scholarly journals Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4257-4265 ◽  
Author(s):  
Francesca Romana Santoni de Sio ◽  
Paolo Cascio ◽  
Anna Zingale ◽  
Mauro Gasparini ◽  
Luigi Naldini
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Cell Cycle Progression ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Proteasome Activity ◽  
Hematopoietic Stem ◽  
Vector Integration ◽  
Cytokine Stimulation

AbstractThe therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture, they display low permissivity to the vector, requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation, we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays, we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer, allowing the reach of very high levels of vector integration in their progeny in vivo. Thus, LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly, cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors, highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy.

scholarly journals Correction of canine X-linked severe combined immunodeficiency by in vivo retroviral gene therapy

Blood ◽  
2006 ◽  
Vol 107 (8) ◽  
pp. 3091-3097 ◽  
Author(s):  
Suk See Ting–De Ravin ◽  
Douglas R. Kennedy ◽  
Nora Naumann ◽  
Jeffrey S. Kennedy ◽  
Uimook Choi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Large Animal Model ◽  
Large Animal ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem

AbstractX-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency and early mortality, the only potential cure being hematopoietic stem cell (HSC) transplantation or gene therapy. Current clinical gene therapy protocols targeting HSCs are based upon ex vivo gene transfer, potentially limited by the adequacy of HSC harvest, transduction efficiencies of repopulating HSCs, and the potential loss of their engraftment potential during ex vivo culture. We demonstrate an important proof of principle by showing achievement of durable immune reconstitution in XSCID dogs following intravenous injection of concentrated RD114-pseudotyped retrovirus vector encoding the corrective gene, the interleukin-2 receptor γ chain (γc). In 3 of 4 dogs treated, normalization of numbers and function of T cells were observed. Two long-term–surviving animals (16 and 18 months) showed significant marking of B lymphocytes and myeloid cells, normalization of IgG levels, and protective humoral immune response to immunization. There were no adverse effects from in vivo gene therapy, and in one dog that reached sexual maturity, sparing of gonadal tissue from gene transfer was demonstrated. This is the first demonstration that in vivo gene therapy targeting HSCs can restore both cellular and humoral immunity in a large-animal model of a fatal immunodeficiency.

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.

scholarly journals Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model

2021 ◽  
Vol 18 ◽  
pp. 347-354
Author(s):  
Masashi Noda ◽  
Kohei Tatsumi ◽  
Hideto Matsui ◽  
Yasunori Matsunari ◽  
Takeshi Sato ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Canine Model ◽  
Gene Transfer Techniques

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Prolonged High-Level Detection of Retrovirally Marked Hematopoietic Cells in Nonhuman Primates after Transduction of CD34+ Progenitors Using Clinically Feasible Methods.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1137-1137
Author(s):  
Tong Wu ◽  
Hyeoung Joon Kim ◽  
Stephanie E. Sellers ◽  
Kristin E. Meade ◽  
Brian A. Agricola ◽  
...  
Keyword(s):  
Stem Cell ◽  
Gene Transfer ◽  
Nonhuman Primates ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Colony Pcr ◽  
High Level

Abstract Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell(HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells(STR), or the fibronectin fragment CH-296(FN), and various cytokines such as flt3 ligand(FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor(SCF), and FLT(our best cytokine combination in prior studies)with a combination of megakaryocyte growth and development factor(MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10–15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5143-5143
Author(s):  
Liesbeth De Waele ◽  
Kathleen Freson ◽  
Chantal Thys ◽  
Christel Van Geet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Phosphoglycerate Kinase ◽  
Lentiviral Vectors ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2839-2848 ◽  
Author(s):  
Hitoshi Hibino ◽  
Kenzaburo Tani ◽  
Kenji Ikebuchi ◽  
Ming-Shiuan Wu ◽  
Hajime Sugiyama ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Nonhuman Primate ◽  
Peripheral Blood ◽  
Common Marmoset ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
The Common

Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (<1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting hematopoietic stem cells.

scholarly journals Thalassemia Gene Therapy By In Vivo Transduction of Mobilized Hematopoietic Stem Cells (HSCs) with an Integrating Hybrid Adenovirus Vector System

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2193-2193
Author(s):  
Afrodite Georgakopoulou ◽  
Hongjie Wang ◽  
Chrysi Kapsali ◽  
Nikoleta Psatha ◽  
Angeliki Koufali ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematological Parameters ◽  
Sleeping Beauty ◽  
Vector System ◽  
Good Tolerability ◽  
Hematopoietic Stem

Abstract To overcome the cost and complexity of current thalassemia ex vivogene therapy protocols, we developed a minimally invasive and readily translatable approach for in vivo HSC gene delivery which abrogates the need for HSC leukapheresis, CD34+ cell selection, ex vivo HSC culture, myeloablation and ultimately, transplantation. Our approach involves HSC mobilization with G-CSF/AMD3100 andintravenous injections of a hybrid vector system consisting ofa CD46-targeting, helper-dependent adenoviral vector and the hyperactive Sleeping Beauty transposase (SB100x) that mediates integration of thevector-encoded γ-globin and mgmtP140K genes. Pretreatment with glucocorticoids before virus injectionsis used to blockthe release of pro-inflammatory cytokines andimmunosuppression is applied in order to avoid responses against human g-globin- and MGMT protein-expressing cells. We tested our approach in a mouse model recapitulating the phenotypeof human β-thalassemia intermedia (Hbbth-3/hCD46++ mice). At week 8 post transduction, hCD46+/+/Hbbth-3 mice expressed HbF in 31.2±2.7% of circulating erythrocytes. Due to a significant drop in HbF expression by week 16 (11.9±3.0%), a 4-dose O6BG/BCNU treatment was administeredin order to in vivo select forgene corrected hematopoietic progenitors, thus recovering the HbF expression in76.0±5.7% of the circulating erythrocytes, by week 29 post in vivo transduction. With an average vector copy number of 1.4/cell, the human γ-globin to mouse α-globin expression was ~10% by HPLC and the human γ-globin to mouse β-globin mRNA ratio ~10%, by qRT-PCR. Hematological parameters (RBCs, Ht, Hb, MCV, RDW, Reticulocytes) at week 29 post in vivo transduction, were significantly improved over baseline or were indistinguishable from normal values, suggesting near to complete phenotypic correction. Treated mice showed significant reduction of spleen size, extramedullary erythropoiesis and parenchymal hemosiderosis. After secondary transplantation and without in vivo selection, more than 90% of donor-derived erythrocytes (CD46+) were g-globin-positive, up to 20 weeks post-transplant. Safety was demonstrated by the good tolerability of treatment, the absence of alterations in hematopoiesis, the normal colony-forming potential of bone marrow cells and the random integration pattern of our vector system. Overall, we present a simplified platform for gene therapy of thalassemia, which can serve as a cost-efficient and "portable" approach to make gene therapy accessible even to resource-poor regions where thalassemia major is endemic but only minimally complex strategies could be adopted. Disclosures No relevant conflicts of interest to declare.

scholarly journals Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Blood ◽  
2018 ◽  
Vol 131 (26) ◽  
pp. 2915-2928 ◽  
Author(s):  
Chang Li ◽  
Nikoletta Psatha ◽  
Pavel Sova ◽  
Sucheol Gil ◽  
Hongjie Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Binding Site ◽  
Genome Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Cell Genome ◽  
Hsc Transplantation

Key Points CRISPR/Cas9-mediated disruption of a BCL11A binding site in HSCs of β-YAC mice results in the reactivation of γ-globin in erythrocytes. Our approach for in vivo HSC genome editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.

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