Minicircle and Miniplasmid DNA Vectors E-Book

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 463

Veröffentlichungsjahr: 2013

Beschreibung

This first title on the topic provides complete coverage, including the molecular basis, production and possible biomedical applications. Written by the most prominent academic researchers in the field as well as by researchers at one of the world's leading companies in industrial production of minicircle DNA, this practical book is aimed at everyone who is directly or indirectly involved in the development of gene therapies.

Leseprobe

Contents

Cover

Related Titles

Title Page

Copyright

Dedication

List of Contributors

Preface

Chapter 1: Minicircle Patents: A Short IP Overview of Optimizing Nonviral DNA Vectors

References

Chapter 2: Operator–Repressor Titration: Stable Plasmid Maintenance without Selectable Marker Genes

2.1 Introduction

2.2 Antibiotics and Metabolic Burden

2.3 The Mechanism of ORT

2.4 ORT Strain Development

2.5 ORT Miniplasmids

2.6 DNA Vaccine and Gene Therapy Vectors

2.7 ORT-VAC: Plasmid-Based Vaccine Delivery Using Salmonella enterica

2.8 Recombinant Protein Expression

2.9 Conclusions and Future Developments

References

Chapter 3: Selection by RNA–RNA Interaction: Maximally Minimized Antibiotic Resistance-Free Plasmids

3.1 Gene Therapy and DNA Vaccines: Emerging Technologies

3.2 Therapeutic Plasmids: Novel Design and the Problem of Selection

3.3 Conclusions

Acknowledgments

References

Chapter 4: Plasmid-Based Medicinal Products – Focus on pFAR: A Miniplasmid Free of Antibiotic Resistance Markers

4.1 Introduction: Rationale for the Development of Biosafe DNA Plasmid Vectors

4.2 Specific Requirements for the Use of DNA Product as Medicines

4.3 Nonviral Gene Vectors Devoid of Antibiotic Resistance Markers

4.4 The pFAR Plasmid Family

4.5 Concluding Remarks and Perspectives

Acknowledgments

References

Chapter 5: Plasmid DNA Concatemers: Influence of Plasmid Structure on Transfection Efficiency

5.1 Introduction

5.2 Plasmid DNA Topology and Size

5.3 Plasmid DNA Concatemers

5.4 Conclusions

Acknowledgments

References

Chapter 6: Analytical Tools in Minicircle Production

6.1 Introduction

6.2 Production of Minicircles

6.3 Analytics of Minicircle Production

6.4 Future Goals

Acknowledgments

References

Chapter 7: Utilizing Minicircle Vectors for the Episomal Modification of Cells

7.1 Introduction

7.2 Studies that Show Passive Episomal Maintenance of Minicircles In Vivo

7.3 Principles of Generating Minicircle Vectors Able to Support Episomal Maintenance

7.4 Episomal Maintenance of S/MAR Minicircles In Vivo

7.5 Potential of Episomal Replication of S/MAR Minicircle Vectors

7.6 Possible Mechanisms Promoting the Episomal Maintenance of Minicircle Vectors

7.7 Conclusions

References

Chapter 8: Replicating Minicircles: Overcoming the Limitations of Transient and Stable Expression Systems

8.1 Gene Therapy: The Advent of Novel Vector Vehicles

8.2 Replicating Nonviral Episomes

8.3 Minimalization Approaches

8.4 Summary and Outlook

Acknowledgments

References

Chapter 9: Magnetofection of Minicircle DNA Vectors

9.1 Introduction

9.2 Overview of Magnetofection Principles

9.3 Cellular Uptake

9.4 Diffusion through the Cytoplasm

9.5 Transgene Expression

9.6 Conclusions

References

Chapter 10: Minicircle-Based Vectors for Nonviral Gene Therapy: In Vitro Characterization and In Vivo Application

10.1 Minicircle Technology for Nonviral Gene Therapy

10.2 Current Status of In Vivo Application of Minicircle Vectors

10.3 Jet Injection Technology for In Vivo Transfer of Naked DNA

10.4 Comparative Performance Analyses of Minicircle Vectors

10.5 In Vivo Application of Minicircle DNA by Jet Injection

References

Chapter 11: Episomal Expression of Minicircles and Conventional Plasmids in Mammalian Embryos

11.1 Introduction

11.2 Fate of Plasmids and Minicircles After Injection into Mammalian Embryos

11.3 Discussion

References

Chapter 12: Tissue-Targeted Gene Electrodelivery of Minicircle DNA

12.1 Introduction

12.2 Plasmid DNA Electrotransfer: From Principle to Technical Design

12.3 Implementation for Efficient Tissue-Targeted Gene Delivery

12.4 Conclusions

Acknowledgments

References

Chapter 13: Increased Efficiency of Minicircles Versus Plasmids Under Gene Electrotransfer Suboptimal Conditions: an Influence of the Extracellular Matrix

13.1 Introduction

13.2 Methods

13.3 Results

13.4 Discussion

13.5 Conclusions

Acknowledgments

References

Index

Related Titles

Schleef, M. (ed.)

DNA Pharmaceuticals

2005

ISBN: 978-3-527-31187-3

Prazeres, D.M.F.

Plasmid Biopharmaceuticals

Basics, Applications, and Manufacturing

2011

ISBN: 978-0-470-23292-7, also available in digital formats

CIBA Foundation Symposium

Bacterial Episomes and Plasmids

2009

ISBN: 978-0470-71503-1 (E-Book)

Biotechnology Journal

www.biotechnology-journal.com

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

Library of Congress Card No.: applied for

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche Nationalbibliothek

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This book is dedicated to Marion & Mariella

List of Contributors

Franck M. André

CNRS

Laboratoire de Vectorologie et

Thérapeutiques Anticancéreuses

UMR 8203

91405 Orsay

France

and

Université Paris-Sud

Laboratoire de Vectorologie et

Thérapeutiques Anticancéreuses

UMR 8203

91405 Orsay

France

and

Institut Gustave Roussy

Laboratoire de Vectorologie et

Thérapeutiques Anticancéreuses

UMR8203, 114 rue Edouard Vaillant

94805 Villejuif

France

Dario Anselmetti

Bielefeld University

Physics Faculty

Experimental Biophysics and Applied Nanoscience

Universitätsstr. 25

33615 Bielefeld

Germany

Orestis Argyros

Imperial College London

National Heart and Lung Institute

Section of Molecular Medicine

Gene Therapy Group

Imperial College Road

South Kensington

London SW7 2AZ

UK

Jutta Aumann

Charité University Medicine Berlin

Experimental and Clinical Research Center

Robert-Rössle-Str. 10

13125 Berlin

Germany

Ruth Baier

PlasmidFactory GmbH & Co. KG

Dept. DNA Production

Meisenstr. 96

33607 Bielefeld

Germany

Elodie Bertosio

OZ Biosciences R&D department

Parc Scientifique de Luminy, Zone Luminy Entreprise

163 Avenue de Luminy, Case 922

13288 Marseille Cedex 9

France

Melanie Bertuzzi

OZ Biosciences R&D department

Parc Scientifique de Luminy, Zone Luminy Entreprise

163 Avenue de Luminy, Case 922

13288 Marseille Cedex 9

France

Stefanie Binius

Helmholtz Center for Infection Research

Department Molecular Biotechnology

Inhoffenstraße 7

38124 Braunschweig

Germany

Markus Blaesen

PlasmidFactory GmbH & Co. KG

Dept. DNA Production

Meisenstr. 96

33607 Bielefeld

Germany

Jürgen Bode∗

Hannover Medical School (MHH)

Institute for Experimental Haematology

OE 6960, Carl-Neuberg-Strasse 1

30625 Hannover

Germany

Sandra Broll

Helmholtz Center for Infection Research

Department Molecular Biotechnology

Inhoffenstraße 7

38124 Braunschweig

Germany

and

Leibniz Universität Hannover

Dezernat 4 – Forschung undTechnologietransfer/NationaleForschungsförderung

Schloßwender Str. 1

30159 Hannover

Germany

Sophie Chabot

Centre National de la RechercheScientifique

Institut de Pharmacologie et deBiologie Structurale

BP 64182, 205 route de Narbonne

31077 Toulouse

France

and

Université de Toulouse

UPS, IPBS

31077 Toulouse

France

Bronwen Connor

University of Auckland

Faculty of Medical and Health Sciences

Centre for Brain Research

Department of Pharmacology & Clinical Pharmacology

Auckland

New Zealand

Charles Coutelle

Imperial College London

National Heart and Lung Institute

Section of Molecular Medicine

Gene Therapy Group

Imperial College Road

South Kensington

London SW7 2AZ

UK

Rocky M. Cranenburgh∗

Cobra Biologics Ltd and Prokarium Ltd

Stephenson Building

Keele Science Park

Keele, Staffordshire ST5 5SP

UK

George Dickson

Royal Holloway University of London

School of Biological Sciences

Egham, Surrey TW20 0EX

UK

Mareike Dieding

Bielefeld University

Physics Faculty

Experimental Biophysics and Applied Nanoscience

Universitätsstr. 25

33615 Bielefeld

Germany

Helen Foster

Royal Holloway University of London

School of Biological Sciences

Egham, Surrey TW20 0EX

UK

Wiebke Garrels

Friedrich Loeffler Institute

Institute of Farm Animal Genetics

Department of Biotechnology

Mariensee

Höltystr. 10

31535 Neustadt

Germany

Muriel Golzio

Centre National de la Recherche Scientifique

Institut de Pharmacologie et de Biologie Structurale

BP 64182, 205 route de Narbonne

31077 Toulouse

France

and

Université de Toulouse

UPS, IPBS

31077 Toulouse

France

Reingard Grabherr

University of Natural Resources and Life Sciences

Department of Biotechnology

Muthgasse 18

1190 Vienna

Austria

Martin Grund∗

GRUND Intellectual Property Group

Nikolaistrasse 15

80802 München

Germany

Richard P. Harbottle∗

Imperial College London

National Heart and Lung Institute

Section of Molecular Medicine

Gene Therapy Group

Imperial College Road

South Kensington

London SW7 2AZ

UK

Markus Heine

Rentschler Biotechnologie GmbH

“Bioprocess Development”/“Virus-based Biologics”

Erwin-Rentschler-Strasse 21

88471 Laupheim

Germany

Niels Heinz

Hannover Medical School (MHH)

Institute for Experimental Haematology

OE 6960, Carl-Neuberg-Strasse 1

30625 Hannover

Germany

Khursheed Iqbal

Beckman Research Institute of City of Hope

Arnold and Mabel Beckman Research CenterDepartment of Molecular and Cellular BiologyDuarte, CA 91010

USA

Vanessa Joubert

CERPEMMaison de la Technopole6 rue Leonard de Vinci53000 LavalFrance

Raju Kandimalla

Erasmus MC

Josephine Nefkens Institute

Department of Pathology

Dr. Molewaterplein 50

3000 CA Rotterdam

The Netherlands

Dennis Kobelt

Max Delbrück Center for Molecular Medicine

Robert-Rössle-Str. 10

13125 Berlin

Germany

Wilfried A. Kues∗

Friedrich Loeffler Institute

Institute of Farm Animal Genetics

Department of BiotechnologyMarienseeHöltystr. 10

31535 Neustadt

Germany

Nicolas Laurent

OZ Biosciences R&D department

Parc Scientifique de Luminy, Zone Luminy Entreprise

163 Avenue de Luminy, Case 922

13288 Marseille Cedex 9

France

Jürgen Mairhofer∗

University of Natural Resources and Life Sciences

Department of Biotechnology

Muthgasse 18

1190 Vienna

Austria

Corinne Marie

Université Paris Descartes

Faculté de Pharmacie

Unité de Pharmacologie Chimique et Génétique et d'Imagerie

Ecole Nationale Supérieure de Chimie de Paris

CNRS UMR8151, INSERM U1022

Paris

France

Christof Maucksch∗

University of Auckland

Faculty of Medical and Health Sciences

Centre for Brain Research

Department of Pharmacology & Clinical Pharmacology

Auckland

New Zealand

Lluis M. Mir∗

CNRS

Laboratoire de Vectorologie etThérapeutiques Anticancéreuses

UMR 8203

91405 Orsay

France

and

Université Paris-Sud

Laboratoire de Vectorologie etThérapeutiques Anticancéreuses

UMR 8203

91405 Orsay

France

and

Institut Gustave Roussy

Laboratoire de Vectorologie etThérapeutiques Anticancéreuses

UMR 8203114 rue Edouard Vaillant94805 Villejuif

France

Kristina Nehlsen

Helmholtz Center for Infection Research

Department Molecular Biotechnology

Inhoffenstraße 7

38124 Braunschweig

Germany

Mickaël Quiviger

Université Paris Descartes

Faculté de Pharmacie

Unité de Pharmacologie Chimique etGénétique et d'Imagerie

Ecole Nationale Supérieure de Chimie de Paris

CNRS UMR8151, INSERM U1022

Paris

France

Anja Rischmüller

PlasmidFactory GmbH & Co. KG

Dept. R & D

Meisenstr. 96

33607 Bielefeld

Germany

and

Bielefeld University

Physics Faculty

Experimental Biophysics and AppliedNanoscience

Universitätsstr. 25

33615 Bielefeld

Germany

Carsten Rudolph

Ludwig-Maximilians UniversityDepartment of PediatricsLindwurmstrasse 2a80337 MunichGermany

and

ethris GmbHLochhamerstr. 1182152 MartinsriedGermany

Cedric Sapet

OZ Biosciences R&D department

Parc Scientifique de Luminy, Zone Luminy Entreprise

163 Avenue de Luminy, Case 922

13288 Marseille Cedex 9

France

Axel Schambach

Hannover Medical School (MHH)

Institute for ExperimentalHaematology

OE 6960, Carl-Neuberg-Strasse 1

30625 Hannover

Germany

Daniel Scherman∗

Université Paris Descartes

Faculté de Pharmacie

Unité de Pharmacologie Chimique etGénétique et d'Imagerie

Ecole Nationale Supérieure deChimie de Paris

CNRS UMR8151, INSERM U1022

Paris

France

Peter M. Schlag

Charité Comprehensive Cancer Center

Invalidenstr. 80

10117 Berlin

Germany

Martin Schleef∗

PlasmidFactory GmbH & Co. KG

Dept. R & D

Meisenstr. 96

33607 Bielefeld

Germany

Marco Schmeer

PlasmidFactory GmbH & Co. KG

Dept. Process Development

Meisenstr. 96

33607 Bielefeld

Germany

Flavie Sicard

OZ Biosciences R&D department

Parc Scientifique de Luminy, ZoneLuminy Entreprise

163 Avenue de Luminy, Case 922

13288 Marseille Cedex 9

France

Ulrike Stein

Max Delbrück Center for MolecularMedicine

Robert-Rössle-Str. 10

13125 Berlin

Germany

and

Charité University Medicine Berlin

Experimental and Clinical Research Center

Robert-Rössle-Str. 10

13125 Berlin

Germany

Justin Teissié∗

Centre National de la Recherche Scientifique

Institut de Pharmacologie et deBiologie Structurale

BP 64182, 205 route de Narbonne

31077 Toulouse

France

and

Université de Toulouse

UPS, IPBS

31077 Toulouse

France

Martina Viefhues

Bielefeld University

Physics Faculty

Experimental Biophysics and AppliedNanoscience

Universitätsstr. 25

33615 Bielefeld

Germany

Wolfgang Walther∗

Max Delbrück Center for MolecularMedicine

Robert-Rössle-Str. 10

13125 Berlin

Germany

and

Charité University Medicine Berlin

Experimental and Clinical Research Center

Robert-Rössle-Str. 10

13125 Berlin

Germany

Suet-Ping Wong

Imperial College London

National Heart and Lung Institute

Section of Molecular Medicine

Gene Therapy Group

Imperial College Road

South Kensington

London SW7 2AZ

UK

Olivier Zelphati∗

OZ Biosciences R&D department

Parc Scientifique de Luminy, ZoneLuminy Entreprise

163 Avenue de Luminy, Case 922

13288 Marseille Cedex 9

France

Preface

After significant improvements in the field of non-viral vector development, we compiled the status of those “new plasmids” within this book. The tools for gene- or cell therapy and DNA vaccination are available in form of pure genetic material (DNA, RNA) or within more complex units (viral vectors, VLP, aggregates containing chemical substances or “simply” cells).

We gave an overview on initial non-viral approaches in 2001 with “Plasmids for Therapy and Vaccination” (edited by M. Schleef, E-Book ISBN 978-3-527-61284-0, Wiley-VCH) summarizing the different types of plasmid vectors to be used, their structure and functionality including regulatory aspects and those of making them. Later we focussed on the route of administration, pharmaceutical DNA and specific features (e.g. CpG motifs) of such: “DNA Pharmaceuticals” (edited by M. Schleef, ISBN 978-3-527-31187-3, Wiley-VCH).

Although plasmids were classified as not potentially risky due to their non-integration into the host chromosome, they were not always the first choice due to their lower efficiency compared to viral vectors. As shown within certain chapters of this book, the efficiency for mini plasmids and minicircle DNA is significantly higher simply because of their smaller size and in some cases also due to contained sequence elements. The safety aspect of plasmids without any antibiotic resistance marker (“mini plasmids”) or – even smaller and with less CpG motifs – in addition without the bacterial backbone with the origin of replication (“mincircles”) is a major advantage for any application where such sequences need to be avoided. For all those who like to further discuss these aspects I look forward to do so at any time ([email protected]).

I wish to thank all colleagues who never gave up in believing in the idea of curing at the place where the initial change from “healthy” (or should I say “wildtype”?) to “not healthy” happened. Also I thank all non-scientists who continued to ask for what we are doing – you should go on with this – we are highly motivated even by difficult questions. Finally, my special thank goes to all Authors participating in this book, the teams of the publisher, the PlasmidFactory team for support and discussion and all friends.

Bielefeld, January 2013

Martin Schleef

Chapter 1

Minicircle Patents: A Short IP Overview of Optimizing Nonviral DNA Vectors

Martin Grund and Martin Schleef

The use of nonviral vectors for gene and cell therapy and especially for vaccination started with the observation of Wolff et al. [1] that the direct application of plasmid DNA containing the expression cassette for a protein into animal muscle led to the expression of this and – subsequently – to the appearance of antibodies against this protein – the idea of a DNA vaccine. While this was initially done with standard cloning or gene expression plasmids typically driven by a CMV promoter, its use in pharmaceutical context required the improvement of the structure of the plasmid with respect to the coding sequence (e.g., codon usage) and also concerning the total molecule: starting from the removal of abundant sequences (e.g., multiple cloning site residues) and the replacement of the antibiotic resistance gene bla (for ampicillin resistance) by a kanamycin resistance up to the removal of CpG motifs from the coding and backbone sequence [2]. Also, the physical structure of plasmid vectors was modulated by using process technology to obtain exclusively ccc-supercoiled DNA through specific cultivation technology [3] or purification processes [4], resulting in the depletion of toxic bacterial chromosomal DNA (with CpG motifs) as recently published [5].

The first major improvement was the removal of any resistance marker sequence from the plasmid (resulting in so-called miniplasmids); many are described in this book (see Chapters 6–13). However, a selection marker was still present on the plasmid, and also the large sequence element responsible for the plasmid replication (bacterial origin of replication – ori [6]) was still there.

The major improvements to further reducing the size and – by the way – removing the nonintended backbone sequences, including the ori, were made by approaches to reducing the DNA molecules carrying the pharmaceutically required expression unit to (mainly) circular structures with almost no other sequence than the sequence of interest, the so-called minicircles.

The first minicircle patent application to be filed was an international application by the US Department of Health with Adhya and Choy as inventors, priority date October 16, 1992, and published as WO 94/09127. The application was subsequently withdrawn in November 1994, and no patents were granted. Claim 1 referred to a DNA construct comprising attB and attP sites with a multiple cloning site (in a later application, by Bigger et al. in 2001 (US application 11/249929), also called “multicloning site sequence”) and a transcription terminator in between. This DNA was to be introduced into a host cell expressing the lambda Int protein, leading to site-specific recombination and excision of a circular construct. Since the construct thus formed was not supposed to contain a resistance gene or an origin of replication, it can be regarded as a minicircle, and the very term was in fact coined in this application. The intention of the inventors was, however, quite different from the gene vector and therapeutic approaches that have characterized later minicircle applications. In fact, the aim of these first constructs was to study the kinetics of promoters, to which end a construct containing only a single promoter with a reporter gene was needed.

It then took several years until the potentially superior properties of minicircles as vectors for gene transfer and therapeutic approaches were exploited in the field of patents. A further approach was submitted by Seeber and Krüger, with priority date August 11, 1994, and published as WO 96/05297. The application led to the grant of patents in Europe (EP 0775203) and the United States (US Patent 6,573,100), which are still in force and directed to the use of minicircles in therapy. The inventors intended to remove the resistance gene bla from a circular plasmid vector by site-specific recombinase (SSR) systems by dividing the circular plasmid into two circles – one containing the gene cassette and the other the residual portion including bla. The growth of the plasmid was performed under selective pressure and the two circles were separated by chromatography. The recombination system proposed was, for example, FLP/FRT. The major field of intended application was the gene therapy of cystic fibrosis.

The first patents to minicircles as such were obtained by the CNRS in France, who had filed an international application published as WO 96/26270 with priority date February 23, 1995, and Cameron et al. as inventors. The application resulted in granted patents in Europe (EP 0815214), the United States (US Patents 6,143,530 and 6,492,164), and Canada (CA 2211427), which are still in force. Claim 1 referred to a double-stranded DNA molecule characterized in that (a) it is circular and supercoiled; (b) it contains an expression cassette under control of a mammalian promoter; (c) it does not contain an origin of replication; (d) it does not contain a marker gene; and (e) it contains a region resulting from the site-specific recombination between two sequences, which is not present in the expression cassette. The introduction of therapeutic genes and the use of minicircles in gene therapy were expressly stated points of the application. The patent emphasizes that the absence of marker and resistance genes and other prokaryotic sequences (e.g., the origin of replication) affords a high genetic purity and low risk of transmission of undesired sequences and proliferation of antibiotics resistance.

Within these patents, methods for the production of such constructs were also provided. In particular, a preferred method involved the generation of minicircles from a precursor plasmid with two recombination sites, which are to be recombined by the coexpression of a recombinase. Recombinases from the lambda integrase family and from the Tn3 family were suggested. The presence of the recombinase gene on the precursor plasmid itself is also contemplated. For the purification of the resulting constructs, several methods are suggested. In particular, specific affinity binding of a ligand to a recognition sequence in the miniplasmid is mentioned and illustrated by the example of triple-helix formation with a specific binding oligonucleotide. This interaction was also used to immobilize minicircles on a chromatography column during purification.

The next minicircle application that has left rights outstanding is US application 11/249929 by Bigger et al., priority date April 10, 2001. The corresponding European application has been abandoned, but the US case is still pending. The novel aspect of this invention is the use of modified recombination sequences for minicircle production. In each of the two sequences that are to be recombined, one half-site is mutated so that the affinity of the recombinase is decreased. While a site consisting of one mutated and one wild-type half-site is still capable of binding the recombinase and being recombined, the resulting new site consisting of two mutated half-sites is no longer active. In this way, unidirectionality can be imparted to the recombination process. If the mutated half-sites are designed to lie on the minicircle after recombination, the yield of minicircle can be increased. Furthermore, the application also discloses the use of a restriction endonuclease to digest specifically the miniplasmid or nonrecombined parental plasmid after recombination. In addition, an additional treatment with an exonuclease for the removal of restriction fragments deriving from miniplasmid or nonrecombined parental plasmid is disclosed there. With this strategy, the yield of minicircles can be increased.

A further minicircle patent has been granted in the United States as US Patent 7,897,380 to Kay and Chen based on an application with priority date August 29, 2002. The corresponding European application (EP 03749280.8) has been refused and is currently under appeal. The claims are directed to minicircles that provide for persistent and high expression levels when present in the cell. The minicircles of the disclosed embodiments are produced by recombination with ΦC31 integrase and subsequent restriction of the miniplasmid, as disclosed already in 2001 by Bigger et al. (US application 11/249929).

In Europe, a patent has been granted to Mayrhofer et al. as EP 1620559, priority date May 5, 2003. The corresponding US application (10/556069) is still pending. The subject matter refers to parent plasmids for the production of minicircles that also encode the required recombinase in the region outside of the recombination sequences [7], as initially disclosed by Cameron et al. (WO 96/26270, see above). Furthermore, a method for purification of the minicircle product is described in EP 1620559, wherein the minicircle is immobilized in the plasma membrane of the producing bacteria upon lysis and can be isolated in this manner.

Immediately thereafter, Schroff and Smith submitted their application with priority date June 10, 2003, which was published as WO 2004/111247. The application has been abandoned in the United States and granted in Europe (EP 1631672). Here, the authors present a method to obtain circular gene expression cassettes by specific restriction digestion of plasmid DNA leading to two linear fragments, one containing the expression cassette and the other the nonintended sequences of the plasmid (e.g., backbone, resistance gene). The fragments are recircularized (but not supercoiled) by ligation. Subsequently, a second digestion step with a different restriction enzyme, cutting exclusively the nonintended molecule (the backbone sequences) at least once, results in a mixture of linear fragments deriving from the backbone molecule and an intact circular GOI molecule. Thereafter, the linear fragments are removed by digestion with an exonuclease, so that the circular GOI molecule is further purified, as disclosed already in 2001 by Bigger et al. (US application 11/249929).

US Patent 7,622,252, granted to Zechiedrich, priority date June 10, 2005, is directed to the production of minicircles in topoisomerase IV-deficient cells. This is said to result in a higher yield of supercoiled minicircles, although these are formed as catenates and have to be decatenated before use. A more recent application by Zechiedrich et al., priority date October 16, 2009, is pending in the United States (12/905612) and in Europe (EP 10824202.5). It is directed to the use of “minivectors” (essentially minicircles) in gene therapy for the continuous expression of shRNA and miRNA in a target (see also Ref. [8]).

Finally, two more recent applications are pending by Kay et al. One, by Chen and Kay, priority date July 3, 2008, is pending in Europe (EP 09773923.9) and has been granted in the United States (US Patent 8,236,548). A method is disclosed for the production of minicircles with a high purity. A precursor plasmid is cleaved in a host cell by site-specific recombination into a minicircle and a miniplasmid, the latter then being digested by an endonuclease encoded in the same host, similarly to the method of Bigger et al. (see above).

The other application, by Wu and Kay, is pending in the United States as 12/925483 with priority date October 23, 2009. It refers to the treatment of ischemic cardiovascular disease by transducing muscle cells of the patient with HIF-1-encoding minicircles.

It is important to note that according to general principles of patent law, a patent may depend on another patent. This means that if the teaching of a later patent is a further development of the teaching of an earlier patent that is still in force, the proprietor of the later patent can use its teaching only with the consent of the proprietor of the earlier patent. In the field of minicircle patents, numerous such dependences exist. For example, most minicircle patents are dependent on the patent of Cameron et al., which covers all minicircles having a gene expression cassette. Another example is the use of restriction enzyme and/or exonuclease treatment of nonminicircle molecules during minicircle production, already disclosed in 2001 by Bigger et al. (US application 11/249929).

Figure 1.1 gives an overview of minicircle patent applications depending on their filing date.

Figure 1.1 Overview of minicircle patents according to their filing date.

The general idea of reducing the size of a circular DNA vector was shown to be successful (see certain examples within this book). The removal of CpG elements as initially described for plasmids and patented by Drocourt et al. as EP 1366176 was a significant improvement with respect to the state-of-the-art plasmids used before as shown for “zero-CpG plasmids” later [2]. However, this CpG-free backbone could be surrounded by the use of minicircle DNA, since the latter is – as long as the sequence of interest is CpG-free – in total almost free of any CpG.

Various applications for the use of minicircle DNA have been presented since their invention (see Tables 7.1 and 10.1). Since the size of minicircles cannot be reduced further, we expect the positive modulation of their functionality.

References

1. Wolff, J.A., Malone, R.W., Williams, P., Chong, G., Acsadi, A., Jani, A., and Felgner, P.L. (1990) Direct gene transfer into mouse muscle in vivo. Science, 247, 1465–1458.

2. Hyde, S.C., Pringle, I.A., Abdullah, S. et al. (2008) CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression. Nature Biotechnology, 26, 549–551.

3. Schmidt, T., Friehs, K., Flaschel, E., and Schleef, M. (1998) Method for the isolation of ccc plasmid DNA. EP 1144656.

4. Schleef, M. and Schmidt, T. (2004) Animal-free production of ccc-supercoiled plasmids for research and clinical applications. The Journal of Gene Medicine, 6, 45–53.

5. Wooddell, C.I., Subbotin, V.M., Sebestyén, M.G., Griffin, J.B., Zhang, G., Schleef, M., Braun, S., Huss, T., and Wolff, J.A. (2011) Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent. Human Gene Therapy, 22, 225–235.

6. Schumann, W. (2001) The biology of plasmids, in Plasmids for Therapy and Vaccination (ed. M. Schleef), Wiley-VCH Verlag GmbH, Weinheim, pp. 1–28.

7. Mayrhofer, P., Blaesen, M., Schleef, M., and Jechlinger, W. (2008) Minicircle-DNA production by site specific recombination and protein–DNA interaction chromatography. The Journal of Gene Medicine, 10, 1253–1269.

8. Zhao, N. et al. (2011) Transfection of shRNA-encoding minivector DNA of a few hundred base pairs to regulate gene expression in lymphoma cells. Gene Therapy, 18, 220–224.

Chapter 2

Operator–Repressor Titration: Stable Plasmid Maintenance without Selectable Marker Genes

Rocky M. Cranenburgh

2.1 Introduction

Operator–repressor titration (ORT®) is the only technology for plasmid selection and maintenance without the requirement for the expression of a selectable marker gene such as an antibiotic resistance gene, requiring instead only a single copy of an operator sequence on the plasmid. It was invented by Prof. David Sherratt at the University of Oxford and developed by Cobra Biologics Ltd. ORT enables the high-yield production in Escherichia coli of antibiotic resistance gene-free DNA vaccine and gene therapy plasmids, which have been used in numerous clinical trials. Once an ORT miniplasmid is generated, it can be used in the same way as a conventional plasmid with no special growth media, additives, or procedures required, which has enabled the technology to be applied to recombinant protein production in E. coli and extended to attenuated strains of Salmonella to enable oral delivery of recombinant vaccines.

2.2 Antibiotics and Metabolic Burden

The use of antibiotics and their resistance genes for the production of biologics has been discouraged by regulatory authorities such as the Food and Drug Administration (FDA) in the United States and the European Medicines Agency (EMA). The β-lactam antibiotics such as ampicillin are not permitted – the primary reasons for this are the safety risk of immune sensitization to patients from residual antibiotics during manufacture and the risk of antibiotic resistance gene transfer to bacteria in the environment that could lead to the spread of antibiotic-resistant pathogens. EMA guidelines for gene therapy products state that “Use of antibiotic resistance genes as selection markers in the vector is generally discouraged. If unavoidable, studies should be performed before first clinical studies addressing inadvertent expression of the resistance gene in human somatic cells.” [1]. However, there are other drawbacks of using antibiotics in the production of biopharmaceuticals. Residual antibiotics represent a contaminant in the manufacturing process that has to be removed, with assays required to demonstrate removal. This leads to manufacturers typically omitting antibiotics from the fermentation, which can lead to a reduction in plasmid yield as cells that have lost their plasmids are at a selective advantage due to a lower metabolic burden and can therefore proliferate in the culture [2]. Even if antibiotics are added, their selection pressure decreases in high biomass fermentations due to degradation.

There are numerous alternative approaches (see also Chapters 3 and 4) to replace antibiotic resistance genes with other selectable marker genes, such as those that complement a chromosomal mutation by the inclusion of the functional gene on the plasmid [3, 4]. Other strategies compensate for chromosomal mutations [5] or repress toxic chromosomal genes [6], but are limited to specific growth media or the presence of specific metabolites. However, as with antibiotic resistance, these all require plasmid-based gene expression in addition to the replication of additional DNA, and a significant factor in plasmid loss is the constitutive expression of the selectable marker gene, which is a principal cause of the metabolic burden to bacterial cells [7].

Approaches to enhance stability include postsegregational killing (PSK) systems such as hok-sok and ccdA-ccdB, where a stable toxin and labile antitoxin are both expressed [8]. When the plasmid is lost from the cell, the antitoxin rapidly degrades, leaving the toxin to kill the cell. However, this mechanism did not evolve to stabilize plasmids – there is no advantage to plasmid stability in killing the bacterial cell once it has been lost. Instead, there is evidence that PSK systems protect a plasmid in a population of bacteria from invasion by a foreign plasmid [9]. If a plasmid possessing PSK genes is displaced from a bacterium by another plasmid, that cell will be killed before the invading plasmid has a chance to spread to other cells in the population. As with the complementation approach, the PSK genes constitute a metabolic burden, in addition to mild toxicity.

With all these mechanisms, there is no inherent stabilization mechanism to retain the plasmid – plasmid-free segregants are killed to prevent them taking over a culture, giving the impression of plasmid stabilization, but if the rate of loss is high this will inevitably result in a high proportion of dead cells in the culture and a corresponding lower growth rate of those cells that retain the plasmid.

2.3 The Mechanism of ORT

ORT relies on the phenomenon of repressor titration. Several genes and operons in bacteria are regulated by feedback control systems: repressor protein produced from a chromosomal gene binds to a chromosomal operator sequence and prevents expression of a second chromosomal gene or operon by blocking the binding of RNA polymerase to the promoter, or blocking transcription, unless an inducer is present to alter the conformation of the repressor and thus prevent it from binding. The binding kinetics and equilibrium of the operator–repressor interaction will determine the extent to which a low level of gene expression from the promoter will be possible. If the cell is transformed with a plasmid that contains an operator sequence that also binds the repressor, then the higher plasmid copy number enables the repressor to be titrated, binding to the multiple plasmidic operators as well as the single-copy chromosomal operator. Repressor titration therefore results in the chromosomal operator being free for longer periods, enabling greater expression levels for the gene(s) being regulated. Examples of practical uses of repressor titration include identification of operator mutations and natural operator sequences [10, 11].

For ORT to function, a genetically modified cell is required wherein a chromosomal gene that is essential for cell survival under the conditions used is regulated by an inducible promoter–operator. This cell can then be grown either under nonselective conditions or in the presence of the inducer that prevents the repressor from switching off the essential gene. When this cell is transformed with a multicopy plasmid containing an operator sequence, the repressor is titrated and the transformants identified by their growth on the selective medium (Figure 2.1).

Figure 2.1 The mechanism of ORT. The ORT strain has the promoter of an essential chromosomal gene replaced with an inducible promoter–operator, and a chromosomal repressor protein gene is also integrated. In the absence of an inducer (a), the repressor will bind to the operator and prevent the expression of the essential gene, thus stalling growth or killing the cell. The addition of an inducer alters the conformation of the repressor protein, preventing it from binding to the operator and enabling expression of the essential gene (b). When the ORT strain is transformed with a plasmid that possesses an operator sequence that binds to the repressor (c), the repressor is titrated and the essential gene expression is induced, thus enabling plasmid selection and maintenance.

The first example of ORT featured an E. coli strain with an inducer-insensitive mutation in the lacI gene encoding the repressor of the lactose operon. This lacIs mutation meant that the mutant strain YA694 produced a lac repressor protein that remained bound to the operator even in the presence of the inducer lactose, making it unable to metabolize this sugar [12]. When YA694 was transformed with a plasmid (pUC18) that possessed the lac operator sequences, it could be grown on a minimal medium containing lactose as a sole carbon source, as ORT enabled expression of the lac operon. Culturing of YA694(pUC18) over multiple generations in minimal medium containing either glucose or lactose as sole carbon sources resulted in the plasmid being lost from the former (where lactose metabolism is not necessary) but retained in the latter [13].

2.4 ORT Strain Development

The first engineered ORT strain was designed to function in the complex media that enable high plasmid yields. A kanamycin resistance gene under the control of the lac promoter–operator was inserted into the chromosome of E. coli DH1 to generate DH1lackan [13]. This ORT strain was not antibiotic-free itself, but did enable the selection of antibiotic resistance gene-free plasmids on nutrient agar plates containing kanamycin.

To develop a completely antibiotic-free ORT strain, the gene dapD was chosen as the conditionally essential gene. dapD encodes the enzyme tetrahydrodipicolinate N-succinyltransferase, which catalyzes a stage in the lysine–diaminopimelate biosynthetic pathway. Lysine is present in bacterial growth media, but diaminopimelic acid (DAP) is not, as it is an exclusively bacterial amino acid. DAP cross-links with peptidoglycan in the cell wall and is essential for maintaining the cell wall structure. Therefore, dapD mutants must be supplemented with DAP to prevent cell lysis. First, the dapD gene was deleted from the E. coli DH1 chromosome by insertion of a kanamycin resistance gene. Then a dapD gene under the control of the lac promoter–operator was inserted in a different locus, which created the strain DH1lacdapD that could be grown in the presence of the LacI inducer isopropyl-β-d-thiogalactopyranoside (IPTG) [14]. This strain enabled plasmid selection following transformation on nutrient agar plates with no additives required.

Untransformed DH1lacdapD was not able to form single colonies on nutrient agar plates – if it did, then selection by ORT would not have been possible. However, it was able to grow in nutrient broth without a plasmid, due to a low level of basal expression from the uninduced lac promoter, which raised the possibility that plasmids could be lost in theory. To address this, a second strain was constructed with a 2 bp mutation in the lac promoter that downregulated expression [14]. This strain DH1lacP2dapD was unable to grow on plates or in broth without a plasmid or inducer, resulting in stable plasmid maintenance. However, plasmids were also stably maintained in DH1lacdapD, perhaps due to the beneficial effect of a higher level of dapD expression than from any plasmid-free segregants. A reduction in plasmid copy number that precedes plasmid loss would result in an immediate reduction in dapD expression, which may place cells with higher plasmid copy at a selective advantage, unlike in competing systems where only complete plasmid loss leads to cell death.

The previously engineered strains all possess chromosomally integrated kanamycin resistance genes, so a new generation of E. coli ORT strains was constructed to be completely free of antibiotic resistance markers. These were created by simultaneous replacement of the wild-type dapD with a gene cassette containing lacI with dapD under the control of Plac and lacO. The second copy of lacI (additional to the wild-type copy in the lac operon) reduces the probability of a lacI mutation, which could prevent it from binding to lacO. Figure 2.2 shows replication of a lacO-containing plasmid in a standard laboratory strain, E. coli DH1, and its ORT derivative DH1-ORT that has the new chromosomal ORT composition. The plasmid constitutively expresses the chloramphenicol resistance gene to provide a constant metabolic burden, and the bacteria were inoculated into LB broth, cultured overnight, and transferred to fresh broth the following day, all in the absence of the antibiotic. This plasmid was rapidly lost from DH1, but maintained in DH1-ORT over the 4-day subculture study, illustrating the significant metabolic burden from selectable marker gene expression and the ability of ORT to stabilize plasmids that are unstable in other strains.

Figure 2.2 Plasmid stabilization by ORT. A high-copy-number plasmid expressing only the chloramphenicol resistance gene and possessing a lac operator sequence was replicated in E. coli strain DH1 and its ORT derivative DH1-ORT over 4-day subculture in LB broth. Plasmid DNA was extracted, subjected to agarose gel electrophoresis, and visualized under UV light.

The chromosome of DH1-ORT was free of antibiotic resistance genes as the Xer-cise™ technology was used to excise the chloramphenicol resistance gene cat that was present on the gene cassette to enable selection of integrant clones [15]. This was accomplished by flanking cat with dif sites, which are recombined by the natural recombinases XerC and XerD, thus deleting the intervening cat gene. The mechanism of ORT strain creation by Xer-cise is illustrated in Figure 2.3.

Figure 2.3 Construction of ORT strains using Xer-cise. New ORT strains are generated by replacement of the 5′-regulatory elements of the native essential gene with the repressor and promoter–operator sequences. These genes are constructed on a plasmid, adjacent to an antibiotic resistance gene flanked by dif sites and flanked by homology to the chromosomal target site. The Xer-cise cassette is cut out from the plasmid by a restriction digest (or is amplified by PCR) and is transformed into the bacterial cell, where the ORT cassette replaces the native sequences by homologous recombination, with integrant colonies selected on LB agar plates containing the antibiotic. Subsequent culturing in the absence of the antibiotic results in dif site-specific recombination and the deletion of the antibiotic resistance gene by native Xer recombinases.

2.5 ORT Miniplasmids

An ORT miniplasmid requires only two elements in addition to the transgene cassette: a bacterial origin of replication (ori) and an operator sequence. The removal of the antibiotic resistance gene typically reduces the size of the original plasmid by ~1.3 kb. The ubiquitous pUC-type pMB1 ori (a ColEI-like ori present in all high-copy-number plasmids) is approximately 600 bp, which restricts the bacterial DNA to a minimum in an ORT plasmid. For the most common clinical applications of plasmids – as DNA vaccines and precursors to viral vectors – immunostimulatory sequences of bacterial origin such as CpG islands are sometimes included to increase DNA vaccine efficacy. ORT plasmids have consistently provided potent immune responses to their transgenes in clinical trials (as described below), but are safe and well tolerated.

The operator sequence required on an ORT plasmid is a short, nonexpressed binding site for the repressor protein (the only selectable marker that does not require gene expression). The original lac operator sequences used in ORT plasmids were lacO1 and lacO3, each 21 bp and separated by 71 bp. The LacI repressor protein forms a tetramer and binds both sequences, with the intervening DNA forming a repression loop. The lacO1–lacO3 conformation was present on a number of pUC-derived plasmids used as early gene therapy or DNA vaccine vectors. To further reduce the plasmid size, a tight-binding, perfectly palindromic 20 bp variant of lacO1 (Ideal lacO) had been identified [16], and this was compared with the wild-type lacO1 and lacO1–lacO3 to determine if it could be used for ORT selection. This was done in both high-copy-number pUC-based and medium-copy-number pBR322-based plasmids, the latter being useful for recombinant protein production [17]. This study showed that medium-copy-number plasmids can be selected and stably replicated by ORT and that the transformation efficiency and plasmid maintenance was equivalent for all three sequence variants. Using expression of the natural chromosomal lacZ gene as a measure of repressor titration, it was demonstrated that the Ideal lacO produced a higher level of repressor titration than lacO1 on both medium- and high-copy-number plasmids.

To generate an ORT plasmid from a conventional antibiotic-selected plasmid, restriction endonuclease digestion is used to excise the antibiotic resistance gene and the remaining plasmid is separated from it by agarose gel electrophoresis. The plasmid DNA is then extracted from the gel, purified, and either self-ligated (if it already contains a lac operator) or ligated to a 20 bp Ideal lacO linker assembled by annealing complementary oligonucleotides. The ligation is then transformed into the ORT strain, with transformants selected on standard LB agar plates.

2.6 DNA Vaccine and Gene Therapy Vectors

ORT plasmids have been used as DNA vaccines in numerous preclinical studies and clinical trials. The ORT HIV-1 DNA vaccine pTHr.HIVA carries a synthetic, multiepitope HIV gene and was constructed by excision of the ampicillin resistance gene from pTH.HIVA, with a key advantage of ORT being that “DNA vaccination does not introduce into the human organism 1012 copies of an antibiotic resistance gene per each 1 μg of DNA delivered” [18]. Studies on plasmid biodistribution and persistence showed that pTHr.HIVA vaccine DNA could not be detected (other than at the injection site) 5 weeks after vaccination, indicating that it is a nonpersisting vector [19]. Phase I trials in the United Kingdom, Uganda, and Kenya demonstrated that the ORT plasmid was safe and well tolerated [20, 21]. In a phase I/IIa clinical trial when used as a prime to an MVA.HIVA boost, pTHr.HIVA induced multifunctional HIV-1-specific T cells in the majority of vaccinees, with the plasmid prime being superior to MVA alone [22]. These IAVI trials represented the first application of a prime-boost approach for an HIV vaccine, and are reviewed in Ref. [19]. A second synthetic HIV-1 immunogen, RENTA, was designed in ORT plasmid pTHr.RENTA and shown to broaden immune responses in combination with pTHr.HIVA and MVA antigen boosts in mice and rhesus macaques [23].

The Eurovacc Consortium tested an ORT HIV DNA vaccine, “DNA-C,” with a poxvirus (NYVAC-C) boost in a phase I clinical trial (EV02). An intramuscular dose of 4 mg ORT plasmid DNA was safe and well tolerated, and generated antigen-specific T-cell responses in 90% of vaccinees, with the DNA-C plasmid prime producing a significantly better response than NYVAC-C alone [24]. ORT has also been used to generate lentiviral vectors due to its ability to stabilize large plasmids that were unstable in conventional E. coli strains by removal of the antibiotic resistance gene. These smaller ORT plasmids also increase the transfection efficiency in mammalian packaging cell lines.

2.7 ORT-VAC: Plasmid-Based Vaccine Delivery Using Salmonella enterica

The use of live bacterial vaccines (LBVs) is an approach that enables oral vaccine delivery to the mucosal immune system. Conventional needle-based vaccination has a number of drawbacks, such as the cost and time taken to purify recombinant protein antigens, the public dislike of needles, the logistical challenges of organizing vaccination programs in developing countries, and the fact that many potential antigens do not produce a sufficient immune response. Therefore, an oral vaccine delivery system is highly desirable, particularly one with reduced costs and timescales. With the LBV approach, the bacteria are simply fermented, centrifuged, washed, lyophilized, and formulated to produce the final vaccine capsule, regardless of the antigen being delivered.

There is a growing body of work that describes a range of attenuated bacteria for vaccine delivery, with S. enterica being the most widely used vector [25]. S. enterica is a good choice as a vector for a number of reasons: it has a broad host range, attenuated strains are available that have been safely used in preclinical and clinical studies, and the invasion and colonization of S. enterica serovar Typhimurium in mice is equivalent to that of serovar Typhi in humans. Figure 2.4 shows how enteric bacteria such as Salmonella and Shigella are able to invade the lining of the gastrointestinal tract via microfold cells and enter the lymphatic nodules called Peyer's patches. Here they invade (or are phagocytosed by) antigen-presenting cells. Shigella can escape into the cytoplasm, but Salmonella remains in the phagosome where it alters the membrane and can survive and multiply until it is eventually digested by fusion with a lysozome. This strategy enables MHC class I and II presentation of antigens expressed from plasmids (Figure 2.4) [26].

Figure 2.4 Oral delivery of recombinant vaccines using attenuated bacteria. The live bacteria carrying the recombinant antigen are delivered orally, transit the stomach, and enter the small intestine. Here they invade the lining of the intestinal tract via microfold (M) cells, enter Peyer's patches, and invade antigen-presenting cells. Salmonella remain within the phagosome, while Shigella and Listeria can escape to the cytoplasm. Antigens are released by secretion and/or cell lysis for MHC presentation.

S. enterica is able to replicate E. coli protein or DNA vaccine plasmids, but plasmid instability is a major problem – plasmids are often lost within a few generations. Even if antibiotics could be applied to the host, the use of all antibiotic resistance genes with LBVs is banned by the FDA and other regulators. To solve this instability problem, the genes enabling ORT to function in E. coli were transferred to S. enterica serovar Typhimurium, with the application of this technology to LBVs termed “ORT-VAC.” The first ORT-VAC strain, SLDAPD, was constructed from the Typhimurium strain SL3261, an aroA mutant unable to synthesize aromatic amino acids and related compounds [27]. To demonstrate plasmid maintenance, a high-copy-number plasmid pUC18I containing the ampicillin resistance gene was transformed into both the unmodified SL3261 and ORT-VAC SLDAPD, and approximately 109colony-forming units (CFU) orally administered to Balb/c mice, in addition to untransformed SL3261. Mice were sacrificed periodically and bacteria extracted from the spleens and Peyer's patches. The proportion of cells containing the plasmid was determined by growth in the presence of ampicillin. This revealed that plasmids were retained in 97% of spleen- and 90% of Peyer's patches-derived SLDAPD compared to 58% of spleen- and 3% of Peyer's patches-derived SL3261 (mean values from three time points), illustrating the greater plasmid stability in the ORT-VAC strain. Figure 2.5 shows the plasmid retention in Peyer's patches over a 16-day period. An ampicillin resistance gene was included to facilitate the identification of cells containing the plasmid, but the effects of the resultant metabolic burden were apparent from a significantly lower CFU in the spleen from both plasmid-bearing strains compared to the control of untransformed SL3261 [27].

Figure 2.5In vivo plasmid stability. The S. enterica serovar Typhimurium strains SL3261 and ORT-VAC derivative strain SLDAPD, both containing the plasmid pUC18I, were orally administered to mice at 109 CFU. The proportion of plasmid-containing bacterial cells from extracted Peyer's patches was recorded at 8, 12, and 16 days postinoculation.

To investigate the vaccine potential of ORT-VAC, an ORT (i.e., antibiotic resistance gene-free, lacO-containing) plasmid pAHL was constructed that expressed the F1 antigen from Yersinia pestis on the surface of SLDAPD [27]. The promoter was regulated by temperature, with no expression at 28 °C (enabling culture at this temperature with a reduced metabolic burden) and induction at around 37 °C for in vivo F1 expression. This strain was orally administered to mice in one or two doses, with mice challenged after 50 days with a low (against the single immunization) or high (against the double immunization) median lethal dose of Y. pestis. The protection conveyed by the oral ORT-VAC immunization was in the range of 83–100%. While all the naïve mice succumbed to the challenge, a low level of protection was seen in mice given the Salmonella without a vaccine plasmid, demonstrating the effectiveness of the inherent adjuvanticity of the vector alone in stimulating an innate immune response.

When delivering a gene expressing a recombinant protein in an LBV, the alternative approach to using a plasmid is to insert the gene into the chromosome. This has clear benefits of stability for a single copy, but tandem copies can be unstable due to homologous recombination or replication slippage, and introducing multiple insertions at defined locations is a lengthy process, with integrated genes vulnerable to the accumulation of mutations. We therefore sought to test the efficacy of an antigen expressed as a single-copy, chromosomally integrated cassette against the same antigen replicated on a multicopy plasmid. The antigen chosen for this was the protective antigen (PA) of Bacillus anthracis, which is a component of the tripartite anthrax toxin (in addition to the lethal and edema factors). A gene cassette was created with the protective antigen gene pagA fused at its 3′-end to the hemolysin gene hlyA, with the genes of the hemolysin export system downstream to enable secretion of the PA-HlyA protein via pore formation [28]. Gene expression is regulated by the PpagC promoter [29], which is part of the PhoP/Q two-component regulator system. This is only induced in vivo when the Salmonella are in the phagosome of the antigen-presenting cell. This environment enables expression from PpagC due to low concentrations of magnesium ions, which is a condition that can be replicated in vitro. Therefore, the metabolic burden of antigen expression is reduced to a minimum.

This cassette was integrated into the chromosome of the S. enterica serovar Typhimurium strain Zoosaloral H and cloned into a plasmid with the pSC101 origin of replication, giving a copy number of approximately 15 per cell in a Zoosaloral H-derived ORT-VAC strain. In vitro expression studies showed that, as expected, more PA was produced from the ORT-VAC plasmid than the single-copy integrated gene (Figure 2.6a). Following a three-dose oral immunization of mice with the unmodified, single-copy, and ORT-VAC strains (and all boosted with 5 μg PA protein), the mice were challenged with a 50 median lethal dose of B. anthracis spores on day 70. None of the naïve mice survived the challenge, but ORT-VAC provided 90% protection, corresponding to the higher PA-specific IgG levels from this strain. The single-copy PA cassette was equivalent to the unmodified bacterium, with protection in the range of 30–40% (Figure 2.6b). This illustrates that the plasmid-based ORT-VAC approach is superior to chromosomal integration for antigen production and in the case of PA gave superior protection [28].

Figure 2.6 Immunization and protection against an anthrax challenge. The relative concentration of secreted anthrax-protective antigen was determined by Western blotting from equal cell numbers of S. enterica serovar Typhimurium integrant (single chromosomal gene) and ORT-VAC (plasmid-bearing) strains from cell pellet and supernatant samples (a). Mice were orally immunized with the unmodified, integrant, and ORT-VAC strains and challenged with a lethal dose of anthrax spores, and percentage survival is displayed (b).

ORT-VAC miniplasmids are effective recombinant protein antigen vectors, but there are antigens for which bacterial expression is not suitable, for instance, if glycosylation or secondary structure is important. The solution to this is to use a DNA vaccine. These are all based on the high-copy-number pUC-type pMB1 ori, and while Salmonella can replicate these plasmids, they are frequently unstable as shown in Figure 2.5. The application of ORT-VAC stabilizes these plasmids, so experiments were performed to test if DNA vaccine delivery is possible using ORT-VAC Salmonella. The disease model chosen was tuberculosis, with the antigen being a secreted protein of unknown function called Mpt64. This was cloned into an ORT DNA vaccine plasmid, transformed into SLDAPD, and orally administered to mice in a single dose at 107–109 CFU. As a positive control, 100 μg of plasmid DNA (produced in E. coli) was injected intramuscularly [30]. The T-cell response is the most important factor for a TB vaccine, and measurements by IFN-γ ELISA and ELISpot showed that the ORT-VAC-vectored vaccine produced significantly greater responses than the plasmid DNA (Figure 2.7). This resulted in a statistically significant reduction in the Mycobacterium tuberculosis CFU in the lungs of mice infected by an aerogenic challenge from a single-dose ORT-VAC oral immunization, which was not seen with the intramuscular plasmid DNA.

Figure 2.7 TB DNA vaccine delivery using ORT-VAC. Mice were orally immunized with three concentrations of ORT-VAC strain SLDAPD carrying a plasmid designed to express the TB antigen gene mpt64 from the PCMV promoter. A positive control group was immunized using 100 μg plasmid DNA by intramuscular injection. Following stimulation of extracted splenocytes with the medium (negative control) or the MPT64 protein, the levels of antigen-specific T-cell responses were quantified by IFN-γ ELISpot.

There have been other reports of Salmonella being used with DNA vaccines, but there is currently no clear mechanism describing how this can occur. From the accepted model of antigen presentation using LBVs in Figure 2.4, Salmonella cannot escape from the phagosome into the cytoplasm like Shigella or Listeria can, so any plasmid DNA that they carry should be digested upon fusion with the lysozome. However, sufficient DNA must enter the cytoplasm, and then the nucleus, for the antigen to be expressed in sufficient quantities to stimulate an immune response.

2.8 Recombinant Protein Expression

The advantages that ORT brings to DNA production are also beneficial in microbial recombinant protein expression. Plasmid loss during fermentation leads to a reduction in protein yield, with the metabolic burden of constitutive selectable marker gene replication and expression being significant contributing factors in plasmid instability through metabolic burden. In addition to selectable marker genes, protein expression plasmids frequently contain repressor genes that are constitutively expressed. ORT strains have been used to manufacture several recombinant proteins, including the enzyme fuculose-1-phosphate aldolase, which was produced in high-density fermentations to 30% of dry cell mass with no plasmid loss or significant reduction in growth rate compared to the uninduced strain [31].

2.9 Conclusions and Future Developments

ORT has been evaluated extensively in the enteric bacteria E. coli and S. enterica