg, Austria
| | - Rouven Schulz
- Institute of Science and Technology (IST) Austria, Am Campus 1, 3400 Klosterneuburg, Austria
| | - Sandra Siegert
- Institute of Science and Technology (IST) Austria, Am Campus 1, 3400 Klosterneuburg, Austria.
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24
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Rossi A, Dupaty L, Aillot L, Zhang L, Gallien C, Hallek M, Odenthal M, Adriouch S, Salvetti A, Büning H. Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity. Sci Rep 2019; 9:3631. [PMID: 30842485 PMCID: PMC6403382 DOI: 10.1038/s41598-019-40071-1] [Citation(s) in RCA: 59] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2018] [Accepted: 02/05/2019] [Indexed: 12/28/2022] Open
Abstract
AAV vectors poorly transduce Dendritic cells (DC), a feature invoked to explain AAV's low immunogenicity. However, the reason for this non-permissiveness remained elusive. Here, we performed an in-depth analysis using human monocyte-derived immature DC (iDC) as model. iDC internalized AAV vectors of various serotypes, but even the most efficient serotype failed to transduce iDC above background. Since AAV vectors reached the cell nucleus, we hypothesized that AAV's intracellular processing occurs suboptimal. On this basis, we screened an AAV peptide display library for capsid variants more suitable for DC transduction and identified the I/VSS family which transduced DC with efficiencies of up to 38%. This property correlated with an improved vector uncoating. To determine the consequence of this novel feature for AAV's in vivo performance, we engineered one of the lead candidates to express a cytoplasmic form of ovalbumin, a highly immunogenic model antigen, and assayed transduction efficiency as well as immunogenicity. The capsid variant clearly outperformed the parental serotype in muscle transduction and in inducing antigen-specific humoral and T cell responses as well as anti-capsid CD8+ T cells. Hence, vector uncoating represents a major barrier hampering AAV vector-mediated transduction of DC and impacts on its use as vaccine platform.
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Affiliation(s)
- Axel Rossi
- International Center for Research in Infectiology (CIRI), INSERM U1111 - Université claude Bernard Lyon 1, CNRS UMR5308, Ecole Normale Supérieur de Lyon, Université de Lyon, Lyon, France
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Léa Dupaty
- Normandie Univ, UNIROUEN, INSERM, U1234, Physiopathologie et biothérapies des maladies inflammatoires et autoimmunes (PANTHER), 76000, Rouen, France
| | - Ludovic Aillot
- International Center for Research in Infectiology (CIRI), INSERM U1111 - Université claude Bernard Lyon 1, CNRS UMR5308, Ecole Normale Supérieur de Lyon, Université de Lyon, Lyon, France
- Cancer Research Center of Lyon, INSERM U1052, CNRS UMR5206, Lyon, France
| | - Liang Zhang
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
| | - Célia Gallien
- International Center for Research in Infectiology (CIRI), INSERM U1111 - Université claude Bernard Lyon 1, CNRS UMR5308, Ecole Normale Supérieur de Lyon, Université de Lyon, Lyon, France
| | - Michael Hallek
- Clinic I of Internal Medicine, University Hospital Cologne, Cologne, Germany
| | | | - Sahil Adriouch
- Normandie Univ, UNIROUEN, INSERM, U1234, Physiopathologie et biothérapies des maladies inflammatoires et autoimmunes (PANTHER), 76000, Rouen, France.
| | - Anna Salvetti
- International Center for Research in Infectiology (CIRI), INSERM U1111 - Université claude Bernard Lyon 1, CNRS UMR5308, Ecole Normale Supérieur de Lyon, Université de Lyon, Lyon, France.
- Cancer Research Center of Lyon, INSERM U1052, CNRS UMR5206, Lyon, France.
| | - Hildegard Büning
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
- German Center for Infection Research (DZIF), partner site Hannover-Braunschweig, Hannover, Germany.
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Goyvaerts C, Breckpot K. The Journey of in vivo Virus Engineered Dendritic Cells From Bench to Bedside: A Bumpy Road. Front Immunol 2018; 9:2052. [PMID: 30254636 PMCID: PMC6141723 DOI: 10.3389/fimmu.2018.02052] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2018] [Accepted: 08/20/2018] [Indexed: 12/13/2022] Open
Abstract
Dendritic cells (DCs) are recognized as highly potent antigen-presenting cells that are able to stimulate cytotoxic T lymphocyte (CTL) responses with antitumor activity. Consequently, DCs have been explored as cellular vaccines in cancer immunotherapy. To that end, DCs are modified with tumor antigens to enable presentation of antigen-derived peptides to CTLs. In this review we discuss the use of viral vectors for in situ modification of DCs, focusing on their clinical applications as anticancer vaccines. Among the viral vectors discussed are those derived from viruses belonging to the families of the Poxviridae, Adenoviridae, Retroviridae, Togaviridae, Paramyxoviridae, and Rhabdoviridae. We will further shed light on how the combination of viral vector-based vaccination with T-cell supporting strategies will bring this strategy to the next level.
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An Adeno-Associated Viral Vector Capable of Penetrating the Mucus Barrier to Inhaled Gene Therapy. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2018; 9:296-304. [PMID: 30038933 PMCID: PMC6054694 DOI: 10.1016/j.omtm.2018.03.006] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/22/2018] [Accepted: 03/19/2018] [Indexed: 01/08/2023]
Abstract
Diffusion of the viral vectors evaluated in inhaled gene therapy clinical trials to date are largely hindered within airway mucus, which limits their access to, and transduction of, the underlying airway epithelium prior to clearance from the lung. Here, we discovered that adeno-associated virus (AAV) serotype 6 was able to rapidly diffuse through mucus collected from cystic fibrosis (CF) patients, unlike previously tested AAV serotypes. A point mutation of the AAV6 capsid suggests a potential mechanism by which AAV6 avoids adhesion to the mucus mesh. Significantly greater transgene expression was achieved with AAV6 compared to a mucoadhesive serotype, AAV1, in air-liquid interface cultures of human CF bronchial epithelium with naturally secreted mucus or induced mucus hypersecretion. In addition, AAV6 achieved superior distribution and overall level of transgene expression compared to AAV1 in the airways and whole lungs, respectively, of transgenic mice with airway mucus obstruction. Our findings motivate further evaluation and clinical development of AAV6 for inhaled gene therapy.
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Chen M, Maeng K, Nawab A, Francois RA, Bray JK, Reinhard MK, Boye SL, Hauswirth WW, Kaye FJ, Aslanidi G, Srivastava A, Zajac-Kaye M. Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors. Hum Gene Ther Methods 2017; 28:49-59. [PMID: 28125909 DOI: 10.1089/hgtb.2016.089] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to KrasG12D+/-, KrasG12D+/-/Pten+/-, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45-70%) compared with WT-AAV8 (7%; p < 0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared with WT-AAV8 (10,285 vs. 1,500 vector copies/μg DNA respectively, p < 0.05). In addition, we observed that intraperitoneal injection of the double-mutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding ∼14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/μg DNA respectively, p < 0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular.
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Affiliation(s)
- Min Chen
- 1 Department of Anatomy and Cell Biology, University of Florida College of Medicine , Gainesville, Florida
| | - Kyungah Maeng
- 1 Department of Anatomy and Cell Biology, University of Florida College of Medicine , Gainesville, Florida
| | - Akbar Nawab
- 1 Department of Anatomy and Cell Biology, University of Florida College of Medicine , Gainesville, Florida
| | - Rony A Francois
- 1 Department of Anatomy and Cell Biology, University of Florida College of Medicine , Gainesville, Florida
| | - Julie K Bray
- 1 Department of Anatomy and Cell Biology, University of Florida College of Medicine , Gainesville, Florida
| | - Mary K Reinhard
- 2 Department of Veterinary Medicine, University of Florida College of Medicine , Gainesville, Florida
| | - Sanford L Boye
- 3 Department of Ophthalmology, University of Florida College of Medicine , Gainesville, Florida
| | - William W Hauswirth
- 3 Department of Ophthalmology, University of Florida College of Medicine , Gainesville, Florida
| | - Frederic J Kaye
- 4 Department of Medicine, University of Florida College of Medicine , Gainesville, Florida
| | - Georgiy Aslanidi
- 5 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine , Gainesville, Florida
| | - Arun Srivastava
- 5 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine , Gainesville, Florida
| | - Maria Zajac-Kaye
- 1 Department of Anatomy and Cell Biology, University of Florida College of Medicine , Gainesville, Florida
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Kanaan NM, Sellnow RC, Boye SL, Coberly B, Bennett A, Agbandje-McKenna M, Sortwell CE, Hauswirth WW, Boye SE, Manfredsson FP. Rationally Engineered AAV Capsids Improve Transduction and Volumetric Spread in the CNS. MOLECULAR THERAPY. NUCLEIC ACIDS 2017; 8:184-197. [PMID: 28918020 PMCID: PMC5503098 DOI: 10.1016/j.omtn.2017.06.011] [Citation(s) in RCA: 57] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/20/2016] [Revised: 06/14/2017] [Accepted: 06/15/2017] [Indexed: 12/13/2022]
Abstract
Adeno-associated virus (AAV) is the most common vector for clinical gene therapy of the CNS. This popularity originates from a high safety record and the longevity of transgene expression in neurons. Nevertheless, clinical efficacy for CNS indications is lacking, and one reason for this is the relatively limited spread and transduction efficacy in large regions of the human brain. Using rationally designed modifications of the capsid, novel AAV capsids have been generated that improve intracellular processing and result in increased transgene expression. Here, we sought to improve AAV-mediated neuronal transduction to minimize the existing limitations of CNS gene therapy. We investigated the efficacy of CNS transduction using a variety of tyrosine and threonine capsid mutants based on AAV2, AAV5, and AAV8 capsids, as well as AAV2 mutants incapable of binding heparan sulfate (HS). We found that mutating several tyrosine residues on the AAV2 capsid significantly enhanced neuronal transduction in the striatum and hippocampus, and the ablation of HS binding also increased the volumetric spread of the vector. Interestingly, the analogous tyrosine substitutions on AAV5 and AAV8 capsids did not improve the efficacy of these serotypes. Our results demonstrate that the efficacy of CNS gene transfer can be significantly improved with minor changes to the AAV capsid and that the effect is serotype specific.
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Affiliation(s)
- Nicholas M Kanaan
- Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI 49503, USA; Mercy Health Saint Mary's, Grand Rapids, MI 49503, USA
| | - Rhyomi C Sellnow
- Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI 49503, USA
| | - Sanford L Boye
- Department of Ophthalmology, University of Florida, Gainesville, FL 32610, USA
| | - Ben Coberly
- Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI 49503, USA; Neuroscience Program, Michigan State University, East Lansing, MI 48825, USA
| | - Antonette Bennett
- Department of Biochemistry and Molecular Biology, The McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, FL 32610, USA
| | - Mavis Agbandje-McKenna
- Department of Biochemistry and Molecular Biology, The McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, FL 32610, USA
| | - Caryl E Sortwell
- Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI 49503, USA; Mercy Health Saint Mary's, Grand Rapids, MI 49503, USA
| | - William W Hauswirth
- Department of Ophthalmology, University of Florida, Gainesville, FL 32610, USA
| | - Shannon E Boye
- Department of Ophthalmology, University of Florida, Gainesville, FL 32610, USA
| | - Fredric P Manfredsson
- Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI 49503, USA; Mercy Health Saint Mary's, Grand Rapids, MI 49503, USA.
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Gernoux G, Wilson JM, Mueller C. Regulatory and Exhausted T Cell Responses to AAV Capsid. Hum Gene Ther 2017; 28:338-349. [PMID: 28323492 PMCID: PMC5399736 DOI: 10.1089/hum.2017.022] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2017] [Accepted: 03/16/2017] [Indexed: 12/31/2022] Open
Abstract
Recombinant adeno-associated viruses (AAVs) are quickly becoming the preferred viral vector for viral gene delivery for the treatment of a wide variety of genetic disorders. However, since their use in a clinical trial targeting hemophilia B patients 10 years ago, immune responses to the AAV capsid appear to have hampered some of the early clinical gene transfer efficacy. Indeed, AAV-based gene transfer has been shown to reactivate capsid-specific memory T cells, which have correlated with a decline in AAV-transduced tissue in some patients. Importantly, clinical trials have also shown that this reactivation can be quelled by administering time-course taper of glucocorticoid steroids before or after dosing. More recently, two clinical studies have shown that AAV gene transfer is not only able to induce a deleterious immune response, but also can result in the initiation of a tolerance to the AAV capsid mediated by regulatory T cells and exhausted T cells. This article reviews clinical trials describing immune responses to AAV, as well as the mechanisms responsible for immune tolerance in chronic infections and how it could apply to AAV-based gene transfer. A better understanding of both cytotoxic and tolerogenic immune responses to recombinant AAV will lead to safer gene transfer protocols in patients.
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Affiliation(s)
- Gwladys Gernoux
- Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts
| | - James M. Wilson
- Gene Therapy Program, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
| | - Christian Mueller
- Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts
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30
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van Diemen FR, Lebbink RJ. CRISPR/Cas9, a powerful tool to target human herpesviruses. Cell Microbiol 2016; 19. [PMID: 27860066 DOI: 10.1111/cmi.12694] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2016] [Revised: 11/07/2016] [Accepted: 11/14/2016] [Indexed: 12/11/2022]
Abstract
Over 90% of the adult population is infected with one or multiple herpesviruses. These viruses are characterized by their ability to establish latency, where the host is unable to clear the invader from infected cells resulting in a lifelong infection. Herpesviruses cause a wide variety of (recurrent) diseases such as cold sores, shingles, congenital defects and several malignancies. Although the productive phase of a herpesvirus infection can often be efficiently limited by nucleoside analogs, these drugs are ineffective during a latent herpesvirus infection and are therefore unable to clear herpesviruses from the human host. Advances in genome engineering using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 facilitates virus research and may hold potential to treat or cure previously incurable herpesvirus infections by directly targeting these viruses within infected cells. Here, we review recent applications of the CRISPR/Cas9 system for herpesviral research and discuss the therapeutic potential of the system to treat, or even cure, productive and latent herpesviral infections.
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Affiliation(s)
- Ferdy R van Diemen
- Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.,Utrecht Cornea Research Group, Department of Ophthalmology, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Robert Jan Lebbink
- Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands
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31
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Effect of Cytotoxic T Lymphocytes Induced by Recombinant Adeno-Associated Virus on Different Hepatitis B Virus Genes. HEPATITIS MONTHLY 2016. [DOI: 10.5812/hepatmon.42685] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
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Santiago-Ortiz JL, Schaffer DV. Adeno-associated virus (AAV) vectors in cancer gene therapy. J Control Release 2016; 240:287-301. [PMID: 26796040 PMCID: PMC4940329 DOI: 10.1016/j.jconrel.2016.01.001] [Citation(s) in RCA: 157] [Impact Index Per Article: 17.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Revised: 12/08/2015] [Accepted: 01/02/2016] [Indexed: 02/06/2023]
Abstract
Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field.
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Affiliation(s)
- Jorge L Santiago-Ortiz
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA
| | - David V Schaffer
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA; Department of Bioengineering, University of California, Berkeley, CA, USA; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA; The Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA.
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Reprogramming Immune Response With Capsid-Optimized AAV6 Vectors for Immunotherapy of Cancer. J Immunother 2016; 38:292-8. [PMID: 26261893 DOI: 10.1097/cji.0000000000000093] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
In the current studies we generated novel capsid-optimized adeno-associated virus (AAV) serotype 6 (AAV6) vectors expressing a tumor-associated antigen, and assessed their ability to activate a protective T-cell response in an animal model. First, we showed that specific mutations in the AAV6 capsid increase the transduction efficiency of these vectors in mouse bone marrow-derived dendritic cells in vitro for approximately 5-fold compared with the wild-type (WT) AAV6 vectors. Next, we evaluated the ability of the mutant AAV6 vectors to initiate specific T-cell clone proliferation in vivo. Our data indicate that the intramuscular administration of AAV6-S663V+T492V vectors expressing ovalbumin (OVA) led to a strong activation (approximately 9%) of specific T cells in peripheral blood compared with AAV6-WT treated animals (<1%). These OVA-specific T cells have a superior killing ability against mouse prostate cancer cell line RM1 stably expressing the OVA antigen when propagated in vitro. Finally, we evaluated the ability of capsid-optimized AAV6-S663V+T492V vectors to initiate a protective anticancer immune response in vivo. Our results document the suppression of subcutaneous tumor growth in animals immunized with AAV6-S663V+T492V vectors expressing prostatic acid phosphatase (PAP) for approximately 4 weeks in comparison with 1 week and 2 weeks for the negative controls, AAV6-EGFP, and AAV6-WT-PAP treated mice, respectively. These studies suggest that successful inhibition of tumor growth in an animal model would set the stage for potential clinical application of the capsid-optimized AAV6-S663V+T492V vectors.
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Ling C, Yin Z, Li J, Zhang D, Aslanidi G, Srivastava A. Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2016; 3:16029. [PMID: 27200382 PMCID: PMC4856060 DOI: 10.1038/mtm.2016.29] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/01/2016] [Revised: 03/17/2016] [Accepted: 03/18/2016] [Indexed: 02/08/2023]
Abstract
Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs) from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ~10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492) were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of homologous Rep proteins, ITRs, and capsids should also lead to more efficacious other AAV serotype vectors for their optimal use in human gene therapy.
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Affiliation(s)
- Chen Ling
- Division of Cellular and Molecular Therapy, Department of Pediatrics; University of Florida College of Medicine, Gainesville, Florida, USA; Powell Gene Therapy Center; University of Florida College of Medicine, Gainesville, Florida, USA; Shands Cancer Center; University of Florida College of Medicine, Gainesville, Florida, USA
| | - Zifei Yin
- Division of Cellular and Molecular Therapy, Department of Pediatrics; University of Florida College of Medicine , Gainesville, Florida, USA
| | - Jun Li
- Division of Cellular and Molecular Therapy, Department of Pediatrics; University of Florida College of Medicine , Gainesville, Florida, USA
| | - Daniel Zhang
- Division of Cellular and Molecular Therapy, Department of Pediatrics; University of Florida College of Medicine , Gainesville, Florida, USA
| | - George Aslanidi
- Division of Cellular and Molecular Therapy, Department of Pediatrics; University of Florida College of Medicine, Gainesville, Florida, USA; Powell Gene Therapy Center; University of Florida College of Medicine, Gainesville, Florida, USA
| | - Arun Srivastava
- Division of Cellular and Molecular Therapy, Department of Pediatrics; University of Florida College of Medicine, Gainesville, Florida, USA; Powell Gene Therapy Center; University of Florida College of Medicine, Gainesville, Florida, USA; Shands Cancer Center; University of Florida College of Medicine, Gainesville, Florida, USA; Genetics Institute; University of Florida College of Medicine, Gainesville, Florida, USA; Department of Molecular Genetics & Microbiology; University of Florida College of Medicine, Gainesville, Florida, USA
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Microglia-specific targeting by novel capsid-modified AAV6 vectors. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2016; 3:16026. [PMID: 27308302 PMCID: PMC4909093 DOI: 10.1038/mtm.2016.26] [Citation(s) in RCA: 95] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/15/2015] [Revised: 02/29/2016] [Accepted: 02/29/2016] [Indexed: 01/14/2023]
Abstract
Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy
applications for central nervous system diseases. Though rAAV can efficiently target
neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are
refractile to rAAV. To identify AAV capsids with microglia-specific transduction
properties, we initially screened the most commonly used serotypes, AAV1–9 and
rh10, on primary mouse microglia cultures. While these capsids were not permissive, we
then tested the microglial targeting properties of a newly characterized set of modified
rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized
rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a
self-complementary genome and microglia-specific promoters (F4/80 or CD68) could
efficiently and selectively transduce microglia in vitro. Delivery of these
constructs in mice brains resulted in microglia-specific expression of green fluorescent
protein, albeit at modest levels. We further show that CD68 promoter–driven
expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to
increased astrogliosis in the brains of wild-type mice. Our study describes the first
instance of AAV-targeted microglial gene expression leading to functional modulation of
the innate immune system in mice brains. This provides the rationale for utilizing these
unique capsid/promoter combinations for microglia-specific gene targeting for modeling or
functional studies.
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36
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Zhou J, Ma P, Li J, Cui X, Song W. Improvement of the cytotoxic T lymphocyte response against hepatocellular carcinoma by transduction of cancer cells with an adeno-associated virus carrying the interferon-γ gene. Mol Med Rep 2016; 13:3197-205. [PMID: 26936017 DOI: 10.3892/mmr.2016.4884] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2015] [Accepted: 12/11/2015] [Indexed: 11/06/2022] Open
Abstract
Dendritic cell (DC)-based antigen-targeted immunotherapy may offer effective adjuvant therapy for hepatocellular carcinoma (HCC), in which cytotoxic T lymphocytes (CTLs) are key. However, in a number of cases, the activity of CTLs is completely inhibited due to the downregulated expression of major human leukocyte antigen (HLA) class I molecules by HCC cells. The aim of the present study was to overcome this issue. Hep3B cells were transduced by HCC‑specific recombinant adeno‑associated virus (rAAV) carrying human α‑fetoprotein promoter (AFPp) and the interferon‑γ (IFN‑γ) gene (rAAV/AFPp‑IFN‑γ). rAAV carrying the cytomegalovirus promoter (CMVp) and human α‑fetoprotein (AFP) gene (rAAV/CMVp‑AFP) was used to transduce professional antigen‑presenting DCs for the purpose of stimulating a CTL response. It was observed that transduction of DCs with rAAV/CMVp‑AFP resulted in: (i) AFP and interleukin‑12 expression; (ii) high expression levels of cluster of differentiation (CD)80, CD83, CD86, CD40, HLA‑death receptor and CD1a; (iii) T cell populations with marked IFN‑γ expression; (iv) a high percentage of CD69+/CD8+ T cells; and (v) the activity of CTLs against HLA‑A2‑expressing Hep3B cells. The transduction of Hep3B cells with rAAV/AFPp‑IFN‑γ resulted in: (i) IFN‑γ expression; (ii) upregulated expression of HLA‑A2; and (iii) an improved CTL response against HLA‑A2‑deficient Hep3B cells. rAAV/CMVp‑AFP‑transduced DCs elicited an AFP‑specific and HLA‑class I‑restricted CTL response against Hep3B cells. In conclusion, it was shown that the transduction of Hep3B with rAAV/AFPp-IFN-γ upregulated the expression of HLA-A2 and improved the sensitivity to CTL response.
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Affiliation(s)
- Jun Zhou
- Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
| | - Ping Ma
- Department of Ophthalmology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
| | - Jun Li
- Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
| | - Xiaonan Cui
- Department of Oncology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, P.R. China
| | - Wei Song
- Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
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Selenica MLB, Reid P, Pena G, Alvarez J, Hunt JB, Nash KR, Morgan D, Gordon MN, Lee DC. Adeno associated viral-mediated intraosseous labeling of bone marrow derived cells for CNS tracking. J Immunol Methods 2016; 432:51-6. [PMID: 26784524 DOI: 10.1016/j.jim.2016.01.008] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2015] [Revised: 01/13/2016] [Accepted: 01/14/2016] [Indexed: 12/24/2022]
Abstract
Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseous impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9-GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the body following insult or injury. Alternatively, this method might find utility in delivering therapeutic genes for neuroinflammatory conditions.
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Affiliation(s)
- Maj-Linda B Selenica
- Dept of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA
| | - Patrick Reid
- Dept of Molecular Pharmacology & Physiology, Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
| | - Gabriela Pena
- Dept of Molecular Pharmacology & Physiology, Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
| | - Jennifer Alvarez
- Dept of Molecular Pharmacology & Physiology, Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
| | - Jerry B Hunt
- Dept of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA
| | - Kevin R Nash
- Dept of Molecular Pharmacology & Physiology, Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
| | - Dave Morgan
- Dept of Molecular Pharmacology & Physiology, Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
| | - Marcia N Gordon
- Dept of Molecular Pharmacology & Physiology, Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
| | - Daniel C Lee
- Dept of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA.
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Wang YG, Huang PP, Zhang R, Ma BY, Zhou XM, Sun YF. Targeting adeno-associated virus and adenoviral gene therapy for hepatocellular carcinoma. World J Gastroenterol 2016; 22:326-337. [PMID: 26755879 PMCID: PMC4698495 DOI: 10.3748/wjg.v22.i1.326] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2015] [Revised: 09/14/2015] [Accepted: 09/30/2015] [Indexed: 02/06/2023] Open
Abstract
Human hepatocellular carcinoma (HCC) heavily endangers human heath worldwide. HCC is one of most frequent cancers in China because patients with liver disease, such as chronic hepatitis, have the highest cancer susceptibility. Traditional therapeutic approaches have limited efficacy in advanced liver cancer, and novel strategies are urgently needed to improve the limited treatment options for HCC. This review summarizes the basic knowledge, current advances, and future challenges and prospects of adeno-associated virus (AAV) and adenoviruses as vectors for gene therapy of HCC. This paper also reviews the clinical trials of gene therapy using adenovirus vectors, immunotherapy, toxicity and immunological barriers for AAV and adenoviruses, and proposes several alternative strategies to overcome the therapeutic barriers to using AAV and adenoviruses as vectors.
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Development of novel AAV serotype 6 based vectors with selective tropism for human cancer cells. Gene Ther 2015; 23:18-25. [PMID: 26270885 DOI: 10.1038/gt.2015.89] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2015] [Accepted: 07/30/2015] [Indexed: 12/24/2022]
Abstract
Viral vectors-based gene therapy is an attractive alternative to common anti-cancer treatments. In the present studies, AAV serotype 6 vectors were identified to be particularly effective in the transduction of human prostate (PC3), breast (T47D) and liver (Huh7) cancer cells. Next, we developed chimeric AAV vectors with Arg-Gly-Asp (RGD) peptide incorporated into the viral capsid to enable specific targeting of integrin-overexpressing malignant cells. These AAV6-RGD vectors improved transduction efficiency approximately 3-fold compared with wild-type AAV6 vectors by enhancing the viral entry into the cells. We also observed that transduction efficiency significantly improved, up to approximately 5-fold, by the mutagenesis of surface-exposed tyrosine and threonine residues involved in the intracellular trafficking of AAV vectors. Therefore, in our study, the AAV6-Y705-731F+T492V vector was identified as the most efficient. The combination of RGD peptide, tyrosine and threonine mutations on the same AAV6 capsid further increased the transduction efficiency, approximately 8-fold in vitro. In addition, we mutated lysine (K531E) to impair the affinity of AAV6 vectors to heparan sulfate proteoglycan. Finally, we showed a significant increase in both specificity and efficiency of AAV6-RGD-Y705-731F+T492V+K531E vectors in a xenograft animal model in vivo. In summary, the approach described here can lead to the development of AAV vectors with selective tropism to human cancer cells.
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Chen H. Adeno-associated virus vectors for human gene therapy. World J Med Genet 2015; 5:28-45. [DOI: 10.5496/wjmg.v5.i3.28] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/08/2015] [Revised: 04/08/2015] [Accepted: 05/18/2015] [Indexed: 02/06/2023] Open
Abstract
Adeno-associated virus (AAV) is a small, non-enveloped virus that contains a single-stranded DNA genome. It was the first gene therapy drug approved in the Western world in November 2012 to treat patients with lipoprotein lipase deficiency. AAV made history and put human gene therapy in the forefront again. More than four decades of research on AAV vector biology and human gene therapy has generated a huge amount of valuable information. Over 100 AAV serotypes and variants have been isolated and at least partially characterized. A number of them have been used for preclinical studies in a variety of animal models. Several AAV vector production platforms, especially the baculovirus-based system have been established for commercial-scale AAV vector production. AAV purification technologies such as density gradient centrifugation, column chromatography, or a combination, have been well developed. More than 117 clinical trials have been conducted with AAV vectors. Although there are still challenges down the road, such as cross-species variation in vector tissue tropism and gene transfer efficiency, pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients, the gene therapy community is forging ahead with cautious optimism. In this review I will focus on the properties and applications of commonly used AAV serotypes and variants, and the technologies for AAV vector production and purification. I will also discuss the advancement of several promising gene therapy clinical trials.
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Ling C, Wang Y, Zhang Y, Ejjigani A, Yin Z, Lu Y, Wang L, Wang M, Li J, Hu Z, Aslanidi GV, Zhong L, Gao G, Srivastava A, Ling C. Selective in vivo targeting of human liver tumors by optimized AAV3 vectors in a murine xenograft model. Hum Gene Ther 2015; 25:1023-34. [PMID: 25296041 DOI: 10.1089/hum.2014.099] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Current challenges for recombinant adeno-associated virus (rAAV) vector-based cancer treatment include the low efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to liver damage. Our research provides a novel means to achieve not only targeted delivery but also the potential for gene therapy of human liver cancer.
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Affiliation(s)
- Chen Ling
- 1 Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine , Gainesville, FL 32611
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Mahon BP, Pinard MA, McKenna R. Targeting carbonic anhydrase IX activity and expression. Molecules 2015; 20:2323-48. [PMID: 25647573 PMCID: PMC6272707 DOI: 10.3390/molecules20022323] [Citation(s) in RCA: 101] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2014] [Accepted: 12/25/2014] [Indexed: 12/12/2022] Open
Abstract
Metastatic tumors are often hypoxic exhibiting a decrease in extracellular pH (~6.5) due to a metabolic transition described by the Warburg Effect. This shift in tumor cell metabolism alters the tumor milieu inducing tumor cell proliferation, angiogenesis, cell motility, invasiveness, and often resistance to common anti-cancer treatments; hence hindering treatment of aggressive cancers. As a result, tumors exhibiting this phenotype are directly associated with poor prognosis and decreased survival rates in cancer patients. A key component to this tumor microenvironment is carbonic anhydrase IX (CA IX). Knockdown of CA IX expression or inhibition of its activity has been shown to reduce primary tumor growth, tumor proliferation, and also decrease tumor resistance to conventional anti-cancer therapies. As such several approaches have been taken to target CA IX in tumors via small-molecule, anti-body, and RNAi delivery systems. Here we will review recent developments that have exploited these approaches and provide our thoughts for future directions of CA IX targeting for the treatment of cancer.
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Affiliation(s)
- Brian P Mahon
- Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32611, USA
| | - Melissa A Pinard
- Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32611, USA
| | - Robert McKenna
- Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32611, USA.
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Weber ND, Aubert M, Dang CH, Stone D, Jerome KR. DNA cleavage enzymes for treatment of persistent viral infections: recent advances and the pathway forward. Virology 2014; 454-455:353-61. [PMID: 24485787 DOI: 10.1016/j.virol.2013.12.037] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2013] [Accepted: 12/26/2013] [Indexed: 12/21/2022]
Abstract
Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application.
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Affiliation(s)
- Nicholas D Weber
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109, USA; Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA.
| | - Martine Aubert
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109, USA.
| | - Chung H Dang
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109, USA.
| | - Daniel Stone
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109, USA.
| | - Keith R Jerome
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109, USA; Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA; Department of Microbiology, University of Washington, Seattle, WA 98195, USA.
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Efficient lysis of epithelial ovarian cancer cells by MAGE-A3-induced cytotoxic T lymphocytes using rAAV-6 capsid mutant vector. Vaccine 2014; 32:938-43. [PMID: 24406390 DOI: 10.1016/j.vaccine.2013.12.049] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Revised: 12/11/2013] [Accepted: 12/18/2013] [Indexed: 11/23/2022]
Abstract
MAGE-A3 is highly expressed in epithelial ovarian cancer (EOC), making it a promising candidate for immunotherapy. We investigated whether dendritic cells (DCs) transduced with a rAAV-6 capsid mutant vector Y445F could elicit effective MAGE-A3-specific anti-tumor cytotoxic T lymphocyte (CTL) responses in vitro. MAGE-A3 was cloned and rAAV-6-MAGE-A3 purified, followed by proviral genome detection using real-time PCR. Immunofluorescence detection of rAAV-6-Y445F-MAGE-A3-transduced DCs demonstrated 60% transduction efficiency. Fluorescent in situ hybridization analysis confirmed chromosomal integration of rAAV vectors. Flow cytometric analysis of transduced DCs showed unaltered expression of critical monocyte-derived surface molecules with retention of allo-stimulatory activity. Co-culture of autologous T lymphocytes with MAGE-A3-expressing DCs produced CTLs that secreted IFN-γ, and efficiently killed MAGE-A3+ EOC cells. This form of rAAV-based DC immunotherapy, either alone or more likely in combination with other immune-enhancing protocols, may prove useful in the clinical setting for management of EOC.
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