Identification of parameters required for efficient lentiviral vector transduction and engraftment of human cord blood CD34+ NOD/SCID-repopulating cells

Liu, Ying; Hangoc, Giao; Campbell, Timothy B.; Goodman, M. N.; Wen, Tao; Pollok, Karen E.; Srour, Edward F.; Broxmeyer, Hal E.

doi:10.1016/j.exphem.2008.06.005

Cited by 22 publications

(23 citation statements)

References 30 publications

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“…Ideally, we would prefer to perform injections earlier than day 65 to access a higher proportion of stem/progenitor cells, although this is technically challenging. In addition, as transduction time is related to transduction efficiency of LVs, 45 we performed two fetal tracheal occlusion maneuvers to maximize the time of exposure of the epithelium to viral vector; (1) distal to the injection site to prevent vector escaping through the larynx during injection and (2) proximal to the injection site to prevent leakage of vector after removing the angiocatheter. Without tracheal occlusion, lung fluid continually secreted by the pulmonary epithelium would flush vector from the airways into amniotic fluid.…”

Section: Discussionmentioning

confidence: 99%

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

et al. 2011

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Viral vector-mediated gene transfer to the postnatal respiratory epithelium has, in general, been of low efficiency due to physical and immunological barriers, non-apical location of cellular receptors critical for viral uptake and limited transduction of resident stem/progenitor cells. These obstacles may be overcome using a prenatal strategy. In this study, HIV-1-based lentiviral vectors (LVs) pseudotyped with the envelope glycoproteins of Jaagsiekte sheep retrovirus (JSRV-LV), baculovirus GP64 (GP64-LV), Ebola Zaire-LV or vesicular stomatitis virus (VSVg-LV) and the adeno-associated virus-2/6.2 (AAV2/6.2) were compared for in utero transfer of a green fluorescent protein (GFP) reporter gene to ovine lung epithelium between days 65 and 78 of gestation. GFP expression was examined on day 85 or 136 of gestation (term is B145 days). The percentage of the respiratory epithelial cells expressing GFP in fetal sheep that received the JSRV-LV (3.18Â10 8 -6.85Â10 9 viral particles per fetus) was 24.6 ± 0.9% at 3 weeks postinjection (day 85) and 29.9±4.8% at 10 weeks postinjection (day 136). Expression was limited to the surface epithelium lining fetal airways o100 mm internal diameter. Fetal airways were amenable to VSVg-LV transduction, although the percentage of epithelial expression was low (6.6 ± 0.6%) at 1 week postinjection. GP64-LV, Ebola Zaire-LV and AAV2/6.2 failed to transduce the fetal ovine lung under these conditions. These data demonstrate that prenatal lung gene transfer with LV engineered to target apical surface receptors can provide sustained and high levels of transgene expression and support the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

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Section: Discussionmentioning

confidence: 99%

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

et al. 2011

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“…18 Immune-deficient mouse assay for human CB donor chimerism was as reported, 11 except that recipients were NOD/SCID/IL2Rg null (NSG). 19 …”

Section: Methodsmentioning

confidence: 99%

Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Broxmeyer

Lee

Hangoc

et al. 2011

Blood

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162

139

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IntroductionThe first cord blood (CB) transplantation saved the life of a young patient with Fanconi anemia using HLA-matched sibling CB cells, 1 a procedure made possible by identification and cryopreservation of transplantable hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) in CB. 2 More than 20 000 CB transplantations have treated the same malignant and nonmalignant disorders as bone marrow (BM). 3-8 CB transplantation is possible because of CB banks, and how long CB can be stored in a cryopreserved state with efficient recovery of HSCs and HPCs is critical for CB banking. We reported highly efficient recovery of CB HPCs after 5, 9 10, 10 and 15 11 years, and recovery of HSCs after 15 years. 11 We now report efficient recovery of functional HPCs up to 21-23.5 years, with more in depth studies on CB HSC engraftment in immune deficient mice, recovery of responsive T cells, generation of induced pluripotent stem (iPS) cells, 12-14 and detection of endothelial colony forming cells (ECFCs). 15 MethodsCB cells were scheduled for discard. 2 The study was approved by the Institutional Review Board of Indiana University (IU). Cryopreservation, thawing, and plating were as reported. 2,9-11 CB was assessed within 36 hours of collection. Cells were either separated into a mononuclear (MNC) fraction (Ficoll-Hypaque; Pharmacia) and aliquoted into cryotubes (Nalge Nunc) or left unseparated and aliquoted into cryo-freezer bags, 2,16,17 in 10% Dimethylsulfoxide and 10% autologous plasma for eventual analysis of HPC recovery. Percent recovery from MNC or unseparated cryopreserved cells was based on total prefreeze cells per volume of the exact same CB unit. 2,9-11 After thaw of unseparated cells, CD34 ϩ cells were magnetic-bead separated 11 for HSC engraftment and iPS cell generation studies. CD4 ϩ and CD8 ϩ T lymphocytes were separated from the CD34 ϩ -depleted cells and stimulated on plates precoated with anti-CD3 (OKT3, 0.5 g/mL) and anti-CD28 (clone CD28.2, 1 g/mL) with 10% FBS, 50M 2ME and 10ng/mL IL-15 as described. 18 Immune-deficient mouse assay for human CB donor chimerism was as reported, 11 except that recipients were NOD/SCID/IL2Rg null (NSG). 19 iPS cell generationAt IU, CD34 ϩ cells isolated from thawed, unseparated cells were grown with 10% FBS, 10 ng/mL human (h) SCF, 10 ng h Flt3-ligand, and 10 ng h Thrombopoietin/mL for 3 days. At day 4, cells were spin-infected (2200 rpm; 45 minutes) with concentrated lentiviral vectors Sox2-Oct4-EGFP and cMyc-Klaf4 (pc DNA-HIV-CS-CGW, provided by Dr P. Zoltick, Children's Hospital, Philadelphia; supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) in ␣-MEM medium with polybrene (Sigma-Aldrich). Medium was replaced at 6 days with the cytokines noted in this paragraph. At day 7, cells were transferred to mitotically inactivated murine embryonic fibroblasts (MEFs) and cultured as for human embryonic stem cells (hESCs). 20 iPS cells were also generated at Johns Hopkins using retro...

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“…Previously reported methods for enhancing transduction of CD34+ HSPCs with viral vectors include duration of prestimulation, number of transductions, multiplicity of infection, centrifugation, use of ABC transport inhibitors, fragments of fibronectin, culture media, and time [8][9][10][11][12][13]. Most of these methods, however, result in only incremental effects on overall transduction efficiency, are not scalable (centrifugation), are highly dependent on vector quality (infectious titer), and are subject to patient-specific variability.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Genetic Modification of Adult Growth Factor Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells With Rapamycin

Torres-Coronado

et al. 2014

Stem Cells Translational Medicine

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Genetic modification of adult human hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors leads to long‐term gene expression in the progeny of the HSPCs and has been used to successfully treat several monogenic diseases. In some cases, the gene‐modified cells have a selective growth advantage over nonmodified cells and eventually are the dominant engrafted population. However, in disease indications for which the gene‐modified cells do not have a selective advantage, optimizing transduction of HSPC is paramount to successful stem cell‐based gene therapy. We demonstrate here that transduction of adult CD34+ HSPCs with lentiviral vectors in the presence of rapamycin, a widely used mTORC1 inhibitor, results in an approximately threefold increase in stable gene marking with minimal effects on HSPC growth and differentiation. Using this approach, we have demonstrated that we can enhance the frequency of gene‐modified HSPCs that give rise to clonogenic progeny in vitro without excessive increases in the number of vector copies per cell or changes in integration pattern. The genetic marking of HSPCs and expression of transgenes is durable, and transplantation of gene‐modified HSPCs into immunodeficient mice results in high levels of gene marking of the lymphoid and myeloid progeny in vivo. The prior safe clinical history of rapamycin in other applications supports the use of this compound to generate gene‐modified autologous HSPCs for our HIV gene therapy clinical trials.

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Identification of parameters required for efficient lentiviral vector transduction and engraftment of human cord blood CD34+ NOD/SCID-repopulating cells

Cited by 22 publications

References 30 publications

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Enhanced Genetic Modification of Adult Growth Factor Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells With Rapamycin

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