Woods, NB, Muessig, A, Schmidt, M, Flygare, J, Olsson, K, Salmon, P et al.. Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis. Blood 101: 1284-1289

Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Baden-Württemberg, Germany
Blood (Impact Factor: 10.45). 03/2003; 101(4):1284-9. DOI: 10.1182/blood-2002-07-2238
Source: PubMed


Efficient vector transduction of hematopoietic stem cells is a requirement for successful gene therapy of hematologic disorders. We asked whether human umbilical cord blood CD34(+)CD38(lo) nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cells (SRCs) could be efficiently transduced using lentiviral vectors, with a particular focus on the average number of vector copies integrating into these primitive progenitor cells. Mouse bone marrow was analyzed by fluorescence-activated cell-sorter scanner and by semiquantitative polymerase chain reaction (PCR) to determine the transduction efficiency into SRCs. Lentiviral vector transduction resulted in an average of 22% (range, 3%-90%) of the human cells expressing green fluorescent protein (GFP), however, multiple vector copies were present in human hematopoietic cells, with an average of 5.6 +/- 3.3 (n = 12) copies per transduced cell. To confirm the ability of lentiviral vectors to integrate multiple vector copies into SRCs, linear amplification mediated (LAM)-PCR was used to analyze the integration site profile of a selected mouse showing low-level engraftment and virtually all human cells expressing GFP. Individually picked granulocyte macrophage colony-forming unit colonies derived from the bone marrow of this mouse were analyzed and shown to have the same 5 vector integrants within each colony. Interestingly, one integration site of the 5 that were sequenced in this mouse was located in a known tumor-suppressor gene, BRCA1. Therefore, these findings demonstrate the ability of lentiviral vectors to transduce multiple copies into a subset of NOD/SCID repopulating cells. While this is efficient in terms of transduction and transgene expression, it may increase the risk of insertional mutagenesis.

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Available from: Johan Flygare, Mar 08, 2014
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    • "Integration-proficient lentiviral vectors (IPLVs) are among the most widely utilized gene transfer vectors because of their efficiency in transducing many cell types, including postmitotic cells, as well as the longevity of transgene expression from an integrated transgene (Naldini et al., 1996a, b). In clinical situations, however, integration events may compromise biosafety because of the potential for insertional oncogenesis (Woods et al., 2003). We investigated the possibility of generating equally efficient and long-lasting transgene expression using safer nonintegrating vectors. "
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    ABSTRACT: Non-integrating gene delivery vectors have an improved safety profile compared to integrating vectors, but transgene retention is problematic as non-replicating episomes are progressively and rapidly diluted out through cell division. We have developed an integration-deficient lentiviral vector (IDLV) system generating mitotically stable episomes capable of long-term transgene expression. We found that a transient cell cycle arrest at the time of transduction with IDLVs resulted in 13-45% of CHO cells expressing the transgene for over 100 cell generations in the absence of selection. The use of a Scaffold/Matrix Attachment Region (S/MAR) did not result in improved episomal retention in this system, and episomes did not form following transduction with AAV or minicircle vectors under the same conditions. Investigations into the episomal status of the vector genome using (i) linear amplification–mediated PCR (LAM-PCR) followed by deep sequencing of vector-genome junctions, (ii) Southern blotting and (iii) fluorescent in situ hybridization (FISH) strongly suggest that the vector is not integrated in the vast majority of cells. In conclusion, we have developed an IDLV procedure generating mitotically stable episomes capable of long-term transgene expression. The application of this approach to stem cell populations could significantly improve the safety profile of a range of stem and progenitor cell gene therapies.
    Human Gene Therapy 05/2014; 25(5):428-442. DOI:10.1089/hum.2013.172 · 3.76 Impact Factor
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    • "However, viral vectors are capable of inducing adverse immunogenic response (Hollon 2000). They can either inhibit tumour suppressor genes or activate oncogenes through ectopic chromosomal integration of viral DNA within the host chromosomes (Schröder et al. 2002; Woods et al. 2003; Li et al. 2002; Hacein-Bey-Abina et al. 2003). Thus, efforts are now being directed towards developing alternative non-viral transfection vectors, mainly cationic liposomes and cationic polymers. "
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    ABSTRACT: Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ~2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10 percent (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10 percent FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway.
    Journal of Biosciences 03/2014; 39(1):43-52. DOI:10.1007/s12038-014-9415-2 · 2.06 Impact Factor
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    • "Third-generation lentiviruses are capable of integrating into the host genome of primitive human repopulating cells multiple times, initially seeming to limit the practicality of using these viruses for reprogramming (for personalized cellular therapeutics) and warranting the need for new reprogramming methodologies that yield transductions with fewer copies per cell [44]. Optimization of the multiplicity of infection to between 0.1 and 10, however, recently demonstrated that over 94% of iPSC colonies had a single stable integration [16]. "
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    ABSTRACT: The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic "stem cell cassette" (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming cassette influences those genes and whether these cells still have clinical potential are not yet known. In this study, we used both microarray and sensitive real-time polymerase chain reaction to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using non-restrictive linear amplification polymerase chain reaction. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. We found that a STEMCCA derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared to post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions by using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. For the first time these studies provide proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.
    Stem Cell Research & Therapy 07/2013; 4(4):87. DOI:10.1186/scrt246 · 3.37 Impact Factor
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