A lentiviral vector‐based adenovirus fiber‐pseudotyping approach for expedited functional assessment of candidate retargeted fibers

Uil, Taco G.; Vrij, Jeroen de; Vellinga, Jort; Rabelink, Martijn J. W. E.; Cramer, Steve; Chan, On Ying A.; Pugnali, Margherita; Magnusson, Maria K.; Lindholm, Leif; Boulanger, Pierre; Hoeben, Rob C.

doi:10.1002/jgm.1395

Cited by 9 publications

(6 citation statements)

References 70 publications

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“…The 293-derived, fiber-trans-complementing cell line, abbreviated 293-Fiber, was obtained from Transgene SA (Strasbourg, France). 293-Fiber cells were grown in the same medium as 293 cells, supplemented with hygromycin at 350 µg/ml [70], [83]. Chinese hamster ovary cells CHO-K1, and proteoglycan-deficient CHO-2241 cells (pgsB-618, ATCC code CRL-2241) were obtained from the European Collection of Cultured Cells via the Institut de Biologie Structurale, Grenoble, France [79].…”

Section: Methodsmentioning

confidence: 99%

Cell Entry and Trafficking of Human Adenovirus Bound to Blood Factor X Is Determined by the Fiber Serotype and Not Hexon:Heparan Sulfate Interaction

et al. 2011

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Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon∶FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.

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Section: Methodsmentioning

confidence: 99%

Cell Entry and Trafficking of Human Adenovirus Bound to Blood Factor X Is Determined by the Fiber Serotype and Not Hexon:Heparan Sulfate Interaction

et al. 2011

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“…The vesicular stomatitis virus G protein-pseudotyped SIN-LVs LV.FLPe NLS+ .PurR, LV.FLPe NLS− .PurR, LV.PurR (negative control vector), LV.GS.GpLuc.v1, LV.GS.PpLuc and LV.GS.GpLuc.v6 were generated in 293T cells with the aid of the LV shuttle plasmids pLV.hCMV-IE.FLPe NLS+ .IRES.PurR.hHBVPRE, pLV.hCMV-IE.FLPe NLS− .IRES.PurR.hHBVPRE, pLV.CMV.IRES.PURO ([14], hereinafter referred to as pLV.hCMV-IE.IRES.PurR.hHBVPRE; Fig. 1C), pLV.GS.GpLuc.v1, pLV.GS.PpLuc and pLV.GS.GpLuc.v6, respectively.…”

Section: Methodsmentioning

confidence: 99%

Development of a Lentivirus Vector-Based Assay for Non-Destructive Monitoring of Cell Fusion Activity

et al. 2014

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Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS−) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS− both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS− while at low acceptor-to-donor cell ratios FLPeNLS− was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for quantifying cell-to-cell fusion progression based on GpLuc has been developed.

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“…All lentivirus (LV) plasmids were based on pRRL-cPPT-CMV-X-PRE-SIN (35) or its derivative pLV.CMV.IRES.PURO (36), the latter of which carries an internal ribosomal entry site (IRES) followed by a puromycin resistance gene. LV vectors generated for this study were LV.AdPol, LV.PP and LV.pol-HA, which respectively encode for Ad5 pol, Ad5 pol bicistronically with puromycin and ‘pol-HA’, a C-terminally HA-tagged version of Ad5 pol.…”

Section: Methodsmentioning

confidence: 99%

Directed adenovirus evolution using engineered mutator viral polymerases

Uil

Vellinga

Vrij

et al. 2010

Nucleic Acids Research

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Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad’s intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, ‘accelerated-evolution’ approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad’s intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing.

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A lentiviral vector‐based adenovirus fiber‐pseudotyping approach for expedited functional assessment of candidate retargeted fibers

Cited by 9 publications

References 70 publications

Cell Entry and Trafficking of Human Adenovirus Bound to Blood Factor X Is Determined by the Fiber Serotype and Not Hexon:Heparan Sulfate Interaction

Cell Entry and Trafficking of Human Adenovirus Bound to Blood Factor X Is Determined by the Fiber Serotype and Not Hexon:Heparan Sulfate Interaction

Development of a Lentivirus Vector-Based Assay for Non-Destructive Monitoring of Cell Fusion Activity

Directed adenovirus evolution using engineered mutator viral polymerases

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