Translational and clinical immunology
Efficacy of lentivirus-mediated gene therapy in an Omenn syndrome recombination-activating gene 2 mouse model is not hindered by inflammation and immune dysregulation

https://doi.org/10.1016/j.jaci.2017.11.015Get rights and content

Background

Omenn syndrome (OS) is a rare severe combined immunodeficiency associated with autoimmunity and caused by defects in lymphoid-specific V(D)J recombination. Most patients carry hypomorphic mutations in recombination-activating gene (RAG) 1 or 2. Hematopoietic stem cell transplantation is the standard treatment; however, gene therapy (GT) might represent a valid alternative, especially for patients lacking a matched donor.

Objective

We sought to determine the efficacy of lentiviral vector (LV)–mediated GT in the murine model of OS (Rag2R229Q/R229Q) in correcting immunodeficiency and autoimmunity.

Methods

Lineage-negative cells from mice with OS were transduced with an LV encoding the human RAG2 gene and injected into irradiated recipients with OS. Control mice underwent transplantation with wild-type or OS-untransduced lineage-negative cells. Immunophenotyping, T-dependent and T-independent antigen challenge, immune spectratyping, autoantibody detection, and detailed tissue immunohistochemical analyses were performed.

Results

LV-mediated GT allowed immunologic reconstitution, although it was suboptimal compared with that seen in wild-type bone marrow (BM)−transplanted OS mice in peripheral blood and hematopoietic organs, such as the BM, thymus, and spleen. We observed in vivo variability in the efficacy of GT correlating with the levels of transduction achieved. Immunoglobulin levels and T-cell repertoire normalized, and gene-corrected mice responded properly to challenges in vivo. Autoimmune manifestations, such as skin infiltration and autoantibodies, dramatically improved in GT mice with a vector copy number/genome higher than 1 in the BM and 2 in the thymus.

Conclusions

Our data show that LV-mediated GT for patients with OS significantly ameliorates the immunodeficiency, even in an inflammatory environment.

Section snippets

LV production

The 2.6kbUCOE-RAG2co LV, in which the human codon-optimized RAG2 cDNA was driven by a 2.6kbUCOE, was produced as previously described.26 For the 2.2kb UCOE-RAG2co LV, the 2.6kbUCOE was replaced by a shorter 2.2kbUCOE form.28

Animals

Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of San Raffaele Hospital and Italian Ministry of Health (Institutional Animal Care and Use Committee no. 710). C57Bl/6 wild-type (WT) mice were obtained from Charles River (Bar

GT improves peripheral B- and T-cell counts in mice with OS

Mice with OS were lethally irradiated and transplanted with Lin cells that were previously transduced with LV 2.6kbUCOE-RAG2co or 2.2kbUCOE-RAG2co (GT mice) to study the efficacy of GT.26, 28 Additional mice were treated with untransduced WT or OS Lin cells as positive (BMT WT mice) and negative (BMT OS mice) controls, respectively. Starting at 6 weeks after treatment, we observed the appearance of B lymphocytes in the peripheral blood of GT mice with both vectors in marked contrast to BMT OS

Discussion

Mutations in RAG genes are responsible for a broad spectrum of clinical manifestations. Allogeneic HSCT is the standard of care, ideally performed before onset of life-threatening complications and organ damage, but its overall outcome remains unsatisfactory and can be recommended only when a matched donor is available.12, 17, 18, 40 In this scenario GT might represent a powerful alternative approach, as demonstrated by encouraging results obtained in recent clinical primary immunodeficiency

References (57)

  • N.P. van Til et al.

    Correction of murine Rag2 severe combined immunodeficiency by lentiviral gene therapy using a codon-optimized RAG2 therapeutic transgene

    Mol Ther

    (2012)
  • V. Marrella et al.

    Anti-CD3e mAb improves thymic architecture and prevents autoimmune manifestations in a mouse model of Omenn syndrome: therapeutic implications

    Blood

    (2012)
  • M. Chiriaco et al.

    Dual-regulated lentiviral vector for gene therapy of X-linked chronic granulomatosis

    Mol Ther

    (2014)
  • F. Marangoni et al.

    Evidence for long-term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models

    Mol Ther

    (2009)
  • M. Bosticardo et al.

    Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome

    J Allergy Clin Immunol

    (2011)
  • H. Takaba et al.

    Fezf2 orchestrates a thymic program of self-antigen expression for immune tolerance

    Cell

    (2015)
  • B. Neven et al.

    Long-term outcome after hematopoietic stem cell transplantation of a single-center cohort of 90 patients with severe combined immunodeficiency

    Blood

    (2009)
  • C. Booth et al.

    Treating immunodeficiency through HSC gene therapy

    Trends Mol Med

    (2016)
  • P.L. Poliani et al.

    Early defects in human T-cell development severely affect distribution and maturation of thymic stromal cells: possible implications for the pathophysiology of Omenn syndrome

    Blood

    (2009)
  • R.H. Buckley

    B-cell function in severe combined immunodeficiency after stem cell or gene therapy: a review

    J Allergy Clin Immunol

    (2010)
  • E. Haddad et al.

    B-cell reconstitution for SCID: should a conditioning regimen be used in SCID treatment?

    J Allergy Clin Immunol

    (2013)
  • A.J. Thrasher et al.

    Evolving gene therapy in primary immunodeficiency

    Mol Ther

    (2017)
  • M.G. Seidel

    Autoimmune and other cytopenias in primary immunodeficiencies: pathomechanisms, novel differential diagnoses, and treatment

    Blood

    (2014)
  • D.B. Kohn et al.

    New frontiers in the therapy of primary immunodeficiency: from gene addition to gene editing

    J Allergy Clin Immunol

    (2017)
  • L.D. Notarangelo et al.

    Human RAG mutations: biochemistry and clinical implications

    Nat Rev Immunol

    (2016)
  • T. Kato et al.

    RAG1 deficiency may present clinically as selective IgA deficiency

    J Clin Immunol

    (2015)
  • D. Buchbinder et al.

    Identification of patients with RAG mutations previously diagnosed with common variable immunodeficiency disorders

    J Clin Immunol

    (2015)
  • C. Cifaldi et al.

    Late-onset combined immune-deficiency due to LIGIV mutations in a 12 years old patient

    Pediatr Allergy Immunol

    (2017)
  • Cited by (25)

    • Gene Therapy for Inborn Errors of Immunity

      2023, Journal of Allergy and Clinical Immunology: In Practice
    • Gene Therapy for Inborn Errors of Immunity: Severe Combined Immunodeficiencies

      2022, Hematology/Oncology Clinics of North America
      Citation Excerpt :

      Residual RAG function partially permits T cell development leading to immune dysregulation. The efficacy of lentiviral-mediated gene therapy was tested in a murine model of Omenn syndrome.64 There was significant amelioration of immunodeficiency in treated mice, with the normalization of immunoglobulin levels and T-cell repertoire, good response to in vivo challenges, and dramatic improvement of autoimmune manifestations.

    • Efficacy and safety of anti-CD45–saporin as conditioning agent for RAG deficiency

      2021, Journal of Allergy and Clinical Immunology
      Citation Excerpt :

      The levels of the liver enzymes alanine aminotransferase and aspartate aminotransferase were measured as described in the Methods section of the Online Repository (at www.jacionline.org). In vivo challenge with the T-dependent antigen 2,4,6-trinitrophenyl (TNP)-conjugated keyhole limpet hemocyanin was performed 4 months after transplantation as previously described.26 TNP-specific antibody titers were measured in serum by ELISA (see the Methods section of the Online Repository).

    • Intrathymic adeno-associated virus gene transfer rapidly restores thymic function and long-term persistence of gene-corrected T cells

      2020, Journal of Allergy and Clinical Immunology
      Citation Excerpt :

      The TCR repertoire was evaluated by means of deep sequencing, and data were analyzed with R Studio software. Screening for IgM/IgG reactivity against autoantigens was performed by using autoantibody arrays.63,64 The detailed methods are available in this article's Online Repository at www.jacionline.org.

    • Severe combined immune deficiency

      2020, Stiehm's Immune Deficiencies: Inborn Errors of Immunity
    View all citing articles on Scopus

    Supported by the Italian Telethon foundation (Telethon Core TGT E2 Grant to A.V.); by the European Commission's 5th, 6th, and 7th and Horizon 2020 Framework Programs (contracts QLK3-CT-2001-00427-INHERINET, LSHB-CT-2004-005242-CONSERT, grant agreement 261387 CELL-PID to A.V. and grant 666908-SCIDNET to A.V.); and by The Netherlands Health Research and Development Organization ZonMw, Translational Gene Therapy Program (project 43100016 to G.W.) and by Italian Ministry of Health (grant GR-2011-02349759 to B.C.).

    Disclosure of potential conflict of interest: L. D. Notarangelo is Editor of Frontiers in Immunology, Associate Editor for the Journal of Clinical Immunology, and Associate Editor for Clinical Immunology; is employed by the National Institutes of Health and the Department of Health and Human Services; and has received royalties from UpToDate. G. Wagemaker has received grants and travel support from The Netherlands Organization for Health Research ZonMw and the European Commission. A. Villa has received a grant from the European Commission. The rest of the authors declare that they have no relevant conflict of interest.

    View full text