Minicircle and Miniplasmid DNA Vectors

Edited by Martin Schleef

The Future of Non-Viral and Viral Gene Transfer

Edited by

Martin Schleef

Minicircle and MiniplasmidDNA Vectors

Related Titles

Schleef, M. (ed.)

DNA Pharmaceuticals2005

ISBN: 978-3-527-31187-3

Prazeres, D.M.F.

Plasmid BiopharmaceuticalsBasics, Applications, and Manufacturing

2011

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

CIBA Foundation Symposium

Bacterial Episomes and Plasmids2009

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

Biotechnology Journalwww.biotechnol ogy-journal.com

Edited by Martin Schleef

Minicircle and Miniplasmid DNA Vectors

The Future of Nonviral and Viral Gene Transfer

The Editor

Dr. Martin SchleefPlasmidFactory GmbH & Co. KGMeisenstra�e 9633607 BielefeldGermany

Limit of Liability/Disclaimer of Warranty:While the publisherand author have used their best efforts in preparing this book,they make no representations or warranties with respect to theaccuracy or completeness of the contents of this book andspecifically disclaim any implied warranties of merchantabilityor fitness for a particular purpose. No warranty can be createdor extended by sales representatives or written sales materials.The Advice and strategies contained herein may not besuitable for your situation. You should consult with aprofessional where appropriate. Neither the publisher norauthors shall be liable for any loss of profit or any othercommercial damages, including but not limited to special,incidental, consequential, or other damages.

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This book is dedicated to Marion & Mariella

Contents

List of Contributors XIIIPerface XXI

1 Minicircle Patents: A Short IP Overview of Optimizing Nonviral DNAVectors 1Martin Grund and Martin SchleefReferences 6

2 Operator–Repressor Titration: Stable Plasmid Maintenance withoutSelectable Marker Genes 7Rocky M. Cranenburgh

2.1 Introduction 72.2 Antibiotics and Metabolic Burden 72.3 The Mechanism of ORT 82.4 ORT Strain Development 92.5 ORT Miniplasmids 122.6 DNA Vaccine and Gene Therapy Vectors 132.7 ORT-VAC: Plasmid-Based Vaccine Delivery Using Salmonella

enterica 142.8 Recombinant Protein Expression 182.9 Conclusions and Future Developments 19

References 19

3 Selection by RNA–RNA Interaction: Maximally Minimized AntibioticResistance-Free Plasmids 23Juergen Mairhofer and Reingard Grabherr

3.1 Gene Therapy and DNAVaccines: Emerging Technologies 233.1.1 Therapeutic Plasmids: General Design Principles 243.2 Therapeutic Plasmids: Novel Design and the Problem of Selection 253.2.1 Replication Control of ColE1-Type Plasmids as an Alternative

Selection Marker 263.2.2 The MINIback Concept: Selection by RNA–RNA Interaction 283.2.3 Improved Production Processes by MINIback Plasmids 30

jVII

3.2.4 Improving Sequence Composition 323.2.5 Efficient Gene Transfer 323.3 Conclusions 33

Acknowledgments 33References 33

4 Plasmid-Based Medicinal Products – Focus on pFAR:A Miniplasmid Free of Antibiotic Resistance Markers 37Corinne Marie, Micka€el Quiviger, Helen Foster,George Dickson, and Daniel Scherman

4.1 Introduction: Rationale for the Development of Biosafe DNA PlasmidVectors 37

4.2 Specific Requirements for the Use of DNA Product as Medicines 394.2.1 Requirements for Plasmid Quality and Purity 394.2.2 Requirements for the Removal of Antibiotic Resistance Markers from

Plasmid DNA 404.2.2.1 Requirements for Biosafe Plasmids 404.2.2.2 Positive Impact on the Removal of Antibiotic Resistance Markers 414.2.2.3 Effect of Plasmid Size on Gene Transfer Efficiency In Vitro and

In Vivo 414.3 Nonviral Gene Vectors Devoid of Antibiotic Resistance Markers 434.3.1 Generalities 434.3.2 Selection Systems Devoid of Antibiotic Resistance Markers 434.3.2.1 Complementation of Host Auxotrophy by a Function-Encoded

Plasmid 434.3.2.2 The Operator–Repressor Titration (ORT) System 444.3.2.3 Protein-Based Antidote/Poison Selection Systems 444.3.2.4 RNA-Based Selection Marker 444.3.2.5 Suppression of a Nonsense Mutation 454.4 The pFAR Plasmid Family 464.4.1 Description of the Antibiotic-Free Selection System 464.4.2 pFAR Vectors Promote Efficient Expression in Several Types

of Mammalian Cells 494.4.2.1 In Vitro Transfection Study 494.4.2.2 In Vivo Transfection Studies 504.4.3 Concluding Remarks on the pFAR4 Biosafe Miniplasmid 514.5 Concluding Remarks and Perspectives 52

Acknowledgments 53References 53

5 Plasmid DNA Concatemers: Influence of Plasmid Structureon Transfection Efficiency 59Christof Maucksch, Bronwen Connor, and Carsten Rudolph

5.1 Introduction 595.2 Plasmid DNATopology and Size 60

VIIIj Contents

5.3 Plasmid DNA Concatemers 625.4 Conclusions 67

Acknowledgments 67References 68

6 Analytical Tools in Minicircle Production 71Anja Rischm€uller, Martina Viefhues, Mareike Dieding, Markus Blaesen,Marco Schmeer, Ruth Baier, Dario Anselmetti, and Martin Schleef

6.1 Introduction 716.1.1 Gene Transfer for Therapy, Vaccination, and Stem Cells 716.1.2 Plasmids 726.1.3 Minicircle Systems 736.2 Production of Minicircles 746.2.1 The Parental Plasmid 746.2.2 Cultivation and Induction 746.2.3 Minicircle Preparation 776.3 Analytics of Minicircle Production 796.3.1 In-Process Control 796.3.1.1 Atomic Force Microscopy 796.3.1.2 Capillary Gel Electrophoresis 816.3.1.3 Continuous Flow Separation in Microfluidic Channels 826.3.2 Finished Product Control 866.4 Future Goals 88

Acknowledgments 88References 89

7 Utilizing Minicircle Vectors for the Episomal Modification of Cells 93Orestis Argyros, Suet-Ping Wong, Charles Coutelle, and Richard P. Harbottle

7.1 Introduction 937.2 Studies that Show Passive Episomal Maintenance of Minicircles

In Vivo 947.3 Principles of Generating Minicircle Vectors Able to Support Episomal

Maintenance 977.3.1 Episomal Maintenance of Minicircle S/MAR Vectors Generated by Flp

Recombinase In Vitro 987.3.2 Episomal Maintenance of Minicircle S/MAR Vectors Generated Using

Cre Recombinase In Vitro 997.3.3 Episomal Maintenance of S/MAR Vectors in Bovine and Murine

Zygotes 1017.4 Episomal Maintenance of S/MAR Minicircles In Vivo 1027.5 Potential of Episomal Replication of S/MAR Minicircle Vectors 1047.6 Possible Mechanisms Promoting the Episomal Maintenance

of Minicircle Vectors 1057.6.1 Histone Modifications 1067.6.2 CpG Dinucleotide Content Reduction 106

Contents jIX

7.6.3 Vector Establishment in the Correct Nuclear Compartment 1077.6.4 Access to Replication Machinery by S/MARs 1077.7 Conclusions 110

References 110

8 Replicating Minicircles: Overcoming the Limitations of Transient andStable Expression Systems 115Kristina Nehlsen, Sandra Broll, Raju Kandimalla, Niels Heinz, MarkusHeine, Stefanie Binius, Axel Schambach, and J€urgen Bode

8.1 Gene Therapy: The Advent of Novel Vector Vehicles 1158.1.1 Nonviral Vectors Avoiding Genomic Disturbances 1168.1.2 Independent Expression Units: Chromatin Domains 1168.1.2.1 S/MARs: a Unifying Principle 1188.1.2.2 S/MAR Actions Are Multifold and Context Dependent 1198.1.2.3 Stress-Induced Duplex Destabilization: a Unifying Property

of S/MARs 1218.1.2.4 Chromosome-Based Expression Strategies: Episomes and/or

Predetermined Integration Sites (RMCE) 1238.2 Replicating Nonviral Episomes 1238.2.1 Can the Yeast ARS Principle Be Verified for

Mammalian Cells? 1258.2.2 ARS and S/MARs: Common (SIDD-) Properties 1258.2.3 S/MAR Plasmids: Verification of the Concept 1268.2.3.1 Transcription into the S/MAR: Directionality and Rate 1268.2.3.2 Cell and Nuclear Permeation 1288.2.3.3 Nuclear Association Sites 1298.2.3.4 RMCE-Based Elaboration Following Establishment 1308.2.4 Remaining Shortcomings and Their Solution 1328.2.4.1 Establishment and Maintenance: the EBV Paradigm 1328.2.4.2 Vector Size Limitations 1368.3 Minimalization Approaches 1378.3.1 Oligomerizing S/MAR Modules: pMARS and Its Properties 1398.3.2 Replicating Minicircles: a Solution with Great Promise 1408.3.2.1 Establishment and Maintenance Parameters 1418.3.2.2 Clonal Behavior 1418.3.2.3 Bi-MC Systems 1438.3.2.4 MC Size Reduction: “In Vivo Evolution” 1448.3.2.5 Transcriptional Termination and Polyadenylation: an Intricate

Interplay 1468.3.2.6 Episomal Status: Proof and Persistence 1478.3.3 Emerging Extensions and Refinements 1498.3.3.1 Combination of Excision and RMCE Strategies 1518.3.3.2 MC Withdrawal at Will 1538.3.3.3 Pronuclear Injection and Somatic Cell Nuclear Transfer 1558.3.3.4 From Cells to Organs 155

Xj Contents

8.4 Summary and Outlook 156Acknowledgments 157References 158

9 Magnetofection of Minicircle DNA Vectors 165Flavie Sicard, Cedric Sapet, Nicolas Laurent, Elodie Bertosio, MelanieBertuzzi, and Olivier Zelphati

9.1 Introduction 1659.2 Overview of Magnetofection Principles 1679.3 Cellular Uptake 1689.4 Diffusion through the Cytoplasm 1699.5 Transgene Expression 1699.6 Conclusions 172

References 173

10 Minicircle-Based Vectors for Nonviral Gene Therapy: In VitroCharacterization and In Vivo Application 177Dennis Kobelt, Jutta Aumann, Martin Schleef, Marco Schmeer,Ulrike Stein, Peter M. Schlag, and Wolfgang Walther

10.1 Minicircle Technology for Nonviral Gene Therapy 17710.2 Current Status of In Vivo Application of Minicircle Vectors 17810.3 Jet Injection Technology for In Vivo Transfer of Naked DNA 18010.4 Comparative Performance Analyses of Minicircle Vectors 18310.5 In Vivo Application of Minicircle DNA by Jet Injection 185

References 186

11 Episomal Expression of Minicircles and Conventional Plasmidsin Mammalian Embryos 189Wiebke Garrels, Khursheed Iqbal, and Wilfried A. Kues

11.1 Introduction 18911.2 Fate of Plasmids and Minicircles After Injection into Mammalian

Embryos 19111.2.1 Minicircle- and Plasmid-Mediated Expression in Early Embryos

and Fetuses 19111.2.2 Expression of Functional Genes in Preimplantation Embryos 19511.3 Discussion 198

References 199

12 Tissue-Targeted Gene Electrodelivery of Minicircle DNA 203Sophie Chabot, Muriel Golzio, and Justin Teissi�e

12.1 Introduction 20312.2 Plasmid DNA Electrotransfer: From Principle to Technical Design 20412.2.1 Mechanism of Gene Electrotransfer 20412.2.2 Preclinical Applications 20512.3 Implementation for Efficient Tissue-Targeted Gene Delivery 206

Contents jXI

12.3.1 Design of DNA Vector 20612.3.2 In VitroMinicircle Electrotransfer 20612.3.3 In VivoMC Electrotransfer 20712.3.3.1 Muscle 20712.3.3.2 Tumor 20812.3.3.3 Skin 20912.4 Conclusions 209

Acknowledgments 209References 210

13 Increased Efficiency of Minicircles Versus Plasmids Under GeneElectrotransfer Suboptimal Conditions: an Influence of the ExtracellularMatrix 215Vanessa Joubert, Franck M. Andr�e, Marco Schmeer, Martin Schleef,and Lluis M. Mir

13.1 Introduction 21513.2 Methods 21513.2.1 Cell Culture and Animals 21513.2.2 Minicircle and Plasmid 21613.2.3 Electrotransfer 21613.2.4 Determination of the Reporter Gene (Luciferase) Activity 21613.2.5 Data Analysis 21713.3 Results 21713.3.1 In Vitro 21713.3.2 In Vivo 21813.4 Discussion 22013.5 Conclusions 223

Acknowledgments 224References 224

Index 227

XIIj Contents

List of Contributors

Franck M. Andr�eCNRSLaboratoire de Vectorologie etTh�erapeutiques Anticanc�ereusesUMR 820391405 OrsayFrance

and

Universit�e Paris-SudLaboratoire de Vectorologie etTh�erapeutiques Anticanc�ereusesUMR 820391405 OrsayFrance

and

Institut Gustave RoussyLaboratoire de Vectorologie etTh�erapeutiques Anticanc�ereusesUMR8203, 114 rue Edouard Vaillant94805 VillejuifFrance

Dario AnselmettiBielefeld UniversityPhysics FacultyExperimental Biophysics and AppliedNanoscienceUniversit€atsstr. 2533615 BielefeldGermany

Orestis ArgyrosImperial College LondonNational Heart and Lung InstituteSection of Molecular MedicineGene Therapy GroupImperial College RoadSouth KensingtonLondon SW7 2AZUK

Jutta AumannCharit�e University Medicine BerlinExperimental and Clinical ResearchCenterRobert-R€ossle-Str. 1013125 BerlinGermany

Ruth BaierPlasmidFactory GmbH & Co. KGDept. DNA ProductionMeisenstr. 9633607 BielefeldGermany

Elodie BertosioOZ Biosciences R&D departmentParc Scientifique de Luminy, ZoneLuminy Entreprise163 Avenue de Luminy, Case 92213288 Marseille Cedex 9France

jXIII

Melanie BertuzziOZ Biosciences R&D departmentParc Scientifique de Luminy, ZoneLuminy Entreprise163 Avenue de Luminy, Case 92213288 Marseille Cedex 9France

Stefanie BiniusHelmholtz Center for InfectionResearchDepartment MolecularBiotechnologyInhoffenstra�e 738124 BraunschweigGermany

Markus BlaesenPlasmidFactory GmbH & Co. KGDept. DNA ProductionMeisenstr. 9633607 BielefeldGermany

J€urgen Bode�

Hannover Medical School (MHH)Institute for ExperimentalHaematologyOE 6960, Carl-Neuberg-Strasse 130625 HannoverGermany

Sandra BrollHelmholtz Center for InfectionResearchDepartment MolecularBiotechnologyInhoffenstra�e 738124 BraunschweigGermany

and

Leibniz Universit€at HannoverDezernat 4 – Forschung undTechnologietransfer/NationaleForschungsf€orderungSchlo�wender Str. 130159 HannoverGermany

Sophie ChabotCentre National de la RechercheScientifiqueInstitut de Pharmacologie et deBiologie StructuraleBP 64182, 205 route de Narbonne31077 ToulouseFrance

and

Universit�e de ToulouseUPS, IPBS31077 ToulouseFrance

Bronwen ConnorUniversity of AucklandFaculty of Medical and HealthSciencesCentre for Brain ResearchDepartment of Pharmacology &Clinical PharmacologyAucklandNew Zealand

Charles CoutelleImperial College LondonNational Heart and Lung InstituteSection of Molecular MedicineGene Therapy GroupImperial College RoadSouth KensingtonLondon SW7 2AZUK

�Corresponding author

XIVj List of Contributors

Rocky M. Cranenburgh�

CobraBiologics Ltd andProkariumLtdStephenson BuildingKeele Science ParkKeele, Staffordshire ST5 5SPUK

George DicksonRoyal Holloway University of LondonSchool of Biological SciencesEgham, Surrey TW20 0EXUK

Mareike DiedingBielefeld UniversityPhysics FacultyExperimental Biophysics and AppliedNanoscienceUniversit€atsstr. 2533615 BielefeldGermany

Helen FosterRoyal Holloway University of LondonSchool of Biological SciencesEgham, Surrey TW20 0EXUK

Wiebke GarrelsFriedrich Loeffler InstituteInstitute of Farm Animal GeneticsDepartment of BiotechnologyMarienseeHöltystr. 1031535 NeustadtGermany

Muriel GolzioCentre National de la RechercheScientifiqueInstitut de Pharmacologie et deBiologie StructuraleBP 64182, 205 route de Narbonne31077 ToulouseFrance

and

Universit�e de ToulouseUPS, IPBS31077 ToulouseFrance

Reingard GrabherrUniversity of Natural Resources andLife SciencesDepartment of BiotechnologyMuthgasse 181190 ViennaAustria

Martin Grund�

GRUND Intellectual Property GroupNikolaistrasse 1580802 M€unchenGermany

Richard P. Harbottle�

Imperial College LondonNational Heart and Lung InstituteSection of Molecular MedicineGene Therapy GroupImperial College RoadSouth KensingtonLondon SW7 2AZUK

Markus HeineRentschler Biotechnologie GmbH“Bioprocess Development”/“Virus-based Biologics”Erwin-Rentschler-Strasse 2188471 LaupheimGermany

Niels HeinzHannover Medical School (MHH)Institute for ExperimentalHaematologyOE 6960, Carl-Neuberg-Strasse 130625 HannoverGermany�Corresponding author

List of Contributors jXV

Khursheed IqbalBeckman Research Institute of Cityof HopeArnold andMabel Beckman ResearchCenterDepartment of Molecular andCellular BiologyDuarte, CA 91010USA

Vanessa JoubertCERPEMMaison de la Technopole6 rue Leonard de Vinci53000 LavalFrance

Raju KandimallaErasmus MCJosephine Nefkens InstituteDepartment of PathologyDr. Molewaterplein 503000 CA RotterdamThe Netherlands

Dennis KobeltMax Delbr€uck Center for MolecularMedicineRobert-R€ossle-Str. 1013125 BerlinGermany

Wilfried A. Kues�

Friedrich Loeffler InstituteInstitute of Farm Animal GeneticsDepartment of BiotechnologyMarienseeHöltystr. 1031535 NeustadtGermany

Nicolas LaurentOZ Biosciences R&D departmentParc Scientifique de Luminy, ZoneLuminy Entreprise163 Avenue de Luminy, Case 92213288 Marseille Cedex 9France

J€urgen Mairhofer�

University of Natural Resources andLife SciencesDepartment of BiotechnologyMuthgasse 181190 ViennaAustria

Corinne MarieUniversit�e Paris DescartesFacult�e de PharmacieUnit�e de Pharmacologie Chimique etG�en�etique et d’ImagerieEcole Nationale Sup�erieure deChimie de ParisCNRS UMR8151, INSERM U1022ParisFrance

Christof Maucksch�

University of AucklandFaculty of Medical and HealthSciencesCentre for Brain ResearchDepartment of Pharmacology &Clinical PharmacologyAucklandNew Zealand

Lluis M. Mir�

CNRSLaboratoire de Vectorologie etTh�erapeutiques Anticanc�ereusesUMR 820391405 OrsayFrance

�Corresponding author

XVIj List of Contributors

and

Universit�e Paris-SudLaboratoire de Vectorologie etTh�erapeutiques Anticanc�ereusesUMR 820391405 OrsayFrance

and

Institut Gustave RoussyLaboratoire de Vectorologie etTh�erapeutiques Anticanc�ereusesUMR 8203114 rue Edouard Vaillant94805 VillejuifFrance

Kristina NehlsenHelmholtz Center for InfectionResearchDepartment MolecularBiotechnologyInhoffenstra�e 738124 BraunschweigGermany

Micka€el QuivigerUniversit�e Paris DescartesFacult�e de PharmacieUnit�e de Pharmacologie Chimique etG�en�etique et d’ImagerieEcole Nationale Sup�erieure deChimie de ParisCNRS UMR8151, INSERM U1022ParisFrance

Anja Rischm€ullerPlasmidFactory GmbH & Co. KGDept. R & DMeisenstr. 9633607 BielefeldGermany

and

Bielefeld UniversityPhysics FacultyExperimental Biophysics and AppliedNanoscienceUniversit€atsstr. 2533615 BielefeldGermany

Carsten RudolphLudwig-Maximilians UniversityDepartment of PediatricsLindwurmstrasse 2a80337 MunichGermany

and

ethris GmbHLochhamerstr. 1182152 MartinsriedGermany

Cedric SapetOZ Biosciences R&D departmentParc Scientifique de Luminy, ZoneLuminy Entreprise163 Avenue de Luminy, Case 92213288 Marseille Cedex 9France

Axel SchambachHannover Medical School (MHH)Institute for ExperimentalHaematologyOE 6960, Carl-Neuberg-Strasse 130625 HannoverGermany

List of Contributors jXVII

Daniel Scherman�

Universit�e Paris DescartesFacult�e de PharmacieUnit�e de Pharmacologie Chimique etG�en�etique et d’ImagerieEcole Nationale Sup�erieure deChimie de ParisCNRS UMR8151, INSERM U1022ParisFrance

Peter M. SchlagCharit�e Comprehensive CancerCenterInvalidenstr. 8010117 BerlinGermany

Martin Schleef �

PlasmidFactory GmbH & Co. KGDept. R & DMeisenstr. 9633607 BielefeldGermany

Marco SchmeerPlasmidFactory GmbH & Co. KGDept. Process DevelopmentMeisenstr. 9633607 BielefeldGermany

Flavie SicardOZ Biosciences R&D departmentParc Scientifique de Luminy, ZoneLuminy Entreprise163 Avenue de Luminy, Case 92213288 Marseille Cedex 9France

Ulrike SteinMax Delbr€uck Center for MolecularMedicineRobert-R€ossle-Str. 1013125 BerlinGermany

and

Charit�e University Medicine BerlinExperimental and Clinical ResearchCenterRobert-R€ossle-Str. 1013125 BerlinGermany

Justin Teissi�e�

Centre National de la RechercheScientifiqueInstitut de Pharmacologie et deBiologie StructuraleBP 64182, 205 route de Narbonne31077 ToulouseFrance

and

Universit�e de ToulouseUPS, IPBS31077 ToulouseFrance

Martina ViefhuesBielefeld UniversityPhysics FacultyExperimental Biophysics and AppliedNanoscienceUniversit€atsstr. 2533615 BielefeldGermany

Wolfgang Walther�

Max Delbr€uck Center for MolecularMedicineRobert-R€ossle-Str. 1013125 BerlinGermany

�Corresponding author

XVIIIj List of Contributors

and

Charit�e University Medicine BerlinExperimental and Clinical ResearchCenterRobert-R€ossle-Str. 1013125 BerlinGermany

Suet-Ping WongImperial College LondonNational Heart and Lung InstituteSection of Molecular MedicineGene Therapy Group

Imperial College RoadSouth KensingtonLondon SW7 2AZUK

Olivier Zelphati�

OZ Biosciences R&D departmentParc Scientifique de Luminy, ZoneLuminy Entreprise163 Avenue de Luminy, Case 92213288 Marseille Cedex 9France

�Corresponding author

List of Contributors jXIX

Preface

After significant improvements in the field of non-viral vector development, wecompiled the status of those “new plasmids” within this book. The tools for gene- orcell therapy and DNA vaccination are available in form of pure genetic material(DNA, RNA) or within more complex units (viral vectors, VLP, aggregates contain-ing 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, theirstructure and functionality including regulatory aspects and those of making them.Later we focussed on the route of administration, pharmaceutical DNA and specificfeatures (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 totheir lower efficiency compared to viral vectors. As shown within certain chapters ofthis book, the efficiency for mini plasmids and minicircle DNA is significantlyhigher simply because of their smaller size and in some cases also due to containedsequence elements. The safety aspect of plasmids without any antibiotic resistancemarker (“mini plasmids”) or – even smaller and with less CpG motifs – in additionwithout the bacterial backbone with the origin of replication (“mincircles”) is amajor advantage for any application where such sequences need to be avoided. Forall those who like to further discuss these aspects I look forward to do so at any time(martin.schleef@plasmidfactory.com).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 forwhat we are doing – you should go on with this – we are highly motivated even bydifficult questions. Finally, my special thank goes to all Authors participating in thisbook, the teams of the publisher, the PlasmidFactory team for support and discus-sion and all friends.

Bielefeld, January 2013 Martin Schleef

jXXI

1Minicircle Patents: A Short IP Overview of OptimizingNonviral DNA VectorsMartin Grund and Martin Schleef

The use of nonviral vectors for gene and cell therapy and especially for vaccinationstarted with the observation of Wolff et al. [1] that the direct application of plasmidDNA containing the expression cassette for a protein into animal muscle led to theexpression of this and – subsequently – to the appearance of antibodies against thisprotein – the idea of a DNA vaccine. While this was initially done with standardcloning or gene expression plasmids typically driven by a CMV promoter, its use inpharmaceutical context required the improvement of the structure of the plasmidwith respect to the coding sequence (e.g., codon usage) and also concerning the totalmolecule: starting from the removal of abundant sequences (e.g., multiple cloningsite residues) and the replacement of the antibiotic resistance gene bla (forampicillin resistance) by a kanamycin resistance up to the removal of CpG motifsfrom the coding and backbone sequence [2]. Also, the physical structure of plasmidvectors was modulated by using process technology to obtain exclusively ccc-supercoiled DNA through specific cultivation technology [3] or purificationprocesses [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 thisbook (see Chapters 6–13). However, a selection marker was still present on theplasmid, and also the large sequence element responsible for the plasmid replica-tion (bacterial origin of replication – ori [6]) was still there.Themajor improvements to further reducing the size and – by the way – removing

the nonintended backbone sequences, including the ori, weremade by approaches toreducing the DNA molecules carrying the pharmaceutically required expressionunit to (mainly) circular structures with almost no other sequence than the sequenceof 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 dateOctober 16, 1992, and published as WO 94/09127. The application was subse-quently withdrawn in November 1994, and no patents were granted. Claim 1

Minicircle and Miniplasmid DNA Vectors: The Future of Nonviral and Viral Gene Transfer,First Edition. Edited by Martin Schleef# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

j1

referred to a DNA construct comprising attB and attP sites with a multiple cloningsite (in a later application, by Bigger et al. in 2001 (US application 11/249929), alsocalled “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. Sincethe construct thus formed was not supposed to contain a resistance gene or anorigin of replication, it can be regarded as aminicircle, and the very termwas in factcoined in this application. The intention of the inventors was, however, quitedifferent from the gene vector and therapeutic approaches that have characterizedlater minicircle applications. In fact, the aim of these first constructs was to studythe kinetics of promoters, to which end a construct containing only a singlepromoter 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 ofpatents. A further approach was submitted by Seeber and Kr€uger, with priority dateAugust 11, 1994, and published as WO 96/05297. The application led to the grant ofpatents in Europe (EP 0775203) and the United States (US Patent 6,573,100), whichare still in force and directed to the use of minicircles in therapy. The inventorsintended to remove the resistance gene bla from a circular plasmid vector by site-specific recombinase (SSR) systems by dividing the circular plasmid into twocircles – one containing the gene cassette and the other the residual portionincluding bla. The growth of the plasmid was performed under selective pressureand the two circles were separated by chromatography. The recombination systemproposed was, for example, FLP/FRT. The major field of intended application wasthe 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 withpriority date February 23, 1995, and Cameron et al. as inventors. The applicationresulted in granted patents in Europe (EP 0815214), the United States (US Patents6,143,530 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 iscircular and supercoiled; (b) it contains an expression cassette under control of amammalian promoter; (c) it does not contain an origin of replication; (d) it doesnot contain a marker gene; and (e) it contains a region resulting from the site-specific recombination between two sequences, which is not present in theexpression cassette. The introduction of therapeutic genes and the use ofminicircles in gene therapy were expressly stated points of the application.The patent emphasizes that the absence of marker and resistance genes andother prokaryotic sequences (e.g., the origin of replication) affords a high geneticpurity and low risk of transmission of undesired sequences and proliferation ofantibiotics resistance.Within these patents, methods for the production of such constructs were also

provided. In particular, a preferred method involved the generation of minicirclesfrom a precursor plasmid with two recombination sites, which are to be recombinedby the coexpression of a recombinase. Recombinases from the lambda integrase

2j 1 Minicircle Patents: A Short IP Overview of Optimizing Nonviral DNA Vectors