Abstract: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 si… Show more
“…Reports analyzing integration sites of murine retrovirus and lentivirus-based gene vectors into HeLa cells, human peripheral blood cells, and CD34 + cells have recently revealed a tendency to integrate in actively transcribed genes. [81][82][83] CONCLUSIONS Current treatment for severe PIs is not satisfactory. Many children affected with these previously fatal 25 diseases are now being rescued with allogeneic BMT.…”
Section: Insertional Mutagenesis and Leukemiamentioning
“…Reports analyzing integration sites of murine retrovirus and lentivirus-based gene vectors into HeLa cells, human peripheral blood cells, and CD34 + cells have recently revealed a tendency to integrate in actively transcribed genes. [81][82][83] CONCLUSIONS Current treatment for severe PIs is not satisfactory. Many children affected with these previously fatal 25 diseases are now being rescued with allogeneic BMT.…”
Section: Insertional Mutagenesis and Leukemiamentioning
“…From each of the mice, 20-30 progenitor colonies were picked and then subjected to LM-PCR as described previously [41] with minor variations. The restriction enzyme used here was Hae III (New England Biolabs, Frankfurt, Germany).…”
“…The biotinylated primer 5′biotin-GTACAATCTAGGTGACCACTTTC-3′ (407) was used in a one step extension at 94°C, 15 sec; 58°C, 2 minutes; 72°C, 10 minutes; 2 cycles. The two internal primers for the nested PCR were 5′-TCTCATCCCAGGTACGTCTATGA-3′ (404) and AP2 as previously described [41]. The DNA from excised bands were cloned into pCR2.1 using the TOPO cloning kit (Invitrogen) and then sequenced on an ABI Gene Amp 3170 System.…”
Objective-Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Lin neg BM) could be genetically modified with a FV vector that expresses the DNA repair protein, O 6 -methylguanine DNA methyltransferase (MGMT P140K ) and selected in vivo with submyeloablative versus myeloablative alkylator therapy.Methods-Lin neg BM was transduced at a low multiplicity-of-infection (MOI), with the FV vector, MD9-P140K, that co-expresses MGMT P140K and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K-transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals.Results-Following submyeloablative therapy, 55% of the mice expressed MGMT P140K in the BM. Proviral integration was observed in ∼50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to 2 integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, following delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced since BM cells from the surviving animals reconstituted secondary recipients with MGMT P140K positive cells for 5-6 months. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. of transduced-stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against the acute toxicity associated with myeloablative therapy.
Conclusions-In
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“…5,6 The other approach is a ligation-mediated PCR (LM-PCR), which involves ligating a short adapter oligonucleotide to the unknown flanking DNA allowing PCR amplification. [7][8][9] Both these methods require numerous DNA preparation steps before amplification, which may limit their sensitivity. Therefore, the aim of this study was to establish a sensitive and rapid method to characterize retroviral vector integration sites.…”
We have developed a highly sensitive polymerase chain reaction (PCR)-based technique termed two-step PCR, which uses arbitrary primers to identify proviral integration sites in retrovirally marked human colony-forming cells. The two-step PCR was established on cell line clones transduced with the SF1m retroviral vector and independently validated by demonstrating identical integration sites with ligationmediated PCR, a different technique requiring restriction enzyme digestion and adapter ligation for amplifying unknown DNA flanking the provirus. Two-step PCR was performed on peripheral blood progenitor cell (PBPC) colonies that contained as few as 75 cells, which was estimated by quantitative real-time PCR. We were able to amplify and directly sequence proviral integration sites in 35 % of PBPC colonies (25/72, five donors). Identity to the vector long-terminal repeat was confirmed and flanking DNA was found to match with human database sequences, reaffirming specificity. Two-step PCR is a valuable new tool for rapid analysis of genomic target sites for viral vectors, and will aid significantly in understanding clonal development of hematopoiesis and other cell types. Our protocol has the potential for general applicability as the arbitrary primers described here bind to genomic DNA and are thus independent of the vector backbone used.
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