Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models.
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ABSTRACT:
SUBMITTER: Uchida N
PROVIDER: S-EPMC8080237 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundHematopoietic stem cell (HSC) transplantation has been widely applied to the treatment of malignant blood diseases. However, limited number of functional HSCs hinders successful transplantation. The purpose of our current study is to develop a new and cost-efficient medium formulation that could greatly enhance the expansion of HSCs while retaining their long-term repopulation and hematopoietic properties for effective clinical transplantation.MethodsEnriched human CD34+ cells and mobilized nonhuman primate peripheral blood CD34+ cells were expanded with a new, cost-efficient expansion medium formulation, named hematopoietic expansion medium (HEM), consisting of various cytokines and nutritional supplements. The long-term repopulation potential and hematologic-lineage differentiation ability of expanded human cells were studied in the non-obese diabetic/severe combined immunodeficiency mouse model. Furthermore, the efficacy and safety studies were performed by autologous transplantation of expanded primate cells in the nonhuman primate model.ResultsHEM could effectively expand human CD34+ cells by up to 129 fold within 9 days. Expanded HSCs retained long-term repopulation potential and hematologic-lineage differentiation ability, as indicated by (1) maintenance (over unexpanded HSCs) of immunophenotypes of CD38-CD90+CD45RA-CD49f+ in CD34+ cells after expansion; (2) significant presence of multiple human hematopoietic lineages in mouse peripheral blood and bone marrow following primary transplantation; (3) enrichment (over unexpanded HSCs) in SCID-repopulating cell frequency measured by limiting dilution analysis; and (4) preservation of both myeloid and lymphoid potential among human leukocytes from mouse bone marrow in week 24 after primary transplantation or secondary transplantation. Moreover, the results of autologous transplantation in nonhuman primates demonstrated that HEM-expanded CD34+ cells could enhance hematological recovery after myelo-suppression. All primates transplanted with the expanded autologous CD34+ cells survived for over 18 months without any noticeable abnormalities.ConclusionsTogether, these findings demonstrate promising potential for the utility of HEM to improve expansion of HSCs for clinical application.
| S-EPMC6567473 | biostudies-literature
Project description:BackgroundPoor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34+ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo.MethodsHuman MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34+ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34+ cells. In addition, CD34+ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34+ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice.ResultsWe did not observe significant differences in viability, cell cycle and ROS expression between CD34+ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34+ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34+ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34+ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34+ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively).ConclusionsCo-transplantation of human CD34+ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.
| S-EPMC8650423 | biostudies-literature
Project description:The search is ongoing for new anticancer therapeutics that would overcome resistance to chemotherapy. This includes chronic myeloid leukemia, particularly suitable for the studies of novel anticancer compounds due to its homogenous and well-known genetic background. Here we show anticancer efficacy of novel dicarboximide denoted BK124.1 (C31H37ClN2O4) in a mouse CML xenograft model and in vitro in two types of chemoresistant CML cells: MDR1 blasts and in CD34+ patients' stem cells (N = 8) using immunoblotting and flow cytometry. Intraperitoneal administration of BK124.1 showed anti-CML efficacy in the xenograft mouse model (N = 6) comparable to the commonly used imatinib and hydroxyurea. In K562 blasts, BK124.1 decreased the protein levels of BCR-ABL1 kinase and its downstream effectors, resulting in G2/M cell cycle arrest and apoptosis associated with FOXO3a/p21waf1/cip1 upregulation in the nucleus. Additionally, BK124.1 evoked massive apoptosis in multidrug resistant K562-MDR1 cells (IC50 = 2.16 μM), in CD34+ cells from CML patients (IC50 = 1.5 µM), and in the CD34+/CD38- subpopulation consisting of rare, drug-resistant cancer initiating stem cells. Given the advantages of BK124.1 as a potential chemotherapeutic and its unique ability to overcome BCR-ABL1 dependent and independent multidrug resistance mechanisms, future development of BK124.1 could offer a cure for CML and other cancers resistant to present drugs.
| S-EPMC9332833 | biostudies-literature
Project description:P-cadherin is overexpressed in various cancers and can be a target for radioimmunotherapy. We investigated the preclinical pharmacokinetics and pharmacology of FF-21101, an 111In- or 90Y-conjugated monoclonal antibody against P-cadherin, to evaluate its clinical applications. Methods: The radiochemical purity, binding affinity, and in vitro serum stability of 111In or 90Y-labeled FF-21101 were evaluated. The pharmacokinetics of 111In or 90Y-FF-21101 were compared in normal mice. Tumor accumulation after 111In-FF-21101 administration was investigated in mice bearing subcutaneous tumors with high (NCI-H1373), moderate (EBC-1), or no (A549) P-cadherin expression. The tumor suppression effect after a single intravenous injection of 90Y-FF-21101 was assessed in NCI-H1373 and EBC-1 mouse xenograft models. The relationship between antibody dose and tumor accumulation was investigated in the NCI-H1373 mouse xenograft model. The absorbed radiation dose in humans after injection of 90Y-FF-21101 was estimated using γ-camera images of cynomolgus monkeys. Results: The radiochemical purities of 111In- and 90Y-FF-21101 were 98.2% ± 2.5% (n = 9) and 99.3% ± 0.6% (n = 5), respectively. The dissociation constants were 1.083 nM for 111In-FF-21101 and 1.367 nM for 90Y-FF-21101. Both 111In- and 90Y-FF-21101 were stable in human serum after 96 h of incubation and exhibited similar pharmacokinetics in normal mice. The tumor accumulation of 111In-FF-21101 was closely related to the intensity of P-cadherin expression in the cells. 90Y-FF-21101 showed significant tumor growth inhibition, indicating that NCI-H1373 and EBC-1 recurrence was not observed after intravenous administration of 3.7 and 7.4 MBq, respectively of 90Y-FF-21101 per animal. Tumor uptake in the mouse xenograft model and estimated absorbed radiation doses in the spleen of monkeys decreased with increasing antibody doses of 111In-FF-21101. Conversely, the estimated absorbed radiation dose in the red marrow increased with increasing antibody dose. An antibody dose of 4.8 mg/m2 was considered appropriate for humans, on the basis of efficacy and safety. The maximum tolerated administered activity of 90Y-FF-21101 was estimated to be 2,886 MBq/human. Conclusion: FF-21101 radioimmunotherapy exhibited high antitumor affinity and antitumor efficacy in mouse xenograft models. Extrapolation of the pharmacokinetics in monkeys to humans suggests the potential for clinical application of FF-21101 for treating P-cadherin-expressing tumor.
| S-EPMC8679590 | biostudies-literature
Project description:Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.
| S-EPMC8963924 | biostudies-literature
Project description:PurposePatient-derived tumor xenografts (PDXs) can provide more reliable information about tumor biology than cell line models. We developed PDXs for epithelial ovarian cancer (EOC) that have histopathologic and genetic similarities to the primary patient tissues and evaluated their potential for use as a platform for translational EOC research.Materials and methodsWe successfully established PDXs by subrenal capsule implantation of primary EOC tissues into female BALB/C-nude mice. The rate of successful PDX engraftment was 48.8% (22/45 cases). Hematoxylin and eosin staining and short tandem repeat analysis showed histopathological and genetic similarity between the PDX and primary patient tissues.ResultsPatients whose tumors were successfully engrafted in mice had significantly inferior overall survival when compared with those whose tumors failed to engraft (p=0.040). In preclinical tests of this model, we found that paclitaxel-carboplatin combination chemotherapy significantly deceased tumor weight in PDXs compared with the control treatment (p=0.013). Moreover, erlotinib treatment significantly decreased tumor weight in epidermal growth factor receptor-overexpressing PDX with clear cell histology (p=0.023).ConclusionPDXs for EOC with histopathological and genetic stability can be efficiently developed by subrenal capsule implantation and have the potential to provide a promising platform for future translational research and precision medicine for EOC.
| S-EPMC5654149 | biostudies-literature
Project description:BackgroundNeuroblastoma (NB) is one of the most frequently diagnosed tumors in infants. NB is a neuroendocrine tumor type with various characteristics and features, and with diverse outcome. The most malignant NBs have a 5-year survival rate of only 40-50%, indicating the need for novel and improved treatment options. 177Lu-octreotate is routinely administered for treatment of neuroendocrine tumors overexpressing somatostatin receptors (SSTR). The aim of this study was to examine the biodistribution of 177Lu-octreotate in mice bearing aggressive human NB cell lines, in order to evaluate the potential usefulness of 177Lu-octreotate for treatment of NB.MethodsBALB/c nude mice bearing CLB-BAR, CLB-GE or IMR-32 tumor xenografts (n = 5-7/group) were i.v. injected with 0.15 MBq, 1.5 MBq or 15 MBq 177Lu-octreotate and sacrificed 1 h, 24 h, 48 h and 168 h after administration. The radioactivity concentration was determined for collected tissue samples, tumor-to-normal-tissue activity concentration ratios (T/N) and mean absorbed dose for each tissue were calculated. Immunohistochemical (IHC) staining for SSTR1-5, and Ki67 were carried out for tumor xenografts from the three cell lines.ResultsHigh 177Lu concentration levels and T/N values were observed in all NB tumors, with the highest for CLB-GE tumor xenografts (72%IA/g 24 h p.i.; 1.5 MBq 177Lu-octreotate). The mean absorbed dose to the tumor was 6.8 Gy, 54 Gy and 29 Gy for CLB-BAR, CLB-GE and IMR-32, respectively, p.i. of 15 MBq 177Lu-octreotate. Receptor saturation was clearly observed in CLB-BAR, resulting in higher concentration levels in the tumor when lower activity levels where administered. IHC staining demonstrated highest expression of SSTR2 in CLB-GE, followed by CLB-BAR and IMR-32.ConclusionT/N values for all three human NB tumor xenograft types investigated were high relative to previously investigated neuroendocrine tumor types. The results indicate a clear potential of 177Lu-octreotate as a therapeutic alternative for metastatic NB.
| S-EPMC8386073 | biostudies-literature
Project description:Adoptive therapies with genetically modified somatic T cells rendered HIV resistance have shown promise for AIDS therapy. A renewable source of HIV-resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian cynomolgus macaques (MCM), using CRISPR-Cas9 technology. We found that CCR5 editing does not affect hematopoietic and T cell differentiation potentials of fib-iPSCs. However, T-iPSCs with edited CCR5 lost their capacity to differentiate into CD4+CD8+ T cells while maintaining myeloid differentiation potential. T cells and macrophages produced from CCR5-edited MCM iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T cell tropic) and SIVmac316 (macrophage-tropic). Overall, these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in a nonhuman primate model.
| S-EPMC9023799 | biostudies-literature
Project description:Endometrial cancer (EC) is the second most frequent gynecological cancer worldwide. Although improvements in EC classification have enabled an accurate establishment of disease prognosis, women with a high-risk or recurrent EC face a dramatic situation due to limited further treatment options. Therefore, new strategies that closely mimic the disease are required to maximize drug development success. Patient-derived xenografts (PDXs) are widely recognized as a physiologically relevant preclinical model. Hence, we propose to molecularly and histologically validate EC PDX models. To reveal the molecular landscape of PDXs generated from 13 EC patients, we performed histological characterization and whole-exome sequencing analysis of tumor samples. We assessed the similarity between PDXs and their corresponding patient's tumor and, additionally, to an extended cohort of EC patients obtained from The Cancer Genome Atlas (TCGA). Finally, we performed functional enrichment analysis to reveal differences in molecular pathway activation in PDX models. We demonstrated that the PDX models had a well-defined and differentiated molecular profile that matched the genomic profile described by the TCGA for each EC subtype. Thus, we validated EC PDX's potential to reliably recapitulate the majority of histologic and molecular EC features. This work highlights the importance of a thorough characterization of preclinical models for the improvement of the success rate of drug-screening assays for personalized medicine.
| S-EPMC9181722 | biostudies-literature
Project description:The translocation t(4;11)(q21;q23) is the hallmark genetic abnormality associated with infant pro-B acute lymphoblastic leukemia (B-ALL) and has the highest frequency of rearrangement in Mixed-lineage leukemia (MLL) leukemias. Unlike other MLL translocations, MLL-AF4-induced proB-ALL is exceptionally difficult to model in mice/humans. Previous work has investigated the relevance of the reciprocal translocation fusion protein AF4-MLL for t(4;11) leukemia, finding that AF4-MLL is capable of inducing proB-ALL without requirement for MLL-AF4 when expressed in murine hematopoietic stem/progenitor cells (HSPCs). Therefore, AF4-MLL might represent a key genetic lesion contributing to t(4;11)-driven leukemogenesis. Here, we aimed to establish a humanized mouse model by using AF4-MLL to analyze its transformation potential in human cord blood-derived CD34+ HSPCs. We show that AF4-MLL-expressing human CD34+ HSPCs provide enhanced long-term hematopoietic reconstitution in primary immunodeficient recipients but are not endowed with subsequent self-renewal ability upon serial transplantation. Importantly, expression of AF4-MLL in primary neonatal CD34+ HSPCs failed to render any phenotypic or hematological sign of disease, and was therefore not sufficient to initiate leukemia within a 36-week follow-up. Species-specific (epi)-genetic intrinsic determinants may underlie the different outcome observed when AF4-MLL is expressed in murine or human HSPCs.
| S-EPMC5669860 | biostudies-literature