Miguel Calero-Garcia

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T-cell gene therapy for perforin deficiency corrects cytotoxicity defects and prevents hemophagocytic lymphohistiocytosis manifestations

Journal of Allergy and Clinical Immunology (2018)

Sujal Ghosh Marlene Carmo Miguel Calero-Garcia Ida Ricciardelli Juan Carlos Bustamante Ogando

BackgroundMutations in the perforin 1 (PRF1) gene account for up to 58% of familial hemophagocytic lymphohistiocytosis syndromes. The resulting defects in effector cell cytotoxicity lead to hypercytokinemia and hyperactivation with inflammation in various organs.ObjectiveWe sought to determine whether autologous gene-corrected T cells can restore cytotoxic function, reduce disease activity, and prevent hemophagocytic lymphohistiocytosis (HLH) symptoms in in vivo models.MethodsWe developed a gammaretroviral vector to transduce murine CD8 T cells in the Prf−/− mouse model. To verify functional correction of Prf−/− CD8 T cells in vivo, we used a lymphocytic choriomeningitis virus (LCMV) epitope–transfected murine lung carcinoma cell tumor model. Furthermore, we challenged gene-corrected and uncorrected mice with LCMV. One patient sample was transduced with a PRF1-encoding lentiviral vector to study restoration of cytotoxicity in human cells.ResultsWe demonstrated efficient engraftment and functional reconstitution of cytotoxicity after intravenous administration of gene-corrected Prf−/− CD8 T cells into Prf−/− mice. In the tumor model infusion of Prf−/− gene–corrected CD8 T cells eliminated the tumor as efficiently as transplantation of wild-type CD8 T cells. Similarly, mice reconstituted with gene-corrected Prf−/− CD8 T cells displayed complete protection from the HLH phenotype after infection with LCMV. Patients' cells showed correction of cytotoxicity in human CD8 T cells after transduction.ConclusionThese data demonstrate the potential application of T-cell gene therapy in reconstituting cytotoxic function and protection against HLH in the setting of perforin deficiency. Mutations in the perforin 1 (PRF1) gene account for up to 58% of familial hemophagocytic lymphohistiocytosis syndromes. The resulting defects in effector cell cytotoxicity lead to hypercytokinemia and hyperactivation with inflammation in various organs. We sought to determine whether autologous gene-corrected T cells can restore cytotoxic function, reduce disease activity, and prevent hemophagocytic lymphohistiocytosis (HLH) symptoms in in vivo models. We developed a gammaretroviral vector to transduce murine CD8 T cells in the Prf−/− mouse model. To verify functional correction of Prf−/− CD8 T cells in vivo, we used a lymphocytic choriomeningitis virus (LCMV) epitope–transfected murine lung carcinoma cell tumor model. Furthermore, we challenged gene-corrected and uncorrected mice with LCMV. One patient sample was transduced with a PRF1-encoding lentiviral vector to study restoration of cytotoxicity in human cells. We demonstrated efficient engraftment and functional reconstitution of cytotoxicity after intravenous administration of gene-corrected Prf−/− CD8 T cells into Prf−/− mice. In the tumor model infusion of Prf−/− gene–corrected CD8 T cells eliminated the tumor as efficiently as transplantation of wild-type CD8 T cells. Similarly, mice reconstituted with gene-corrected Prf−/− CD8 T cells displayed complete protection from the HLH phenotype after infection with LCMV. Patients' cells showed correction of cytotoxicity in human CD8 T cells after transduction. These data demonstrate the potential application of T-cell gene therapy in reconstituting cytotoxic function and protection against HLH in the setting of perforin deficiency.

Hemophagocytic Lymphohistiocytosis

10.1016/j.jaci.2017.11.050

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Citations (48)

Microfluidic transfection of mRNA into human primary lymphocytes and hematopoietic stem and progenitor cells using ultra-fast physical deformations

Scientific Reports (2021)

Jocelyn Loo Ian Sicher Ailin Goff Ockchul Kim Nicole Clary

Abstract Messenger RNA (mRNA) delivery provides gene therapy with the potential to achieve transient therapeutic efficacy without risk of insertional mutagenesis. Amongst other applications, mRNA can be employed as a platform to deliver gene editing molecules, to achieve protein expression as an alternative to enzyme replacement therapies, and to express chimeric antigen receptors (CARs) on immune cells for the treatment of cancer. We designed a novel microfluidic device that allows for efficient mRNA delivery via volume exchange for convective transfection (VECT). In the device, cells flow through a ridged channel that enforces a series of ultra-fast and large intensity deformations able to transiently open pores and induce convective transport of mRNA into the cell. Here, we describe efficient delivery of mRNA into T cells, natural killer (NK) cells and hematopoietic stem and progenitor cells (HSPCs), three human primary cell types widely used for ex vivo gene therapy applications. Results demonstrate that the device can operate at a wide range of cell and payload concentrations and that ultra-fast compressions do not have a negative impact on T cell function, making this a novel and competitive platform for the development of ex vivo mRNA-based gene therapies and other cell products engineered with mRNA.

Ex vivo

10.1038/s41598-021-00893-4

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Citations (24)

140. Point Mutation Correction for ADA SCID

Molecular Therapy (2016)

Miguel Calero-Garcia Marlene Carmo Adrian J. Thrasher H. Bobby Gaspar

Adenosine deaminase (ADA) deficiency is a rare genetic disease characterised by the absence of the ADA enzyme (EC 3.5.4.4) involved in purine salvage and degradation. Patients lacking ADA build up adenosine and deoxyadenosine metabolites in the organism, something that in turn leads to an accumulation of other deoxynucleotides and gives rise to functional defects in all lymphocyte lineages and severe combined immunodeficiency (ADA SCID). This SCID phenotype can be corrected by hematopoietic stem cell transplant, enzyme replacement therapy, and integrative gene therapy protocols employing autologous CD34+ cells. ADA deficiency confers a positive selective advantage to corrected lymphocytes over deficient cells, thus offering a great system for the study of novel molecular therapies such as gene editing. The advent of the CRISPR/Cas9 system has allowed for the swift tailoring of novel gene editing strategies in laboratories without access to advanced cloning platforms. We believe this democratisation of gene editing has also made more practicable the therapeutic reversal of genetic mutations into wild type sequences, the ultimate form of genetic correction. We have designed a CRISPR/Cas9 strategy that can target the nonsense Q3X (ADA c7C>T) point mutation endemic to ADA SCID patients of Somali origin residing in the UK, as well as its equivalent site in wild type sequences. In the present work we show evidence of the Q3X site non-homologous end joining (NHEJ) gene ablation in Jurkat cells by means of DNA sequencing and ADA protein readouts (approximately 70% efficiency). Moreover, we have designed a donor for the Q3X repair step that contains an adjacent traceable silent mutation in order to track any homology directed repair (HDR) when working with wild type cells. Here we show preliminary evidence of Q3X site HDR in wild type Jurkat cells in DNA sequencing results by detection of the aforementioned traceable mutation, albeit with low efficiency (less than 3% efficiency). We are currently attempting the translation of this approach into human CD34+ cells, the ultimate target cell population for the correction of the SCID phenotype in ADA deficient patients.

Adenosine deaminase deficiency

Nonsense mutation

10.1016/s1525-0016(16)32949-5

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Citations (2)

Acute Abdomen and Intestinal Necrosis Produced by Varicella-Zoster Virus in an Immunocompromised Host

Clinical Infectious Diseases (1996)

J. S. Moreno Marina López‐Rubio Miguel Calero-Garcia T. Ratia-Jimenez Alberto Arranz-Caso

Varicella zoster virus

Acute abdomen

10.1093/clinids/22.5.857

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The Landscape of Early Clinical Gene Therapies outside of Oncology

Molecular Therapy (2019)

Laure Rittié Takis Athanasopoulos Miguel Calero-Garcia Marie L. Davies David J. Dow

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward in vivo therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.

Anticipation (artificial intelligence)

10.1016/j.ymthe.2019.09.002

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Citations (21)

Gene-ectomy: Gene Ablation with CRISPR/Cas9 in Human Hematopoietic Cells

Cell stem cell (2014)

Miguel Calero-Garcia H. Bobby Gaspar

Hematopoietic stem cell

10.1016/j.stem.2014.10.014

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Citations (3)

CRISPR/Cas9 mutation-specific gene editing for ADA SCID