Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

Awedikian, Rafi; François, Achille; Guilbaud, Mickaël; Moullier, Philippe; Salvetti, Anna

doi:10.1016/j.virol.2005.02.024

Cited by 6 publications

(5 citation statements)

References 34 publications

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“…We hypothesized that infectious AAV8ΔVP1 particles could be explained by the relatively high AAV multiplicity used and the presence of Ad, known to be an intracellular carrier for various biological molecules, especially through its endosomolytic activity. 29 , 30 Thus, the high Ad multiplicity (500 IUs/cell) used in the TCID 50 and ICA may help non-infectious AAV particles to escape the endosomes and reach the cell nucleus. Importantly, in the case of AAV8ΔVP1, the difference in the VG:IU ratio calculated with the TCID 50 (2.9 × 10 3 ) was about 4-log 10 higher compared to that calculated with the ICA (3.1 × 10 7 ).…”

Section: Resultsmentioning

confidence: 99%

“…To this end, for all samples prepared by procedures 1–4, protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) were added and samples underwent 3 freeze/thaw cycles to disrupt membranes. Purified His-tagged Rep68 protein (500 ng) 30 was then spiked in each sample, before the addition of 4 sample volumes of ice-cold acetone and overnight precipitation at −20°C. Proteins were then pelleted at 16,000 × g for 15 min at 4°C and resuspended in 150 μL of 1× Laemmli buffer.…”

Section: Methodsmentioning

confidence: 99%

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Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls

François

Bouzelha

Lecomte

et al. 2018

Molecular Therapy - Methods & Clinical Development

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Although the clinical use of recombinant adeno-associated virus (rAAV) vectors is constantly increasing, the development of suitable quality control methods is still needed for accurate vector characterization. Among the quality criteria, the titration of infectious particles is critical to determine vector efficacy. Different methods have been developed for the measurement of rAAV infectivity in vitro, based on detection of vector genome replication in trans-complementing cells infected with adenovirus, detection of transgene expression in permissive cells, or simply detection of intracellular vector genomes following the infection of indicator cells. In the present study, we have compared these methods for the titration of infectious rAAV8 vector particles, and, to assess their ability to discriminate infectious and non-infectious rAAV serotype 8 particles, we have generated a VP1-defective AAV8-GFP vector. Since VP1 is required to enter the cell nucleus, the lack of VP1 should drastically reduce the infectivity of rAAV particles. The AAV8 reference standard material was used as a positive control. Our results demonstrated that methods based on measurement of rAAV biological activity (i.e., vector genome replication or transgene expression) were able to accurately discriminate infectious versus non-infectious particles, whereas methods simply measuring intracellular vector genomes were not. Several cell fractionation protocols were tested in an attempt to specifically measure vector genomes that had reached the nucleus, but genomes from wild-type and VP1-defective AAV8 particles were equally detected in the nuclear fraction by qPCR. These data highlight the importance of using suitable controls, including a negative control, for the development of biological assays such as infectious unit titration.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls

François

Bouzelha

Lecomte

et al. 2018

Molecular Therapy - Methods & Clinical Development

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“…In vitro evidences have suggested that Tat/PTD-fusion proteins or Tat peptides enter via an energy-dependent endocytotic (including caveolae) process [110][111][112][113][114], because the membrane inhibitor sodium azide inhibits ATP production and impairs endocytosis [115,116]. Further, protein transduction was strongly inhibited by energy depletion in cells at low temperature [110,112].…”

Section: Mechanism Of Ptd Internalizationmentioning

confidence: 99%

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

Chauhan

Tikoo²,

Kapur

et al. 2007

Journal of Controlled Release

156

136

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Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV-Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. Inspite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences.

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“…[242][243][244][245] Here it was subsequently shown that several recombinaseswhich incidentally are cationic proteins-actually contain innate membrane-translocation properties. [246][247][248] Several accounts have now appeared where attempts were made to compare the delivery efficiency of various vectors (e.g., Refs. 249,250).…”

Section: Delivery Efficiencymentioning

confidence: 99%

Cellular uptake mechanisms and potential therapeutic utility of peptidic cell delivery vectors: Progress 2001–2006

Fischer

2006

Medicinal Research Reviews

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Cell delivery vectors (CDVs) are short amphipathic and cationic peptides and peptide derivatives, usually containing multiple lysine and arginine residues. They possess inherent membrane activity and can be conjugated or complexed with large impermeable macromolecules and even microscopic particles to facilitate cell entry. Various mechanisms have been proposed but it is now becoming clear that the main port of entry into cells of such CDV constructs involves adsorptive-mediated endocytosis rather than direct penetration of the plasma membrane. It is still unclear, however, how and to what extent CDV constructs are capable of exiting endosomal compartments and reaching their intended cellular site of action, usually the cytosol or the nucleus. Furthermore, although many CDVs can mediate cellular uptake of their cargo and appear comparatively non-toxic to cells in tissue culture, the utility of CDVs for in vivo applications remains poorly understood. Whatever the mechanisms of cell entry and disposition, the overriding question as far as potential pharmacological application of CDV conjugates is concerned is whether or not a therapeutic margin can be achieved by their administration. Such a margin will only result if the intracellular concentration in the target tissues necessary to elicit the biological effect of the CDV cargo can be achieved at systemic CDV exposure levels that are non-toxic to both target and bystander cells. It is proposed that the focus of CDV research now be shifted from mechanistic in vitro studies with labeled but otherwise unconjugated CDVs to in vivo pharmacological and toxicological studies using CDV-derivatized and other cationized forms of inherently non-permeable macromolecules of true therapeutic interest.

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Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

Cited by 6 publications

References 34 publications

Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls

Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

Cellular uptake mechanisms and potential therapeutic utility of peptidic cell delivery vectors: Progress 2001–2006

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