Produção e Purificação de Adenovírus não replicativa Canino Tipo 2 vectores derivados

Summary

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Abstract

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10¹⁰ infectious particles per ml and can be directly administrated in vivo.

Introduction

Over the past decades, adenoviruses (Ads) derived vectors have proven their efficacy for gene therapy and vaccination, as well as in oncolytic virotherapy ¹. Ads are nonenveloped icosahedral viruses of the Adenoviridae family, which have been isolated from mammals, birds, reptiles, amphibians and fish. Since their discovery in 1953, Ads have been intensively studied as models to study virus/cell interactions and, more recently, as vector-based gene delivery systems ². Indeed, Ads-based vectors offer many advantages, such as their broad host range and well-characterized biology, together with the ease with which they can be genetically manipulated and amplified for large-scale production.

In order to overcome clinical difficulties related to pre-existing immunity in human populations towards vectors derived from human Ads ³, we and others began to derive vectors from nonhuman Ads ⁴. In addition, nonhuman adenoviral vectors appear to be more adapted for mass vaccination in veterinary medicine than the classically used adenovirus type 5 (Ad5), since they should be more compliant with safety and security requirements during the risk assessment by the national regulatory authorities. In the late 1990s, we described the first recombinant CAV2 as a nonhuman alternative to vectors derived from Ad5 ⁵. Since then, numerous studies have confirmed the potential of CAV2 vectors for therapeutic or antigenic gene transfer (for reviews ^6,7). Like other Ads, CAV2-derived vectors are stable and can be produced at high titers, which facilitates their in vivo use. They are also safe, since they are not integrative, although they allow long lasting transgene expression in vivo (>1 year) ⁸. Remarkably, and better than human Ads serotypes, CAV2 vectors have been shown to be highly neurotropic, with a very efficient retrograde transport in axons ^9,10. This intrinsic property brought the idea of using recombinant CAV2 vectors to transduce neurons of specific brain areas that are hardly accessible to lentiviral vectors, so far commonly used for gene transfer into the central nervous system ¹¹.

Here, we describe a simple and classical protocol to construct, produce and purify nonreplicative CAV2 vectors derived from the Manhattan strain. Recombinant CAV2 genomes are constructed by cloning the desired gene of interest (GOI) in a cassette downstream of the cytomegalovirus (CMV) early promoter, which provides a ubiquitous expression. CAV2 vectors have a cloning capacity of ~4.2 kbp, which allows expressing large cDNA. In a second step, this expression cassette is inserted by homologous recombination in place of the E1 region of the CAV2 genome, leading to a nonreplicative virus (dlE1), as described initially by Chartier et al.¹² Finally, viral particles are produced upon transfection of the genomic plasmid into a CAV2-E1-expressing canine kidney cell line (DK-E1) and are serially amplified prior to concentration and purification of viral stocks. The method described here uses a two-step ultracentrifugation in cesium chloride (CsCl) gradients, which enables enrichment of infectious particles with a high final titer (usually over 10¹⁰ infectious particles per ml). The viral suspension is then desalted by chromatography through disposable Sephadex G-25 columns so that it is ultimately pure enough to be directly used in vivo.

Protocol

1. Construction of a Nonreplicative CAV2 Genome Plasmid Note: all plasmids described in this protocol have been described previously7 and are available upon request. Clone the GOI into the pShuttle plasmid, using appropriate restriction enzymes, to generate pShuttle-GOI (Figure 1A). Digest 2 μg of pShuttle-GOI by AscI/PacI. The digestion releases the Shuttle-GOI fragment (~2.9 kbp + GOI ORF sequen…

Representative Results

Production of recombinant CAV2 vectors relies on basic but important molecular biology techniques. Prior to the production of viral vectors, it is indeed necessary to construct a recombinant CAV2 genome bearing an expression cassette for the transgene. This construction involves two steps: first, the gene of interest (GOI) is cloned in a shuttle plasmid as an expression cassette (i.e. with promoter and polyadenylation signal). This functional element is flanked by two CAV2-derived genomic sequences, represe…

Discussion

The simple method described herein, adapted from the well-documented Ad5 technology, allows the efficient production of nonreplicative CAV2 vectors with a typical cloning capacity of ~4.2 kbp. Regarding time considerations, a recombinant CAV2 genome is usually generated in two weeks, whereas it will take an additional 5 weeks to amplify, purify and titer viral suspensions.

Although the production of the first batch of CAV2-derived vectors may appear time-consuming, it should be reminded that C…

Materials

Restriction enzymes	New England Biolabs	Various
Jet Prime transfection kit	Polyplus	114-01
DMEM	Gibco (Life Technologies)	10567014
E. coli BJ5183 bacteria	Agilent	200154
PD-10 desalting column	GE Healthcare	17-0851-01
EmeraldAmp polymerase	Takara	RR320A
Nucleospin plasmid kit	Macherey-Nagel	740558.250
NucleoSpin Gel and PCR Clean-up	Macherey-Nagel	740609.50
14 ml tubes, Ultra Clear	Beckman	344060