Evidence for Efficacy and Safety of Lentiviral Mediated Gene Transfer in T Cells and CD34+ Cells from Wiskott-Aldrich Syndrome Patients.

Abstract Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by eczema, recurrent infections, severe hemorrhages and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Therapy based on the transplant of genetically correct autologous stem cells could represent a valid alternative approach. We investigated the efficacy and the safety of WAS gene transfer using HIV-based lentiviral vector encoding for WAS cDNA under the control of an autologous promoter (1.6 kb). T cells obtained from WAS patients showed normal level of WAS expression after lentiviral transduction. Transduced T cells showed a correction in TCR-driven proliferation and IL-2 production. Furthermore, a selective growth advantage of transduced T cells was observed in long-term in vitro cultures. Studies in T cell clones generated from transduced WAS CD4+ T cells revealed that 1–2 vector copies were necessary and sufficient to correct T cells function. CD34+ cells, isolated from mobilized peripheral blood and bone marrow of healthy donors, were transduced using WASP or GFP-encoding lentiviral vectors. Cells were cultured in the presence of different cytokines to investigate if WAS gene transfer could have any effect on short and long-term differentiation (CFU-C, LTC-IC and B/NK assays). Transduction resulted in a comparable number of CFU-C and LTC-IC colonies and normal B and NK cells differentiation with respect to untransduced cells. Furthermore, transduction of CD34+ cells isolated from the bone marrow of a WAS patient was performed under optimized culture conditions. Lentiviral gene transfer led to restoration of WASP expression in differentiated cells with copy number ranging from 1 to 5 copies per cell. In conclusion, our data demonstrate that the WAS promoter/cDNA-containing lentiviral vector can efficiently transduce and restore WASP expression in CD34+ cells and T cells from WAS patients. Experiments in the Rag2−/−/γchain- murine model are ongoing to test the efficacy and safety of the WASP transduced CD34+ cells. Together, our studies provide a preclinical basis for the implementation of a gene therapy trial for WAS patients.

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Role of Osteoclasts in Regulation of Human Hematopoietic Stem Cell Niche and Fate.

Blood ◽

10.1182/blood.v104.11.4253.4253 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4253-4253

Author(s):

Shmuel Yaccoby ◽

Kenichiro Yata ◽

Yun Ge ◽

Bart Barlogie ◽

Joshua Epstein ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cord Blood ◽

Scid Mice ◽

Common Mechanism ◽

Hematopoietic Stem ◽

Variable Expression ◽

Cd34 Cells ◽

Rabbit Bone

Abstract Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (>95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (>90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after >6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

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

Blood ◽

10.1182/blood.v106.11.1289.1289 ◽

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.

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

Blood ◽

10.1182/blood.v108.11.3249.3249 ◽

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.

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Increased Aldehyde Dehydrogenase Activity Indentifies a Subset of Human Leukemic Blasts with Stem Cell Characteristics and Correlates with Poor Prognosis.

Blood ◽

10.1182/blood.v112.11.1345.1345 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1345-1345

Author(s):

Dan Ran ◽

Mario Schubert ◽

Larissa Pietsch ◽

Isabel Taubert ◽

Christian Wallenwein ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Prognostic Factors ◽

Aldehyde Dehydrogenase ◽

Protein Level ◽

Aldh Activity ◽

Cd34 Cells ◽

Dividing Cells

Abstract INTRODUCTION: Normal hematopoietic stem cells (HSC) are characterized by their ability to self-renew, to generate multiple cell-lineages, and show slow divisional kinetics. Leukemic stem cells (LSC) have been reported to show similar characteristics but their identification has been elusive. We have studied various methods and have identified aldehyde dehydrogenase (ALDH) staining as an optimal method for the enrichment of primary human LSC. MATERIAL&METHODS: Bone marrow samples were obtained from patients with newly diagnosed AML after informed consent. Mononuclear cells were stained with Aldefluor and sorted by flow cytometry according to their forward/side scatter characteristics and ALDH activity (ALDH+/ALDH−). Alternatively, primary AML samples were being enriched for CD34+ cells by magnetic column, then double-stained with CD34-antibodies and Aldefluor and sorted for the co-expression of CD34+ and ALDH+, respectively for CD34+ alone. Human Mesenchymal Stromal Cells (MSC), isolated from human bone marrow, were used as a surrogate model for the cellular microenvironment of the hematopoietic niche. Adhesion of various AML cell lines and subpopulations of primary leukemic cells (ALDH+, ALDH−, CD34+, CD34+/ALDH+, all blasts) to MSC was tested in the adhesion chamber assay. Semi-quantitative RT-PCR was used to analyze the gene expression of various adhesion molecules and Western- Blot analysis was performed to validate the PCR-results on protein level. The generation of secondary leukemic colonies was evaluated in a semi-solid methylcellulose medium, as well as in a long term co-culture system (LSC-IC assay; in analogy to the LTC-IC assay). RESULTS: The percentage of ALDH+ cells ranged from 0.01% to 13.2% with a median of 1.47% (n=55). Adhesion significantly differed in the ALDH+ and ALDH− subpopulations: 85±4% of ALDH+ cells but only 61±8% of ALDH− cells were adherent (n=11, p<0.001). Adhesion molecules, such as CXCR4 and CD44, were highly expressed on the ALDH+ subpopulation both on mRNA level and protein level, in contrast to the ALDH− subpopulation. Analysis of the initial divisional kinetics on single cell base showed that the ALDH+ subpopulation contained more slow dividing cells whereas the majority of the ALDH− subpopulation consisted of fast-dividing cells (n=3; p<0.01). The frequency of long term leukemic colony initiating cells (LSC-IC) was 3.82% in the ALDH+ but only 0.01% in the ALDH− (n=21; p<0.01). In the CD34+ the LSC-IC frequency was 1.96% versus 0.01% in the CD34− (n=5, p<0.01). The highest LSC-IC frequency could be monitored in ALDH+/CD34+ cells: 6.1% generated secondary leukemic colonies (n=5). These colonies, harvested after 7 weeks of cultivation, were examined for their immune phenotype and screened for cytogenetic aberrations by fluorescent in situ hybridization (FISH) and the chromosomal aberrations were consistent with the AML samples taken at diagnosis. Furthermore, the frequency of ALDH+ cells correlated significantly with adverse prognostic factors: patients with a high-risk karyotype had a mean of 2.9% ALDH+ cells (n=21); in contrast, patients with a normal karyotype had a mean of 0.4% ALDH+ cells in their bone marrow (n=34; p<0.001). The ability of ALDH+ versus ALDH− subsets to generate secondary leukemia in the animal model is concurrently examined. DISCUSSION: In summary, measurement of the ALDH activity provides a useful tool for the isolation of a distinct AML-blast subpopulation with stem-cell like features (LSC). The ALDH+ subsets showed higher affinity to the surrogate niche (MSC), elevated expression of CD44, Cadherin-2, and CXCR4 and were associated with an increased frequency of secondary leukemic colonies in vitro (LSC-IC). Above all, the frequency of ALDH+ blasts correlated with clinical prognostic factors, which substanciates LSC as a relevant therapeutic target.

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Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽

10.1182/blood.v122.21.1999.1999 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1999-1999

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Benedetta Nicolini ◽

Nadim Mahmud ◽

Elisa Bonetti ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

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Gene Therapy for Fanconi Anemia in Seattle: Clinical Experience and Next Steps

Blood ◽

10.1182/blood.v128.22.3510.3510 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3510-3510 ◽

Cited By ~ 4

Author(s):

Jennifer E Adair ◽

Pamela S. Becker ◽

Devikha Chandrasekaran ◽

Grace Choi ◽

Ann E Woolfrey ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Gene Transfer ◽

Blood Cell ◽

Phase I ◽

Fanconi Anemia ◽

Magnetic Bead ◽

Cd34 Cells ◽

Equity Ownership

Abstract One hallmark of the rare, monogenic disorder called Fanconi anemia (FA) is an accelerated decline in hematopoietic stem cells (HSCs) leading to bone marrow (BM) failure. Long-term treatment requires successful bone marrow transplant (BMT) from an unaffected donor. However, BMT success is limited if the donor is not a matched sibling and ~70% of FA patients lack such donors. Gene therapy could be an alternative, correcting the genetic defect in the patient's own HSCs, and negating the need for a BMT donor. Based on lessons learned in previous FA gene therapy studies, we developed an optimized protocol for lentivirus (LV)-mediated FANCA gene transfer into HSCs from FA-A patients. This phase I clinical trial incorporates vector recommendations from the International FA Gene Therapy Working Group. Two patients have been treated on this trial to date (National Clinical Trials Registry ID: NCT01331018). This protocol harvests BM to collect unmanipulated HSC and does not include conditioning prior to cell infusion. Patient 1 is a 22-year old male with confirmed FA-A resulting from a splice variant in exon 22 of the FANCA gene (c. 1827-1 G>A). Baseline ANC averaged 0.5-1.0 K/mcL and baseline platelet counts averaged 40 K/mcL. A total of 3.2 x 107 CD34+ cells were present in 1.1L of BM, but only 9.4 x 106 total CD34+ cells were successfully isolated by magnetic bead separation, due to low level CD34 expression. LV transduction at 10 infectious units (IU)/cell resulted in a vector copy number (VCN) of 0.33 per cell and 18.4% of colony-forming cells transduced. Patient 2 is a 10-year old male with confirmed FA-A resulting from gross deletion of exons 6-31 of the FANCA gene. Baseline ANC and platelets declined over a 4.5-year interval prior to gene therapy and were 0.67 K/mcL and 82 K/mcL, respectively, in the 6 months prior to treatment. A total of 400mL of BM was collected, containing a total of 30.6 x 106 CD34+ cells. To avoid excessive CD34+ cell loss, the CD34+ cell magnetic bead purification step was omitted and the entire red blood cell depleted BM product was subjected to LV transduction at 10 IU/cell. We observed a VCN of 1.83 per cell and 43% of colony-forming cells transduced, suggesting more efficient transduction of the mixed cell population. Both patients tolerated the harvest and infusion procedures but displayed low and declining levels of transduced cells in peripheral blood after infusion. For future subjects, use of pre-infusion conditioning may be required to achieve long-term engraftment in vivo. Interestingly, both patients have maintained stable blood cell counts since gene therapy. These data demonstrate that LV gene therapy in FA patients is safe and suggest that avoidance of direct CD34 selection is advantageous for transduction and gene transfer. However, one complicating factor is the volume of concentrated LV vector required to transduce the non-purified cell product infused in Patient 2. To address these barriers, we developed a modified clinical protocol which utilizes depletion of mature cell lineages including CD3+, CD14+, CD16+ and CD19+ cells. Using healthy donor bone marrow we demonstrate that this protocol efficiently depletes >85% of cells expressing each lineage marker, reducing the volume of LV vector required for gene transfer by 60-70%. Most importantly, we demonstrate that this protocol preserves >90% of CD34+ cells present in the starting bone marrow product, and that these cells are efficiently transduced and capable of engrafting in a xenotransplant model. This protocol is currently being implemented for subjects in the ongoing phase I trial. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

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

Blood ◽

10.1182/blood-2002-01-0025 ◽

2003 ◽

Vol 101 (1) ◽

pp. 112-118 ◽

Cited By ~ 71

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.

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Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression

Blood ◽

10.1182/blood.v78.2.310.bloodjournal782310 ◽

1991 ◽

Vol 78 (2) ◽

pp. 310-317 ◽

Cited By ~ 1

Author(s):

JF Apperley ◽

BD Luskey ◽

DA Williams

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Bone Marrow Cells ◽

Selectable Marker ◽

Retroviral Vector ◽

G418 Selection ◽

Marrow Cells

Retroviral-mediated gene transfer of human adenosine deaminase (hADA) provides a model system for the development of somatic gene therapy as a therapy for diseases of bone marrow-derived cells. We have previously demonstrated that hADA can be observed in all hematopoietic lineages in a minority of mice transplanted with bone marrow cells infected with a simplified retroviral vector, ZipPGK-ADA. Here we report a majority of mice (six of eight) demonstrate expression of hADA in the peripheral blood at least 6 months after transplantation with bone marrow infected with this simplified retroviral vector, which contains no selectable marker. The failure to express hADA in two of eight mice was associated with the absence of the recombinant retroviral provirus in DNA prepared from bone marrow cells of these mice apparently due to failure to efficiently infect the reconstituting hematopoietic stem cell. In an effort to preselect bone marrow stem cells containing proviral integrations, we incorporated the selectable marker neo phosphotransferase (NEO) into a retroviral vector encoding hADA, N2/ZipPGK-ADATKNEO, and used G418 selection of infected bone marrow cells before transplantation. In contrast to the simplified retroviral vector, hADA expression in these recipients was short lived (less than 8 weeks), despite the continued presence of intact provirus in DNA prepared from bone marrow of these mice. To determine whether the preselection of bone marrow using G418 was responsible for the lack of sustained hADA expression, we repeated the infection with the N2/ZipPGK- ADATKNEO vector but omitted the G418 selection step. Again, the majority of recipient mice failed to express hADA long term, although the continued presence of provirus in DNA prepared from peripheral blood cell mononuclear cells was clearly demonstrated. Finally, we demonstrate clonal fluctuation of infected stem cells, and observe a temporal correlation between cessation of expression of hADA and the emergence of a dominant stem cell clone between 14 and 20 weeks posttransplantation in one recipient. These data suggest that inclusion of a second transcriptional unit that includes neo phosphotransferase sequences in this simplified vector is associated with decreased expression of the nonselectable ADA sequences.

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Long-Term Follow-Up of Serially Transplanted Mice Receiving Extended Reduced Intensity Selection of MGMT-P140K Expressing Murine Repopulating Stem Cells Demonstrates Stable Oligo- to Polyclonal Hematopoiesis without Clonal Exhaustion.

Blood ◽

10.1182/blood.v106.11.1286.1286 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1286-1286

Author(s):

Claudia Ball ◽

Manfred Schmidt ◽

Ingo Pilz ◽

Monika Schrempp ◽

Christof von Kalle ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Blood Cells ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Reduced Intensity ◽

Selection Of

Abstract In vivo selection of gene modified hematopoietic stem cells permanently increases the relative proportion of blood cells that carry a therapeutic transgene despite initially low gene transfer efficiency, thereby decreasing the likelihood of insertional mutagenesis and avoiding the need of myeloablative conditioning regimens. P140K Mutant O6-methylguanine-DNA methyltransferase (MGMT) enzyme confers resistance to the combination of the MGMT inhibitor O(6)-benzylguanine (O(6)BG) and nitrosourea drugs such as 1,3-bis-(2 chloroethyl)-1-nitrosourea (BCNU). We have previously shown that reduced intensity and toxicity BCNU/O6-BG selection allows efficient selection of MGMT-P140K expressing oligoclonal murine hematopoiesis. Nevertheless, whether long-term selection and the associated proliferative stress impairs long-term differentiation and proliferation of MGMT-P140K expressing stem cell clones is currently unknown and remains a major concern in the clinical application of MGMT selection. To address this question, serial transplantations of murine MGMT-P140K expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were done. After ex vivo gene transfer of an MGMT/IRES/eGFP encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and primary, secondary and tertiary recipient mice were subsequently treated every four weeks in order to exaggerate potential effects on long-term clonal behaviour. Lineage contribution of the transduced hematopoiesis was monitored by FACS over a total of 14 rounds of selection and clonality by LAM-PCR over a total of 12 rounds of selection. In primary mice the percentage of transduced blood cells increased from 4.7 ± 0.8 % to 36.4 ± 9.8 % (n=12) and in secondary mice from 29.9 ± 7.2 % to 65.1 ± 8.7 % (n=18) after selection without persisting peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential of transduced cells in 1st, 2nd and 3rd generation animals. LAM PCR analysis of peripheral blood samples revealed stable oligo- to polyclonal hematopoiesis in primary and secondary mice. Evidence for predominant clones or clonal exhaustion was not observed despite up to 12 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice that received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis, indicating extensive self-renewal of transplantable stem cells in the primary mice resulting in a net symmetric refilling of the stem cell compartment. In summary, we demonstrate that even extended selection of MGMT-P140K expressing hematopoietic stem cells by repetitive chemotherapy does not affect their differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies for the amplification of a limited number of gene corrected clones in clinical gene therapy.

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Long-Term Safety and Efficacy of Stem Cell Gene Therapy for ADA-SCID.

Blood ◽

10.1182/blood.v108.11.200.200 ◽

2006 ◽

Vol 108 (11) ◽

pp. 200-200

Author(s):

Alessandro Aiuti ◽

Ulrike Benninghoff ◽

Barbara Cassani ◽

Federica Cattaneo ◽

Luciano Callegaro ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Gene Transfer ◽

Severe Combined Immunodeficiency ◽

Hematopoietic Stem ◽

Enzyme Replacement ◽

Cell Functions ◽

Cd34 Cells

Abstract Severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency is a fatal congenital disorder of the immune system associated with systemic toxicity due to accumulation of purine metabolites. We previously showed that retroviral-mediated ADA gene transfer into autologous hematopoietic stem/progenitor cells (HSC) allowed restoration of immune and metabolic functions. We have now enrolled eight ADA-SCID children (age: 7–67 months) in our phase I/II gene therapy trial in which HSC are combined with low intensity conditioning with busulfan (total dose 4 mg/Kg i.v.). Previous treatment included haploidentical bone marrow transplant (n=3) or long-term (>1 year) enzyme replacement therapy (PEG-ADA) (n=4) associated with insufficient immune reconstitution or severe autoimmunity. In the latter case, PEG-ADA was discontinued to favour the growth advantage for gene corrected cells. The patients received a median dose of 8.8x106/Kg bone marrow CD34+ cells (range 0.9–10.8), containing on average 26.2±9.6% transduced CFU-C. Five patients experienced ANC <0.5x109/L, which was extended beyond day +30 in two patients. With a median follow up of 3.1 years (range 0.4–5.9), no adverse events related to gene transfer have been observed. Long-term engraftment of transduced HSC was demonstrated by stable multilineage marking, persisting more than 5 years from gene therapy. The average proportion of transduced cells in the peripheral blood at one year post-gene therapy (n=6) was 5% for granulocytes, 95% for T cells, 56% for B cells and 62% for NK cells. Comparison of the insertion sites retrieved ex vivo from patients with those identified in pre-transplant transduced CD34+ cells showed no skewing in the profile of genome distributions or in the gene families hit by the vector, and no clonal expansion. In the six children with a follow-up >1 year after gene therapy, we observed a progressive increase in lymphocyte counts which was sustained over time (median at 1.5 years 1.6x109/L), polyclonal thymopoiesis and normalization of T-cell functions in vitro. Serum Ig levels improved and evidence of antigen-specific antibodies was obtained, leading to IVIG discontinuation in five patients. All the children are currently healthy and thriving, and none of them showed severe infections. Sustained ADA activity in lymphocytes and RBC resulted in a dramatic reduction of RBC purine toxic metabolites (dAXP<30 nmoles/ml in 5 patients) and amelioration of children’s growth and development. In summary, these data confirm that gene therapy is safe and efficacious in correcting both the immune and metabolic defect in ADA-SCID, with proven clinical benefit.

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