Effects of busulfan dose escalation on engraftment of infant rhesus monkey hematopoietic stem cells after gene marking by a lentiviral vector

2006 ◽  
Vol 34 (3) ◽  
pp. 369-381 ◽  
Author(s):  
Christoph A. Kahl ◽  
Alice F. Tarantal ◽  
Chang I. Lee ◽  
Daniel F. Jimenez ◽  
Christopher Choi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Rhesus Monkey ◽  
Hematopoietic Stem Cells ◽  
Dose Escalation ◽  
Lentiviral Vector ◽  
Hematopoietic Stem ◽  
Infant Rhesus

Long-Term In Vivo Expression from a Self-Inactivating Lentiviral Vector Flanked by a Chromatin Insulator Element.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1289-1289
Author(s):  
Ping Xia ◽  
Richard Emmanuel ◽  
Kuo Isabel ◽  
Malik Punam
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vector ◽  
Gfp Expression ◽  
Hematopoietic Stem ◽  
Insulator Element ◽  
Gfp Fluorescence

Abstract We have previously shown that self-inactivating lentiviral vectors infect quiescent hematopoietic stem cells (HSC), express long-term, resist proviral silencing in HSC and express in a lineage specific manner. However, their random integration into the host chromosome results in variable expression, dependent upon the flanking host chromatin (Mohamedali et al, Mol. Therapy 2004). Moreover, the recent occurrence of leukemogenesis from activation of a cellular oncogene by the viral enhancer elements calls for safer vector designs, with expression cassettes that can be ‘insulated’ from flanking cellular genes. We analyzed the role of the chicken β-globin locus hypersensitive site 4 insulator element (cHS4) in a self-inactivating (SIN) lentiviral vector in the RBC progeny of hematopoietic stem cells (HSC) in long term in vivo. We designed an erythroid-specific SIN-lentiviral vector I8HKGW, expressing GFP driven by the human ankyrin gene promoter and containing two erythroid-specific enhancer elements and compared it to an analogous vector I8HKGW-I, where the cHS4 insulator was inserted in the SIN deletion to flank the I8HKGW expression cassette at both ends upon integration. First, murine erythroleukemia (MEL) cells were transduced at <5% transduction efficiency and GFP+ cells were sorted to generate clones. Single copy MEL clones showed no difference in the mean GFP fluorescence intensity (MFI) between the I8HKGW+ and the I8HKGW-I+ MEL clones. However, there was a reduction in the chromatin position effect variegation (PEV), reflected by reduced coefficient of variation of GFP expression (CV) in I8HKGW-I clones (n=115; P<0.01), similar to in vitro results reported by Ramezani et al (Blood 2003). Next, we examined for expression and PEV in the RBC progeny of HSC, using the secondary murine bone marrow transplant model. Lethally irradiated C57Bl6 (CD45.2) mice were transplanted with I8HKGW and I8HKGW-I transduced B6SJL (CD45.1) Sca+Lin- HSC and 4–6 months later, secondary transplants were performed. Mice were analyzed 3–4 months following secondary transplants (n=43). While expression from both I8HKGW and I8HKGW-I vectors appeared similar in secondary mice (46±6.0% vs. 48±3.6% GFP+ RBC; MFI 31±2.6 vs. 29±1.4), there were 0.37 vs. 0.22 copies/cell in I8HKGW and I8HKGW-I secondary recipients, respectively (n=43), suggesting that the probability of GFP expression from I8HKGW-I vectors was superior when equalized for vector copy. The CV of GFP fluorescence in RBC was remarkably reduced to 55±1.7 in I8HKGW-I vs. 196±32 in I8HKGW RBC (P<0.001). We therefore, analyzed these data at a clonal level in secondary CFU-S and tertiary CFU-S. The I8HKGW-I secondary CFU-S had more GFP+ cells (32.4±4.4%) vs. I8HKGW CFU-S (8.1±1.2%, n=143, P<0.1x10E-11). Similarly, I8HKGW-I tertiary CFU-S also had more GFP+ cells (25±1.8%) vs. I8HKGW CFU-S (6.3±0.8%, n=166, P<0.3x10E-10). We also plated bone marrow from secondary mice in methylcellulose and analyzed GFP expression in individual BFU-E. The I8HKGW-I tertiary BFU-E had more GFP+ cells (28±3.9%) vs. I8HKGW BFU-E (11±5%, n=50, P<0.03) with significantly reduced CV (67 vs 125, n=50, P<6.6X10E-7). Taken together, the ‘insulated’ erythroid-specific SIN-lentiviral vector increased the probability of expression of proviral integrants and reduced PEV in vivo, resulting in higher, consistent transgene expression in the erythroid cell progeny of HSC. In addition, the enhancer blocking effect of the cHS4, although not tested here, would further improve bio-safety of these vectors for gene therapy for RBC disorders.

Successful treatment of murine β-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 506-513 ◽  
Author(s):  
Derek A. Persons ◽  
Esther R. Allay ◽  
Nobukuni Sawai ◽  
Phillip W. Hargrove ◽  
Thomas P. Brent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Lentiviral Vector ◽  
Drug Resistant ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

AbstractSuccessful gene therapy of β-thalassemia will require replacement of the abnormal erythroid compartment with erythropoiesis derived from genetically corrected, autologous hematopoietic stem cells (HSCs). However, currently attainable gene transfer efficiencies into human HSCs are unlikely to yield sufficient numbers of corrected cells for a clinical benefit. Here, using a murine model of β-thalassemia, we demonstrate for the first time that selective enrichment in vivo of transplanted, drug-resistant HSCs can be used therapeutically and may therefore be a useful approach to overcome limiting gene transfer. We used an oncoretroviral vector to transfer a methylguanine methyltransferase (MGMT) drug-resistance gene into normal bone marrow cells. These cells were transplanted into β-thalassemic mice given nonmyeloablative pretransplantation conditioning with temozolomide (TMZ) and O6-benzylguanine (BG). A majority of mice receiving 2 additional courses of TMZ/BG demonstrated in vivo selection of the drug-resistant cells and amelioration of anemia, compared with untreated control animals. These results were extended using a novel γ-globin/MGMT dual gene lentiviral vector. Following drug treatment, normal mice that received transduced cells had an average 67-fold increase in γ-globin expressing red cells. These studies demonstrate that MGMT-based in vivo selection may be useful to increase genetically corrected cells to therapeutic levels in patients with β-thalassemia.

Inhibition of Let-7 Processing in Adult Murine Hematopoietic Stem Cells Induces a Fetal-Like High Self-Renewal Pattern in Their Progeny

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 45-45 ◽  
Author(s):  
Michael R. Copley ◽  
David G. Kent ◽  
Claudia Benz ◽  
Stefan Wohrer ◽  
Keegan M. Rowe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Mirna Biogenesis ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Control Virus ◽  
Elevated Expression

Abstract Abstract 45 Fetal hematopoietic stem cells (HSCs) in mice differ from their adult counterparts in a number of key properties. These include a higher cycling activity, an ability to more rapidly reconstitute the HSC compartment of irradiated recipient mice, a higher output of myeloid as compared to lymphoid progeny, and a greater sensitivity to the self-renewal promoting activity of Steel factor. We have previously shown that most of these features of fetal HSCs are sustained until 3 weeks after birth at which time they are rapidly (within 1 week), completely and permanently replaced with the corresponding properties of adult HSCs. A candidate regulator of this transition, Hmga2, was identified based on its greater expression in highly purified fetal versus adult HSCs (CD45+EPCR+CD48−CD150+; E-SLAM cells) with persistence of this difference in the matching lineage-negative (lin−) compartments. Experiments in which Hmga2 was overexpressed by lentiviral transduction of purified adult HSCs which were then transplanted into irradiated mice provided evidence that this chromatin remodeling factor can activate a fetal-like HSC program in these cells; i.e., more rapidly reconstitute the HSC compartment (increased self-renewal response) and produce clones with a higher proportion of myeloid cells. Based on the known ability of the let-7 family of microRNAs (miRNAs) to target Hmga2 transcripts resulting in their degradation and/or translational repression, we next hypothesized that let-7 miRNAs might be involved in controlling HSC developmental programs. A comparison of the levels of expression of 6 members of the let-7 family in purified fetal and adult HSCs, as well as in lin− hematopoietic cells, showed that transcripts for all of these are higher in the adult subsets, although this difference was significant only for let-7b (p<0.05). Since Lin28 is a natural inhibitor of let-7 miRNA biogenesis we proposed that overexpression of this protein might be used to simultaneously inhibit all let-7 miRNA species and therefore modulate let-7-mediated effects in HSCs. Transduction of BA/F3 cells with a Lin28-YFP lentiviral vector led to an elevated expression of Lin28 and a significant decrease in multiple let-7 miRNAs. To investigate the influence of Lin28 overexpression on adult HSC self-renewal activity in vivo, we used the same Lin28 lentiviral vector (or a control YFP vector) to transduce highly purified HSCs (40 E-SLAM cells, i.e. ∼20 HSCs/group/experiment, 3 experiments) in a 3–4-hour exposure protocol and then transplanted all of the cells directly into irradiated mice (total of 3–4 mice/group). The number of HSCs regenerated 6 weeks later was subsequently measured by performing limiting-dilution transplants in secondary mice (total of 12–16 secondary mice/group/experiment). Interestingly, analysis of the secondary recipients showed that the Lin28-overexpressing adult HSCs had expanded in the primary recipients ∼6-fold more than the control-virus transduced HSCs (p<0.001). These findings support our thesis that alterations in let-7 miRNA levels play a key role in regulating the developmental switch from fetal to adult HSCs programs that occurs between 3 and 4 weeks after birth in mice. Disclosures: No relevant conflicts of interest to declare.

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