Gespeichert in:
| Titel: | Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells |
|---|---|
| Von: |
presented by Maria Silvia Roman Azcona
|
| Person: |
Roman Azcona, Maria Silvia
Verfasser aut |
| Hauptverfasser: | |
| Format: | Abschlussarbeit Buch |
| Sprache: | Englisch |
| Veröffentlicht: |
Freiburg im Breisgau
November 2022
|
| Schlagworte: | |
| Online-Zugang: | https://d-nb.info/1294772767/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034572368&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| Beschreibung: | ix, 119 Seiten Illustrationen, Diagramme 30 cm |
Internformat
MARC
| LEADER | 00000nam a2200000 c 4500 | ||
|---|---|---|---|
| 001 | BV049311257 | ||
| 003 | DE-604 | ||
| 005 | 00000000000000.0 | ||
| 007 | t| | ||
| 008 | 230904s2022 gw a||| m||| 00||| eng d | ||
| 015 | |a 23,H09 |2 dnb | ||
| 016 | 7 | |a 1294772767 |2 DE-101 | |
| 020 | |c Broschur | ||
| 035 | |a (OCoLC)1375567140 | ||
| 035 | |a (DE-599)DNB1294772767 | ||
| 040 | |a DE-604 |b ger |e rda | ||
| 041 | 0 | |a eng | |
| 044 | |a gw |c XA-DE | ||
| 049 | |a DE-355 | ||
| 084 | |8 1\p |a 616.994 |2 23ksdnb | ||
| 084 | |8 2\p |a 610 |2 23sdnb | ||
| 084 | |8 3\p |a 570 |2 23sdnb | ||
| 100 | 1 | |a Roman Azcona, Maria Silvia |e Verfasser |0 (DE-588)1286003733 |4 aut | |
| 245 | 1 | 0 | |a Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells |c presented by Maria Silvia Roman Azcona |
| 264 | 1 | |a Freiburg im Breisgau |c November 2022 | |
| 300 | |a ix, 119 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Albert-Ludwigs-University Freiburg, Germany |d 2022 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1294772767/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034572368&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 883 | 0 | |8 1\p |a emakn |c 0,10604 |d 20230710 |q DE-101 |u https://d-nb.info/provenance/plan#emakn | |
| 883 | 2 | |8 2\p |a dnb |d 20230809 |q DE-101 |u https://d-nb.info/provenance/plan#dnb | |
| 883 | 2 | |8 3\p |a dnb |d 20230809 |q DE-101 |u https://d-nb.info/provenance/plan#dnb | |
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-034572368 | |
Datensatz im Suchindex
| DE-BY-UBR_katkey | 6741048 |
|---|---|
| _version_ | 1835079654436765696 |
| adam_text | CONTENTS
LIST
OF
ABBREVIATIONS
.............................................................................................VII
ABSTRACT
....................................................................................................................
IX
1.
INTRODUCTION
.........................................................................................................
1
1.1
THE
ROLE
OF
T
CELLS
IN
THE
IMMUNE
SYSTEM
.......................................................................
1
1.1.1
THE
IMMUNE
SYSTEM
...................................................................................................
1
1.1.2
T
CELL
BIOLOGY
.............................................................................................................
2
1.1.3
THE
PROCESS
OF
T
CELL
ACTIVATION
...............................................................................
2
1.1.4
REGULATION
OF
T
CELL
ACTIVITY
......................................................................................
5
1.1.4.1
PD-1
AS
A
T
CELL
REGULATOR
..................................................................................
6
1.1.4.2
LAG-3
AS
A
T
CELL
REGULATOR
...............................................................................
7
1.1.5
T
CELL
EXHAUSTION
........................................................................................................
8
1.2
CAR
T
CELL-BASED
CANCER
IMMUNOTHERAPY
....................................................................
8
1.2.1
CANCER
IMMUNOTHERAPY
.............................................................................................
8
1.2.2
CHIMERIC
ANTIGEN
RECEPTOR
(CAR)
T
CELLS
............................................................
10
1.2.3
CLINICAL
SUCCESS
AND
MANUFACTURING
OF
CAR
T
CELLS
.............................................
13
1.2.4
LIMITATIONS
OF
CAR
T
CELL
THERAPY
..........................................................................
16
1.2.4.1
LIMITATIONS
IN
HEMATOLOGICAL
MALIGNANCES
.....................................................
16
1.2.4.2
CHALLENGES
TO
TREAT
SOLID
TUMORS
WITH
CAR
T
CELLS
........................................
17
1.2.5
STRATEGIES
TO
OVERCOME
THE
LIMITATIONS
OF
CAR
T
CELL
THERAPY
..............................
19
1.3
EPIGENOME
EDITING
TECHNOLOGY
......................................................................................
21
1.3.1
THE
EPIGENOME
........................................................................................................
21
1.3.1.1
DNA
METHYLATION
AS
EPIGENETIC
MARK
............................................................
22
1.3.1.2
HISTONE
MODIFICATIONS
AS
EPIGENETIC
MARKS
....................................................
23
I
1.3.2
TARGETED
EPIGENOME
EDITING
...................................................................................24
1.3.2.1
DNA
BINDING
MODULES
....................................................................................
25
1.3.2.2
EFFECTOR
DOMAINS
.............................................................................................
27
1.3.3
DESIGNER
EPIGENOME
MODIFIERS
(DEMS)
..............................................................29
1.3.4
LIMITATIONS
AND
ADVANTAGES
OF
TARGETED
EPIGENOME
EDITING
.................................
30
1.4
AIMS
OF
THE
PHD
THESIS
..................................................................................................
32
2.
MATERIALS
AND
METHODS
....................................................................................
33
2.1
MOLECULAR
BIOLOGY
METHODS
..........................................................................................
33
2.1.1
PLASMID-DNA
EXTRACTION
.........................................................................................
33
2.1.2
GENOMIC
DNA
EXTRACTION
........................................................................................
33
2.1.3
RNA
EXTRACTION
........................................................................................................
33
2.1.4
NUCLEIC
ACID
QUANTIFICATION
AND
QUALITY
ASSESSMENT
.............................................
33
2.1.5
PCR
PRODUCT
PURIFICATION
........................................................................................
34
2.1.6
BACTERIA
TRANSFORMATION
...........................................................................................
34
2.1.7
AGAROSE
GEL
ELECTROPHORESIS
OF
DNA
.....................................................................34
2.1.8
IN
VITRO
TRANSCRIBED
(IVT)
MRNA
PRODUCTION
.......................................................
35
2.1.8.1
PLASMID
LINEARIZATION
.......................................................................................
35
2.1.8.2
T
RANSCRIPTION
REACTION
......................................................................................
35
2.1.8.3
QUALITY
CONTROL
OF
IVT-MRNA
......................................................................
36
2.2
CELL
CULTURE
METHODS
.......................................................................................................
36
2.2.1
CELLS
CULTURING
CONDITIONS
.......................................................................................
36
2.2.2
CELLS
DENSITY
AND
VIABILITY
MEASUREMENT
................................................................
37
2.2.3
CELLS
THAWING
AND
CRYOPRESERVATION
......................................................................
38
2.2.4
PURIFICATION
OF
HUMAN
PBMCS
FROM
LRS
CHAMBER
...............................................
38
II
2.2.5
CD3*
CELLS
PURIFICATION
.............................................................................................
40
2.2.6
PRIMARY
T
CELLS
ACTIVATION
.......................................................................................40
2.2.7
ELECTROPORATION
OF
PBMCS
AND
CD3
+
CELLS
............................................................
41
2.2.8
PACKAGING
OF
Y-RETROVIRUS
........................................................................................
43
2.2.8.1
CAR-ENCODING
VIRUS
TITRATION
..........................................................................44
2.2.9
COMBINATION
OF
T
CELLS
TRANSDUCTION
PLUS
ELECTROPORATION
...................................
45
2.2.10
CELLS
PREPARATION
FOR
FLOW
CYTOMETRY
......................................................................
45
2.2.10.1
FLOW
CYTOMETRY
DATA
ANALYSIS
..........................................................................
45
2.3
GENE
EXPRESSION
ANALYSIS
................................................................................................46
2.3.1
REVERSE
TRANSCRIBED
REAL
TIME
PCR
(RT-QPCR)
...................................................
46
2.3.1.1
REVERSE
TRANSCRIPTION
(RT)
...............................................................................46
2.3.1.2
REAL
TIME
PCR
(QPCR)
....................................................................................
47
2.3.1.3
RELATIVE
QUANTIFICATION
OF
GENE
EXPRESSION
.....................................................47
2.4
EPIGENETICS
STUDIES
.........................................................................................................48
2.4.1
BISULFITE
SEQUENCING
................................................................................................48
2.4.1.1
BISULFITE
CONVERSION
.........................................................................................48
2.4.1.2
BISULFITE
TREATED
DNA
AMPLIFICATION
...............................................................
48
2.4.1.3
AMPLICON
CLONING
.............................................................................................49
2.4.1.4
SCREENING
OF
POSITIVE
CLONES
............................................................................
49
2.4.1.5
PREPARATION
FOR
SANGER
SEQUENCING
.................................................................
50
2.4.1.6
BISULFITE
SEQUENCING
DATA
ANALYSIS
..................................................................
50
2.5
IN
VITRO
SPECIALIZED
CAR-RELATED
FUNCTIONAL
ASSAYS
......................................................
50
2.5.1
ACUTE
STIMULATION
OF
EPI-CAR
T
CELLS
..................................................................
50
2.5.1.1
EPI-CAR
T
CELLS
DIFFERENTIATION
...................................................................
51
III
2.5.1.2
EPI-CAR
T
CELLS
ACTIVATION
AND
SILENCING
POST
STIMULATION
.............................
51
2.5.1.3
QUANTIFICATION
OF
CYTOKINES
RELEASED
..............................................................
51
2.5.1.4
EPI-CAR
T
CELLS
CYTOTOXICITY
...........................................................................
52
2.5.2
CHRONIC
STIMULATION
OF
EPI-CAR
T
CELLS
...............................................................
52
2.5.2.1
EPI-CAR
T
CELLS
PROLIFERATION
..........................................................................
53
2.5.2.2
S
ILENCING
PERSISTENCE
ASSESSMENT
......................................................................
53
2.6
STATISTICAL
ANALYSIS
..................................................................................................................
53
3.
RESULTS
..................................................................................................................
54
3.1
ESTABLISHING
SILENCING
VIA
EPIGENOME
EDITING
IN
HUMAN
T
CELLS
..................................
54
3.1.1
SCREENING
OF
PDCD1
TARGETING
DEMS
..................................................................
54
3.1.2
PDCD1
SILENCING
ESTABLISHMENT
IN
PRIMARY
T
CELLS
.............................................
56
3.1.2.1
CELLS
RECOVERY
POST
ELECTROPORATION
...............................................................
57
3.1.2.2
EVALUATION
OF
SILENCING
EFFICIENCY
...................................................................
57
3.1.2.3
CORROBORATION
OF
INCREASED
DNA
METHYLATION
...............................................
58
3.1.3
DESIGN
OF
LAG3
TARGETING
DEMS
...........................................................................60
3.1.4
LAG3
SILENCING
ESTABLISHMENT
IN
PRIMARY
T
CELLS
.............................................
61
3.1.4.1
CELLS
RECOVERY
POST
ELECTROPORATION
.................................................................
61
3.1.4.2
EVALUATION
OF
SILENCING
EFFICIENCY
...................................................................
61
3.1.4.3
CORROBORATION
OF
INCREASED
DNA
METHYLATION
...............................................
62
3.1.5
PDCD1
AND
LAG3
DUAL
SILENCING
ESTABLISHMENT
.................................................64
3.2
GENERATION
OF
EPIGENETICALLY
EDITED
CAR
T
CELLS
FOR
PDCD1
SILENCING
....................
66
3.2.1
PDCD1
SILENCING
UPON
ACUTE
STIMULATION
.............................................................67
3.2.2
PDCD1
SILENCING
UPON
CHRONIC
STIMULATION
..........................................................
69
3.3
IN
VITRO
FUNCTIONAL
CHARACTERIZATION
OF
EPI-CAR
T
CELLS
.............................................
71
IV
3.3.1
CHARACTERIZATION
OF
RESPONSE
TO
ACUTE
STIMULATION
.................................................
71
3.3.2
CHARACTERIZATION
OF
RESPONSE
TO
CHRONIC
STIMULATION
.............................................
73
3.4
GENERATION
OF
DUAL
EPIGENETICALLY
EDITED
CAR
T
CELLS
FOR
PDCD1
AND
LAG3
SILENCING
.......................................................................................................................................
75
3.4.1
DUAL
SILENCING
UPON
ACUTE
AND
CHRONIC
STIMULATION
..............................................
76
3.4.2
FUNCTIONAL
CHARACTERIZATION
OF
DUAL-EPI-CAR
T
CELLS
...........................................
79
4.
DISCUSSION
........................................................................................................
83
4.1
TARGETING
IMMUNE
CHECKPOINTS
TO
PREVENT
CAR
T
CELL
EXHAUSTION
.............................
83
4.2
THE
CHOICE
OF
EPIGENOME
EDITING
FOR
DUAL
TARGETING
OF
IMMUNE
CHECKPOINTS
............
85
4.3
THE
IMPACT
OF
DEMS
TARGETING
PDCD1
AND
LAG3
ON
THEIR
EXPRESSION
LEVELS
..........
88
4.4
CONSEQUENCES
OF
EPIGENOME
EDITING
IN
CAR
T
CELLS
..................................................
90
4.5
CONCLUSIONS
AND
FUTURE
PERSPECTIVES
.............................................................................
92
5.
REFERENCES
........................................................................................................94
6.
APPENDIX
........................................................................................................
113
6.1
PREPARATION
OF
REAGENTS
.................................................................................................
113
6.1.1
LB
MEDIUM
.............................................................................................................
113
6.1.2
LB-AGAR
PLATES
........................................................................................................
113
6.1.3
MAMMALIAN
CELLS
FREEZING
MEDIUM
......................................................................
113
6.1.4
RESUSPENSION
BUFFER
FOR
PBMCS
PURIFICATION
.....................................................
113
6.1.5
CELL
CULTURE
GROWTH
MEDIUM
..................................................................................
114
6.1.6
PEI
TRANSFECTION
REAGENT
........................................................................................
114
6.1.7
PRE-TREATMENT
OF
CULTURE
PLATES
WITH
PDL
.............................................................
114
6.1.8
FACS
BUFFER
...........................................................................................................
114
6.2
PRIMER
PAIR
S
EFFICIENCY
TEST
FOR
QPCR
........................................................................
115
6.3
IRRADIATION
OF
TARGET
CELLS
................................................................................................
115
6.4
LIST
OF
TABLES
...................................................................................................................
116
6.5
LIST
OF
FIGURES
.................................................................................................................
116
7.
ACKNOWLEDGEMENTS
........................................................................................
119
VI
|
| any_adam_object | 1 |
| author | Roman Azcona, Maria Silvia |
| author_GND | (DE-588)1286003733 |
| author_facet | Roman Azcona, Maria Silvia |
| author_role | aut |
| author_sort | Roman Azcona, Maria Silvia |
| author_variant | a m s r ams amsr |
| building | Verbundindex |
| bvnumber | BV049311257 |
| ctrlnum | (OCoLC)1375567140 (DE-599)DNB1294772767 |
| format | Thesis Book |
| fullrecord | <?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01770nam a2200409 c 4500</leader><controlfield tag="001">BV049311257</controlfield><controlfield tag="003">DE-604</controlfield><controlfield tag="005">00000000000000.0</controlfield><controlfield tag="007">t|</controlfield><controlfield tag="008">230904s2022 gw a||| m||| 00||| eng d</controlfield><datafield tag="015" ind1=" " ind2=" "><subfield code="a">23,H09</subfield><subfield code="2">dnb</subfield></datafield><datafield tag="016" ind1="7" ind2=" "><subfield code="a">1294772767</subfield><subfield code="2">DE-101</subfield></datafield><datafield tag="020" ind1=" " ind2=" "><subfield code="c">Broschur</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(OCoLC)1375567140</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-599)DNB1294772767</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-604</subfield><subfield code="b">ger</subfield><subfield code="e">rda</subfield></datafield><datafield tag="041" ind1="0" ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="044" ind1=" " ind2=" "><subfield code="a">gw</subfield><subfield code="c">XA-DE</subfield></datafield><datafield tag="049" ind1=" " ind2=" "><subfield code="a">DE-355</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="8">1\p</subfield><subfield code="a">616.994</subfield><subfield code="2">23ksdnb</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="8">2\p</subfield><subfield code="a">610</subfield><subfield code="2">23sdnb</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="8">3\p</subfield><subfield code="a">570</subfield><subfield code="2">23sdnb</subfield></datafield><datafield tag="100" ind1="1" ind2=" "><subfield code="a">Roman Azcona, Maria Silvia</subfield><subfield code="e">Verfasser</subfield><subfield code="0">(DE-588)1286003733</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells</subfield><subfield code="c">presented by Maria Silvia Roman Azcona</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="a">Freiburg im Breisgau</subfield><subfield code="c">November 2022</subfield></datafield><datafield tag="300" ind1=" " ind2=" "><subfield code="a">ix, 119 Seiten</subfield><subfield code="b">Illustrationen, Diagramme</subfield><subfield code="c">30 cm</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="b">n</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="b">nc</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="502" ind1=" " ind2=" "><subfield code="b">Dissertation</subfield><subfield code="c">Albert-Ludwigs-University Freiburg, Germany</subfield><subfield code="d">2022</subfield></datafield><datafield tag="655" ind1=" " ind2="7"><subfield code="0">(DE-588)4113937-9</subfield><subfield code="a">Hochschulschrift</subfield><subfield code="2">gnd-content</subfield></datafield><datafield tag="856" ind1="4" ind2="2"><subfield code="m">B:DE-101</subfield><subfield code="q">application/pdf</subfield><subfield code="u">https://d-nb.info/1294772767/04</subfield><subfield code="3">Inhaltsverzeichnis</subfield></datafield><datafield tag="856" ind1="4" ind2="2"><subfield code="m">DNB Datenaustausch</subfield><subfield code="q">application/pdf</subfield><subfield code="u">http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034572368&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA</subfield><subfield code="3">Inhaltsverzeichnis</subfield></datafield><datafield tag="883" ind1="0" ind2=" "><subfield code="8">1\p</subfield><subfield code="a">emakn</subfield><subfield code="c">0,10604</subfield><subfield code="d">20230710</subfield><subfield code="q">DE-101</subfield><subfield code="u">https://d-nb.info/provenance/plan#emakn</subfield></datafield><datafield tag="883" ind1="2" ind2=" "><subfield code="8">2\p</subfield><subfield code="a">dnb</subfield><subfield code="d">20230809</subfield><subfield code="q">DE-101</subfield><subfield code="u">https://d-nb.info/provenance/plan#dnb</subfield></datafield><datafield tag="883" ind1="2" ind2=" "><subfield code="8">3\p</subfield><subfield code="a">dnb</subfield><subfield code="d">20230809</subfield><subfield code="q">DE-101</subfield><subfield code="u">https://d-nb.info/provenance/plan#dnb</subfield></datafield><datafield tag="943" ind1="1" ind2=" "><subfield code="a">oai:aleph.bib-bvb.de:BVB01-034572368</subfield></datafield></record></collection> |
| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV049311257 |
| illustrated | Illustrated |
| indexdate | 2024-12-20T20:06:13Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-034572368 |
| oclc_num | 1375567140 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | ix, 119 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2022 |
| publishDateSearch | 2022 |
| publishDateSort | 2022 |
| record_format | marc |
| spellingShingle | Roman Azcona, Maria Silvia Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells |
| subject_GND | (DE-588)4113937-9 |
| title | Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells |
| title_auth | Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells |
| title_exact_search | Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells |
| title_full | Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells presented by Maria Silvia Roman Azcona |
| title_fullStr | Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells presented by Maria Silvia Roman Azcona |
| title_full_unstemmed | Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells presented by Maria Silvia Roman Azcona |
| title_short | Epigenome editing to induce sustained modulation of Immune checkpoints' expression in CAR T cells |
| title_sort | epigenome editing to induce sustained modulation of immune checkpoints expression in car t cells |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1294772767/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034572368&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT romanazconamariasilvia epigenomeeditingtoinducesustainedmodulationofimmunecheckpointsexpressionincartcells |
An UBR ausleihen
Ausleihen an der UBR sind mit eigenem Ausweis möglich.