Project Details
Projekt
Modeling the reconstitution dynamics of hematopoiesis after stem cell transplantation using neutral multicolor barcode marking for high-resolution cell tracking
Subject Area
Hematology, Oncology
Bioinformatics and Theoretical Biology
Bioinformatics and Theoretical Biology
Term
since 2016
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 319089048
Hematopoietic stem cell transplantation (HSCT) is often the only curative therapy for various, life-threatening blood disorders. In addition to transplantation with cells from a foreign donor (allogeneic), the transplantation of the body's own cells (autologous) is becoming increasingly important. Especially for gene therapy treatments, autologous stem cells are collected and, after correction of the genetic defect with the help of stably integrating viral vectors, are returned to the patient. The central aim of this treatment is to ensure that these genetically modified cells engraft and that their progeny permanently contributes to polyclonal hematopoiesis. Although the random but distinct integration site of the viral vector (VIS) represents an identifiable marker for the detection of undesired clonal expansion, the exact quantification of clonal sizes remains a major challenge. Furthermore, it is not understood on which time scale genetically modified cells will expand in a recipient and, above all, how strongly this process can be influenced by the pre-treatment and composition of the graft. Based on our joint preliminary work on clonal quantification using genetic barcodes and previous studies on the contribution and clonality of different hematopoietic subpopulations after HSCT, we address two clinically relevant questions in the context of this follow-up proposal: (I) We complement our existing barcode system by the use of unique molecular identifiers (UMIs), which allow to decouple clonal quantification from PCR-inherent amplification biases. We transfer this technology to VIS-based clonal quantification and are thus able to compare this clinically relevant methodology with the barcode method used experimentally. (II) We use the optimized methodology to investigate in a suitable preclinical mouse model of Fanconi anemias (FA) the time scale on which genetically corrected cells are able to displace mutant cells and how this process can be influenced by myeloablative conditioning, composition of the graft and additional stress induction. Especially in FA, permanent polyclonal displacement of mutated cells is of paramount importance as it is the only way to reduce the risk of secondary, potentially cancer initiating mutations. We complement these analyses with mathematical models that allow to quantify influencing factors of clonal development and are suitable to estimate the potential risk arising from remaining "uncorrected" hematopoiesis also in the human situation. The long-standing, successful cooperation and the project-specific preparatory work of the applicants ensure that the proposed work program is technically feasible and promises successful project completion. Hematopoietic stem cell transplantation (HSCT) is often the only curative therapy for various, life-threatening blood disorders. In addition to transplantation with cells from a foreign donor (allogeneic), the transplantation of the body's own cells (autologous) is becoming increasingly important. Especially for gene therapy treatments, autologous stem cells are collected and, after correction of the genetic defect with the help of stably integrating viral vectors, are returned to the patient. The central aim of this treatment is to ensure that these genetically modified cells engraft and that their progeny permanently contributes to polyclonal hematopoiesis. Although the random but distinct integration site of the viral vector (VIS) represents an identifiable marker for the detection of undesired clonal expansion, the exact quantification of clonal sizes remains a major challenge. Furthermore, it is not understood on which time scale genetically modified cells will expand in a recipient and, above all, how strongly this process can be influenced by the pre-treatment and composition of the graft. Based on our joint preliminary work on clonal quantification using genetic barcodes and previous studies on the contribution and clonality of different hematopoietic subpopulations after HSCT, we address two clinically relevant questions in the context of this follow-up proposal: (I) We complement our existing barcode system by the use of unique molecular identifiers (UMIs), which allow to decouple clonal quantification from PCR-inherent amplification biases. We transfer this technology to VIS-based clonal quantification and are thus able to compare this clinically relevant methodology with the barcode method used experimentally. (II) We use the optimized methodology to investigate in a suitable preclinical mouse model of Fanconi anemias (FA) the time scale on which genetically corrected cells are able to displace mutant cells and how this process can be influenced by myeloablative conditioning, composition of the graft and additional stress induction. Especially in FA, permanent polyclonal displacement of mutated cells is of paramount importance as it is the only way to reduce the risk of secondary, potentially cancer initiating mutations. We complement these analyses with mathematical models that allow to quantify influencing factors of clonal development and are suitable to estimate the potential risk arising from remaining "uncorrected" hematopoiesis also in the human situation. The long-standing, successful cooperation and the project-specific preparatory work of the applicants ensure that the proposed work program is technically feasible and promises successful project completion.
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