Impact of Chemoselective Pressure on Integration Site Patterns of Lentivirally Transduced Human Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4622-4622
Author(s):  
Nadja Grund ◽  
Patrick Maier ◽  
Uwe Appelt ◽  
Heike Allgayer ◽  
Frederik Wenz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
In Silico ◽  
Integration Site ◽  
General Distribution ◽  
Cytostatic Drug ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Hematologic side effects of cancer chemotherapy like myelosuppression are frequently dose-limiting. Lentiviral gene therapy with cytostatic drug resistance gene transfer to human hematopoietic stem cells (CD34+) is a promising approach to overcome this problem. In this context it is of interest if chemotherapy mediated selection has an impact on lentiviral integration site patterns of transduced hematopoietic stem cells (CD34+). Concerning this issue, human CD34+ cells transduced with a lentiviral self-inactivating (SIN) vector encoding MGMTP140K (the O6-BG resistant mutant of O6-methylguanine- DNA methyltransferase) were in vitro treated with the alkylating agent BCNU. For integration site analysis LM-PCR was performed and integration patterns of the treated and untreated CD34+ cells were analyzed and compared with an in silico set of 106 random integrations. We found different integration preferences of the lentiviral vector between either the treated (82 integrations) or the untreated (30 integrations) CD34+ cells and the in silico set: both groups showed chromosomal preferences, a significant bias for integrations in genes (74,4% in the treated, respectively 70% in the untreated to 40% in the in silico group), especially by favouring introns, a random integration distribution regarding transcription start sites (TSS), and most importantly no significant differences concerning the number of integrations in or near cancer genes. Concerning all integration characteristics we could not find significant differences when comparing the untreated with the treated group. In conclusion, the general distribution of lentiviral integrations in either untreated or treated human CD34+ cells showed no distinct differences between both groups but significant differences compared to the in silico integration set. These results suggest that chemoselection of cells lentivirally overexpressing a specific chemoresistence gene might not influence the integration pattern. Therefore chemotherapy pressure seems not to hamper the safety of lentiviral vectors in gene transfer studies.

Gene transfer into murine hematopoietic stem cells with helper-free foamy virus vectors

Blood ◽  
2001 ◽  
Vol 98 (3) ◽  
pp. 604-609 ◽  
Author(s):  
George Vassilopoulos ◽  
Grant Trobridge ◽  
Neil C. Josephson ◽  
David W. Russell
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Integration Site ◽  
Foamy Virus ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Human Cd34

Abstract Gene transfer into hematopoietic stem cells (HSCs) is an ideal treatment strategy for many genetic and hematologic diseases. However, progress has been limited by the low HSC transduction rates obtained with retroviral vectors based on murine leukemia viruses. This study examined the potential of vectors derived from the nonpathogenic human foamy virus (HFV) to transduce human CD34+ cells and murine HSCs. More than 80% of human hematopoietic progenitors present in CD34+ cell preparations derived from cord blood were transduced by a single overnight exposure to HFV vector stocks. Mice that received transduced bone marrow cells expressed the vector-encoded transgene long term in all major hematopoietic cell lineages and in over 50% of cells in some animals. Secondary bone marrow transplants and integration site analysis confirmed that gene transfer occurred at the stem cell level. Transgene silencing was not observed. Thus vectors based on foamy viruses represent a promising approach for HSC gene therapy.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

scholarly journals Efficient Gene Transfer into Human CD34+ Cells by a Retargeted Adenovirus Vector

2000 ◽  
Vol 74 (6) ◽  
pp. 2567-2583 ◽  
Author(s):  
Dmitry M. Shayakhmetov ◽  
Thalia Papayannopoulou ◽  
George Stamatoyannopoulos ◽  
André Lieber
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Fluorescent Protein ◽  
Transduction Efficiency ◽  
Limiting Factor ◽  
Stable Gene ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Specific Subset

ABSTRACT Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and αv integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34+ cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34+ cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an αv integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34+ cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34+cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34+ cells expressing αv integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34+ cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34+ cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34+ c-Kit+cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34+ c-Kit+ cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.

scholarly journals Generation of Dyskeratosis Congenita-like Hematopoietic Stem Cells through the Stable Inhibition of DKC1

2021 ◽  
Author(s):  
Carlos Carrascoso-Rubio ◽  
Hidde A. Zittersteijn ◽  
Laura Pintado-Berninches ◽  
Beatriz Fernández-Varas ◽  
M. Luz Lozano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Failure ◽  
Clinical Manifestations ◽  
Dyskeratosis Congenita ◽  
Bone Marrow Failure Syndrome ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34 + cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1 -interfered CD34 + cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence and apoptosis. Moreover, DKC1 -interfered human CD34 + cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome, and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.

Novel Lentiviral Vectors Displaying ‘Early-Acting-Cytokines’ Preferentially Promote the Survival and Transduction of NOD/SCID Repopulating Human Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2107-2107
Author(s):  
E.L.S. Verhoeyen ◽  
Maciej Wiznerowicz ◽  
Delphine Olivier ◽  
Brigitte Izac ◽  
Didier Trono ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vectors ◽  
Hematopoietic Stem ◽  
Efficient Gene ◽  
Cd34 Cells

Abstract A major limitation of current generation lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent target cells which hampers their application for hematopoietic stem cell gene therapy. Human CD34+ cells that reside into G0 phase of the cell cycle and thus are quiescent, are indeed higly enriched in hematopoietic stem cells. Here, we designed novel lentiviral vectors that overcome this type of restriction by displaying early-acting-cytokines on their surface. Presentation of a single cytokine, thrombopoietin (TPO), or co-presentation of TPO and stem cell factor (SCF) on the lentiviral vector surface improved gene transfer into quiescent CD34+ cord blood cells by 45-fold and 77-fold, respectively, as compared to conventional lentiviral vectors. Moreover, these new LVs preferentially transduced and promoted the survival of immature resting cells rather than cycling CD34+ cells. Most importantly, the new early-cytokine-displaying lentiviral vectors allowed highly efficient gene transfer in CD34+ immature cells with long-term in vivo NOD/SCID mice repopulating capacity, a hallmark of bona fide HSCs. In conclusion, the novel ‘early-acting cytokines’ displaying LVs described here provide simplified, reproducible gene transfer protocols that ensure efficient gene transfer in hematopoietic stem cells. As such, these novel reagents bring us one step closer to selective in vivo gene therapy.

Interleukin-27 directly induces differentiation in hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 1903-1912 ◽  
Author(s):  
Jun Seita ◽  
Masayuki Asakawa ◽  
Jun Ooehara ◽  
Shin-ichiro Takayanagi ◽  
Yohei Morita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Extramedullary Hematopoiesis ◽  
Multiple Roles ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Common Signal

Interleukin (IL)-27, one of the most recently discovered IL-6 family cytokines, activates both the signal transducer and activator of transcription (STAT)1 and STAT3, and plays multiple roles in pro- and anti-inflammatory immune responses. IL-27 acts on various types of cells including T, B, and macrophage through the common signal-transducing receptor gp130 and its specific receptor WSX-1, but the effect of IL-27 on hematopoietic stem cells (HSCs) remains unknown. Here, we show that IL-27 together with stem cell factor (SCF) directly acts on HSCs and supports their early differentiation in vitro and in vivo. CD34−/lowc-Kit+Sca-1+lineage marker− (CD34−KSL) cells, a population highly enriched in mouse HSCs, were found to express both IL-27 receptor subunits. In vitro cultures of CD34−KSL cells with IL-27 and SCF resulted in an expansion of progenitors including short-term repopulating cells, while some of their long-term repopulating activity also was maintained. To examine its in vivo effect, transgenic mice expressing IL-27 were generated. These mice exhibited enhanced myelopoiesis and impaired B lymphopoiesis in the bone marrow with extramedullary hematopoiesis in the spleen. Moreover, IL-27 similarly acted on human CD34+ cells. These results suggest that IL-27 is one of the limited cytokines that play a role in HSC regulation.

Novel lentiviral vectors displaying “early-acting cytokines” selectively promote survival and transduction of NOD/SCID repopulating human hematopoietic stem cells

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3386-3395 ◽  
Author(s):  
Els Verhoeyen ◽  
Maciej Wiznerowicz ◽  
Delphine Olivier ◽  
Brigitte Izac ◽  
Didier Trono ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Surface Display ◽  
Lentiviral Vectors ◽  
Hematopoietic Stem ◽  
Efficient Gene ◽  
Cd34 Cells

AbstractA major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells, such as human CD34+ cells, that reside in the G0 phase of the cell cycle and that are highly enriched in hematopoietic stem cells. This hampers their application for gene therapy of hematopoietic cells. Here, we designed novel LVs that overcome this restriction by displaying “early-acting cytokines” on their surface. Display of thrombopoietin, stem cell factor, or both cytokines on the LV surface allowed efficient gene delivery into quiescent cord blood CD34+ cells. Moreover, these surface-engineered LVs preferentially transduced and promoted survival of resting CD34+ cells rather than cycling cells. Finally, and most importantly, these novel LVs allowed superior gene transfer in the most immature CD34+ cells as compared to conventional LVs, even when the latter vectors were used to transduce cells in the presence of recombinant cytokines. This was demonstrated by their capacity to promote selective transduction of CD34+ cell in in vitro derived long-term culture-initiating cell (LTC-IC) colonies and of long-term NOD/SCID repopulating cells (SRCs) in vivo.

Clonal Analysis of Hematopoietic Stem Cells after Serial Transplantation of Animals Receiving MGMTP140K Gene Transduced Cells Following Chemotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1182-1182
Author(s):  
Stephanie Laufs ◽  
Ursula Sorg ◽  
Veronika Kleff ◽  
Laila Gao ◽  
Michael Flasshove ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Integration Site ◽  
Hematopoietic Stem ◽  
Vector Integration ◽  
Serial Transplantation ◽  
Selection Of

Abstract Gene transfer of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) into hematopoietic stem cells has been shown to protect hematopoiesis from the toxic side effects of O6-guanine alkylating drugs such as BCNU, ACNU or temozolomide (TMZ). In addition, MGMT gene transfer allows efficient in vivo selection of transduced hematopoietic stem cells and enrichment of genetically corrected cells in the context of gene therapy for monogenetic diseases. We here have analysed the long-term effect of MGMT gene transfer on the hematopoietic stem cell compartment using an in vivo murine transplantation/gene therapy model and a retroviral vector carrying the gene for MGMTP140K, a mutant resistant to the wtMGMT-specific inhibitor O6-benzylguanine (BG). Serial transplants were performed and primary, secondary as well as tertiary recipients were treated with combined BG/ACNU, BG/BCNU or BG/TMZ chemotherapy at doses myeloablative in non-MGMT-protected hematopoiesis. Serial transplantation was performed with 1.8 – 3.0 x 106 mononuclear bone marrow cells and 2 to 3 animals were transplanted per primary or secondary animal. While initial gene transfer efficiency was low (1–5% of cells engrafted at week four) chemotherapy resulted in efficient selection of transduced cells in primary animals (70–90% transgene expression in peripheral blood). Secondary and tertiary recipients showed 40–80% transgene expression even before CTX. Efficient stem cell engraftment and protection from CTX was demonstrated in > 90% of secondary animals, while tertiary recipients clearly demonstrated compromised engraftment and a substantial number of animals did not survive CTX treatment. Retroviral vector integration site analysis to study the clonality of hematopoiesis of stem cells by ligation mediated PCR (LM-PCR) was performed in the serially transplanted mice. In three mice of the secondary transplantation cohort we detected 3, 0, and 6 clones, respectively. In three mice of the tertiary transplantation cohort 7, 2, and 2 clones, respectively, were found. Thus, an exhaustion of transduced hematopoiesis following regenerative stress by high dose chemotherapy was not evident. Of the total 20 detected clones one could not be mapped to the mouse genome, while the others could be blasted against the mouse genome (assembly 2004, http://genome.ucsc.edu/; >99.5% identity). It turned out that 5 of 8 integrations landed in RefSeq in the tertiary transplantation cohort, while 3 of 8 integrations occurred in RefSeq genes in the secondary transplantation cohort. This suggests that clones profit from the transcription machinery of their integration site. Thus, our LM-PCR results indicate that the multiclonality of hematopoiesis is conserved after serial transplants which may be considered a safety feature for drug-resistance gene therapy. Furthermore, vector integration in highly resistant stem cells is favored in actively transcribed genomic regions.

Characterization of Human CD34+ Hematopoietic Stem Cells Following Administration of G-CSF or Plerixafor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3476-3476 ◽  
Author(s):  
Michael P. Rettig ◽  
William D. Shannon ◽  
Julie Ritchey ◽  
Matthew Holt ◽  
Kyle McFarland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Hematopoietic Stem Cells ◽  
B Lymphocytes ◽  
Gene Signature ◽  
Drug Clearance ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Background: Current clinical protocols use granulocyte colony-stimulating factor (G-CSF) to mobilize normal hematopoietic stem cells (HSCs) from the bone marrow (BM) to the peripheral blood. Unfortunately, this process requires from 4 to 6 days of G-CSF injection and is associated with significant morbidity, most notably bone pain. We are evaluating a novel method for the mobilization of HSCs using a direct antagonist of the CXCR4/SDF-1 interaction called Plerixafor (AMD3100). Methods: Human CD34+ cells were collected from three different studies at our institution. In the first study, fifteen healthy allogeneic related donors were initially mobilized with increasing doses of intravenous (IV) AMD3100 (80, 160, 240, or 320 μg/kg). After 4 days of drug clearance, the same donors were then mobilized with a single subcutaneous (s.c.) dose of 240 μg/kg AMD3100 and collected cells were used as a source of HSCs for transplantation. In the second study, ten healthy donors were mobilized with 5 days of s.c. injection of G-CSF (10 μg/kg/day), and leukapheresed on day 5. In the final study, eight individual normal donors were mobilized sequentially with AMD3100 and G-CSF. Donors received 1 s.c. injection of 240 μg/kg AMD3100, followed by leukaphersis beginning 4 h after drug treatment. After 10 days of drug clearance, the same donors were mobilized with 5 days s.c. injection of 10 μg/kg/day G-CSF, and leukapheresed on day 5. Human CD34+ cells were purified by positive selection with a Magnetic Affinity Cell Selection (MACS) CD34 isolation kit and total RNA was isolated using RNeasy Mini Kit columns (Qiagen). The purity (>85% for all experiments) and phenotype of isolated CD34+ cells was quantified by flow cytometry. RNA profiling analyses were performed using Affymatrix U133+2 arrays. Results: Peak mobilization of CD34+ cells occurred 4 to 6 hours after both IV and s.c. dosing, however, patients given IV doses had higher peak levels of CD34/μl at every time point. There was a clear doseresponse relationship of IV AMD3100 on mobilization of CD34+ HSCs in normal donors, with the 320 μg/kg dose yielding a maximum increase in circulating CD34+ cells from 3.3 ± 1.8 CD34+/μl at baseline to 28.8 ± 4.7 CD34+/μl at 6 h after injection. Although the magnitude of neutrophil, monocyte, and T lymphocyte mobilization by IV AMD3100 was less than that observed for CD34+ cells (2 to 3 fold increase over baseline), the kinetics of their mobilization were similar to the CD34+ HSCs (peak mobilization 4 to 6 h after AMD3100). In contrast, B-lymphocytes were mobilized more rapidly (4.5 ± 1.7-fold at 15 min post-AMD3100) and efficiently (6.6 ± 2.6-fold at 2 h post-AMD3100) by IV AMD3100. This rapid mobilization of B-lymphocytes correlates with our pharmacokinetic studies, which showed that peak levels of AMD3100 occur between 15 and 30 minutes after IV infusion. The gene signature of AMD3100-mobilized human CD34+ HSCs is distinct from that of G-CSF-mobilized CD34+ cells. Of note, EMR1, GIMAP8, PIM1, S100A8, SOCS3, and TMEM49 were expressed more abundantly in all GCSF-mobilized CD34+ cells while BCL-2, CLC, CXCR4, C200rf118, DNTT, IRF8, PRG2, RASD1, RNASE6, and UHRF1 were more abundantly expressed in all AMD3100-mobilized CD34+ cells. Interestingly, the RNA profile of CD34+ HSCs obtained from the BM of three healthy donors clustered with AMD3100-mobilized CD34+ HSCs rather than GCSF mobilized HSCs. Using flow cytometry, we identified a CD34dimCD45RA+ hematopoietic precursor cell that is uniquely enriched in nearly 60% of the AMD3100 products evaluated to date (6/10 patients). In contrast to G-CSF mobilized products, where <2% of CD34+ cells are CD34dimCD45RA+, up to 20% of CD34+ cells in AMD3100 treated donors are CD34dimCD45RA+. Preliminary flow cytometry data suggest that this CD34dimCD45RA+ population represents a pro-DC2 (for progenitor of pre-dendritic cell type 2) progenitor compartment as indicated by their IL-3RαbrightCD62Lbrightα4β7dimCD4dimCD25−c-kit−CD13− phenotype. Conclusions: These observations suggest that IV AMD3100 may be a more effective mobilization agent with a low side effect profile. The gene signature and phenotype of AMD3100-mobilized CD34+ HSCs is distinct from that of G-CSF-mobilized HSCs and resemble CD34+ HSCs present in an unmanipulated BM.

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