{"id":{"repo_id":"freiburg-diss","oai_identifier":"oai:freidok.uni-freiburg.de:1534"},"canonical_url":"https://search.dev.ndltd.org/etd/freiburg-diss/oai:freidok.uni-freiburg.de:1534","repository":{"repo_id":"freiburg-diss","name":"University of Freiburg","base_url":"https://freidok.uni-freiburg.de/oai/oai2.php"},"display":{"title":"Insights into the clonal inventory of gene corrected human SCID-XI hematopoiesis","abstract":"The semi-random nature of retroviral integration introduces a stable molecular marker into the cellular genome in the form of a genomic-proviral fusion sequence which can be passed on unmodified to all the progenies of a transduced cell. Retroviral integration site analysis provides a powerful method to study the clonal composition of in vivo hematopoiesis after hematopoietic stem cell transplantation.
In the present study, a highly sensitive and specific LAM-PCR method was employed to characterize the in vivo hematopoiesis on DNA material from patient No. 4 of the French SCID-X1 gene therapy clinical trial, who has developed a T-cell leukemia like disease at 31 months after gene therapy. Multiple integration sites could be identified in posttransplantation samples, demonstrating multiple transduced clones contributing to hematopoiesis after transplantation. LAM-PCR analysis performed on 37 months posttransplantation samples, however, displayed an oligoclonal repopulation profile, suggesting that only a few predominant transduced clones were contributing to post-chemotherapy hematopoiesis. Two clones identified from 37 months post-chemotherapy samples could be traced back in 6 months and 24 months post-transplantation samples, which provided the first
molecular evidence of prolonged activity of individual transduced clones even after clinical interventions such as chemotherapy. Altogether 148 RISs (97 appable RISs) could be identified in this patient. To address the question whether human gene coding regions are favored targets for retroviral integration, all the identified RISs were analyzed with relationship to human RefSeq genes. 64.7% (22/34) of the pre-transplantation RISs as well as 38.1% of the post-transplantation RISs had landed within RefSeq genes. This finding is significant different from a set of 10,000 computer simulated random integration, whereby only 22.4% of integrations has landed within RefSeq genes, indicating a strong tendency of retroviral integration towards gene coding regions of the human genome. Further development of safer designed vectors to be used in human
hematopoietic stem cell gene therapy trials is therefore justified.","abstract_html":"The semi-random nature of retroviral integration introduces a stable molecular marker into the cellular genome in the form of a genomic-proviral fusion sequence which can be passed on unmodified to all the progenies of a transduced cell. Retroviral integration site analysis provides a powerful method to study the clonal composition of in vivo hematopoiesis after hematopoietic stem cell transplantation. <br>In the present study, a highly sensitive and specific LAM-PCR method was employed to characterize the in vivo hematopoiesis on DNA material from patient No. 4 of the French SCID-X1 gene therapy clinical trial, who has developed a T-cell leukemia like disease at 31 months after gene therapy. Multiple integration sites could be identified in posttransplantation samples, demonstrating multiple transduced clones contributing to hematopoiesis after transplantation. LAM-PCR analysis performed on 37 months posttransplantation samples, however, displayed an oligoclonal repopulation profile, suggesting that only a few predominant transduced clones were contributing to post-chemotherapy hematopoiesis. Two clones identified from 37 months post-chemotherapy samples could be traced back in 6 months and 24 months post-transplantation samples, which provided the first <br>molecular evidence of prolonged activity of individual transduced clones even after clinical interventions such as chemotherapy. Altogether 148 RISs (97 appable RISs) could be identified in this patient. To address the question whether human gene coding regions are favored targets for retroviral integration, all the identified RISs were analyzed with relationship to human RefSeq genes. 64.7% (22/34) of the pre-transplantation RISs as well as 38.1% of the post-transplantation RISs had landed within RefSeq genes. This finding is significant different from a set of 10,000 computer simulated random integration, whereby only 22.4% of integrations has landed within RefSeq genes, indicating a strong tendency of retroviral integration towards gene coding regions of the human genome. Further development of safer designed vectors to be used in human <br>hematopoietic stem cell gene therapy trials is therefore justified.","abstract_has_math":false,"creators":["Hu, Jingqing"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Kalle, Christof von"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":null,"date_issued":"","date_published":null,"updated_at":"2026-07-24T02:22:16Z","subjects":["SCID-X1","Integrationsstellenanalyse","integration site analysis"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://freidok.uni-freiburg.de/data/1534","outbound_label":"Repository record","outbound_source":"source_url"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Kalle, Christof von"]},{"key":"dc:creator","label":"Author","values":["Hu, Jingqing"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:type","label":"Dc Type","values":["DoctoralThesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["SCID-X1","Integrationsstellenanalyse","integration site analysis"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The semi-random nature of retroviral integration introduces a stable molecular marker into the cellular genome in the form of a genomic-proviral fusion sequence which can be passed on unmodified to all the progenies of a transduced cell. Retroviral integration site analysis provides a powerful method to study the clonal composition of in vivo hematopoiesis after hematopoietic stem cell transplantation.
In the present study, a highly sensitive and specific LAM-PCR method was employed to characterize the in vivo hematopoiesis on DNA material from patient No. 4 of the French SCID-X1 gene therapy clinical trial, who has developed a T-cell leukemia like disease at 31 months after gene therapy. Multiple integration sites could be identified in posttransplantation samples, demonstrating multiple transduced clones contributing to hematopoiesis after transplantation. LAM-PCR analysis performed on 37 months posttransplantation samples, however, displayed an oligoclonal repopulation profile, suggesting that only a few predominant transduced clones were contributing to post-chemotherapy hematopoiesis. Two clones identified from 37 months post-chemotherapy samples could be traced back in 6 months and 24 months post-transplantation samples, which provided the first
molecular evidence of prolonged activity of individual transduced clones even after clinical interventions such as chemotherapy. Altogether 148 RISs (97 appable RISs) could be identified in this patient. To address the question whether human gene coding regions are favored targets for retroviral integration, all the identified RISs were analyzed with relationship to human RefSeq genes. 64.7% (22/34) of the pre-transplantation RISs as well as 38.1% of the post-transplantation RISs had landed within RefSeq genes. This finding is significant different from a set of 10,000 computer simulated random integration, whereby only 22.4% of integrations has landed within RefSeq genes, indicating a strong tendency of retroviral integration towards gene coding regions of the human genome. Further development of safer designed vectors to be used in human
hematopoietic stem cell gene therapy trials is therefore justified.","Retrovirale Vektoren können infolge ihrer stabilen und halb zufälligen Integrationsverteilung in das zelluläre Genom als Klonalitätsmarker verwendet werden. Die retrovirale Integrationsstellenanalyse ermöglicht eine in vivo Klonalitäts Analyse nach Transplantation von hämatopoetischen Stammzellen.
Im Rahmen dieser Arbeit wurde die retrovirale Integrationsstellenanalyse mittels einer hoch sensitiven und spezifischen LAM-PCR (Lineare amplifikationsmediierte PCR) an DNA aus Patient Nr. 4 der Französischen SCID-X1 Gentherapie Studie durchgeführt. Dieser Patient wurde zuerst erfolgreich mit Gentherapie behandelt, bekam aber eine Leukämie-ähnliche Erkrankung 31 Monate nach Transplantation. Die erzielten Ergebnisse legen nahe, dass multiple Vorläufer- oder Stamzellklone an der Hämatopoese dieses Patienten nach Transplantation von autologen hämatopoetischen Stammzellen beteiligt sind. Im Gegensatz dazu, nur ein paar predominante Vorläufer- oder Stammzellklone an der Hämatopoese nach Chemotherapie beteiligt sind. Anschließend wurde eine longitudinale Analyse zum Auffinden
identischer Integrationsflanken durchgeführt. Zwei Klone, die 37 Monate nach
Transplantation auftauchten, konnten retrospektive auch 6 Monate und 24 Monate nach Transplantation wieder detektiert werden. Somit wurde die kontinuierliche Beteilung individueller Klone an der langfristigen lymphoiden Hämatopoese nach Transplantation nachgewiesen werden. Die 148 untersuchten retroviralen Integrationsstellen (RISs), (davon 97 genau kartierte RISs) haben weiterhin mitgeholfen, die Fragen bezüglich einer möglichen Präferenz der RIS für
genomische Kodierungsregionen aufzuklären. 64.7% (22/34) der Vortransplantations RISs bzw. 38.1% (24/68) der Post-transplantation RISs sind innerhalb eines RefSeq Genbereichs gelandet. Diese Ergebnisse unterscheiden sich deutlich von 10,000 Computersimulierten zufälligen Integrationsereignissen, wobei nur 22.4% der RISs in RefSeq Regionen gelandet sind. Dies deutet darauf hin, dass humane genomische Kodierungsregionen bei der retroviralen Integration bevorzugt werden. Die weitere Entwicklung von sicheren retroviralen Vektoren für humane HSCs Gentherapie Studien hat nach diesen Ergebnissen höchste Priorität."]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Insights into the clonal inventory of gene corrected human SCID-XI hematopoiesis","Untersuchung der klonalen Zusammensetzung der Hämatopoese in einer menschlichen SCID-X1 Gentherapie-Studie"]}]}],"canonical_facts":{"dc:contributor":["Kalle, Christof von"],"dc:creator":["Hu, Jingqing"],"dc:description.abstract":["The semi-random nature of retroviral integration introduces a stable molecular marker into the cellular genome in the form of a genomic-proviral fusion sequence which can be passed on unmodified to all the progenies of a transduced cell. Retroviral integration site analysis provides a powerful method to study the clonal composition of in vivo hematopoiesis after hematopoietic stem cell transplantation.
In the present study, a highly sensitive and specific LAM-PCR method was employed to characterize the in vivo hematopoiesis on DNA material from patient No. 4 of the French SCID-X1 gene therapy clinical trial, who has developed a T-cell leukemia like disease at 31 months after gene therapy. Multiple integration sites could be identified in posttransplantation samples, demonstrating multiple transduced clones contributing to hematopoiesis after transplantation. LAM-PCR analysis performed on 37 months posttransplantation samples, however, displayed an oligoclonal repopulation profile, suggesting that only a few predominant transduced clones were contributing to post-chemotherapy hematopoiesis. Two clones identified from 37 months post-chemotherapy samples could be traced back in 6 months and 24 months post-transplantation samples, which provided the first
molecular evidence of prolonged activity of individual transduced clones even after clinical interventions such as chemotherapy. Altogether 148 RISs (97 appable RISs) could be identified in this patient. To address the question whether human gene coding regions are favored targets for retroviral integration, all the identified RISs were analyzed with relationship to human RefSeq genes. 64.7% (22/34) of the pre-transplantation RISs as well as 38.1% of the post-transplantation RISs had landed within RefSeq genes. This finding is significant different from a set of 10,000 computer simulated random integration, whereby only 22.4% of integrations has landed within RefSeq genes, indicating a strong tendency of retroviral integration towards gene coding regions of the human genome. Further development of safer designed vectors to be used in human
hematopoietic stem cell gene therapy trials is therefore justified.","Retrovirale Vektoren können infolge ihrer stabilen und halb zufälligen Integrationsverteilung in das zelluläre Genom als Klonalitätsmarker verwendet werden. Die retrovirale Integrationsstellenanalyse ermöglicht eine in vivo Klonalitäts Analyse nach Transplantation von hämatopoetischen Stammzellen.
Im Rahmen dieser Arbeit wurde die retrovirale Integrationsstellenanalyse mittels einer hoch sensitiven und spezifischen LAM-PCR (Lineare amplifikationsmediierte PCR) an DNA aus Patient Nr. 4 der Französischen SCID-X1 Gentherapie Studie durchgeführt. Dieser Patient wurde zuerst erfolgreich mit Gentherapie behandelt, bekam aber eine Leukämie-ähnliche Erkrankung 31 Monate nach Transplantation. Die erzielten Ergebnisse legen nahe, dass multiple Vorläufer- oder Stamzellklone an der Hämatopoese dieses Patienten nach Transplantation von autologen hämatopoetischen Stammzellen beteiligt sind. Im Gegensatz dazu, nur ein paar predominante Vorläufer- oder Stammzellklone an der Hämatopoese nach Chemotherapie beteiligt sind. Anschließend wurde eine longitudinale Analyse zum Auffinden
identischer Integrationsflanken durchgeführt. Zwei Klone, die 37 Monate nach
Transplantation auftauchten, konnten retrospektive auch 6 Monate und 24 Monate nach Transplantation wieder detektiert werden. Somit wurde die kontinuierliche Beteilung individueller Klone an der langfristigen lymphoiden Hämatopoese nach Transplantation nachgewiesen werden. Die 148 untersuchten retroviralen Integrationsstellen (RISs), (davon 97 genau kartierte RISs) haben weiterhin mitgeholfen, die Fragen bezüglich einer möglichen Präferenz der RIS für
genomische Kodierungsregionen aufzuklären. 64.7% (22/34) der Vortransplantations RISs bzw. 38.1% (24/68) der Post-transplantation RISs sind innerhalb eines RefSeq Genbereichs gelandet. Diese Ergebnisse unterscheiden sich deutlich von 10,000 Computersimulierten zufälligen Integrationsereignissen, wobei nur 22.4% der RISs in RefSeq Regionen gelandet sind. Dies deutet darauf hin, dass humane genomische Kodierungsregionen bei der retroviralen Integration bevorzugt werden. Die weitere Entwicklung von sicheren retroviralen Vektoren für humane HSCs Gentherapie Studien hat nach diesen Ergebnissen höchste Priorität."],"dc:format.medium":["application/pdf"],"dc:subject":["SCID-X1","Integrationsstellenanalyse","integration site analysis"],"dc:title":["Insights into the clonal inventory of gene corrected human SCID-XI hematopoiesis","Untersuchung der klonalen Zusammensetzung der Hämatopoese in einer menschlichen SCID-X1 Gentherapie-Studie"],"dc:type":["DoctoralThesis"]},"updated_at":"2026-07-24T02:22:16Z"}