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1.
Mol Ther ; 31(8): 2472-2488, 2023 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-37147803

RESUMEN

Engineered T cells represent an emerging therapeutic modality. However, complex engineering strategies can present a challenge for enriching and expanding therapeutic cells at clinical scale. In addition, lack of in vivo cytokine support can lead to poor engraftment of transferred T cells, including regulatory T cells (Treg). Here, we establish a cell-intrinsic selection system that leverages the dependency of primary T cells on IL-2 signaling. FRB-IL2RB and FKBP-IL2RG fusion proteins were identified permitting selective expansion of primary CD4+ T cells in rapamycin supplemented medium. This chemically inducible signaling complex (CISC) was subsequently incorporated into HDR donor templates designed to drive expression of the Treg master regulator FOXP3. Following editing of CD4+ T cells, CISC+ engineered Treg (CISC EngTreg) were selectively expanded using rapamycin and maintained Treg activity. Following transfer into immunodeficient mice treated with rapamycin, CISC EngTreg exhibited sustained engraftment in the absence of IL-2. Furthermore, in vivo CISC engagement increased the therapeutic activity of CISC EngTreg. Finally, an editing strategy targeting the TRAC locus permitted generation and selective enrichment of CISC+ functional CD19-CAR-T cells. Together, CISC provides a robust platform to achieve both in vitro enrichment and in vivo engraftment and activation, features likely beneficial across multiple gene-edited T cell applications.


Asunto(s)
Linfocitos T CD4-Positivos , Interleucina-2 , Ratones , Animales , Linfocitos T CD4-Positivos/metabolismo , Interleucina-2/genética , Interleucina-2/farmacología , Interleucina-2/metabolismo , Linfocitos T Reguladores/metabolismo , Sirolimus/farmacología , Receptores de Interleucina-2/metabolismo
2.
JCI Insight ; 9(6)2024 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-38516892

RESUMEN

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving ß cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.


Asunto(s)
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Humanos , Ratones , Animales , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Linfocitos T Reguladores , Autoantígenos , Tolerancia Inmunológica
3.
Methods Mol Biol ; 2429: 103-124, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35507158

RESUMEN

Endothelial-to-hematopoietic transition (EHT) is a unique morphogenic event in which flat, adherent hemogenic endothelial (HE) cells acquire round, non-adherent blood cell morphology. Investigating the mechanisms of EHT is critical for understanding the development of hematopoietic stem cells (HSCs) and the entirety of the adult immune system, and advancing technologies for manufacturing blood cells from human pluripotent stem cells (hPSCs). Here we describe a protocol to (a) generate and isolate subsets of HE from hPSCs, (b) assess EHT and hematopoietic potential of HE subsets in bulk cultures and at the single-cell level, and (c) evaluate the role of NOTCH signaling during HE specification and EHT. The generation of HE from hPSCs and EHT bulk cultures are performed in xenogen- and feeder-free system, providing the unique advantage of being able to investigate the role of individual signaling factors during EHT and the definitive lympho-myeloid cell specification from hPSCs.


Asunto(s)
Hemangioblastos , Células Madre Pluripotentes , Diferenciación Celular , Hematopoyesis , Células Madre Hematopoyéticas , Humanos
4.
Nat Med ; 26(11): 1720-1725, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32929265

RESUMEN

The therapeutic potential of donor-derived mesenchymal stromal cells (MSCs) has been investigated in diverse diseases1, including steroid-resistant acute graft versus host disease (SR-aGvHD)2. However, conventional manufacturing approaches are hampered by challenges with scalability and interdonor variability, and clinical trials have shown inconsistent outcomes3,4. Induced pluripotent stem cells (iPSCs) have the potential to overcome these challenges, due to their capacity for multilineage differentiation and indefinite proliferation5,6. Nonetheless, human clinical trials of iPSC-derived cells have not previously been completed. CYP-001 (iPSC-derived MSCs) is produced using an optimized, good manufacturing practice (GMP)-compliant manufacturing process. We conducted a phase 1, open-label clinical trial (no. NCT02923375) in subjects with SR-aGvHD. Sixteen subjects were screened and sequentially assigned to cohort A or cohort B (n = 8 per group). One subject in cohort B withdrew before receiving CYP-001 and was excluded from analysis. All other subjects received intravenous infusions of CYP-001 on days 0 and 7, at a dose level of either 1 × 106 cells per kg body weight, to a maximum of 1 × 108 cells per infusion (cohort A), or 2 × 106 cells per kg body weight, to a maximum dose of 2 × 108 cells per infusion (cohort B). The primary objective was to assess the safety and tolerability of CYP-001, while the secondary objectives were to evaluate efficacy based on the proportion of participants who showed a complete response (CR), overall response (OR) and overall survival (OS) by days 28/100. CYP-001 was safe and well tolerated. No serious adverse events were assessed as related to CYP-001. OR, CR and OS rates by day 100 were 86.7, 53.3 and 86.7%, respectively. The therapeutic application of iPSC-derived MSCs may now be explored in diverse inflammatory and immune-mediated diseases.


Asunto(s)
Enfermedad Injerto contra Huésped/terapia , Células Madre Pluripotentes Inducidas/trasplante , Trasplante de Células Madre Mesenquimatosas , Esteroides/uso terapéutico , Adolescente , Adulto , Anciano , Resistencia a Medicamentos , Femenino , Enfermedad Injerto contra Huésped/patología , Humanos , Masculino , Persona de Mediana Edad , Inducción de Remisión/métodos , Esteroides/efectos adversos , Tasa de Supervivencia , Adulto Joven
5.
Exp Hematol ; 71: 3-12, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30500414

RESUMEN

Human pluripotent stem cells (hPSCs) have been suggested as a potential source for the de novo production of blood cells for transfusion, immunotherapies, and transplantation. However, even with advanced hematopoietic differentiation methods, the primitive and myeloid-restricted waves of hematopoiesis dominate in hPSC differentiation cultures, whereas cell surface markers to distinguish these waves of hematopoiesis from lympho-myeloid hematopoiesis remain unknown. In the embryo, hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries, but not veins. This observation led to a long-standing hypothesis that arterial specification is an essential prerequisite to initiate the HSC program. It has also been established that lymphoid potential in the yolk sac and extraembryonic vasculature is mostly confined to arteries, whereas myeloid-restricted hematopoiesis is not specific to arterial vessels. Here, we review how the link between arterialization and the subsequent definitive multilineage hematopoietic program can be exploited to identify HE enriched in lymphoid progenitors and aid in in vitro approaches to enhance the production of lymphoid cells and potentially HSCs from hPSCs. We also discuss alternative models of hematopoietic specification at arterial sites and recent advances in our understanding of hematopoietic development and the production of engraftable hematopoietic cells from hPSCs.


Asunto(s)
Arterias , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Neovascularización Fisiológica , Animales , Arterias/embriología , Arterias/metabolismo , Biomarcadores , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Humanos
6.
Nat Commun ; 9(1): 1828, 2018 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-29739946

RESUMEN

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43-CD73-DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs.


Asunto(s)
Arterias/citología , Endotelio Vascular/citología , Hemangioblastos/citología , Hematopoyesis , Neovascularización Fisiológica , Células Madre Pluripotentes/citología , Receptores Notch/metabolismo , Transducción de Señal , Animales , Antígenos CD/inmunología , Arterias/metabolismo , Proteínas de Unión al Calcio , Diferenciación Celular , Línea Celular , Linaje de la Célula , Rastreo Celular/instrumentación , Técnicas de Cocultivo , Embrión de Mamíferos/citología , Endotelio Vascular/metabolismo , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/inmunología , Hemangioblastos/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/inmunología , Células Madre Pluripotentes/inmunología
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