Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

Summary

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

Abstract

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

Introduction

For gene therapeutic applications it is of great importance to avoid cytotoxic and immunogenic side effects caused by expression of viral proteins, the transgene itself, or by incoming viral proteins. Adenovirus vectors (AdV) are widely used to introduce foreign DNA into a wide variety of cells to investigate the impact of transgene expression ^1,2. The most advanced version of AdV is represented by high-capacity adenovirus vectors (HCAdV) lacking all viral coding sequences ^3,4 and thereby offering a packaging capacity up to 35 kb combined with low immunogenicity and low toxicity ^5-8. Due to their high packaging capacity they allow deli....

Protocol

1. Construction of Recombinant HCAdV Genomes based on the Plasmid pAdFTC

Note: All plasmids have been described previously ^11,12 and are available upon request. The cloning procedure is schematically shown in Figure 1.

1. Clone a gene of interest (GOI) including promoter and polyadenylation signal (pA) into the shuttle plasmid pHM5, using a cloning strategy of choice, to generate pHM5-GOI.
   NOTE: Since pAdFTC is a relatively large plasmid, classical plasmid preparation protocols are recommended to avoid sheering of the plasmid DNA by using commercial plasmid purification kits that are based on sili....

Results

Here representative examples for cloning, amplification and purification of HCAdV preparations are shown. An overview of the cloning strategy (Figure 1) and representative examples for cloning and release of the HCAdV genome by restriction enzyme digest are provided (Figure 2). A typical restriction pattern after release of the GOI-expression cassette from pHM5 by PI-SceI and I-CeuI digest and subsequent phenol-chloroform extraction and .......

Discussion

The protocol presented here allows purification of HCAdV vectors based on human adenovirus type 5 based on previously described procedures ^4,12. The HCAdV genome within the pAdFTC plasmid is devoid of all adenovirus genes and only carries the 5'- and 3'- ITRs and the packaging signal. In this strategy the HV AdNG163R-2 ⁴ provides all necessary genes for efficient virus production in trans. This offers a packaging capacity of up to 35 kb, which clearly outcompetes first and second ge.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
I-CeuI	New England Biolabs	R0699S	restriction digest
PI-SceI	New England Biolabs	R0696S	restriction digest
T4 Ligase	New Engand Biolabs	M0202S	ligation
SwaI	New England Biolabs	R0604S	restriction digest
NotI	New England Biolabs	R0189S	restriction digest
Calf Intestinal Alkaline Phosphatase (CIP)	New England Biolabs	M0290S	dephosphorylation of digested plasmids
Hygromycin B	PAN Biotech	P02-015	selection of CRE expressing 116 cells
DMEM	PAN Biotech	P04-03590	Hek293T cell culture medium
Minimal Essential Medium (MEM) Eagle	PAN Biotech	P04-08500	116 cell culture medium
Dulbecco’s phosphate buffer saline (DPBS)	PAN Biotech	P04-36500	washing of cells, resuspension of cells
250-ml storage bottle	Sigma	CLS430281-24EA	infection of 116 cells grown in suspension
500-ml PP CentrifugeTubes	Sigma	CLS431123-36EA	sedimentation of cells from suspension culture
Spinner flask	Bellco	1965-61030	growth of 116 cells in suspension
Ultra Clear Ultracentrifuge tubes	Beckmann Coulter	344059	density gradient centrifugation
Ultracentrifuge	Beckmann Coulter		density gradient centrifugation
SW-41 rotor	Beckmann Coulter		density gradient centrifugation
Spectrum Laboratories Spectrapor Membrane	VWR	132129	dialysis tubing
ready-to-use dialysis cassettes	Thermo	66383	dialysis
one shot DH10B electrocompetent E. coli	invitrogen	C4040-52	transformation of ligation reactions
PureYield Plasmid Midiprep System	Promega	A2495	midiprep
peqGOLD Tissue DNA Mini Kit	Peqlab	12-3396-02	isolation of genomic DNA
SuperFect Transfection Reagent	Qiagen	301305	tranfection of 116 cells
opti MEM (10% FBS)	Gibco	31985-062	transfection of 116 cells
iQ SYBR Green Supermix	BioRad	170-8882	q-PCR
CFX 96 C1000 touch	Biorad		qPCR machine
Phenol/Chloroform/Isoamyl alcohol	Carl Roth	A156.1	purification of DNA
Cesium chloride	Carl Roth	8627.1	density gradient centrifugation
sodium acetate 99%	Carl Roth	6773.2	DNA precipitation
LB medium	Carl Roth	X968.3	bacterial growth medium
ethanol  99.8% pure	Carl Roth	9065.5	DNA precipitation and washing
SDS 99.5%	Carl Roth	2326.2	lysis  buffer
EDTA	Carl Roth	8043.2	lysis  buffer
Tris-HCl 99%	Carl Roth	9090.3	dialysis buffer/ lysis  buffer
glycerol 99.5%	Carl Roth	3783.1	dialysis buffer
MgCl2 98.5%	Carl Roth	KK36.2	dialysis buffer
NaCl	Carl Roth	3957.1	optional dialysis buffer
KH2PO4	Carl Roth	3904.2	optional dialysis buffer
sucrose	Carl Roth	9286.1	optional dialysis buffer
Na2HPO4x2H2O	Carl Roth	4984.2	optional dialysis buffer
1.5-ml tubes	sarstedt	72,730,005	storage of virus preparations at -80 °C