Lack of dominant-negative effects of a truncated γc on retroviral-mediated gene correction of immunodeficient mice

Blood ◽  
2001 ◽  
Vol 97 (6) ◽  
pp. 1618-1624 ◽  
Author(s):  
Makoto Otsu ◽  
Kazuo Sugamura ◽  
Fabio Candotti
Keyword(s):  
Gene Therapy ◽  
Murine Model ◽  
Severe Combined Immunodeficiency ◽  
Dominant Negative ◽  
Interleukin 4 ◽  
Gene Correction ◽  
Negative Effects ◽  
Gamma Chain ◽  
Immunodeficient Mice

A recent clinical trial of gene therapy for X-linked severe combined immunodeficiency (XSCID) has shown that retroviral-mediated gene correction of bone marrow stem cells can lead to the development of normal immune function. These exciting results have been preceded by successful immune reconstitution in several XSCID mouse models, all carrying null mutations of the common gamma chain (γc). One question not formally addressed by these previous studies is that of possible dominant-negative effects of the endogenous mutant γc protein on the activity of the wild-type transferred gene product. The present work was therefore undertaken to study whether corrective gene transfer was applicable to an XSCID murine model with preserved expression of a truncated γc molecule (Δγc+-XSCID). Gene correction of Δγc+-XSCID mice resulted in the reconstitution of lymphoid development, and preferential repopulation of lymphoid organs by gene-corrected cells demonstrated the selective advantage of γc-expressing cells in vivo. Newly developed B cells showed normalization of lipopolysaccharide-mediated proliferation and interleukin-4 (IL-4)–induced immunoglobulin G1 isotype switching. Splenic T cells and thymocytes of treated animals proliferated normally to mitogens and responded to the addition of IL-2, IL-4, and IL-7, indicating functional reconstitution of γc-sharing receptors. Repopulated thymi showed a clear increase of CD4−/CD8− and CD8+fractions, both dramatically reduced in untreated Δγc+-XSCID mice. These improvements were associated with the restoration of Bcl-2 expression levels and enhanced cell survival. These data indicate that residual expression of the endogenous truncated γc did not lead to dominant-negative effects in this murine model and suggest that patient selection may not be strictly necessary for gene therapy of XSCID.

Restoration of Lymphoid Populations in a Murine Model of X-Linked Severe Combined Immunodeficiency by a Gene-Therapy Approach

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3027-3036 ◽  
Author(s):  
Mindy Lo ◽  
Michael L. Bloom ◽  
Kazunori Imada ◽  
Maria Berg ◽  
Julie M. Bollenbacher ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Murine Model ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Intraepithelial Lymphocytes ◽  
Cytokine Receptor ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Deficient Mice

X-linked severe combined immunodeficiency (XSCID) is a life-threatening syndrome in which both cellular and humoral immunity are profoundly compromised. This disease results from mutations in theIL2RG gene, which encodes the common cytokine receptor γ chain, γc. Previously, we generated γc-deficient mice as a murine model of XSCID. We have now used lethally irradiated γc-deficient mice to evaluate a gene therapeutic approach for treatment of this disease. Transfer of the human γc gene to repopulating hematopoietic stem cells using an ecotropic retrovirus resulted in an increase in T cells, B cells, natural killer (NK) cells, and intestinal intraepithelial lymphocytes, as well as normalization of the CD4:CD8 T-cell ratio and of serum Ig levels. In addition, the restored cells could proliferate in response to interleukin-2 (IL-2). Thus, our results provide added support that gene therapy is a feasible therapeutic strategy for XSCID. Moreover, because we used a vector directing expression of human γc to correct a defect in γc-deficient mice, these data also indicate that human γc can cooperate with the distinctive cytokine receptor chains such as IL-2Rβ and IL-7R to mediate responses to murine cytokines in vivo.

Restoration of Lymphoid Populations in a Murine Model of X-Linked Severe Combined Immunodeficiency by a Gene-Therapy Approach

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3027-3036 ◽  
Author(s):  
Mindy Lo ◽  
Michael L. Bloom ◽  
Kazunori Imada ◽  
Maria Berg ◽  
Julie M. Bollenbacher ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Murine Model ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Intraepithelial Lymphocytes ◽  
Cytokine Receptor ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract X-linked severe combined immunodeficiency (XSCID) is a life-threatening syndrome in which both cellular and humoral immunity are profoundly compromised. This disease results from mutations in theIL2RG gene, which encodes the common cytokine receptor γ chain, γc. Previously, we generated γc-deficient mice as a murine model of XSCID. We have now used lethally irradiated γc-deficient mice to evaluate a gene therapeutic approach for treatment of this disease. Transfer of the human γc gene to repopulating hematopoietic stem cells using an ecotropic retrovirus resulted in an increase in T cells, B cells, natural killer (NK) cells, and intestinal intraepithelial lymphocytes, as well as normalization of the CD4:CD8 T-cell ratio and of serum Ig levels. In addition, the restored cells could proliferate in response to interleukin-2 (IL-2). Thus, our results provide added support that gene therapy is a feasible therapeutic strategy for XSCID. Moreover, because we used a vector directing expression of human γc to correct a defect in γc-deficient mice, these data also indicate that human γc can cooperate with the distinctive cytokine receptor chains such as IL-2Rβ and IL-7R to mediate responses to murine cytokines in vivo.

scholarly journals Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 549
Author(s):  
Laura Garcia-Perez ◽  
Anita Ordas ◽  
Kirsten Canté-Barrett ◽  
Pauline Meij ◽  
Karin Pike-Overzet ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Severe Combined Immunodeficiency ◽  
Good Manufacturing Practice ◽  
Proof Of Concept ◽  
Preclinical Development ◽  
Hematopoietic Stem ◽  
Immune Deficiencies ◽  
Suitable Vector

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats

2002 ◽  
Vol 283 (5) ◽  
pp. H2021-H2028 ◽  
Author(s):  
Yasuhiro Ikeda ◽  
Yoshikazu Yonemitsu ◽  
Chu Kataoka ◽  
Shiro Kitamoto ◽  
Terutoshi Yamaoka ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Pulmonary Hypertension ◽  
Right Ventricular ◽  
Mononuclear Cells ◽  
Systolic Pressure ◽  
Signal Pathway ◽  
Dominant Negative ◽  
Monocyte Chemoattractant Protein 1 ◽  
Ventricular Systolic Pressure

Monocyte/macrophage chemoattractant protein-1 (MCP-1), a potent chemoattractant chemokine and an activator for mononuclear cells, may play a role in the initiation and/or progression of pulmonary hypertension (PH). To determine whether blockade of a systemic MCP-1 signal pathway in vivo may prevent PH, we intramuscularly transduced a naked plasmid encoding a 7-NH2terminus-deleted dominant negative inhibitor of the MCP-1 (7ND MCP-1) gene in monocrotaline-induced PH. We also simultaneously gave a duplicate transfection at 2-wk intervals or skeletal muscle-directed in vivo electroporation (EP) to evaluate whether a longer or higher expression might be more effective. The intramuscular reporter gene expression was enhanced 10 times over that by EP than by simple injection, and a significant 7ND MCP-1 protein in plasma was detected only in the EP group. 7ND MCP-1 gene transfer significantly inhibited the progression of MCT-induced PH as evaluated by right ventricular systolic pressure, right ventricular hypertrophy, medial hypertrophy of pulumonary arterioles, and mononuclear cell infiltration into the lung. Differential effects of longer or higher transgene expression were not apparent. Although the in vivo kinetics of 7ND MCP-1 gene therapy should be studied further, these encouraging results suggest that an anti-inflammatory strategy via blockade of the MCP-1 signal pathway may be an alternative approach to treat subjects with PH.

scholarly journals Anti-DKK1 mAb (BHQ880) as a potential therapeutic agent for multiple myeloma

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 371-379 ◽  
Author(s):  
Mariateresa Fulciniti ◽  
Pierfrancesco Tassone ◽  
Teru Hideshima ◽  
Sonia Vallet ◽  
Puru Nanjappa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Murine Model ◽  
Severe Combined Immunodeficiency ◽  
Neutralizing Antibody ◽  
Nuclear Factor Κb ◽  
Bone Lesions ◽  
Wnt Inhibitor

Abstract Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)–hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated β-catenin level while down-regulating nuclear factor-κB (NF-κB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3216-3226 ◽  
Author(s):  
Aisha V. Sauer ◽  
Emanuela Mrak ◽  
Raisa Jofra Hernandez ◽  
Elena Zacchi ◽  
Francesco Cavani ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Severe Combined Immunodeficiency ◽  
Intrinsic Defect ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Enzyme Replacement ◽  
Bone Phenotype

Abstract Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

Modeling Human T Cell Acute Lymphoblastic Leukemia In Vitro and In Vivo with LMO2.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3648-3648
Author(s):  
James A Kennedy ◽  
Sara Berthiaume ◽  
Frederic Barabe
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Progenitor Cell ◽  
Leukemic Cell ◽  
Human Cells ◽  
Immunodeficient Mice ◽  
Human T Cell

Abstract Abstract 3648 The studies identifying gene translocations and mutations in T-ALL cell lines and/or in patients have contributed significantly to the understanding of the genetic abnormalities involved in T-ALL. However, studies on the biology of these genes, the targeted cells, the sequence and the number of hits required to convert a primary human hematopoietic stem cell (HSC)/progenitor cell into a fully transformed leukemic cell require good experimental models of human T cell development both in vivo and in vitro. The only in vivo model of human T cell leukemogenesis came unexpectedly from the gene therapy trial on patients with X-linked severe combined immunodeficiency (SCID-X1). Three to five years after gene therapy, 4 out of 10 patients in the trial developed clonal T-ALL. In these patients, retroviral integrations were found in proximity to the LMO2 promoter in the malignant clones, leading to aberrant expression of the oncogene. However, little is known on the effect of LMO2 overexpression in human cells and how it facilitates the development of T-ALL. We have developed in vivo and in vitro models to study the role of T cell oncogenes in human cells. Using the OP9-DL1 co-culture system to differentiate human HSC into mature T cells in vitro, we culture human HSC transduced with lentiviruses expressing LMO2. LMO2 overexpressing cells are blocked at the double negative stage (CD4-CD8-) of differentiation when co-cultured on OP9-Delta-Like1 stroma and proliferate 50 to 100 times more than control cells. However, these cells are not immortalized and cultures lasted approximately 80 days. LMO2 overexpression have no effect on myeloid differentiation in vitro. In vivo, LMO2 transduced human HSC/progenitor cells engraft the bone marrow of immunodeficient mice to levels comparable to control cells, while normal myeloid and B cell populations 20–24 weeks post-transplantation. LMO2 transduced cells have an increased capacity to generate T cells in the thymus in comparison to control cells (42% engraftment vs 8%, p<0.0001). Surprisingly, thymic and peripheral LMO2 cells are not blocked in their differentiation. LMO2 cells did not engraft secondary mice, confirming that LMO2 doesn't induce self-renewal of human HSC. However, the increase in thymic repopulation by LMO2 cells and the lack of differentiation block in vivo suggest that LMO2 overexpression generates an abnormal T cell population with an increase repopulation advantage (increase proliferation or decrease apoptosis) in the thymus which becomes the substrate for additional genetic/epigenetic events. To test this hypothesis, we tried to immortalize LMO2 cells in vitro with secondary hits. Our preliminary results show that insertional mutagenesis can immortalized LMO2 cells in vitro. However these cells are not able to engraft immunodeficient mice or generate leukemia in vivo. The addition of intracellular NOTCH to one immortalized LMO2 cell line allows these cells to engraft and generate human T-ALL in vivo. Globally, these results show that T cell oncogenes can be studied in primary human hematopoietic cells both in vitro and in vivo. Also, at least three hits are required to transform a human primary HSC/progenitor cell into a leukemic cell able to engraft and generate leukemia in vivo. It also suggests that a non-engrafting cell can be turned into a leukemic cell generating leukemia in vivo, implying that a cell can regain self-renewing properties. Disclosures: No relevant conflicts of interest to declare.

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