Les vecteurs rétroviraux dérivés du virus Moloney de la leucémie murine (MoMLV) ont été utilisés pour livrer des gènes depuis plus de 20 ans et ils continuent d’être le meilleur outil disponible pour transférer de façon stable et efficace des gènes thérapeutiques dans différents types de cellules. Bien que la plupart des études précliniques de thérapie génique utilisent des surnageants bruts ou concentrés de vecteurs rétroviraux, l’étape de purification, pour éliminer le sérum et les impuretés dérivées des cellules hôtes contenus dans ces préparations, est incontournable pour les applications cliniques. Cette thèse décrit le développement de stratégies de purification des vecteurs rétroviraux. Au cours de ce projet, deux procédés complets de purification (à partir d’un surnageant brut de rétrovirus jusqu’au virus de grade clinique) ont été établis, vérifiés, et leurs performances ont été analysées en détail. La filtration sur membrane a contribuée à la clarification, la concentration, à l’échange de tampon et à la purification partielle des particules retrovirales à partir de surnageants à l’état brut sans aucune perte significative d’infectivité virale. Deux nouvelles méthodes de purification, spécifiquement adaptées aux caractéristiques biochimiques et physiques des particules rétrovirales, ont été développées. La première méthode de purification des particules rétrovirales, utilise la chromatographie d’affinité sur colonne d'héparine suivie d’un tamis moléculaire. L’avantage principal d’utiliser les techniques de chromatographie pour la purification des virus, est d’offrir la possibilité de purifier à grande échelle les rétrovirus de façon sélective et efficace. De plus, la chromatographie d’affinité sur colonne d'héparine a donné lieu à des taux de récupération exceptionnels de particules infectieuses et s’est avérée utile pour la purification des vecteurs rétroviraux produits par différentes lignées cellulaires indépendamment de l’enveloppe protéique utilisée pour le pseudo-typage. La deuxième méthode de purification est basée sur la technique de centrifugation zonale transitoire utilisant l’iodixanol comme milieu pour former un gradient. La force de cette technique repose sur les hauts niveaux de pureté obtenus en une seule étape de purification et la capacité à séparer les particules virales des espèces proches telles que les vecteurs défectueux et / ou les vésicules membranaires, qui posent un sérieux défi dans les procédés de purification. Les récupérations finales en particules infectieuses (~ 38%) et le degré de pureté atteint (plus de 95%) étaient comparables avec l’une ou l’autre des stratégies de purification utilisées. Les méthodes décrites dans cette thèse représentent une amélioration significative sur la méthodologie conventionnelle utilisant un gradient de densité de sucrose pour la purification des rétrovirus et contribuera certainement à l’avancement technologique dans le domaine de la thérapie génique.

Retroviral vectors derived from the Moloney murine leukemia virus (MoMLV) have been used as gene delivery vehicles for more than two decades and continue to be the best available tool for stable and efficient transfer of therapeutic genes into various cell types. Although most gene therapy preclinical studies use crude or concentrated retroviral vector supernatants, purification to eliminate serum and host-derived impurities contained in these stocks is a must for clinical applications. This thesis describes the development of downstream processing strategies for retroviral vectors. During the course of this project, two complete multi-step purification schemes (from crude retrovirus supernatant to clinical-grade virus) were designed, tested and their performance analyzed in detail. Membrane filtration contributed to the clarification, concentration, buffer exchange and partial purification of retroviral particles from crude supernatants with essentially no loss in vector infectivity. Two novel purification methods specifically tailored to the biochemical and physical features of retroviral particles were developed. The first method consists of the chromatographic purification of retroviral particles by heparin affinity chromatography followed by size exclusion chromatography. The main advantage of employing chromatography technology for virus purification is that it offers the possibility to selectively and efficiently purify retroviruses on a large-scale. Moreover, heparin affinity chromatography resulted in exceptional recoveries of infective particles and proved to be useful for the purification of retroviral vectors produced by different packaging cell lines independently of the Env-protein used for pseudotyping. The second purification method is based on a rate zonal centrifugation technique using iodixanol as gradient medium. The power of this technique was revealed by the high levels of purity achieved in a single purification step and its potential to separate viral particles from closely-related species such as defective vector forms and/or cell membrane vesicles, all of which pose a serious challenge in downstream processing. The overall yield of infective particles (~38%) and level of purity achieved (over 95%) using either purification strategy was comparable. The methods described in this thesis represent a significant improvement over the conventional sucrose density gradient methodology used for retrovirus purification and will hopefully contribute to the technological progress in the field of gene therapy.

The body of this thesis is composed of four chapters, prefaced by a general introduction and followed by a general conclusion and perspective section. Each chapter is based on a scientific article which at the time of the thesis submission was either published, accepted or in evaluation. The author of this thesis is also the principal writer of these articles and responsible for the planning, execution and analysis of the experimental work presented herein. It wouldn’t have been possible to complete such an endeavor without the constant guidance and supervision of Dr. Alain Garnier at the Université Laval and Dr. Amine Kamen at the Biotechnology Research Institute of National Research Council of Canada (BRI-NRC) who are co-authors in all four articles.

The first chapter consists in a review article that provides all relevant background information about the state of the art in downstream processing of retroviral vectors. The methods currently described in the literature for clarification, concentration and purification of retroviral vectors are presented whereas problems associated with stability and quantitation of retroviral particles are critically analyzed. The review also covers aspects of lentiviral vectors purification given that the structural similarities between both types of particles permit them to share common purification strategies. This article has recently been accepted for publication by Biotechnology Advances (December 6^th 2005).

In the second chapter, a complete scaleable purification strategy for retroviral vectors that utilizes membrane and chromatography technologies is presented. In this article, heparin affinity chromatography is introduced as a novel and convenient technique for the purification of retroviral particles. This article was published in Biotechnology & Bioengineering, 2005, 90: 391-404. Dr. Pierre Trudel, co-author of this article, participated in the analysis of the data and revision of the draft manuscript.

An alternative complete purification strategy is presented in the third chapter of this thesis. This strategy is based on a rate zonal ultracentrifugation using iodixanol as gradient medium. The development of this method as well as the thorough characterization of the purified product is presented in an article that has been accepted for publication by the Journal of Virological Methods (October 6^th 2005). The method is mainly intended for use in laboratories that may lack preparative liquid chromatography systems but are equipped with ultracentrifuges, which is typically the case in academic virology laboratories. The possibility of scaling-up such protocol will depend on the availability of high capacity ultracentrifuge equipment suitable for retrovirus purification purposes.

In the last chapter, the general applicability of heparin affinity chromatography to the purification of retroviral vectors produced by different cell lines and carrying different Env-proteins is assessed. Results obtained from these studies are presented in a fourth article. Co-author of this article Marie-Claude Lavoie, a graduate student at Université Laval, carried out RD114-pseudotyped vector production.

In addition, results from this project were communicated in the following conferences:

“Purification and characterization of recombinant retrovirus” Oral presentation. Segura M.M. , Garnier A., and Kamen A. Seventh Ontario-Quebec CSChE Biotechnology Meeting, Queen’s University, Kingston, June 9-10, 2005. Best Ph.D. oral presentation award.

“Retrovirus vectors-heparin interaction and its implications for gene therapy” Oral presentation. Segura M.M. , Kamen A., Trudel P. and Garnier A. 7ième colloque de l’Association de thérapie génique du Québec, Quebec, October 29, 2004.

“Are host proteins on the viral membrane responsible for retrovirus attachment to target cells?” Oral presentation. Segura M.M ., Kamen A., Pierre T. and Garnier A. 54th Canadian Chemical Engineering Conference, Calgary, October 3-6, 2004.

“Preparation of highly purified retroviral vectors by rate zonal ultracentrifugation” Poster. Segura M.M. , Garnier A. and Kamen A. Bioprocess Perspective Meeting-Biotechnology Research Institute, Montreal, September 24, 2004.

“Développement d’une nouvelle méthode pour la purification à grande échelle de vecteurs rétroviraux” Poster. Segura M.M. , Kamen A., Trudel P., Transfiguracion J. and Garnier A. 4º Symposium annuel du CRESFSIP, Quebec, May 10, 2004.

“A novel scaleable approach for retrovirus vector purification” Poster. Segura M.M. , Kamen A., Trudel P., Transfiguracion J. and Garnier A. Cell Culture Engineering IX, Cancún, March 7-12, 2004.

"Development of a novel protocol for the downstream processing of recombinant retroviral vectors". Poster. Segura M.M. , Kamen A., Trudel P., Transfiguracion J. and Garnier A. 6th colloque de l'Association de Thérapie Génique du Québec, Montreal, October 24, 2003.

“A novel approach for MuLV-derived retrovirus vector purification”. Poster. Segura M.M. , Kamen A., Trudel P., Transfiguracion J. and Garnier A. Stem Cell Network Annual General Meeting, Vancouver, September 18-20, 2003. Best poster award.

“Development of a scaleable method for recombinant retroviral vector purification”. Oral presentation. Segura M.M. , Kamen A., Trudel P., Transfiguracion J. and Garnier A. Fifth Ontario-Quebec Biotechnology Meeting, University of Waterloo, Waterloo, June 12-13, 2003.

“Downstream processing of recombinant retroviral vectors” Poster. Segura M.M. , Kamen A., Trudel P., Transfiguracion J. and Garnier A. Stem Cell Network Annual General Meeting, Toronto, September 26-28, 2002. Best poster award.

This thesis is the result of three years of experimental research at the Biotechnology Research Institute of the National Research Council of Canada (NRC-BRI) in collaboration with the Université Laval.

First and foremost, I would like to express my sincere gratitude to my thesis director, Dr. Alain Garnier, for successfully guiding me through my doctoral studies. I feel privileged to have had the opportunity of working under his supervision and I am deeply indebted to him for his endless encouragement, interest in my research activities and kind support. I am equally grateful to my thesis co-director, Dr. Amine Kamen, my mentor at the NRC-BRI, who opened the doors of the institute for me and closely guided me throughout my studies while allowing me to conduct research independently. I greatly benefited from his wisdom, advice and pertinent criticism.

I would also like to thank Dr. Manuel Caruso, associate professor at the Université Laval, for his encouragement during my graduate courses and helpful discussions about my research work and Dr. Rowe Gerald, expert in the field of downstream processing at the NRC-BRI, who graciously revised the review manuscript. I acknowledge Dr. Angélica Meneses-Acosta, Dr. Yves Durocher and Dr. Parminder Chahal at the NRC-BRI whose expertise and helpful comments enhanced this learning experience.

My most sincere gratitude to Normand Arcand who guided me in my first steps in the laboratory, helped me interpret results and made valuable suggestions in manuscripts drafts. I would also like to acknowledge Alice Bernier for introducing me to the fascinating world of virus purification, for careful review of manuscripts and helpful discussions. Thank to them as well as other members of the viral vector team at the NRC-BRI including Marc Aucoin, Edwige Dormond, Roseanne Tom and Hélène Coehlo my stay at the institute was not only fruitful but enjoyable. Special thanks to Dr. Pierre Trudel and Marie-Claude Lavoie for their participation in this project. The help of Robert Alain with electron microscopy and Andre Migneault with image files was greatly appreciated.

I want to express my gratitude to my family in Argentina for their unconditional love and support, particularly to my mother who taught me the most important lessons and, in spite of the distance, managed to closely accompany me in every step of my career and life. I am forever grateful to my friends, particularly those who I was fortunate enough to have close to me during my studies. Their moral support and encouragement enabled me to complete this thesis and have a wonderful time along the way. Finally, I thank Gavin for the many hours of stimulating discussions and editing during the preparation of this thesis, but above all for being a part of my life.

This work was financially supported by NSERC and the Canadian Stem Cell Network.

AAV: adeno-associated virus

BSA: bovine serum albumin

CA: capsid protein

CaPO₄: calcium phosphate

CsCl: cesium chloride

Da: Dalton

DEAE: diethylaminoethyl

DMEM: Dulbecco’s modified Eagle’s medium

DMSO: dimethyl sulfoxide

DNA: deoxyribonucleic acid

cDNA: complementary DNA

dsDNA: double-stranded deoxyribonucleic acid

DTT: 1,4-dithiotreitol

EBA: expanded bed adsorption

Env-protein: envelope protein

EDTA: ethylene diamine tetraacetic acid

ELISA: enzyme-linked immunosorbent assay

FACS: fluorescence-activated cell sorting

FBS: foetal bovine serum

F-MLV: Friend murine leukemia virus

GAG: glycosaminoglycan

Gag: group specific antigen retroviral protiens

GFP: green fluorescent protein

HCl: hydrochloric acid

HEK 293: Human embryonic kidney 293 cell line

HIV-1: human immunodeficiency virus type 1

HPLC: high pressure liquid chromatography

HRP: horseradish peroxidase

HSC: hematopoietic stem cells

HTLV: human T-cell lymphotropic virus

IMAC: immobilized metal affinity chromatography

IN: integrase

IVP: infective virus particles

LDL: low density lipoprotein

MA: matrix protein

Mab: Monoclonal antibody

MoMLV: Moloney murine leukemia virus

mRNA: messenger ribonucleic acid

MWCO: molecular weight cut-off

NaCl: sodium chloride

NC: nucleocapsid protein

NSEM: negative stain electron microscopy

PBS: phosphate-buffered saline

PCR: polymerase chain reaction

PTA: phosphotungstic acid

RCV: replication-competent virus

RT-PCR: reverse transcriptase polymerase chain reaction

PR: protease

qPCR: quantitative polymerase chain reaction

qRT-PCR: quantitative reverse transcriptase polymerase chain reaction

RNA: ribonucleic acid

mRNA : messenger RNA

RT: reverse transcriptase

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEC: Size-exclusion chromatography

SO3-: sulphoisobutyl chromatography ligands

SU: surface subunit

t_1/2: half-life

TK: thymidine kinase protein

TM: transmembrane subunit

VP: total virus particles

VSV: vesicular stomatitis virus

VSV-G: vesicular stomatitis virus glycoprotein