title: Comparative clonal analysis of reconstitution kinetics after transplantation of hematopoietic stem cells gene marked with a lentiviral SIN or a γ-retroviral LTR vector.
abstract: Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (LAM-PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat (LTR)-driven γ-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus LTR-driven γ-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of LAM-PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for LAM-PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with γ-retroviral LTR vectors.
authors.affiliation: Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Cornils
authors.forename: Cynthia C
authors.initials: CC
authors.lastname: Bartholomae
authors.forename: Lars
authors.initials: L
authors.lastname: Thielecke
authors.forename: Claudia
authors.initials: C
authors.lastname: Lange
authors.forename: Anne
authors.initials: A
authors.lastname: Arens
authors.forename: Ingmar
authors.initials: I
authors.lastname: Glauche
authors.forename: Ulrike
authors.initials: U
authors.lastname: Mock
authors.forename: Kristoffer
authors.initials: K
authors.lastname: Riecken
authors.forename: Sebastian
authors.initials: S
authors.lastname: Gerdes
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Ingo
authors.initials: I
authors.lastname: Roeder
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
mesh_terms: D000818:Animals; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D017988:Gammaretrovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000821:virology*; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction; D020079:Terminal Repeat Sequences / Q000235:genetics*; D014161:Transduction, Genetic