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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Materials

References

Biology

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

Published: October 26th, 2018

DOI:

10.3791/58480

1Center of Biomedical Education and Research, University Witten/Herdecke

The protocol described here enables researchers to specifically modify adenovirus capsids at selected sites by simple chemistry. Shielded adenovirus vectors particles and retargeted gene transfer vectors can be generated, and vector host interactions can be studied.

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

Adenoviruses (Ad), members of the family Adenoviridae, are non-enveloped DNA viruses of which more than 70 types so far have been identified (http://hadvwg.gmu.edu). Depending on hemaglutination properties, genome structure, and sequencing results, the 70 Ad types can be divided into seven species (human adenoviruses A to G)1,2. The human Ad genome is 38 kb in size and encapsulated by an icosahedral nucleocapsid3. Due to their abundance, the capsid protein hexon, penton base, and fiber are all referred to as major capsid proteins. The most abundant and largest capsid protein hexon forms....

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NOTE: In the following, a protocol for geneti-chemical PEGylation of an Ad vector is described to detail. To enable specific coupling of the PEG moiety, an Ad5 vector was beforehand genetically modified by introducing a cysteine residue into the hexon protein at the hypervariable loop 5 as described in a previous publication36, and a maleimide-activated PEG compound is used as coupling compound.

1. Preparation of Buffers for Vector Purification by CsCL Step Gradients

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Figure 2 shows examples of the cytopathic effect (CPE) on 293 (HEK 293) cells that indicates successful vector production. Cells should show morphology (Figure 2C) 40-48 hours after inoculation with the virus vector. The right timepoint for harvesting is crucial for not losing virus particles by cell lysis and preventing oxidation of the genetically introduced thiol groups. If vector particles are released into the medium by cell.......

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The efficiency by which the genetically introduced cysteines can be chemically modified is typically 80-99%, and certain variables influence this efficiency. First, it is paramount that the genetically introduced cysteines do not undergo premature oxidation. While being well-protected in the reducing environment of the producer cells, it is mandatory to provide a non-oxidative environment after releasing vector particles from the producer cells and during chemical modification. To this end, reducing reagents can be used .......

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Name Company Catalog Number Comments
Vector purification and chemical modification
Argon gas Air liquide local gas dealer
Liquid Nitrogen Air liquide local gas dealer
500 mL centrifuge tubes Corning 431123
Stericup Express Plus 0.22 µm Millipore SCGPU02RE
Tris(2-carboxyethyl) phosphine (TCEP) Sigma-Aldrich C4706-10g
2 mL (3mL) Norm Ject (syringes) Henke Sass Wolf 4020.000V0
Fine-Ject needles for single use (yellow 0.9 x 40 mm) Henke Sass Wolf 4710009040
Caesium chloride 99.999% Ultra Quality Roth 8627.1
Silica gel beads Applichem A4569.2500
Methoxypolyethylene glycol maleimide - 750 (PEG mal-750) Iris Biotech store in silica gel beads at -80 °C
13.2 mL Ultra Clear Ultracentrifuge Tubes Beckman Coulter 344059 only open in hood
PD-10 size exclusion chromatography column GE Healthcare 17-0851-01 store at 4 °C
Hepes AppliChem A1069.1000
SDS Ultrapure AppliChem A1112,0500
Glycerol AppliChem A1123.1000
Name Company Catalog Number Comments
Material for cell-culture
DPBS PAN Biotech P04-36500
DMEM PAN Biotech P04-03590
Trypsin/EDTA PAN Biotech P10-0231SP
FBS Good PAN Biotech P40-37500
Penicillin/Streptomycin PAN Biotech P06-07100
Biosphere Filter Tips (various sizes) Sarstedt
Serological Pipettes (various sizes) Sarstedt
reaction tubes (various sizes) Sarstedt
TC plates 15cm Sarstedt 83.3903
Name Company Catalog Number Comments
Material for silver staining protocol
Methanol J.T.Baker 8045
Ethanol absolute AppliChem 1613,2500PE
Acetic Acid AppliChem A0820,2500PE
Formaldehyde 37% AppliChem A0877,0250
Ethanol absolute AppliChem A1613,2500PE
Sodium thiosulfate AppliChem 1,418,791,210
Silver nitrate AppliChem A3944.0025
Sodium carbonate AppliChem A3900,0500
Name Company Catalog Number Comments
Special Lab Equipment
Desiccator Nalgene 5311-0250
Megafuge 40 Heraeus
Roter for Megafuge TX750 + Adapter andLlids for 500 mL tubes Heraeus
Water bath Conventional
Ultracentrifuge e.g. Optima XPN-80 Beckman Coulter
suitable Ultrazentrifuge Rotor e.g. SW41 Beckman Coulter
pH -Meter Conventional
Stand with clamps Conventional
Goose neck lamp Conventional
Over-head rotor Conventional
Thermal Block Conventional
Photometer (OD 260) Conventional

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