La lecture en ligne est gratuite
Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres
Télécharger Lire

Insights into the clonal inventory of gene corrected human SCID-X1 hematopoiesis [Elektronische Ressource] / vorgelegt von Jingqiong Hu

De
107 pages
Aus dem Institut für Molekulare Medizin und Zellforschung der Albert-Ludwigs-Universität Freiburg i. Br. INSIGHTS INTO THE CLONAL INVENTORY OF GENE CORRECTED HUMAN SCID-X1 HEMATOPOIESIS INAUGURAL-DISSERTATION zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br. vorgelegt 2004 von Jingqiong Hu geboren in Shanghai, V.R.China Dekan: Prof. Josef Zentner Erster Gutachter: Prof. Christof von Kalle Zweiter Gutachter: Prof. Fritz von Weizsäcker Jahr der Promotion: 2004 DECLARATION I hereby do solemnly declare that: the work presented in this dissertation has been carried out by me, under the supervision of Professor Christof von Kalle of the Molecular and Gene Therapy Programme Group, Division of Experimental Hematology, Cincinnati Children’s Hospital in USA and Dr. Manfred Schmidt of the Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University of Freiburg in Germany. Signed Name: Jingqiong Hu Table of Contents - I - TABLE OF CONTENTS 1 INTRODUCTION............................................................................1 1.1 Hematopoiesis and Hematopoietic Stem Cells....
Voir plus Voir moins


Aus dem Institut für Molekulare Medizin und Zellforschung
der Albert-Ludwigs-Universität Freiburg i. Br.

INSIGHTS INTO THE CLONAL
INVENTORY OF GENE
CORRECTED HUMAN SCID-X1
HEMATOPOIESIS


INAUGURAL-DISSERTATION
zur
Erlangung des Medizinischen Doktorgrades
der Medizinischen Fakultät
der Albert-Ludwigs-Universität
Freiburg i. Br.













vorgelegt 2004
von Jingqiong Hu
geboren in Shanghai, V.R.China






Dekan: Prof. Josef Zentner
Erster Gutachter: Prof. Christof von Kalle
Zweiter Gutachter: Prof. Fritz von Weizsäcker
Jahr der Promotion: 2004

























DECLARATION

I hereby do solemnly declare that:
the work presented in this dissertation has been carried out by me,
under the supervision of Professor Christof von Kalle of the Molecular
and Gene Therapy Programme Group, Division of Experimental
Hematology, Cincinnati Children’s Hospital in USA and Dr. Manfred
Schmidt of the Institute of Molecular Medicine and Cell Research,
Albert-Ludwigs-University of Freiburg in Germany.


Signed
Name: Jingqiong Hu





















Table of Contents - I -
TABLE OF CONTENTS

1 INTRODUCTION............................................................................1
1.1 Hematopoiesis and Hematopoietic Stem Cells............................................................... 1
1.1.1 Hematopoiesis ......................................................................................................... 1
1.1.2 Hematopoietic Stem Cells....................................................................................... 3
1.2 Retrovirus and Retroviral Vectors.................................................................................. 5
1.2.1 Retrovirus................................................................................................................5
1.2.2 Retroviral Vectors...................................................................................................8
1.2.3 Retroviral Integration Mechanism........................................................................... 9
1.3 Gene Therapy ............................................................................................................... 11
1.3.1 General Aspects of Gene Therapy......................................................................... 11
1.3.2 Hematopoietic Stem Cell Gene Therapy............................................................... 12
1.3.3 Gene Therapy Clinical Trials ................................................................................ 14
1.3.4 Gene Therapy of SCID-X1.................................................................................... 14
1.3.5 Potential Hazards in Retroviral Stem Cell Gene Therapy..................................... 18
1.4 Aim of Study ................................................................................................................ 20
2 MATERIALS AND METHODS......................................................22
2.1 Materials ................................................................................................................. 22
2.1.1 Patients Material....................................................................................................22
2.1.2 Hela Cell Clone ..................................................................................................... 22
2.1.3 LN Vector..............................................................................................................23
2.1.4 Human Umbilical Cord Blood CD34+ Cells ........................................................ 23
2.1.5 Oligonucleotides23
2.1.6 Enzymes................................................................................................................24
2.1.7 Chemicals and Reagents........................................................................................ 24
2.1.9 Cell Culture Medium............................................................................................. 25
2.1.10 Kits………………………………………………………. ................................... 25
2.1.11 Instruments ............................................................................................................ 26
2.1.12 Data Analysis Tools .............................................................................................. 26
2.2 METHODS................................................................................................................. 27 Table of Contents - II -
2.2.1 Transduction of Normal Human Umbilical Cord Blood CD34+ Cells................. 27
2.2.1.1 Purification of Umbilical Blood CD34+Cells................................................ 27
2.2.1.2 Transduction Protocol .................................................................................... 27
2.2.1.3 DNA Isolation from Transduced CD34+ Cells .............................................. 27
2.2.2 Semi-quantitative PCR: Myoglobin- PCR& LTR-PCR........................................ 28
2.2.3 Linear Amplification Mediated PCR (LAM-PCR)............................................... 29
2.2.3.1 Linear PCR..................................................................................................... 31
2.2.3.2 Magnetic Capture ........................................................................................... 31
2.2.3.3 Hexanucleotide-Priming................................................................................. 31
2.2.3.4 Restriction Digestion...................................................................................... 32
2.2.3.5 Ligation .......................................................................................................... 32
2.2.3.6 Denaturation................................................................................................... 32
2.2.3.7 Exponential PCR............................................................................................ 33
2.2.4 Gelelectrophoresis................................................................................................. 33
2.2.4.1 Agarose Gelelectrophoresis............................................................................ 33
2.2.4.2 High Resolution Spreadex Gelelectrophoresis............................................... 33
2.2.5 Picogram Cloning.................................................................................................. 34
2.2.5.1 DNA Extraction from Spreadex Gel .............................................................. 34
2.2.5.2 TA Cloning..................................................................................................... 34
2.2.6 Plasmid DNA Extraction....................................................................................... 34
2.2.6.1 Plasmid DNA Extraction with Qiagen QuickSpin Kits ................................. 34
2.2.6.2 96-well Manifold DNA Extraction ................................................................ 34
2.2.7 Sequencing ............................................................................................................ 35
2.2.7.1 MegaBACE 500 DNA Sequencing from Plasmid DNA ............................... 35
2.2.7.2 Direct Sequencing from Purified PCR Products............................................ 36
2.2.8 Longitudinal Analysis ........................................................................................... 36
2.2.9 Sequence Analysis................................................................................................. 38
3 RESULTS.......................................................................................39
3.1 Feasibility of LAM-PCR to Highly Polyclonal Samples with Low Vector Copy
Numbers .................................................................................................................39
3.1.1 LAM-PCR Analysis on Retrovirally Transduced Human Cord Blood
CD34 + Cells.........................................................................................................39
3.1.2 LAM-PCR Analysis on Low Copy and Highly Polyclonal Pre- Table of Contents - III -
Transplantation SCID-X1 Patient Sample ............................................................ 41
3.2 Retroviral Integration Site Analysis on Pre-Transplantation CD34+ Cells ................. 43
3.2.1 Estimation of DNA Concentration and Transduction Efficiency ......................... 44
3.2.2 LAM-PCR Analysis of Pre-Transplantation Sample ............................................ 45
3.3 Retroviral Integration Site Analysis on Post-Transplantation CD3+
T Lymphocytes............................................................................................................. 46
3.3.1 ite Analysis on 6 Months Post- Transplantation CD3+ T
Lymphocytes ......................................................................................................... 46
3.3.1.1 Estimation of DNA Concentration and Integration Level................................ 47
3.3.1.2 Retroviral Integration Site Analysis on 6 Months Post-Transplantation
CD3+ T Lymphocytes ...................................................................................... 48
3.3.2 Retroviral Integration Site Analysis on 24 Months Post-Transplantation CD3+ T
Lymphocytes ......................................................................................................... 50
3.3.3 ite Analysis on 37 Months and 41 Months
Post-Transplantation Samples...............................................................................51
3.4 Distribution of Retroviral Integration Sites on the Human Genome .......................... 53
3.5 Longitudinal Analysis ................................................................................................ 56
4 DISCUSSION ................................................................................59
4.1 Characterization of Retroviral Integration Sites by LAM-PCR.................................. 60
4.1.1 Different Methods to Characterize Retroviral Integration Sites ........................... 60
4.1.2 High-Sensitivity Detection of Multiple Retroviral Integration Sites
by LAM-PCR........................................................................................................61
4.1.3 Characterization of Retroviral Integration Sites in SCID-X1 Gene Therapy
Clinical Trial by LAM-PCR.................................................................................. 62
4.1.4 Optimization of LAM-PCR to Highly Polyclonal Samples with Low
Vector Copy Numbers...........................................................................................63
4.2 Characterization of in vivo Hematopoiesis in SCID-X1 Trial ................................... 65
4.2.1 Retroviral Integration Site Analysis in Studying in Vivo Hematopoiesis............. 65
4.2.2 Characterization of Post-Transplantation Hematopoiesis in SCID-X1 Trial........ 67
4.2.3 Characterization of Post-Transplantation Malignant Transformation in Patient 4
by LAM-PCR ........................................................................................................ 68
4.2.4 Characterization of Post-Chemotherapy Hematopoiesis....................................... 69 Table of Contents - IV -
4.3 Retroviral Integration Profile in SCID-X1 Gene Therapy Trial ................................. 71
4.3.1 Reassessment of the Risk of Insertional Mutagenesis .......................................... 71
4.3.2 Characterization of Post-Transplantation Retroviral Integration Site Profile in
SCID-X1 Trial....................................................................................................... 72
4.3.3 Characterization of Pre-Transplantation Retroviral Integration Site Profile in
SCID-X1 Trial 74
4.4 Perspectives ................................................................................................................. 76
5 SUMMARY ....................................................................................78
6 APPENDIX80
7 REFERENCES...............................................................................85



























- V -
ABBREVIATIONS

ADA-SCID Adenosine Deaminase Deficient Severe Combined Immunodeficiency
bio biotin
bp basepair
BLAST Basic Local Alignment Search Tool
BLAT BLAST Like Alignment Tool
BM Bone Marrow
BSA Bovine Serum Albumin
CLPs Common Lymphoid Progenitors
CMPs Common Myeloid Progenitors
CD Cluster of Differentiation
CFU Colony Forming Unit
CSF Colony Stimulating Factor
DNA Deoxyribonucleic Acid
DMEM Dulbecco’s Modified Eagle Medium
Env Envelope
FACS Fluorescence Activated Cell Sorter
FCS Fetal Calf Serum
FP Flanking Primer
Gag Group Specific Antigen
GFP Green Fluorescent Protein
GMPs Common Myelomonocytic Progenitors
HSC Hematopoietic Stem Cell
IC Internal Control
kb kilobases
LAM-PCR Linear Amplification Mediated Polymerase Chain Reaction
LC Linker Cassette
LT-HSC Long-Term Hematopoietic Stem Cells
LTR Long Terminal Repeat
MLV Murine Leukemia Virus
MPC Magnetic Particle Concentrator
mRNA messenger Ribonucleic Acid
NCBI National Center for Biotechnology Information
Post T. Post-Transplantation
PB Primer Binding Site
PCR Polymerase Chain Reaction
PICs Preintegration Complexes
Pre T. Pre-Transplantation
RISs Retroviral Integration Sites
RNA Ribonucleic Acid
SCID Severe Combined Immunodeficiency
ST-HSC Short-Term Hematopoietic Stem Cell
Taq Thermus Aquaticus
U3 / U5 Unique Region 3 / 5
UCSC University of California Santa Cruz
- VI -
FIGURES

Figure 1: Hematopoiesis ....................................................................................................... 3
Figure 2: Genomic Structure of Moloney Murine Leukemia Provirus................................. 6
Figure 3: Wild Type Molony Murine Leukemia Virus Replication Cycle .......................... 7
Figure 4: Construction of Retroviral Vector ......................................................................... 8
Figure 5: Defects in T Cell Development That Result in Severe Combined
Immunodeficiencies............................................................................................15
Figure 6: Scheme of γ-Chain Containing Cytokine Receptors and
Cytokine Pathway................................................................................................ 16
Figure 7: LAM-PCR Strategy............................................................................................. 30
Figure 8: Two-Step Tracking PCR Strategy....................................................................... 37
Figure 9: High-Resolution Spreadex Gelelectrophoresis of LAM-PCR Analysis on
LN Vector Transduced CD34+ cells ................................................................... 40
Figure 10: Optimization of LAM-PCR to Low Copy Highly Polyclonal
Pre-Transplantation Sample................................................................................42
Figure 11: Myoglobin-PCR and LTR-PCR of Pre-Transplantation Sample ........................ 45
Figure 12: High Resolution Gelelectrophoresis of LAM-PCR Analysis on DNA
Isolated from Transduced CD34+ Cells Prior to Transplantation
without Optimization (12A), with Optimization (12B)....................................... 46
Figure13: Myoglobin-PCR on 6 Months Post-Transplantation CD3+ T lymphocytes ....... 47
Figure14: LTR-PCR on 6 Months Post-Transplantation CD3+ T Lymphocytes ................ 48
Figure 15: LAM-PCR Product Separated on 2% Agarose Gel............................................. 49
Figure 16: High Resolution Gelelectrophoresis of LAM-PCR Analysis Performed
on 6 Months Post-Transplantation CD3+ T lymphocytes................................... 49
Figure 17: LAM-PCR Analysis Performed on 24 Months Post-Transplantation
CD3+ T Lymphocytes ......................................................................................... 50
Figure 18: med on DNA from Post-Chemotherapy
(37 Months Post-Transplantation) CD3+ T Lymphocytes and
Post-Allotransplantation (41 Months Post-Transplantation)
Peripheral Blood Leukocytes (PBL) Sample... ................................................... 52
Figure 19: Locus Distribution of PreTransplantation RISs .................................................. 54
Figure 20: Locus Distribution of Post-Transplantation RISs................................................ 54
Figure 21: Tracking of Clone 8963 and Clone 8966 ............................................................ 58 - VII -
TABLES


Table 1: Advantages and Disadvantages of Different Vectors Used in Gene Therapy. ..... 12
Table 2: Myoglobin-PCR Parameters.................................................................................. 28
Table 3: LTR-PCR Parameters............................................................................................ 29
Table 4: LAM-PCR: Linear PCR Parameters ..................................................................... 31
Table 5: LAM-PCR: 1. and 2. Exponential PCR Parameters.............................................. 33
Table 6: Sequencing PCR Parameters ................................................................................ 35
Table 7: 1.Nested PCR Parameters...................................................................................... 37
Table 8: 2. Nested PCR Parameters..................................................................................... 37
Table 9: Sequenced Retroviral Integration Sites from LN-Vector Transduced
Human Cord Blood CD34+ Cells ......................................................................... 41
Table 10: Optimization of LAM-PCR Parameters to Low Copy Highly
Polyclonal Samples...............................................................................................43
Table 11: Sequencing Overview of Patient No. 4, Pre-Transplantation Integration Sites .... 81
Table 12: Sequencing Overview of Patient No. 4, Post-Transplantation Integration Sites... 83
Table 13: Overview of Retroviral Integration Sites with Relevance to Refseq Genes (1).... 55
Table 14: efseq Genes (2).... 56
Table 15: Sequencing Results of Tracking PCR ................................................................... 59