RESUMEN
Reports on insertional "genotoxicity" in patients have created intense interest in characterizing retroviral vector integrations on the genomic level. The retroviral vector SF91m3 was used for transduction of human peripheral blood progenitor cells (PBPC). These PBPC were transplanted into nonobese diabetic/severe combined immunodeficient mice. A total of 186 retroviral vector integration sites were isolated by ligation-mediated PCR from chimeric mouse bone marrow of five PBPC donors, sequenced, and blasted against the human genome. Preferred integration near the transcription start regions, within CpG islands, and within Alu regions was observed. Detailed analysis of targeted RefSeq genes showed a favored integration within the first intron. Integrations were most common in genes coding for signaling proteins, transcription factors, and kinases. In all genes targeted independently multiple times the respective orientation of the provirus within the gene was identical, indicating integration hot spot regions and similar steric determinants for integration sites. Possible explanations for these findings could be nonrandom vector integration, clonal selection due to gene expression interference, or engraftment issues related to gene insertion in signaling and cell cycle genes. The low frequency of integrations in exons may be reassuring as to the safety of retroviral gene therapy with normal human PBPC.
Asunto(s)
Vectores Genéticos/genética , Trasplante de Células Madre Hematopoyéticas , Mutagénesis Insercional/genética , Retroviridae/genética , Integración Viral/genética , Elementos Alu/genética , Animales , Cromosomas Humanos/genética , Islas de CpG/genética , Técnicas de Transferencia de Gen , Terapia Genética , Células Madre Hematopoyéticas/virología , Humanos , Intrones/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Sitio de Iniciación de la TranscripciónRESUMEN
Methods to analyze the clonality of an adverse event in preclinical or clinical retroviral stem cell gene therapy protocols are needed. We analyzed the progeny of retrovirally transduced human peripheral blood progenitor cells (PBPCs) after transplantation and engraftment in immune-deficient mice. The integration site of the provirus serves as a unique tag of the individual transduced PBPC. A plasmid library of junctions between proviral and human genomic DNA was generated. We were able to detect individual transduced cell clones that amounted to 0.14%-0.0001% of chimeric bone marrow cells. This is the first report in which the contribution of individual marrow-repopulating cells to human hematopoiesis is directly quantified.
Asunto(s)
Antígenos CD34/sangre , Células de la Médula Ósea/citología , Trasplante de Células Madre/métodos , Trasplante Heterólogo/inmunología , Animales , Terapia Genética/métodos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Increasing use of hematopoietic stem cells for retroviral vector-mediated gene therapy and recent reports on insertional mutagenesis in mice and humans have created intense interest to characterize vector integrations on a genomic level. We studied retrovirally transduced human peripheral blood progenitor cells with bone marrow-repopulating ability in immune-deficient mice. By using a highly sensitive and specific ligation-mediated polymerase chain reaction (PCR) followed by sequencing of vector integration sites, we found a multitude of simultaneously active human stem cell clones 8 weeks after transplantation. Vector integrations occurred with significantly increased frequency into chromosomes 17 and 19 and into specific regions of chromosomes 6, 13, and 16, although most of the chromosomes were targeted. Preferred genomic target sites have previously only been reported for wild-type retroviruses. Our findings reveal for the first time that retroviral vector integration into human marrow-repopulating cells can be nonrandom (P =.000 37).