Mosaic Mutagenesis In Vivo Reveals Mutant Blood Stem Cells Intrinsically Resistant to Inflammatory Mediators in Clonal Hematopoiesis

Clonal hematopoiesis (CH) is a state of clonal dominance of a mutant hematopoietic stem and progenitor cell (HSPC) promoted by unknown mechanisms. To study the competitive behavior and the mechanism of such expansion in a native environment, we used a combinatorial approach of HSPCs labeling and mutagenesis in vivo in a technique we called Tissue editing With Inducible Stem cell Tagging via Recombination (TWISTR). TWISTR utilizes Zebrabow zebrafish to fluorescently label endogenous HSPCs, and allows following of the clonal activity and sorting of labeled HSPCs within the hematopoietic system. CRISPR/Cas9 mutagenesis was used to simultaneously induce mosaic insertions/deletions (indels) in zebrafish orthologs of 12 human CH genes during development, and zebrafish were followed for 8 months. As such, mutant stem cells were directly competed against endogenous wildtype stem cells. We achieved a high degree of mosaic mutagenesis with heterozygous edits, with a median of 5 targeted genes per zebrafish in total marrow cells at variant allele frequency (VAF) of 5% or greater. Serial sampling and sequencing of peripheral blood cells showed selective expansion of clones with frameshift indels in exon 12 of asxl1, while clones with other CH mutations did not show a significant change over the 4 months period evaluated. No changes were noted in control gene indels or in the CH gene indels in non-hematopoietic tissue in the same time period. We identified significant enrichment of frameshift indels in asxl1 in sorted dominant clones compared to sorted smaller clones in the same zebrafish (p<0.01) at 8 months, while indels in DNMT3A ortholog dnmt8 were present in clones of various competitive nature. Introduction of asxl1 mutations singly in a mosaic fashion also resulted in clonally dominant states. To identify gene expression signatures associated with the clonal expansion, we used single cell RNA sequencing of marrow cells in zebrafish with a dominant clone with unique sets of mutations. We identified increased expression of inflammatory signaling genes in these zebrafish compared to controls, consistent with recent findings in other organisms. Within sorted dominant clones, mutant neutrophils and macrophages exhibited more than 4-fold increased expression of cytokines, such as il1b and tnfb. In mutant HSPCs and myeloid-biased progenitor cells, we observed elevated levels of genes involved in suppressing responses to cytokines and inflammatory lipids, like socs3a, nr4a1, atf3 and ier2a. This supports a model in which the mutant HSPCs of the dominant clone express anti-inflammatory genes that dampen their response to the inflammatory mediators produced by mature cells of the same clone, limiting the inflammation-induced HSPC exhaustion and providing a selective mechanism for the mutant stem cells to expand over time. We successfully used the Zebrabow color labeling system and prospective endogenous mosaic mutagenesis to show that mutant asxl1 is a potent inducer of CH, and that CH gene mutations lead to clonal dominance by modifying the response of stem cells to inflammatory cues. Disclosures Zon: Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Amagma Therapeutics: Current equity holder in private company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy.

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Short- and Long-Term Multilineage Repopulating Hematopoietic Stem Cells in Late Fetal and Newborn Mice: Models for Human Umbilical Cord Blood

Blood ◽

10.1182/blood.v90.1.174 ◽

1997 ◽

Vol 90 (1) ◽

pp. 174-181 ◽

Cited By ~ 50

Author(s):

David E. Harrison ◽

Clinton M. Astle

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Umbilical Cord Blood ◽

Functional Abilities ◽

Newborn Mice ◽

Hematopoietic Stem ◽

Short And Long Term ◽

Marrow Cells

Abstract Blood from late fetal and newborn mice is similar to umbilical cord blood obtained at birth in human beings, an important source of stem cells for clinical transplantation. The mouse model is useful because long-term functions can be readily assayed in vivo. To evaluate the functions of hematopoietic precursors in the blood and other tissues of late fetal and newborn mice, short- and long-term multilineage repopulating abilities were measured in vivo by competitive repopulation. Manipulations that might affect cell function, such as enrichment, tissue culture, or retroviral marking, were avoided. Hematopoietic stem cell functions of late fetal or newborn blood, liver, and spleen, were assayed as myeloid and lymphoid repopulating abilities relative to standard adult marrow cells. Donor cells from these tissues as well as adult control donor marrow cells were all of the same genotype. Cells from each donor tissue were mixed with portions from a pool of standard adult “competitor” marrow distinguished from the donors by genetic differences in hemoglobin and glucosephosphate isomerase. After 21 to 413 days, percentages of donor type myeloid and lymphoid cells in recipient blood were measured to assay the functional abilities of donor precursors relative to the standard. These relative measures are expressed as repopulating units, where each unit is equivalent to the repopulating ability found in 100,000 standard adult marrow cells. Thus, measures of repopulating units do not compare single cells but overall repopulating abilities of donor cell populations. Relative functional abilities in 1 million nucleated cells from late fetal or newborn blood were several times less than those found in adult marrow, but far more than in normal adult blood, and appeared to include long-term functional primitive hematopoietic stem cells (PHSC) similar to those in marrow. To estimate functional abilities of individual PHSC, variances among large groups of identical recipients were analyzed using both the binomial model and competitive dilution, a new model based on the Poisson distribution. The data best fit the hypothesis that individual PHSC from adult marrow, late fetal blood, or newborn blood each produce similar fractions of the total lymphoid and erythroid cells found in the recipient for many months.

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Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells

Blood ◽

10.1182/blood-2005-05-2003 ◽

2005 ◽

Vol 106 (12) ◽

pp. 3988-3994 ◽

Cited By ~ 131

Author(s):

H. Jeffrey Lawrence ◽

Julie Christensen ◽

Stephen Fong ◽

Yu-Long Hu ◽

Irving Weissman ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Function ◽

Bone Marrow Cells ◽

Homeobox Gene ◽

Normal Numbers ◽

Hematopoietic Stem ◽

Fold Reduction ◽

Marrow Cells

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells, and overexpression of Hoxa-9 markedly expands hematopoietic stem cells, suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin-c-kit+flk-2-Sca-1+ [KLFS] cells). However, sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia, indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays, Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability, a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures, Hoxa-9-deficient Lin-Sca-1+ cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation, with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo.

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Cellular Barcoding of HSCs during Development Reveals Long-Term Persistence of T Cell Progenitor Clones in the Thymus

Blood ◽

10.1182/blood-2020-136837 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-32

Author(s):

Sara A. Rubin ◽

Chloé S. Baron ◽

Song P. Yang ◽

Aaron McKenna ◽

Leonard I. Zon

Keyword(s):

Stem Cells ◽

T Cell ◽

Blood Cells ◽

Lineage Tracing ◽

Genetic Lineage ◽

Publicly Traded ◽

Private Company ◽

Cell Stage ◽

Hematopoietic Stem ◽

Cellular Barcoding

Developmental hematopoietic stem and progenitor cells (HSPCs) have been thought to be largely replaced by adult hematopoietic stem cells (HSCs) after birth. Here, we performed simultaneous genetic lineage tracing by cellular barcoding and transcriptional profiling of zebrafish hematopoiesis to investigate HSPC clonal dynamics and gain insight into the developmental origin of adult blood cells. We took advantage of a recently developed dynamic lineage tracing system, scGESTALT, a CRISPR-based approach that allows for inducible cellular barcoding at two different time points during zebrafish embryonic development. We induced the first stage of lineage recording in the early embryo via microinjection at the one-cell stage and selected 28 hours post-fertilization, just before the birth of definitive blood stem cells, for additional recording activity. In this way, we were able to barcode HSCs, which will pass their unique barcodes on to their progeny, thereby enabling lineage tracing. To study the clonal dynamics and developmental origins of adult blood cells, we dissected paired adult kidney marrow and thymi tissues from barcoded fish between 3 and 7 months of age. We then performed single-cell RNA sequencing (scRNA-seq) in combination with lineage tracing by specifically amplifying the GESTALT barcode and sequencing both transcriptional and GESTALT barcode libraries. In total, we recovered transcriptional profiles from 71,109 cells across 9 fish. By optimizing promoter choice, we improved barcode recovery by almost 3-fold (17.7% vs. 6.5%) for detection in cells from the zebrafish kidney marrow. In two of these more efficient lineage recording fish, we detected 61 and 72 unique HSPC clones that contributed to adult hematopoiesis. The majority of these HSPC clones did not exhibit significant lineage biases; however, we demonstrated that 6/61 and 4/72 clones from the two fish, respectively, were significantly enriched in the myeloid lineage and 8/61 and 8/72 clones were significantly enriched in the lymphoid lineage (p< 0.05). These findings demonstrate that myeloid and lymphoid biased clones arise during normal development. From our paired thymus and kidney marrow sample, we identified 51 unique kidney marrow clones, 10 shared, and 2 unique thymus clones. This latter finding of unique thymus clones was surprising and suggests that long-lived embryonic T cell progenitors persist and contribute to adult T cell production in the zebrafish. Taken together, we have demonstrated how scGESTALT can uncover complex lineage relationships in blood, mapping the origins and contributions of HSCs and embryonic progenitors to the adult hematopoietic system. Disclosures Zon: Amagma Therapeutics: Current equity holder in private company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder.

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NF-Ya Is Essential for Hematopoieic Stem Cell Proliferation.

Blood ◽

10.1182/blood.v114.22.1507.1507 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1507-1507 ◽

Cited By ~ 1

Author(s):

Gerd Bungartz ◽

Russell Garrett ◽

Stephen G. Emerson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Conflicts Of Interest ◽

Conditional Knockout ◽

Regulatory Subunit ◽

Mouse Development ◽

Potent Inducer ◽

Hematopoietic Stem ◽

M Phase

Abstract Abstract 1507 Poster Board I-530 Proliferation, self-renewal and differentiation of hematopoietic stem cells (HSCs) must be tightly regulated in order to sustain hematopoiesis over a lifetime and to prevent uncontrolled expansion of cells. Several genes have been implicated in the regulation of HSC behavior, such as HoxB4, Notch1, Lef-1 and others. Previous studies from our and other laboratories have demonstrated that the heterotrimeric transcription factor NF-Y is a potent inducer of many of these regulatory genes by over-expressing NF-Ya, the regulatory subunit of NF-Y. Furthermore, Bhattarchaya et al. showed that the deletion of NF-Ya in mice leads to lethality before day E8.5, highlighting its importance in mouse development. While there is no doubt that NF-Y plays a role in the progression of the cell cycle in vitro, different groups – targeting different subunits for deletion or silencing – have obtained different results. In order to comprehensively investigate the in vivo function of NF-Y in the hematopoietic system, we utilized a conditional knockout mouse model. We found that the bone marrow (BM) cellularity decreases sharply starting as soon as one day after the ablation of NF-Ya. Our data indicate that the cell loss can be attributed to a combination of cell cycle arrest in G2/M-phase of the cell cycle and apoptosis at 24 hours after the gene deletion. Since NF-Y has been identified as a master regulator of genes involved in HSCs behavior, we focused on the effects of the NF-Ya deletion within the HSC compartment. We found a down regulation of HoxB4, Notch-1, Lef-1 and Bmi-1 following NF-Ya deletion. However, 24h after induction of NF-Ya deletion the HSC population appeared unaffected and their numbers remained stable, likely due to their predominantly quiescent status. To investigate the capability of stem cells to progress though the cell cycle, we activated HSCs using the interferon inducer poly IC and observed that, once activated, also HSCs accumulate in the G2/M-phase of the cell cycle. Finally, to test whether the deletion of NF-Ya impairs or absolutely abrogates HSC function, we performed long term experiments including competitive BM transplantation and colony formation assays that demonstrate that NF-Y activity is absolutely essential for HSC function. Altogether, our data identify NF-Y plays a pivotal role in the survival of hematopoietic cells and the progression of cells though the G2/M-phase of the cell cycle in vivo. Additionally, while we found that NF-Y ablation leads to reduced expression of many genes important for HSC behavior, this had no immediate effects on the maintenance of these cells due to their quiescent nature. Disclosures No relevant conflicts of interest to declare.

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5-Azacytidine Depletes Hematopoietic Stem Cells and Synergizes with an Anti-CD117 Antibody to Augment Engraftment of Donor Stem Cells in Immunocompetent Mice

Blood ◽

10.1182/blood-2020-139058 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 23-24

Author(s):

Andriyana K Bankova ◽

Wendy W Pang ◽

Brenda J Velasco ◽

Janel R Long-Boyle ◽

Judith A Shizuru

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Single Agent ◽

Private Company ◽

Hematopoietic Stem ◽

Donor Chimerism ◽

Immunocompetent Mice

Monoclonal antibody (mAb)-targeting of CD117 (c-Kit) present on hematopoietic stem cells (HSC) is an emerging conditioning strategy for hematopoietic cell transplantation (HCT) to replace standard-of-care alkylator and radiation therapy used to deplete recipient HSC and permit donor HSC engraftment. In preclinical and clinical studies anti-CD117 mAbs, which inhibit stem cell factor (SCF) from binding to CD117, have shown single agent efficacy in permitting donor HSC engraftment in immunocompromised recipients. However, preclinical data suggest that anti-CD117 mAb, ACK2, used as a single agent is insufficient to enable HSC engraftment in immunocompetent mice. Hence, there is a desire to potentiate the effect of naked anti-CD117 mAbs to permit targeted elimination of host HSC for a range of immune competent bone marrow (BM) states, from monogenic disorders, e.g., hemoglobinopathies, to BM clonal malignant diseases. Here, we show that ACK2 synergizes with the widely used hypomethylating agent, 5-Azacytidine (AZA), to deplete HSC and enable robust long-term HSC engraftment in immunocompetent mice. The cell depleting effect of AZA or ACK2+AZA was first tested on mature and primitive hematopoietic cells of C57BL/6 (B6) mice. Mice received AZA at 5mg/kg i.p. for 5 days either alone or combined with ACK2 at 500 µg i.v. 5 days prior to AZA. BM and spleen were harvested days 6, 10 and 20 after the first AZA dose and analyzed by extended immunophenotype. For HCT studies both congenic [B6 (H-2b, Thy1.1, CD45.1) into B6 recipients (H-2b, Thy1.1, CD45.1/CD45.2)] and allogeneic [B6 (H-2b, Thy1.1, CD45.1) into BALB.B recipients (H-2b, Thy1.2, CD45.2)] transplantation mouse models were tested. Single agent AZA induced rapid depletion of CD117+ cells in the BM, including LT-HSC (Lin-Sca+Kit+[LSK]CD150+CD48-) and ST-HSC (LSKCD150-CD48-). In vivo HSC depletion by AZA has not been previously reported, and was surprising given that most HSC are quiescent, and non-proliferating cells are known to be insensitive to the toxicities of AZA. Examination of HSC populations in the spleen did not suggest that mobilization of LT- or ST-HSC to secondary organs caused the decrease in BM. Rather, AZA-induced myelosuppression was followed by rapid expansion of myeloid-biased MPP and increased HSC proliferation as assessed by Ki67 intracellular protein expression. We hypothesized, that the in vivo HSC-depletive effects of AZA might result from increased sensitivity of proliferating HSC to AZA. Hence, the in vitro effects of different AZA concentrations were tested on proliferating HSC. In both mouse and human HSC cultures, supplemented with SCF and TPO, we observed that AZA reduced the number of cells in a dose dependent manner and decreased cell viability at concentrations of ≥ 0.5 µg/ml. In vivo, combined ACK2+AZA led to more robust HSC depletion and prolonged time to HSC recovery compared to AZA alone. We then evaluated if blockade of CD117 with its ligand SCF by ACK2 was the cause of this prolonged HSC-depletion. 2B8 mAb, which also binds mouse CD117, but only partially inhibits CD117/SCF signaling, did not result in augmented in vivo HSC depletion (Figure 1A). We further tested if immunocompetent mice treated with ACK2+AZA could be "rescued" with exogenous recombinant SCF. SCF-treatment resulted in faster recovery of LT-HSC. Together these studies suggest that the synergy of ACK2+AZA on the depth and duration of HSC-depletion depends of the disruption of CD117/SCF binding. Finally, we tested ACK2+AZA conditioning on donor stem cell engraftment in immunocompetent mice. Transplantation of 15-20x106 WBM cells following ACK2+AZA conditioning led to significantly increased stable mean multilineage donor chimerism of >60% in the congenic (Figure 1B) and >20% in the allogeneic setting. Single agent AZA enabled sustained donor engraftment with levels of myeloid donor chimerism of 10-15%, serving as proof of principle that AZA treatment can indeed clear HSC niches and permit engraftment of true HSC. Similar to WBM grafts, 5x104 purified HSC (LSK) engrafted successfully in the ACK2+AZA group, reaching long-term (26 weeks) mean donor myeloid chimerism >25% in both congenic and allogeneic settings. Our studies reveal a previously unknown effect of AZA on BM HSC and provide a platform for the clinical use of a novel less toxic non-myeloablative conditioning strategy for HCT, which is aimed at the rapid translation into clinical practice. Disclosures Pang: Jasper Therapeutics, Inc: Current Employment, Current equity holder in private company. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

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Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells

Blood ◽

10.1182/blood.v75.2.337.bloodjournal752337 ◽

1990 ◽

Vol 75 (2) ◽

pp. 337-343 ◽

Cited By ~ 2

Author(s):

CA Corey ◽

AD DeSilva ◽

CA Holland ◽

DA Williams

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Retroviral Vectors ◽

Hematopoietic Stem ◽

Genetic Sequences ◽

Marrow Cells

Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate- induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.

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Mild preconditioning and low-level engraftment confer methotrexate resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity

Blood ◽

10.1182/blood.v96.4.1334.h8001334_1334_1341 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1334-1341

Author(s):

Rohaizah I. James ◽

Christopher A. Warlick ◽

Miechaleen D. Diers ◽

Roland Gunther ◽

R. Scott McIvor

Keyword(s):

Stem Cells ◽

Dihydrofolate Reductase ◽

Reductase Activity ◽

Lethal Dose ◽

Hematopoietic Cells ◽

High Dose ◽

Hematopoietic Stem ◽

Low Level ◽

Marrow Cells

Effective engraftment of hematopoietic cells targeted for gene transfer is facilitated by cytoreductive preconditioning such as high-dose total body irradiation (TBI). To minimize the adverse side effects associated with TBI, experiments were conducted to determine whether sublethal doses of TBI would allow sufficient engraftment of MTX-resistant hematopoietic cells to confer survival on recipient mice administered MTX. FVB/N animals were administered 1, 2, or 4 Gy TBI (lethal dose, 8.5 Gy), transplanted with 107 FVB/N transgenic marrow cells expressing an MTX-resistant dihydrofolate reductase (DHFR) transgene, and then administered MTX daily for 60 days. Control mice administered 1 Gy with or without subsequent transplantation of normal marrow cells succumbed to MTX toxicity by day 45. In contrast, nearly all animals transplanted with transgenic marrow survived MTX administration, regardless of the TBI dose used for preconditioning. The donor DHFR transgenic marrow engraftment level was proportional to the preconditioning dose of TBI but was surprisingly reduced in animals given 2 or 4 Gy TBI and subsequently administered MTX when compared with control animals administered phosphate-buffered saline. Animals preconditioned with 1 Gy were also protected from MTX toxicity when transplanted with reduced amounts (5 × 106 and 1 × 106 cells) of DHFR transgenic donor marrow, resulting in low-level (approximately 1%) engraftment. In conclusion, very mild preconditioning allows sufficient low-level engraftment of genetically modified stem cells for in vivo manifestation of the modified phenotype, suggesting the usefulness of mild preconditioning regimens in human gene therapy trials targeting hematopoietic stem cells.

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High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽

10.1182/blood.v104.11.1758.1758 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1758-1758

Author(s):

Axel Schambach ◽

Bernhard Schiedlmeier ◽

Jens Bohne ◽

Dorothee von Laer ◽

Geoff Margison ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgene Expression ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Hiv 1 ◽

Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

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Mild preconditioning and low-level engraftment confer methotrexate resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity

Blood ◽

10.1182/blood.v96.4.1334 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1334-1341 ◽

Cited By ~ 22

Author(s):

Rohaizah I. James ◽

Christopher A. Warlick ◽

Miechaleen D. Diers ◽

Roland Gunther ◽

R. Scott McIvor

Keyword(s):

Stem Cells ◽

Dihydrofolate Reductase ◽

Reductase Activity ◽

Lethal Dose ◽

Hematopoietic Cells ◽

High Dose ◽

Hematopoietic Stem ◽

Low Level ◽

Marrow Cells

Abstract Effective engraftment of hematopoietic cells targeted for gene transfer is facilitated by cytoreductive preconditioning such as high-dose total body irradiation (TBI). To minimize the adverse side effects associated with TBI, experiments were conducted to determine whether sublethal doses of TBI would allow sufficient engraftment of MTX-resistant hematopoietic cells to confer survival on recipient mice administered MTX. FVB/N animals were administered 1, 2, or 4 Gy TBI (lethal dose, 8.5 Gy), transplanted with 107 FVB/N transgenic marrow cells expressing an MTX-resistant dihydrofolate reductase (DHFR) transgene, and then administered MTX daily for 60 days. Control mice administered 1 Gy with or without subsequent transplantation of normal marrow cells succumbed to MTX toxicity by day 45. In contrast, nearly all animals transplanted with transgenic marrow survived MTX administration, regardless of the TBI dose used for preconditioning. The donor DHFR transgenic marrow engraftment level was proportional to the preconditioning dose of TBI but was surprisingly reduced in animals given 2 or 4 Gy TBI and subsequently administered MTX when compared with control animals administered phosphate-buffered saline. Animals preconditioned with 1 Gy were also protected from MTX toxicity when transplanted with reduced amounts (5 × 106 and 1 × 106 cells) of DHFR transgenic donor marrow, resulting in low-level (approximately 1%) engraftment. In conclusion, very mild preconditioning allows sufficient low-level engraftment of genetically modified stem cells for in vivo manifestation of the modified phenotype, suggesting the usefulness of mild preconditioning regimens in human gene therapy trials targeting hematopoietic stem cells.

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Reversible Transplantable Chronic Phase CML-Like Disease in SCLtTA/BCR-ABL Transgenic Mice.

Blood ◽

10.1182/blood.v110.11.1002.1002 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1002-1002

Author(s):

Mirle Schemionek ◽

Jörg Stypmann ◽

Sven Hermann ◽

Christian Elling ◽

Nicole Bäumer ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Small Intestine ◽

Stem Cell ◽

Fdg Pet ◽

Bone Marrow Cells ◽

Chronic Phase ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Chronic myeloid leukemia (CML) is a disorder arising from the transformation of hematopoietic stem cells (HSC). Treatment with kinase inhibitors eradicates BCR-ABL positive progenitors but spares quiescent leukemic HSC (Copland et al. Blood 2006, Hu et al. PNAS 2006). The exact mechanism of this discrepancy is unknown. To better characterize the biology of CML stem cells in vivo, we have previously generated an inducible transgenic mouse model in which stem-cell specific expression of BCR-ABL leads to chronic phase CML-like disease. Here, we followed these mice non-invasively using positron emission tomography (FDG-PET) and abdominal high-resolution ultrasound. Moreover, we performed bone marrow transplants to analyze whether the disease is cell-autonomous and whether the phenotype of the disease is affected by the scheduling of BCR-ABL induction or the donor cell type. Splenomegaly was detectable as early as day 7 in induced double-transgenic SCLtTA/BCR-ABL mice, with an increase of the percentage of Gr-1+/Mac-1+ myeloid cells in spleen and bone marrow. Splenomegaly and myeloid cell proliferation progressed, and there was a close correlation between in vivo ultrasound measurements of the spleen and splenic weights upon autopsy. FDG-PET analysis demonstrated enhanced glucose uptake in the bone marrow suggestive of hyperproliferation. In addition, both FDG-PET and ultrasound revealed abnormalities of the small intestine, characterized by increased FDG uptake and distension of the intestinal wall. Upon autopsy, the small intestine showed an increased infiltration by granulocytic cells. These phenotypic changes were also evident in mice transplanted with cells from the bone marrow of double-transgenic sibling mice and were reversible upon tetracycline re-administration, demonstrating that this abnormality arises from bone marrow cells and is not due to expression of the oncogene outside of the hematopoietic system. We analyzed whether pre-transplant induction of BCR-ABL affected the repopulation potential of HSC or the disease phenotype. When recipient mice receiving unfractionated bone marrow cells from 3-week induced donor mice were compared with non-induced donors, there was no difference in the development of neutrophilia, myeloproliferation, or splenomegaly. However, when FACS-sorted LinnegSca-1+c-kit+ HSC were used as donor cells, the disease latency increased from 8 to 11 weeks post-transplant, and the increase of Gr-1+Mac-1+ cells in the spleen was less pronounced than in mice receiving unfractionated bone marrow. In conclusion, this model reliably and efficiently demonstrates transplantable reversible chronic phase CML-like disease and may thus be valuable for the in vivo analysis of CML stem cell biology and susceptibility to stem-cell directed anti-leukemic therapies.

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