An improved method for high-efficiency gene delivery...

An improved method for high-efficiency gene delivery and expression in mammalian cells

Qiaohua (Josh) Wu Defence R&D Canada – Suffield

Technical Report

DRDC Suffield TR 2005-015

January 2005

Defence Research and Recherche et développement Development Canada pour la défense Canada

An improved method for high-efficiency gene delivery and expression in mammalian cells

Qiaohua (Josh) Wu Defence R&D Canada – Suffield

Defence R&D Canada – Suffield Technical Report DRDC Suffield TR 2005-015 January 2005

DRDC Suffield TR 2005-015 i

Abstract

Through millions of years of evolution, viruses have developed special molecular mechanism that allow them to bind to a host cell and to efficiently deliver their genetic material. To take advantage of these refined mechanisms, viruses have been modified as vectors to deliver desired genes into a large variety of cells. These include both in vitro (e.g. mammalian cell culture) and in vivo (e.g. mice) systems. To broaden DRDC’s existing programs in biotechnology, this report describes the development of an adenovirus-based vector for high-efficiency gene delivery and expression in mammalian cells. To illustrate the feasibility of this vector system, a recombinant adenovirus vector was produced, which contains gene encoding the enhanced green fluorescent protein (EGFP). The robust expression of EGFP was demonstrated in cells infected with the recombinant adenovirus. The adenovirus vector described in this report can be applied to many research areas including: 1) the expression of recombinant proteins in tissue culture cells, 2) the development of live viral vectored vaccines, 3) the expression of therapeutic proteins (e.g. interferon and monoclonal antibody) and nucleic acids (e.g. small interfering RNAs, antisense RNA) in animals against pathogens, and 4) the production of polyclonal antibody by direct injection of animals with recombinant adenovirus expressing antigens.

Résumé

Durant des millions d’années d’évolution, les virus ont développé des mécanismes moléculaires spéciaux qui leur permettent de s’attacher à une cellule hôte et de livrer leur matériel génétique efficacement. Pour bénéficier de ces mécanismes raffinés, les virus ont été modifiés en vecteurs pourvoyant les gènes désirés à une grande variété de cellules. Ces dernières comprennent des systèmes in vitro (par ex. : culture cellulaire mammalienne) et in vivo (par ex. : souris). Ce rapport, qui vise à étendre les programmes de RDDC existants en biotechnologie, décrit le développement d’un adénovirus utilisé comme vecteur qui permet une livraison de gènes et une expression hautement efficace dans les cellules mammaliennes. Pour illustrer la faisabilité de ce système de vecteurs, un adénovirus recombinant utilisé comme vecteur a été produit. Il contient un gène codant la protéine d’un vert fluorescent améliorée (PVFA) L’expression robuste de la PVFA a été démontrée dans les cellules infectées avec l’adénovirus recombinant. L’adénovirus utilisé comme vecteur qui est décrit dans ce rapport peut s’appliquer à beaucoup de domaines de la recherche y compris : 1) l’expression de protéines recombinantes dans le tissus des cultures cellulaires, 2) la mise au point de vaccins antiviraux vivants utilisés comme vecteurs, 3) l’expression de protéines thérapeutiques (par ex. : antigènes interférons et monoclonaux) et acides nucléiques (par ex. : ARN anti-sens, petit ARN à interférence chez les animaux contre des pathogènes et 4) la production d’antigènes polyclonaux en injectant directement des adénovirus recombinants exprimant l’antigène, chez les animaux.

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DRDC Suffield TR 2005-015 iii

Executive Summary

Advances in biotechnology have allowed the transfer of genes into mammalian cells and to express the protein of interest. One of the most important elements for gene transfer and expression is to develop a vector that can efficiently deliver the gene into the cells.

Currently, gene transfer mainly uses two types of vector: non-viral and viral. Non-viral vectors, including polyamines, polycationic lipids, and neutral polymers, can accommodate large size of gene, are less immunogenic and easy to produce. However, gene transfer by non-viral vector varies substantially in efficiency for different cell lines. Viruses have evolved molecular tools for high-efficiency delivery of their genes into the host cell. Because of this, as an alternative, viruses have been modified as vectors to deliver desired gene into a variety of cells, both in tissue culture system and in living animals.

At DRDC Suffield, a non-viral vector system based on polycationic lipids (liposome) has been developed and successfully used for delivering drugs and therapeutic genes. To broaden DRDC’s capability in gene transfer, this report describes the development of a recombinant virus-based vector for high-efficiency gene expression in mammalian cells. The viral vector was derived from adenovirus that had been successfully used as a live viral vaccine to prevent acute respiratory disease in military. To develop a recombinant adenoviral vector expressing the foreign protein, the gene encoding protein of interest was first cloned into a shuttle plasmid. The gene was then inserted into a full-length adenovirus genome by homologous recombinatioin in bacteria. Finally, the recombinant adenovirus vector was generated by transfection of cells with a full-length adenovirus genome containing the foreign gene. To illustrate the feasibility of the procedure, adenovirus vector expressing the autofluorescent protein of EGFP was made and the high-level production of EGFP was demonstrated in mammalian cells infected with the recombinant adenovirus.

The development of adenoviral vector technology will enhance the DRDC R&D programs in biotechnology and vaccine development. A recombinant adenovirus vector can be used in a mammalian cell expression system to produce recombinant proteins as diagnostic agents or therapeutics against biological warfare (BW) agents. In addition, these vectors can be explored to develop safe, single-dose live vaccine against different BW agents.

Wu, Q.J. 2005. An improved method for high-efficiency gene delivery and expression in mammalian cells. DRDC Suffield TR 2005-015. Defence R&D Canada – Suffield.

iv DRDC Suffield TR 2005-015

Sommaire

Les progrès en biotechnologie ont permis de transférer des gènes dans les cellules mammaliennes et d’exprimer la protéine désirée. Un des éléments le plus important dans le transfert de gènes et son expression est de développer un vecteur qui peut livrer efficacement le gène dans les cellules.

Le transfert de gène est actuellement réalisé par deux types de vecteurs : non viral et viral. Les vecteurs non viraux qui comprennent les lipides polycationiques et les polymères neutres, peuvent s’adapter aux gènes de grande taille, sont moins immunogènes et sont faciles à produire. L’efficacité du transfert de gènes par des vecteurs non viraux varie cependant de manière importante selon les lignées cellulaires. Les virus possèdent des outils moléculaires évolués qui réalisent une livraison hautement efficace de leurs gènes dans les cellules hôtes. Pour cette raison et comme alternative, les virus ont été modifiés comme vecteurs livrant les gènes désirés dans une variété de cellules, aussi bien dans des systèmes de culture cellulaire que chez des animaux vivants.

À RDDC Suffield, un système de vecteurs non viraux basé sur les lipides polycationiques (liposomes) a été mis au point et a été utilisé avec succès pour la livraison des médicaments et des gènes thérapeutiques. Pour étendre la capacité de RDDC à transférer les gènes, ce rapport décrit la mise au point du virus recombinant utilisé comme vecteur permettant une expression génétique de haute efficacité dans les cellules mammaliennes. Le vecteur viral a été dérivé d’adénovirus que l’on a réussi à utiliser comme vaccin antiviral vivant pour prévenir les maladies respiratoires aiguës chez les militaires. La protéine de codage de gènes désirée a d’abord été clonée dans un plasmide pour développer un adénovirus recombinant utilisé comme vecteur exprimant la protéine étrangère. Le gène a été inséré dans un adénovirus de génome de pleine longueur par une recombinaison homologue dans la bactérie. Enfin, l’adénovirus recombinant utilisé comme vecteur a été généré par la transfection de cellules par le génome adénoviral de pleine longueur contenant le gène étranger. Pour illustrer la faisabilité de cette méthode, l’adénovirus utilisé comme vecteur exprimant la protéine autofluorescente de la PVFA a été produit et la production hautement efficace de la PVFA a été démontrée dans les cellules mammaliennes infectées de l’adénovirus recombinant.

Le développement de la technologie d’adénovirus utilisé comme vecteur améliorera les programmes de R & D de RDDC dans les domaines de la biotechnologie et de la mise au point des vaccins. Un adénovirus recombinant utilisé comme vecteur peut être utilisé dans un système d’expression de cellule mammalienne pour produire des protéines recombinantes comme agents diagnostiques ou thérapeutiques contre les agents de guerre biologiques (CWB). De plus, ces vecteurs peuvent être explorés pour développer des doses uniques sécuritaires de vaccin vivant contre les différents agents BW.

Wu, Q.J. 2005. An improved method for high-efficiency gene delivery and expression in mammalian cells. DRDC Suffield TR 2005-015. R & D pour la défense Canada – Suffield.

DRDC Suffield TR 2005-015 v

Table of Contents

Abstract........................................................................................................................................ i

Executive Summary................................................................................................................... iii

Sommaire................................................................................................................................... iv

Table of Contents ....................................................................................................................... v

List of Figures............................................................................................................................ vi

Acknowledgements .................................................................................................................. vii

Introduction ................................................................................................................................ 1 Materials and Methods…………………………………………………………………………3

Cells and Cell Culture…………………………………………………………………3 Plasmid Construction………………………………………………………………….3 Generation of Recombinant HAd5 Expressing EGFP………………………………...5 Virus Amplification, Purification, and Titration………………………………………7 Inoculation of Cells with Recombinant Adenovirus Ad5-EGFP……………………...7 Results and Discussion…………………………………………………………………………8 Insertion of Gene Encoding EGFP into HAd5 Genome………………………………8 Production of Recombinant HAd5 Containing EGFP Gene…………………………10 Expression of EGFP in Cells Infected with Ad5-EGFP……………………………..11 Conclusions…………………………………………………………………………………...13

References ................................................................................................................................ 15

List of Symbols/Abbreviations/Acronymes/Initialisms ........................................................... 17

vi DRDC Suffield TR 2005-015

List of Figures

Figure 1. Insertion of gene encoding EGFP into HAd5 genome………………………………..4

Figure 2. Plasmid map of pShuttle-CMV………………………………………………………5 Figure 3. Generation of recombinant adenovirus Ad5-EGFP………………………………….6 Figure 4. Plasmid map of pAd5-EGFPcontaining expression cassette for EGFP……………..9 Figure 5. Analysis of plasmid pAd5-EGFP…………………………………………………..10 Figure 6. Adenovirus-producing foci after transfection cells with pAd5-EGFP……………..11 Figure 7. Adenovirus-mediated EGFP expression in cells…………………………………...12 Figure 8. Steps and time course for the generation of recombinant adenovirus……………..13

List of Tables

Table 1. Calculated sizes of the DNA fragments after digestion of pAdEasy-1 and pAd5 EGFP with restriction enzymes ................................................................................... 9

DRDC Suffield TR 2005-015 vii

Acknowledgements

I thank Dr Les P. Nagata for providing plasmid pEGFP-N3 and Vero cells, and for his support of this project.

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DRDC Suffield TR 2005-015 1

Introduction

Advances in biotechnology have allowed the transfer of a gene into mammalian cells and to express the foreign proteins in the cells. Three basic elements are required for this strategy: a gene that encodes the foreign protein, a promoter that directs the expression of the gene, and perhaps most importantly, a vector system that can deliver the gene into the cells.

Currently, gene transfer mainly uses two types of vectors: non-viral and viral. Non-viral vectors include polyamines, polycationic lipids, and neutral polymers, capable of housing DNA into nanoparticles with radii of 20-100 nm [1]. The non-viral vector system can accommodate large sizes of DNA, is less immunogenic and is easy to produce. However, the use of non-viral vectors for gene transfer has been hampered by low transfection efficiency for most primary cell lines. The other method for gene transfer is that of viral vectors. After millions of years of evolution, viruses have acquired the molecular tools to enter and efficiently deliver their genes into the host cell to achieve virus propagation. Through molecular biology manipulations, a virus (e.g. adenovirus) can be re-engineered to allow it to be used as a tool to deliver desired gene into varieties of cells, both in tissue culture systems and in living animals [2]. To modify a virus as a gene delivery vector, the genes encoding essential viral proteins are deleted. This cripples virus replication to enhance the safety profile of viral vectors. In addition, the deletion allows the insertion of larger size of foreign DNA into viral vectors.

At DRDC Suffield, a polycationic lipid (liposome)-based, non-viral vector system has been developed and successfully used for drug delivery [3, 4]. To broaden DRDC’s capability in gene delivery and biotechnology, this report describes the development of an adenovirus-based vector for high-efficiency gene delivery and expression in mammalian cells.

Adenovirus is usually associated with respiratory infections in humans [5, 6]. Since its isolation in the early 1950s, the molecular biology and pathogenesis of adenovirus have been extensively studied [8]. A live adenovirus vaccine has been developed to prevent acute respiratory disease in military trainees. Clinical vaccination with the vaccine showed no significant side effects [7]. The relative safety of live adenovirus vaccine, its capability to infect a wide variety of cell types , and its ease to produce in large quantities have made adenovirus an excellent candidate for developing into a gene delivery vector [9, 10].

The genome of adenovirus consists of a double-stranded DNA molecule which is contained tightly inside a protein case called capsid. Since the capsid size of adenovirus is fixed, the amount of foreign DNA that can be packed inside the capsid is limited. To solve this problem, two adenoviral genes (E1 and E3) are deleted, which allows to insert up to 7.5 kilobases (kb) foreign gene into adenovirus vector [11, 12]. In addition, because E1 gene encodes viral essential proteins, deletion of E1 renders adenovirus vector replication deficient, which further enhances safety profile of adenovirus vector. To generate and grow the recombinant adenovirus, complemetary cell lines such as human embryonic kidney 293 (HEK293) cells have been developed which can provide E1 function to the recombinant viruses [13-15].

2 DRDC Suffield TR 2005-015

In this report, the method to construct adenovirus vector expressing foreign protein was illustrated by making a recombinant adenovirus virus, Ad5-EGFP, and showing that the EGFP was efficiently expressed in HEK293 and Vero cells. The method described in this report should have potential applications in biotechnology and biodefence vaccine development.


Materials and Methods Cells and Cell Culture HEK293 cells, originally isolated from primary human embryonic kidney cells transformed by sheared DNA from human adenovirus serotype 5 (HAd5) [15], were purchased from the American Type Culture Collection (ATCC CRL 1573, Manassas, VA, USA). HEK 293 cells were used to generate, propagate, and titrate the recombinant HAd5 encoding EGFP. Vero cells (ATCC CCL 81), the stable line of African green monkey kidney cells [16], were kindly provided by Dr L. P. Nagata (DRDC Suffield). These were used to monitor EGFP expression after inoculating the cells with recombinant HAd5 encoding EGFP. Both HEK 293 and Vero cells were grown in Dulbecco’s modified Eagle medium (D-MEM with high glucose, L-glutamine; Invitrogen/GIBCO, Burlington, ON, Canada) supplemented with 10% defined fetal bovine serum (FBS; HyClone, Mississauga, ON, Canada), 1 mM sodium pyruvate (Invitrogen/GIBCO), and antibiotics-antimycotics (Invitrogen/GIBCO). To propagate the cells, trypsin-EDTA (0.05% Trypsin, EDTA-4Na; Invitrogen/GIBCO) was used to detach adherent cells from the flask. The single cell suspension was diluted at a 1:5 ratio with fresh medium and seeded into new flasks. In addition, an HEK 293 cell bank was established by freezing early passage (passage 4) of cells at 1X106 cells/ml in D-MEM containing 40% FBS, 10% dimethyl sulfoxide (DMSO), 1 mM sodium pyruvate, and antibiotics-antimycotics. HEK 293 cells were used within 30 passages.

Plasmid Construction The gene encoding EGFP was isolated from plasmid pEGFP-N3 (BD Biosciences, Mississauga, ON, Canada) by double digestion with restriction enzymes KpnI and NotI (Figure 1). The 748 base pair (bp) DNA fragment containing EGFP gene was separated by 0.8% agarose gel and purified by QIAquick Gel Extraction kit (QIAGEN, Mississauga, ON, Canada). The purified DNA fragment was cloned into KpnI-NotI sites of plasmid vector pShuttle-CMV (Qbiogene, Carlsbad, CA, USA; Figure 2). The resultant plasmid, pSCMV-EGFP, was linearized with restriction enzyme PmeI and co-transformed with pAdEasy-1 plasmid (Qbiogene) into Escherichia coli (E. coli) strain BJ5183 (Qbiogene). The plasmid, pAdEasy-1, contains 33.4 kb HAd5 genome with deletions in the E1 and E3 coding regions [17]. Through homologous recombination in BJ5183 [18], the gene encoding EGFP was inserted into HAd5 genome, generating the plasmid pAd5-EGFP.


Figure 1. Insertion of gene encoding EGFP into HAd5 genome. The Plasmid DNA is indicated by a thin line, HAd5 genomic DNA by a filled box, and EGFP gene by dotted box. ∆E1 and ∆E3 are deletions in

the E1 and E3 coding regions of HAd5; Kan: kanamycin resistance.

NotI KpnI

Plasmid pEGFP-N3

The gene encoding EGFP was isolated from plasmid pEGFP-N3 by digestion of the plasmid with restriction enzymes

Plasmid pSEGFP

EGFP

EGFP

Co-transformed with pAdEasy-1 into E. coli BJ5183 to insert EGFP gene into full-length HAd5 genome through homologous recombination

Plasmid pAd5-EGFP

748 bp DNA fragment containing EGFP

Plasmid vector pShuttle-CMV Ligation of the DNA fragment into

Compatible sites of pShuttle-CMV

Linealization with PmeI

Plasmid pAdEasy-1

EGFP

PmeI PmeI

PacI PacI

PmeI

EGFP

∆E1 ∆E3

PacI PacI

∆E1 ∆E3

NotI KpnI

EGFP

Kan


Figure 2. Plasmid map of pShuttle-CMV. Plasmid pShuttle-CMV is a transfer vector for insertion of a foreign gene into HAd5 genome. The plasmid consists of multiple cloning sites (MCS) flanked by the

cytomegalovirus promoter (CMV promoter) and Simian virus 40 polyadenylation signal (polyA). Surrounding CMV promoter, MCS, and polyA are left and right “arms” which contain the left and right

ends of HAd5 genome. These “arms” allow homologous recombination with pAd-Easy-1 in E. coli. For viral replication in mammalian cells, HAd5 encapsidation signal, and left and right inverted terminal repeat (LITR and RITR) are included in the plasmid. In addition, the plasmid contains a kanamycin

resistance gene and the origin of replication (Ori) for plamid production in E. coli.

Generation of Recombinant HAd5 Virus Expressing EGFP Ad5-EGFP, a recombinant HAd5 virus expressing EGFP, was generated by transfection of HEK 293 cells with plasmid pAd5-EGFP. As shown in Figure 3, 40 µg of plasmid DNA of pAd5-EGFP, prepared by QIAGEN Plasmid Maxi kit (QIAGEN), was digested with restriction enzyme PacI and purified by ethanol precipitation. Then, 8 µg of the digested DNA was incubated with 60 µl of Lipofectamine 2000 (Invitrogen) at room temperature for 20 minutes. The DNA-Lipofectamine 2000 complexes were added dropwise onto HEK 293 cell monolayer seeded in a T25 flask (Corning Inc., Corning, NY, USA). The recombinant virus Ad5-EGFP was generated by incubation of the flask at 370C in a CO2 incubator. The virus was harvested after appearance of cytopathic effect (CPE) in the transfected cells. Lysates from Ad5-EGFP-infected cells were prepared by three cycles of freeze-and-thaw in total of 1 ml of D-MEM supplemented with 2% defined fetal bovine serum, 1 mM sodium pyruvate, and antibiotics-antimycotics.


∆E1 ∆E3 Figure 3. Generation of recombinant adenovirus Ad5-EGFP. PCMV: cytomegalovirus promoter; EGFP: enhanced green fluorescent protein; PA: Simian virus 40 polyadenylation signal; ∆E1 and ∆E3: deletions

in the E1 and E3 coding regions of HAd5.

Ad5-EGFP containing EGFP expression cassette

PCMV EGFP PA

Linealization with PacI

Transfection of PacI-linealized adenovirus DNA into HEK 293 cells

Incubation of the transfected cells at 370C for 5 days

Plasmid pAd5-EGFPEGFP

PacI PacI

∆E1 ∆E3

EGFP PacI PacI

∆E1 ∆E3


Virus Amplification, Purification, and Titration HEK 293 cells were seeded in five T150 flasks (Corning Inc.) and each flask was inoculated with 100 ul of Ad5-EGFP-infected cell lysate in a total of 5 ml of D-MEM supplemented with 2% defined FBS, 1 mM sodium pyruvate, and antibiotics-antimycotics. After one-hour incubation at 370C, an extra 15 ml of culture medium was added to each flask. Both the cell culture medium and the infected cells were harvested when complete CPE appeared in infected cells. The viruses were purified by BD Adeno-X virus purification kit (BD Biosciences) according to manufacturer’s instruction. The purified viruses were aliquoted as 500 µl per tube and stored at -700C.

The concentration of the infectious virus particles (virus titer) present in the above purified virus sample was determined by the assay of the tissue culture infectious dose 50 (TCID50), which is based on the presence of CPE in HEK 293 cells. The TCID50 is defined as that dilution of a virus that will infect 50% of a given batch of inoculated cell monolayers.

To determine virus titer of Ad5-EGFP, HEK 293 cells suspended in D-MEM supplemented with 2% defined fetal bovine serum, 1 mM sodium pyruvate, and antibiotics-antimycotics were seeded into Linbro 96-well flat bottom plates (Flow Laboratories, Inc., McLean, Virginia, USA) at a concentration of 104 cells per well. A serial of 10-fold dilution of the virus stock was made and 100 µl of diluted virus was dispensed into each well. The plate was incubated at 370C in a CO2 incubator for 10 days. The number of wells showing CPE was counted and the ratio of CPE wells per row was calculated. TCID50 was determined using the formula: T = 101+d(S-0.5), d = Log10 of the dilution, S = the sum of ratios in each well. The final titer for the virus stock was converted to plaque forming units (pfu)/ml by multiplying the TCID50 titer by 0.7.

Inoculation of Cells with Recombinant Adenovirus Ad5-EGFP HEK293 and Vero cells were seeded overnight at 2X105 cells per well in 12-well plate (Corning Inc.). These were inoculated with purified recombinant adenovirus Ad5-EGFP at a multiplicity of infection (MOI) of 1 in a total volume of 300 µl of D-MEM supplemented with 2% defined FBS, 1 mM sodium pyruvate, and antibiotics-antimycotics. After one hour incubation at 370C, another 200 ul of medium was added into each well. Twenty-four hours after inoculation, the expression of autofluorescent protein EGFP in infected cells was observed under Nikon ECLIPSE E600W fluorescence microscopy (Nikon Instruments Inc., Melville, NY, USA) and photographs were taken with a Nikon Digital Still Camera DXM1200 (Nikon Instruments Inc.).


Results and Discussion Insertion of Gene Encoding EGFP into HAd5 Genome The direct ligation of a foreign gene fragment into adenoviral genome is not a trivial task. It is often difficult because the adenoviral DNA is a large (36 kb), linear molecule that contains only a few unique and useful sites for restriction enzymes. Although an in vitro ligation method has been developed to clone foreign genes into the HAd5 genome [19-21], the low efficiency of large fragment ligation, and the labour intensive screening of recombinant viruses, have limited the use of this method for construction of recombinant adenoviruses. To improve the cloning efficiency, a homologous recombination method has been developed by Mehtali’s group at Transgene S.A., France [18] and later modified by He et al. [22]. This method uses the highly efficient homologous recombination machinery present in E. coli strain BJ5183 [23, 24] to insert the foreign gene into the adenoviral genome. In this method, the gene of interest is cloned into a transfer vector, which contains part of the adenoviral genome. The transfer vector is then co-transformed with the backbone vector (containing most of the adenoviral genome) into E. coli strain BJ5183.

Using this method, plasmid pAd5-EGFP (Figure 4) was made with the EGFP gene inserted into the E1 coding region of the HAd5 genome. The DNA fragment containing EGFP coding sequence was first cloned into the transfer vector pShuttle-CMV. The resulting plasmid, pSEGFP, was then used for homologous recombination in E. coli BJ5183 with the vector pAdEasy-1 which contains the full-length HAd5 genomic DNA with deletions in both the E1 and E3 viral coding regions. After selection with kanamycin, plasmid DNA was extracted from the smallest bacterial colonies and analyzed by digestion of restriction endonuclease. Compared to backbone plasmid pAdEasy-1, several unique bands correspond to the calculated sizes of digested DNA fragment were appeared in plasmid pAd5-EGFP after digestion with EcoRV, HindIII, and PacI (Table 1 and Figure 5), indicating that the gene encoding EGFP was inserted into the HAd5 genome.


Figure 4. Plasmid map of pAd5-EGFP containing expression cassette for EGFP. Locations of the restriction enzymes EcoRV, HindIII, and PacI sites are indicated.Ori: origin replication for plasmid;

Encap sig: HAd5 encapsidation signal; CMV Prom: cytomegalovirus promoter; EGFP: enhanced green fluorescent protein; poly A: Simian virus 40 polyadenylation signal; E2b, Penton, Hexon, E2a, Fiber, and

E4: genes encoding viral proteins of HAd5.

Table 1. Calculated sizes (bp) of the DNA fragments after digestion of pAdEasy-1 and pAd5-EGFP with restriction enzymes EcoRV, HindIII, and PacI, respectively.

RESTRICTION ENZYME pAdEasy-1 pAd5-EGFP EcoRV 11539 7637 7637 6840* 4546 5936 3806 4546 2623 3806 2052 2623 1238 2052 1238 HindIII 8010 8010 7409 5650 5322 5322 4597 4597 3010 3010 2937 2996 2081 2937 75 2081 75 PacI N/A 31678 3000

• The number in bold face indicates the unique digestion fragment for pAd5-EGFP.


Figure 5. Analysis of plasmid pAd5-EGFP. Plasmid DNA of pAd5-EGFP was purified by QIAGEN Plasmid Maxi Preparation kit. The 0.7 µg DNA was digested for one hour at 370C with restriction enzymes EcoRV (lane 2), HindIII (lane 3), and PacI (lane 4), respectively, and was analyzed by

electrophoresis through an 0.8% agarose gel and ethidium bromide staining. * : the unique DNA fragments after digestion of pAd5-EGFP with respective restriction enzyme. Lane 1: 1 kb DNA ladder (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) with the unique DNA fragment sizes indicated on

the left. Production of Recombinant HAd5 Containing EGFP Gene To produce the recombinant adenovirus Ad5-EGFP, PacI-linearized P plasmid DNA of pAd5-EGF was transfected into HEK293 cells. Adenovirus-producing foci were observed at 5 days post-transfection (Figure 6B). The virus was amplified in HEK293 cells and purified by a kit from BD Biosciences. This kit uses an adsorbent membrane that selectively binds adenoviral particles based on their distinctive surface-associated properties. Compared to commonly used method of cesium chloride (CsCl) density gradient centrifugation for adenovirus purification, the chromatographic method is less labor-intensive and faster (only four hours required for purification as opposed to three days by CsCl method). A total of 3 ml of purified recombinant Ad5-EGFP virus was obtained from 100 ml of cell lysates harvested from five T150 flasks and the titer for the purified virus was 2.5x107 pfu/ml based on the TCID50 assay.

10,000 8,000 6,000 5,000 4,000

3,000 2,500

2,000 1,500

1,000 500

1 2 3 4

*

*

*


Figure 6. Adenovirus-producing foci after transfection of HEK293 cells with PacI-linearized plasmid DNA of pAd5-EGFP. HEK293 cells were transfected either with Lipofectamine only (A) or 8 µg of PacI-digested pAd5-EGFP DNA plus Lipofectamine (B). The pictures were taken five days after transfection

Expression of EGFP in Cells Infected with Ad5-EGFP To demonstrate that EGFP could be expressed from recombinant Ad5-EGFP virus, HEK 293 and Vero cells were infected with the virus at an MOI of 1. The EGFP expression was monitored at 24 hours after virus infection. Uninfected HEK293 and Vero cells were also included as negative controls. As shown in Figure 7 (B and D), the monolayers of HEK293 and Vero cells turned green after infection with Ad5-EGFP, demonstrating that EGFP was expressed at high level in these cells. Experiments are now underway to determine the gene transfer efficiency of Ad5-EGFP in other cell lines.

A B


Mock Ad5-EGFP

Figure 7. Adenovirus-mediated EGFP expression in cells. HEK293 or Vero cells grown in 12-well plate were either infected with recombinant adenovirus Ad5-EGFP at an MOI of 1 (B and D) or mock-infected (A and C). The expression of EGFP in cells was directly visualized by fluorescence microscopy at 24

hours after infection

HEK 293

Vero


Conclusions

In this report, an efficient method for making recombinant adenovirus expressing foreign protein has been described. Using this method, the gene encoding EGFP was cloned into the full-length HAd5 genome and the EGFP was efficiently expressed in cells infected with the recombinant adenovirus. As summarized in Figure 8, the whole procedure consists of three major steps: 1) cloning of the gene of interest into a shuttle plasmid; 2) insertion of the gene of interest into full-length adenovirus genome by homologous recombination in E. coli; and 3) generation of recombinant adenovirus by transfection of HEK 293 cells with HAd5 DNA containing the foreign gene. Using this method, recombinant adenovirus expressing foreign protein can be made in less than two weeks.

Figure 8. Steps and time course for the generation of recombinant adenovirus

Cloning of the gene of interest into shuttle plasmid (e.g. pShuttle-CMV)

Digestion with PmeI and co-transformation with supercoiled plasmid Containing adenoviral genome

(e.g. pAdEasy-1) into bacteria

3 days

1 day

Screening recombinants by restriction enzymes 2 days

Large scale preparation of DNA for transfection 2 days

Digestion of DNA with PacI and transfection of HEK293 cells 1 day

5 days

Step 1

Step 2

Virus generation

Step 3


Adenoviral vector technology reported in this paper should have broad applications for the DRDC R&D programs in biotechnology and development of biodefence vaccines. Recombinant adenovirus vector can be used as a mammalian cell expression system to produce recombinant proteins as diagnostic agents or therapeutics for other BW agents. With diameters ranging from 70 to 100 nm, adenovirus vectors can work as nanoparticles to deliver the payload after fusing with the cell membrane. Particles of adenovirus vector in the nanometer size range can be genetically modified to incorporate targeting moieties or bio-recognition molecules, such as ligands, receptors, and antibodies. The method described in the report can also provide the site-specific delivery of therapeutic genes and vaccines as new countermeasures against the threats of bio-terrorism.


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List of Symbols/Abbreviations/Acronyms/Initialisms

ATCC American Type Culture Collection

bp base pairs

BW biological warfare

CF Canadian Forces

CPE cytopathic effect

CsCl cesium chloride

D-MEM Dulbecco’s modified Eagle medium

DMSO dimethyl sulfoxide

E. coli Escherichia coli

EGFP enhanced green fluorescent protein

FBS fetal bovine serum

HAd5 human adenovirus serotype 5

HEK 293 Human embryonic kidney 293

kb kilobases

MOI multiplicity of infection

pfu plaque forming units

TCID50 tissue culture infectious dose 50

UNCLASSIFIED SECURITY CLASSIFICATION OF FORM (highest classification of Title, Abstract, Keywords)

DOCUMENT CONTROL DATA (Security classification of title, body of abstract and indexing annotation must be entered when the overall document is classified)

1. ORIGINATOR (the name and address of the organization preparing the document. Organizations for who the document was prepared, e.g. Establishment sponsoring a contractor's report, or tasking agency, are entered in Section 8.)

Defence R&D Canada – Suffield PO Box 4000, Station Main Medicine Hat, AB T1A 8K6

2. SECURITY CLASSIFICATION (overall security classification of the document, including special

warning terms if applicable)

UNCLASSIFIED

3. TITLE (the complete document title as indicated on the title page. Its classification should be indicated by the appropriate abbreviation (S, C or U) in parentheses after the title).

An improved method for high-efficiency gene delivery and expression in mammalian cells (U)

4. AUTHORS (Last name, first name, middle initial. If military, show rank, e.g. Doe, Maj. John E.)

Wu, Qiaohua (Josh)

5. DATE OF PUBLICATION (month and year of publication of document)

January 2005

6a. NO. OF PAGES (total containing information, include Annexes, Appendices, etc) 27

6b. NO. OF REFS (total cited in document)

24

7. DESCRIPTIVE NOTES (the category of the document, e.g. technical report, technical note or memorandum. If appropriate, enter the type of report, e.g. interim, progress, summary, annual or final. Give the inclusive dates when a specific reporting period is covered.)

Technical Report

8. SPONSORING ACTIVITY (the name of the department project office or laboratory sponsoring the research and development. Include the address.)

Defence R&D Canada – Suffield

9a. PROJECT OR GRANT NO. (If appropriate, the applicable research and development project or grant number under which the document was written. Please specify whether project or grant.)

WBE 16qb32

9b. CONTRACT NO. (If appropriate, the applicable number under which the document was written.)

10a. ORIGINATOR'S DOCUMENT NUMBER (the official document number by which the document is identified by the originating activity. This number must be unique to this document.)

DRDC Suffield TR 2005-015

10b. OTHER DOCUMENT NOs. (Any other numbers which may be assigned this document either by the originator or by the sponsor.)

11. DOCUMENT AVAILABILITY (any limitations on further dissemination of the document, other than those imposed by security classification)

( x ) Unlimited distribution ( ) Distribution limited to defence departments and defence contractors; further distribution only as approved ( ) Distribution limited to defence departments and Canadian defence contractors; further distribution only as approved ( ) Distribution limited to government departments and agencies; further distribution only as approved ( ) Distribution limited to defence departments; further distribution only as approved ( ) Other (please specify):

12. DOCUMENT ANNOUNCEMENT (any limitation to the bibliographic announcement of this document. This will normally corresponded to the Document Availability (11). However, where further distribution (beyond the audience specified in 11) is possible, a wider announcement audience may be selected).

UNCLASSIFIED SECURITY CLASSIFICATION OF FORM


13. ABSTRACT (a brief and factual summary of the document. It may also appear elsewhere in the body of the document itself. It is highly desirable that the abstract of classified documents be unclassified. Each paragraph of the abstract shall begin with an indication of the security classification of the information in the paragraph (unless the document itself is unclassified) represented as (S), (C) or (U). It is not necessary to include here abstracts in both official languages unless the text is bilingual).

Through millions of years of evolution, viruses have developed special molecular mechanism that allow them to bind to a host cell and to efficiently deliver their genetic material. To take advantage of these refined mechanisms, viruses have been modified as vectors to deliver desired genes into a large variety of cells. These include both in vitro (e.g. mammalian cell culture) and in vivo (e.g. mice) systems. To broaden DRDC’s existing programs in biotechnology, this report describes the development of an adenovirus-based vector for high-efficiency gene delivery and expression in mammalian cells. To illustrate the feasibility of this vector system, a recombinant adenovirus vector was produced, which contains gene encoding the enhanced green fluorescent protein (EGFP). The robust expression of EGFP was demonstrated in cells infected with the recombinant adenovirus. The adenovirus vector described in this report can be applied to many research areas including: 1) the expression of recombinant proteins in tissue culture cells, 2) the development of live viral vectored vaccines, 3) the expression of therapeutic proteins (e.g. interferon and monoclonal antibody) and nucleic acids (e.g. small interfering RNAs, antisense RNA) in animals against pathogens, and 4) the production of polyclonal antibody by direct injection of animals with recombinant adenovirus expressing antigens.

14. KEYWORDS, DESCRIPTORS or IDENTIFIERS (technically meaningful terms or short phrases that characterize a document and could be helpful in cataloguing the document. They should be selected so that no security classification is required. Identifies, such as equipment model designation, trade name, military project code name, geographic location may also be included. If possible keywords should be selected from a published thesaurus, e.g. Thesaurus of Engineering and Scientific Terms (TEST) and that thesaurus-identified. If it is not possible to select indexing terms which are Unclassified, the classification of each should be indicated as with the title.) gene delivery, gene expression, adenovirus vector, enhanced green fluorescent protein


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