RESUMEN
Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing ß-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.
Asunto(s)
Anemia de Células Falciformes , Edición Génica , Terapia Genética , Células Madre Hematopoyéticas , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Anemia de Células Falciformes/terapia , Anemia de Células Falciformes/genética , Edición Génica/métodos , Animales , Células Madre Hematopoyéticas/metabolismo , Humanos , Femenino , Ratones , Terapia Genética/métodos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Trasplante de Células Madre Hematopoyéticas , Globinas beta/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Reparación del ADN , Mutación , Talasemia beta/terapia , Talasemia beta/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de GenRESUMEN
Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive universal CAR T-cells scaffold using precise TALEN-mediated gene editing and DNA matrices vectorized by recombinant adeno-associated virus 6. We simultaneously disrupt and repurpose the endogenous TRAC and B2M loci to generate TCRαß- and HLA-ABC-deficient T-cells expressing the CAR construct and the NK-inhibitor named HLA-E. This highly efficient gene editing process enables the engineered T-cells to evade NK cell and alloresponsive T-cell attacks and extend their persistence and antitumor activity in the presence of cytotoxic levels of NK cell in vivo and in vitro, respectively. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for clinical applications.
Asunto(s)
Edición Génica , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Humanos , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/genética , Linfocitos TRESUMEN
Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their αß T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19(+) tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an "off-the-shelf" manner akin to other biologic immunopharmaceuticals
Asunto(s)
Técnicas de Inactivación de Genes , Inmunoterapia Adoptiva , Linfocitos T/trasplante , Alemtuzumab , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Antígenos CD/genética , Antígenos CD19/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Secuencia de Bases , Antígeno CD52 , Citotoxicidad Inmunológica , Resistencia a Medicamentos , Glicoproteínas/deficiencia , Glicoproteínas/genética , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Activación de Linfocitos , Linfoma/terapia , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , ARN Mensajero , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.