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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. Prepare 400 mL of Adenovirus buffer (Ad-buffer) by adding 50 mM HEPES (4.76 g/400 mL) and 150 mM NaCl (3.504 g/400 mL) to double-distilled w....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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