scholarly journals SUMOylation Targets Adeno-associated Virus Capsids but Mainly Restricts Transduction by Cellular Mechanisms

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Qingxin Chen ◽  
Robin Njenga ◽  
Barbara Leuchs ◽  
Susanna Chiocca ◽  
Jürgen Kleinschmidt ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Host Cell ◽  
Point Mutations ◽  
Host Factors ◽  
Cellular Proteins ◽  
Gene Transduction ◽  
Adeno Associated Virus ◽  
A Genome ◽  
Major Mode ◽  
Sumo Pathway

ABSTRACT Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly. IMPORTANCE Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.

scholarly journals Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells

2021 ◽  
Vol 22 (21) ◽  
pp. 11499
Author(s):  
Lisa Strasser ◽  
Stefano Boi ◽  
Felipe Guapo ◽  
Nicholas Donohue ◽  
Niall Barron ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Host Cell ◽  
Reversed Phase ◽  
Hek293 Cells ◽  
Label Free ◽  
Cellular Mechanisms ◽  
Adeno Associated Virus ◽  
Cellular Processes ◽  
Cell Productivity ◽  
Product Manufacturing

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.

scholarly journals Impact of capsid modifications by selected peptide ligands on recombinant adeno-associated virus serotype 2-mediated gene transduction

2012 ◽  
Vol 93 (10) ◽  
pp. 2131-2141 ◽  
Author(s):  
Matthias Naumer ◽  
Ruth Popa-Wagner ◽  
Jürgen A. Kleinschmidt
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Viral Vectors ◽  
Peptide Ligands ◽  
Gene Transduction ◽  
Cell Binding ◽  
Adeno Associated Virus ◽  
Vector Systems ◽  
Virus Serotype ◽  
Selection Of

Vectors based on adeno-associated virus serotype 2 (AAV2) belong to today’s most promising and most frequently used viral vectors in human gene therapy. Like in many other vector systems, the broad but non-specific tropism limits their use for certain cell types or tissues. One approach to screen for transduction-improved vectors is the selection of random peptide libraries displayed directly on the AAV2 capsid. Although the AAV2 library system has been widely applied for the successful selection of improved gene therapy vectors, it remains unknown which steps of the transduction process are most affected and therefore critical for the selection of targeting peptides. Attachment to the cell surface is the first essential step of AAV-mediated gene transduction; however, our experiments challenge the conventional belief that enhanced gene transfer is equivalent to more efficient cell binding of recombinant AAV2 vectors. A comparison of the various steps of gene transfer by vectors carrying a wild-type AAV2 capsid or displaying two exemplary peptide ligands selected from AAV2 random libraries on different human tumour cell lines demonstrated strong alterations in cell binding, cellular uptake, as well as intracellular processing of these vectors. Combined, our results suggest that entry and post-entry events are decisive for the selection of the peptides NDVRSAN and GPQGKNS rather than their cell binding efficiency.

scholarly journals A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Rutger D. Luteijn ◽  
Ferdy van Diemen ◽  
Vincent A. Blomen ◽  
Ingrid G. J. Boer ◽  
Saravanan Manikam Sadasivam ◽  
...  
Keyword(s):  
Virus Infection ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Host Cell ◽  
Host Factors ◽  
Virus Infectivity ◽  
Sequencing Analysis ◽  
Vaccinia Virus Infection ◽  
Genome Wide ◽  
A Genome

ABSTRACTVaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCEPoxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

scholarly journals A CRISPR Screen Identifies the Cell Polarity Determinant Crumbs 3 as an Adeno-associated Virus Restriction Factor in Hepatocytes

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Victoria J. Madigan ◽  
Tyne O. Tyson ◽  
Julianne A. Yuziuk ◽  
Minakshi Pillai ◽  
Sven Moller-Tank ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Surface ◽  
Cell Polarity ◽  
Cell Types ◽  
Host Factors ◽  
Cellular Polarity ◽  
A Genome ◽  
Organ Systems ◽  
Gene Therapy Vectors ◽  
Crispr Screen

ABSTRACTAdeno-associated viruses (AAV) are helper-dependent parvoviruses that have been developed into promising gene therapy vectors. Many studies, including a recent unbiased genomic screen, have identified host factors essential for AAV cell entry, but no genome-wide screens that address inhibitory host factors have been reported. Here, we utilize a novel CRISPR screen to identify AAV restriction factors in a human hepatocyte cell line. The major hit from our gain-of-function screen is the apical polarity determinant Crumbs 3 (Crb3). Knockout (KO) of Crb3 enhances AAV transduction, while overexpression exerts the opposite effect. Further, Crb3 appears to restrict AAV transduction in a serotype- and cell type-specific manner. Particularly, for AAV serotype 9 and a rationally engineered AAV variant, we demonstrate that increased availability of galactosylated glycans on the surfaces of Crb3 KO cells, but not the universal AAV receptor, leads to increased capsid attachment and enhanced transduction. We postulate that Crb3 could serve as a key molecular determinant that restricts the availability of AAV glycan attachment factors on the cell surface by maintaining apical-basal polarity and tight junction integrity.IMPORTANCEAdeno-associated viruses (AAVs) have recently emerged at the forefront as gene therapy vectors; however, our understanding of host factors that influence AAV transduction in different cell types is still evolving. In the present study, we perform a genome-scale CRISPR knockout screen to identify cellular host factors that restrict AAV infection in hepatocyte cultures. We discover that Crumbs 3, which determines cellular polarity, also influences the distribution of certain carbohydrate attachment factors on the cell surface. This in turn affects the ability of virions to bind and enter the cells. This study underscores the importance of cell polarity in AAV transduction and provides a potential molecular basis for the differential infectious mechanism(s) in cell culture versus organ systems.

Basic Biology of Adeno-Associated Virus (AAV) Vectors Used in Gene Therapy

2014 ◽  
Vol 14 (2) ◽  
pp. 86-100 ◽  
Author(s):  
Balaji Balakrishnan ◽  
Giridhara Jayandharan
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Basic Biology

Comparative Study of Adeno-associated Virus, Adenovirus, Bacu lovirus and Lentivirus Vectors for Gene Therapy of the Eyes

2017 ◽  
Vol 17 (3) ◽  
Author(s):  
Giedrius Kalesnykas ◽  
Emmi Kokki ◽  
Laura Alasaarela ◽  
Hanna P. Lesch ◽  
Timo Tuulos ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Comparative Study ◽  
Adeno Associated Virus ◽  
Lentivirus Vectors

Recombinant adeno-associated virus 9-mediated expression of Kallistatin suppresses lung tumor growth in mice

2020 ◽  
Vol 20 ◽  
Author(s):  
Weihong Qu ◽  
Jianguo Zhao ◽  
Yaqing Wu ◽  
Ruian Xu ◽  
Shaowu Liu
Keyword(s):  
Lung Cancer ◽  
Gene Therapy ◽  
Tumor Growth ◽  
Lung Tumor ◽  
Control Group ◽  
High Dose ◽  
Blank Control Group ◽  
Adeno Associated Virus ◽  
Tumor Tissues ◽  
Subcutaneous Xenograft

Background:: Lung cancer remains the most common cause of cancer-related deaths in China and worldwide. Traditional surgery and chemotherapy do not offer an effective cure although gene therapy may be a promising future alter-native. Kallistatin (Kal) is an endogenous inhibitor of angiogenesis and tumorigenesis. Recombinant adeno-associated virus (rAAV) is considered the most promising vector for gene therapy of many diseases due to persistent and long-term transgen-ic expression. Objective:: The aim of this study was to investigate whether rAAV9-Kal inhibited NCI-H446 subcutaneous xenograft tumor growth in mice. Method:: The subcutaneous xenograft mode were induced by subcutaneous injection of 2×106 H446 cells into the dorsal skin of BALB/c nude mice. The mice were administered with ssrAAV9-Kal (single-stranded rAAV9) or dsrAAV9-Kal (double-stranded rAAV9）by intraperitoneal injection (I.P.). Tumor microvessel density (MVD) was examined by anti-CD34 stain-ing to evaluate tumor angiogenesis. Results:: Compared with the PBS (blank control) group, tumor growth in the high-dose ssrAAV9-Kal group was inhibited by 40% by day 49, and the MVD of tumor tissues was significantly decreased. Conclusion:: The results indicate that this therapeutic strategy is a promising approach for clinical cancer therapy and impli-cate rAAV9-Kal as a candidate for gene therapy of lung cancer.

scholarly journals Intracellular Routing and Recognition of Lipid-Based mRNA Nanoparticles

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 945
Author(s):  
Christophe Delehedde ◽  
Luc Even ◽  
Patrick Midoux ◽  
Chantal Pichon ◽  
Federico Perche
Keyword(s):  
Gene Therapy ◽  
Immune Responses ◽  
Transient Expression ◽  
Messenger Rna ◽  
Lipid Nanoparticles ◽  
Delivery Systems ◽  
Clinical Applications ◽  
Cellular Proteins ◽  
Mrna Sequence ◽  
Cytoplasmic Expression

Messenger RNA (mRNA) is being extensively used in gene therapy and vaccination due to its safety over DNA, in the following ways: its lack of integration risk, cytoplasmic expression, and transient expression compatible with fine regulations. However, clinical applications of mRNA are limited by its fast degradation by nucleases, and the activation of detrimental immune responses. Advances in mRNA applications, with the recent approval of COVID-19 vaccines, were fueled by optimization of the mRNA sequence and the development of mRNA delivery systems. Although delivery systems and mRNA sequence optimization have been abundantly reviewed, understanding of the intracellular processing of mRNA is mandatory to improve its applications. We will focus on lipid nanoparticles (LNPs) as they are the most advanced nanocarriers for the delivery of mRNA. Here, we will review how mRNA therapeutic potency can be affected by its interactions with cellular proteins and intracellular distribution.

scholarly journals Quantitation of Trace Levels of DNA Released from Disrupted Adeno-Associated Virus Gene Therapy Vectors

2021 ◽  
Author(s):  
Jared S. Bee ◽  
Kristin O'Berry ◽  
Yu (Zoe) Zhang ◽  
Megan Kuhn Phillippi ◽  
Akanksha Kaushal ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Virus Gene ◽  
Gene Therapy Vectors
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