In vivo selection of hematopoietic stem cells transduced at a low multiplicity-of-infection with a foamy viral MGMTP140K vector

Abstract

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 NIH Public Access

Magselectofection: an integrated method of nanomagnetic separation and genetic modification of target cells

“…44 Here, magselectofection with low MOIs (Յ 3) yielded up to 50% transduced cells ( Figure 4) compared with only 9.5% with an MOI of 5 to 8 using a standard transduction protocol for Lin Ϫ BM cells as reported previously. 45 Importantly, the magselectofected cells were fully competent in colony-forming assays ( Figure 4B-C), and administration in mice also resulted in long-term stable reconstitution and long-term GFP expression ( Figure 4D-F).…”

Overnight transduction with foamyviral vectors restores the long-term repopulating activity of Fancc−/− stem cells

“…After one week, 20 colonies were picked for each mouse and subjected to standard LM-PCR analysis for identifying the location of the provirus in the genome. As described previously, 46 the position of the transgene from each colony was evaluated using the SeqMap website (http://seqmap.compbio.iupui.edu) and confirmed using the http://genome.ucsc.edu and http://www.ensembl.org websites. From each mouse, one proviral integration was detected (Table 2).…”

“…45 The DNA from excised bands was cloned into pCR2.1 using the TOPO cloning kit (Invitrogen, Frederick, MD) and then sequenced on an ABI Gene Amp 3770 System (Applied Biosystems, Foster City, CA). As described previously, 46 SeqMap (http://seqmap.compbio.iupui.edu, Indiana University School of Medicine) was used to map the sequences against the mouse genome. This was then confirmed by mapping the positions using the Ensembl website (http://www.ensembl.org) and mus musculus database release 42, December 2006, 47 and the UCSC Genome Browser (http://genome.ucsc.edu).…”

Therapeutic Modulation of DNA Damage and Repair Mechanisms in Blood Cells

“…(Beard et al, 2010;Neff et al, 2005) Numerous transplant studies have convincingly proven that long-term repopulating murine HSCs could be selected in vivo with O 6 -BG/BCNU, O 6 -BG /TMZ, or O 6 -BG /CCNU. (Cai et al, 2008;Davis et al, 2004;Jansen et al, 2002;Kreklau et al, 2003;Milsom et al, 2004;Persons et al, 2003;Persons et al, 2004;Ragg et al, 2000;Sawai et al, 2003;Sawai et al, 2001) In regards to modeling of this approach with human HSPCs, we and others previously demonstrated that MGMT P140K -transduced SCID-repopulating cells and their progeny could be selected in vivo in NOD/SCID mice. Human HSPCs derived from umbilical cord blood (UCB) or granulocyte colony-stimulating factormobilized peripheral blood (MPB) that expressed MGMT P140K could be selected in vivo by www.intechopen.com nonmyeloablative doses of O 6 -BG and BCNU.…”