RESUMO
Umbilical cord blood (UCB) T cells exhibit distinct naive ontogenetic profiles and may be an attractive source of starting cells for the production of chimeric antigen receptor (CAR) T cells. Pre-selection of UCB-T cells on the basis of CD62L expression was investigated as part of a machine-based manufacturing process, incorporating lentiviral transduction, CRISPR-Cas9 editing, T-cell expansion and depletion of residual TCReeeT cells. This provided stringent mitigation against the risk of graft versus host disease (GVHD), and was combined with simultaneous knockout of CD52 to enable persistence of edited T cells in combination with preparative lymphodepletion using Alemtuzumab. Under compliant manufacturing conditions, two cell banks were generated with high levels of CAR19 expression and minimal carriage of TCReeeT cells. Sufficient cells were cryopreserved in dose-banded aliquots at the end of each campaign to treat dozens of potential recipients. Molecular characterisation captured vector integration sites and CRISPR editing signatures and functional studies, including in vivo potency studies in humanised mice, confirmed antileukaemic activity comparable to peripheral blood-derived universal CAR19 T cells. Machine manufactured UCB derived T cells banks offer an alternative to autologous cell therapies and could help widen access to CAR T cells.
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BACKGROUND: Cytidine deamination that is guided by clustered regularly interspaced short palindromic repeats (CRISPR) can mediate a highly precise conversion of one nucleotide into another - specifically, cytosine to thymine - without generating breaks in DNA. Thus, genes can be base-edited and rendered inactive without inducing translocations and other chromosomal aberrations. The use of this technique in patients with relapsed childhood T-cell leukemia is being investigated. METHODS: We used base editing to generate universal, off-the-shelf chimeric antigen receptor (CAR) T cells. Healthy volunteer donor T cells were transduced with the use of a lentivirus to express a CAR with specificity for CD7 (CAR7), a protein that is expressed in T-cell acute lymphoblastic leukemia (ALL). We then used base editing to inactivate three genes encoding CD52 and CD7 receptors and the ß chain of the αß T-cell receptor to evade lymphodepleting serotherapy, CAR7 T-cell fratricide, and graft-versus-host disease, respectively. We investigated the safety of these edited cells in three children with relapsed leukemia. RESULTS: The first patient, a 13-year-old girl who had relapsed T-cell ALL after allogeneic stem-cell transplantation, had molecular remission within 28 days after infusion of a single dose of base-edited CAR7 (BE-CAR7). She then received a reduced-intensity (nonmyeloablative) allogeneic stem-cell transplant from her original donor, with successful immunologic reconstitution and ongoing leukemic remission. BE-CAR7 cells from the same bank showed potent activity in two other patients, and although fatal fungal complications developed in one patient, the other patient underwent allogeneic stem-cell transplantation while in remission. Serious adverse events included cytokine release syndrome, multilineage cytopenia, and opportunistic infections. CONCLUSIONS: The interim results of this phase 1 study support further investigation of base-edited T cells for patients with relapsed leukemia and indicate the anticipated risks of immunotherapy-related complications. (Funded by the Medical Research Council and others; ISRCTN number, ISRCTN15323014.).
Assuntos
Transplante de Células-Tronco Hematopoéticas , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Criança , Feminino , Humanos , Antígenos CD19 , Antígenos CD7 , Antígeno CD52 , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imunoterapia Adotiva/efeitos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfócitos T/genética , Recidiva , Transplante de Células-Tronco , Linfócitos TRESUMO
Recessive dystrophic epidermolysis bullosa is a debilitating blistering skin disorder caused by loss-of-function mutations in COL7A1, which encodes type VII collagen, the main component of anchoring fibrils at the dermal-epidermal junction. Although conventional gene therapy approaches through viral vectors have been tested in preclinical and clinical trials, they are limited by transgene size constraints and only support unregulated gene expression. Genome editing could potentially overcome some of these limitations, and CRISPR/Cas9 has already been applied in research studies to restore COL7A1 expression. The delivery of suitable repair templates for the repair of DNA cleaved by Cas9 is still a major challenge, and alternative base editing strategies may offer corrective solutions for certain mutations. We show highly targeted and efficient cytidine deamination and molecular correction of a defined recessive dystrophic epidermolysis bullosa mutation (c.425A>G), leading to restoration of full-length type VII collagen protein expression in primary human fibroblasts and induced pluripotent stem cells. Type VII collagen basement membrane expression and skin architecture were restored with de novo anchoring fibrils identified by electron microscopy in base-edited human recessive dystrophic epidermolysis bullosa grafts recovered from immunodeficient mice. The results show the potential and promise of emerging base editing technologies in tackling inherited disorders with well-defined single nucleotide mutations.
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Genome editing of allogeneic T cells can provide "off-the-shelf" alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor α chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator-like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3' long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 106 to 2.0 × 106 CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy.
Assuntos
Doença Enxerto-Hospedeiro , Leucemia de Células B , Leucemia Linfocítica Crônica de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Criança , Humanos , Alemtuzumab , Antígenos CD19/metabolismo , Ciclofosfamida , Doença Enxerto-Hospedeiro/metabolismo , Imunoterapia Adotiva , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Linfócitos T , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genéticaRESUMO
Chimeric antigen receptor (CAR) T cell therapy is an innovative immunotherapy for treating cancers in both children and adults with proven utility in numerous clinical trials. Significantly, some CAR T cell therapies have now been approved by relevant national regulatory bodies across numerous countries for clinical therapeutic use outside of clinical trials. One such recently licensed product is tisagenlecleucel, a CAR T therapy approved for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) using autologous T cells from the patient. The genetically engineered T cells target a protein called CD19, common to B cells, through a CAR incorporating a 4-1BB costimulatory domain to improve response. Since tisagenlecleucel is now a standard of care treatment for B-ALL, it is clinically essential to be able to accurately monitor these CAR T cells in patients. Assessment of the copy number variant (CNV) of the CAR T cell products allows this within a clinically acceptable timeframe for optimal patient benefit. However, no standardized method with high reproducibility and efficiency has been described within a routine clinical laboratory setting. Here, we demonstrated a novel digital droplet PCR (ddPCR)-based methodology for the study of CNV (ddPCR-CNV) in 4-1BB CD19-specific CAR T cells with universal applicability across clinical diagnostic laboratories.
Assuntos
Imunoterapia Adotiva , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos de Linfócitos T , Adulto , Criança , Variações do Número de Cópias de DNA , Humanos , Imunoterapia Adotiva/métodos , Linfoma de Células B/metabolismo , Reação em Cadeia da Polimerase/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/uso terapêutico , Reprodutibilidade dos Testes , Linfócitos TRESUMO
Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by 'T v T' fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in 'self-enrichment' yielding populations 99.6% TCR-/CD3-/CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic 'off-target' activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.
Assuntos
Antígenos CD7/genética , Complexo CD3/genética , Imunoterapia Adotiva/métodos , Leucemia de Células T/terapia , Linfócitos T/imunologia , Animais , Antígenos CD7/química , Antígenos CD7/metabolismo , Complexo CD3/antagonistas & inibidores , Complexo CD3/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Edição de Genes , Humanos , Leucemia de Células T/imunologia , Leucemia de Células T/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Emerging base editing technology exploits CRISPR RNA-guided DNA modification effects for highly specific C > T conversion, which has been used to efficiently disrupt gene expression. These tools can enhance synthetic T cell immunity by restricting specificity, addressing histocompatibility leukocyte antigen (HLA) barriers, and promoting persistence. We report lentiviral delivery of a hepatitis B-virus (HBV)-specific recombinant T cell receptor (rTCR) and a linked CRISPR single-guide RNA for simultaneous disruption of endogenous TCRs (eTCRs) when combined with transient cytosine deamination. Discriminatory depletion of eTCR and coupled expression of rTCR resulted in enrichment of HBV-specific populations from 55% (SEM, ±2.4%) to 95% (SEM, ±0.5%). Intensity of rTCR expression increased 1.8- to 2.9-fold compared to that in cells retaining their competing eTCR, and increased cytokine production and killing of HBV antigen-expressing hepatoma cells in a 3D microfluidic model were exhibited. Molecular signatures confirmed that seamless conversion of C > T (G > A) had created a premature stop codon in TCR beta constant 1/2 loci, with no notable activity at predicted off-target sites. Thus, targeted disruption of eTCR by cytosine deamination and discriminatory enrichment of antigen-specific T cells offers the prospect of enhanced, more specific T cell therapies against HBV-associated hepatocellular carcinoma (HCC) as well as other viral and tumor antigens.
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RNA polymerase III (Pol III) promoters express short non-coding RNAs and have been adopted for expression of microRNA, interference RNA, and CRISPR single guide RNA (sgRNA). Vectors incorporating H1 and U6 Pol III promoters are being applied for therapeutic genome editing, including multiplexed CRISPR/Cas9 effects. We report a nucleosome-depleted, minimal U6 promoter, which when embedded within lentiviral long terminal repeat (LTR) regions, supports high level transcriptional activity. Furthermore, duplex minimal H1 & U6 promoters transcribed dual sgRNAs for simultaneous disruption of T cell receptor (TCR) and human leukocyte antigen (HLA) molecules, supporting efficient generation of 'universal' CAR T cells.
Assuntos
Sistemas CRISPR-Cas , Nucleossomos , Edição de Genes , Humanos , Nucleossomos/genética , Regiões Promotoras Genéticas , RNA Guia de CinetoplastídeosRESUMO
Analysis of 226 exome-sequenced UK cases of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia identified 2 individuals who harbored a P497H and P506S UBQLN2 mutation, respectively (n = 0.9%). The P506S index case presented with behavioral variant frontotemporal dementia at the age of 54 years then progressed to ALS surviving 3 years. Three sons presented with (1) slowly progressive pure spastic paraplegia with an onset at 25 years and (2) ALS with disease onset of 25 years and survival of 2 years, and (3) ALS presenting symptoms at the age of 26 years, respectively. Analysis of postmortem tissue from the index case revealed frequent neuronal cytoplasmic UBQLN2-positive inclusions in the dentate gyrus and TDP-43-positive neuronal cytoplasmic inclusions in the frontal and temporal cortex and granular cell layer of the dentate gyrus of the hippocampus. Furthermore, a comprehensive analysis of published UBQLN2 mutations demonstrated that only proline-rich domain mutations contribute to a significantly earlier age of onset in male patients (p = 0.0026).
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ciclo Celular/genética , Demência Frontotemporal/genética , Mutação/genética , Paraplegia/genética , Ubiquitinas/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Giro Denteado/metabolismo , Progressão da Doença , Feminino , Lobo Frontal/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Masculino , Pessoa de Meia-Idade , Fenótipo , Fatores Sexuais , Lobo Temporal/metabolismo , Ubiquitinas/metabolismoRESUMO
Recent advances in the era of genetic engineering have significantly improved our ability to make precise changes in the genomes of human cells. Throughout the years, clinical trials based on gene therapies have led to the cure of diseases such as X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID) and Wiskott-Aldrich syndrome. Despite the success gene therapy has had, there is still the risk of genotoxicity due to the potential oncogenesis introduced by utilising viral vectors. Research has focused on alternative strategies like genome editing without viral vectors as a means to reduce genotoxicity introduced by the viral vectors. Although there is an extensive use of RNA-guided genome editing via the clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein-9 (Cas9) technology for biomedical research, its genome-wide target specificity and its genotoxic side effects remain controversial. There have been reports of on- and off-target effects created by CRISPR-Cas9 that can include small and large indels and inversions, highlighting the potential risk of insertional mutagenesis. In the last few years, a plethora of in silico, in vitro and in vivo genome-wide assays have been introduced with the sole purpose of profiling these effects. Here, we are going to discuss the genotoxic obstacles in gene therapies and give an up-to-date overview of methodologies for quantifying CRISPR-Cas9 effects.