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

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).

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

Blood ◽

10.1182/blood-2009-03-209221 ◽

2009 ◽

Vol 114 (15) ◽

pp. 3216-3226 ◽

Cited By ~ 45

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.

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Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽

10.1182/blood.v110.11.5143.5143 ◽

2007 ◽

Vol 110 (11) ◽

pp. 5143-5143

Author(s):

Liesbeth De Waele ◽

Kathleen Freson ◽

Chantal Thys ◽

Christel Van Geet ◽

Désiré Collen ◽

...

Keyword(s):

Gene Therapy ◽

Transgene Expression ◽

Ex Vivo ◽

Phosphoglycerate Kinase ◽

Lentiviral Vectors ◽

Wild Type ◽

Hematopoietic Stem ◽

Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

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Modeling human lymphoid precursor cell gene therapy in the SCID-hu mouse

Blood ◽

10.1182/blood.v84.5.1393.1393 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1393-1398 ◽

Cited By ~ 67

Author(s):

RK Akkina ◽

JD Rosenblatt ◽

AG Campbell ◽

IS Chen ◽

JA Zack

Keyword(s):

Gene Therapy ◽

In Vivo Model ◽

Hematopoietic Stem ◽

Gene Therapies ◽

Human Cd34 ◽

Human T Lymphocyte ◽

Thymocyte Differentiation ◽

Immunodeficiency Syndrome

Abstract Gene therapy of human T-lymphocyte disorders, including acquired immunodeficiency syndrome (AIDS), would be greatly facilitated by the development of an in vivo system in which transduced human hematopoietic stem cells can be used to reconstitute the T-lymphoid compartment. Here we use the SCID-hu mouse as a recipient for human CD34+ hematopoietic progenitor cells transduced in vitro with a retroviral vector carrying the neomycin resistance gene (neoR). The transduced cells engraft and reconstitute the lymphoid compartments of the human thymus implant with as few as 5 x 10(4) CD34+ cells. The neoR gene was expressed at low levels in human thymocytes and there was no apparent effect on thymocyte differentiation as a result of vector transduction. Thus, this SCID-hu mouse system is the first in vivo model showing human thymopoiesis after transduction of exogenous vectors, and should allow preclinical testing of gene therapeutic reagents designed to function in human cells of the T-lymphoid lineage. Because human immunodeficiency virus type 1 infection induces depletion of human thymocytes in SCID-hu mice, this system may be particularly valuable in evaluating efficacy of gene therapies to combat AIDS.

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Human reconstituting hematopoietic stem cells up-regulate Fas expression upon active cell cycling but remain resistant to Fas-induced suppression

Blood ◽

10.1182/blood-2002-07-2286 ◽

2003 ◽

Vol 102 (1) ◽

pp. 118-126 ◽

Cited By ~ 18

Author(s):

Ingunn Dybedal ◽

Liping Yang ◽

David Bryder ◽

Ingbritt Aastrand-Grundstrom ◽

Karin Leandersson ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Severe Combined Immunodeficiency ◽

Constitutive Expression ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Nonobese Diabetic

Abstract The Fas receptor and its ligand have been implicated in mediating the bone marrow (BM) suppression observed in graft-versus-host disease and a number of other BM-failure syndromes. However, previous studies have suggested that Fas is probably not expressed on human hematopoietic stem cells (HSCs), but up-regulated as a consequence of their commitment and differentiation, suggesting that progenitors or differentiated blood cells, rather than HSCs, are the targets of Fas-mediated suppression. The present studies confirm that candidate HSCs in human cord blood and BM lack constitutive expression of Fas, but demonstrate that Fas expression on CD34+ progenitor and stem cells is correlated to their cell cycle and activation status. With the use of recently developed in vitro conditions promoting HSC self-renewing divisions, Fas was up-regulated on virtually all HSCs capable of multilineage reconstituting nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice in vivo, as well as on long-term culture-initiating cells (LTC-ICs). Similarly, in vivo cycling of NOD-SCID repopulating cells upon transplantation, resulted in up-regulation of Fas expression. However, repopulating HSCs expressing high levels of Fas remained highly resistant to Fas-mediated suppression, and HSC function was compromised only upon coactivation with tumor necrosis factor. Thus, reconstituting human HSCs up-regulate Fas expression upon active cycling, demonstrating that HSCs could be targets for Fas-mediated BM suppression. (Blood. 2003;102: 118-126)

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In Vitro and In Vivo Selection of Genetically Modified Cells Using shRNA Against HPRT and Treatment with 6-thioguanine.

Blood ◽

10.1182/blood.v110.11.2590.2590 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2590-2590

Author(s):

Christopher C. Porter ◽

James DeGregori

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Genetically Modified ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Stem ◽

Selection Of

Abstract Inefficient transduction, poor long term expression, and engraftment failure of ex vivo manipulated cells have slowed the practical advancement of gene therapy trials. Thus, the ability to select for or amplify a population of cells that has been modified to express a gene of interest might enhance the effectiveness of gene therapy. Strategies for in vivo expansion of genetically modified cells that have been studied to date have relatively high toxicity or low efficacy in selection of hematopoietic stem cells. We hypothesized that resistance to the purine analog 6-thioguanine (6TG) could be programmed via lentiviruses, and that treatment with 6TG would allow for selection of genetically modified cells in vitro and in vivo. Using short hairpin RNAs, we achieved efficient knockdown of hypoxanthine phosphoribosyl transferase (HPRTkd), the enzyme required for 6TG cytotoxicity, in the murine hematopoietic progenitor cell line FL5.12. In so doing we were able to provide Fl5.12 cells with resistance to 6TG. In the presence of 6TG, HPRTkd cells continued to proliferate for at least 30 days, whereas control transduced cells ceased proliferating after 7-10 days. 6TG treatment of mixed cultures of GFP+-HPRTkd cells and untransduced cells resulted in selective outgrowth of HPRTkd cells. Knockdown of HPRT in FL5.12 cells was found to attenuate the checkpoint activation, cell cycle arrest and apoptosis seen in control transduced cells when treated with 6TG. Knockdown of HPRT in murine primary hematopoietic cells also allowed for selection of transduced cells with 6TG ex vivo. Furthermore, and most importantly, after transduction of whole bone marrow and transplantation into sub-lethally irradiated recipient mice, a single, short course of treatment with 6TG resulted in up to 12 fold greater percentages of circulating transduced granulocytes as compared to untreated controls. These results suggest that genetically modified hematopoietic stem cells can be selected in vivo using 6TG. This strategy may be useful for therapy of a variety of hematopoietic diseases, particularly those that affect hematopoietic progenitors. The benefits of this strategy include the following: 1) the use of a lentivirus with a self inactivating long terminal repeat, 2) a very short cassette encoding drug resistance, making the vector easier to manipulate, and 3) a very well tolerated and relatively non-toxic medication for selection.

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

Blood ◽

10.1182/blood.v94.9.3027.421k11_3027_3036 ◽

1999 ◽

Vol 94 (9) ◽

pp. 3027-3036 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v94.9.3027 ◽

1999 ◽

Vol 94 (9) ◽

pp. 3027-3036 ◽

Cited By ~ 60

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.

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Gene Therapies for Primary Immune Deficiencies

Frontiers in Immunology ◽

10.3389/fimmu.2021.648951 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lisa A. Kohn ◽

Donald B. Kohn

Keyword(s):

Gene Therapy ◽

Leukocyte Adhesion ◽

Severe Combined Immunodeficiency ◽

Retroviral Vectors ◽

Long Terminal Repeats ◽

Hematopoietic Stem ◽

Gene Therapies ◽

Immune Deficiencies ◽

Clinically Significant ◽

Better Than

Gene therapy is an innovative treatment for Primary Immune Deficiencies (PIDs) that uses autologous hematopoietic stem cell transplantation to deliver stem cells with added or edited versions of the missing or malfunctioning gene that causes the PID. Initial studies of gene therapy for PIDs in the 1990–2000's used integrating murine gamma-retroviral vectors. While these studies showed clinical efficacy in many cases, especially with the administration of marrow cytoreductive conditioning before cell re-infusion, these vectors caused genotoxicity and development of leukoproliferative disorders in several patients. More recent studies used lentiviral vectors in which the enhancer elements of the long terminal repeats self-inactivate during reverse transcription (“SIN” vectors). These SIN vectors have excellent safety profiles and have not been reported to cause any clinically significant genotoxicity. Gene therapy has successfully treated several PIDs including Adenosine Deaminase Severe Combined Immunodeficiency (SCID), X-linked SCID, Artemis SCID, Wiskott-Aldrich Syndrome, X-linked Chronic Granulomatous Disease and Leukocyte Adhesion Deficiency-I. In all, gene therapy for PIDs has progressed over the recent decades to be equal or better than allogeneic HSCT in terms of efficacy and safety. Further improvements in methods should lead to more consistent and reliable efficacy from gene therapy for a growing list of PIDs.

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Modeling human lymphoid precursor cell gene therapy in the SCID-hu mouse

Blood ◽

10.1182/blood.v84.5.1393.bloodjournal8451393 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1393-1398 ◽

Cited By ~ 1

Author(s):

RK Akkina ◽

JD Rosenblatt ◽

AG Campbell ◽

IS Chen ◽

JA Zack

Keyword(s):

Gene Therapy ◽

In Vivo Model ◽

Hematopoietic Stem ◽

Gene Therapies ◽

Human Cd34 ◽

Human T Lymphocyte ◽

Thymocyte Differentiation ◽

Immunodeficiency Syndrome

Gene therapy of human T-lymphocyte disorders, including acquired immunodeficiency syndrome (AIDS), would be greatly facilitated by the development of an in vivo system in which transduced human hematopoietic stem cells can be used to reconstitute the T-lymphoid compartment. Here we use the SCID-hu mouse as a recipient for human CD34+ hematopoietic progenitor cells transduced in vitro with a retroviral vector carrying the neomycin resistance gene (neoR). The transduced cells engraft and reconstitute the lymphoid compartments of the human thymus implant with as few as 5 x 10(4) CD34+ cells. The neoR gene was expressed at low levels in human thymocytes and there was no apparent effect on thymocyte differentiation as a result of vector transduction. Thus, this SCID-hu mouse system is the first in vivo model showing human thymopoiesis after transduction of exogenous vectors, and should allow preclinical testing of gene therapeutic reagents designed to function in human cells of the T-lymphoid lineage. Because human immunodeficiency virus type 1 infection induces depletion of human thymocytes in SCID-hu mice, this system may be particularly valuable in evaluating efficacy of gene therapies to combat AIDS.

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A seleno-hormetine protects bone marrow hematopoietic cells against ionizing radiation-induced toxicity

10.1101/432286 ◽

2018 ◽

Author(s):

Bartolini Desirée ◽

Wang Yanzhong ◽

Zhang Jie ◽

Giustarini Daniela ◽

Rossi Ranieri ◽

...

Keyword(s):

Bone Marrow ◽

Ionizing Radiation ◽

Preclinical Development ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Major Player ◽

Stem And Progenitor Cells ◽

Thiol Peroxidase

Abstract2,2’-diselenyldibenzoic acid (DSBA) is a mild thiol peroxidase agent presently in preclinical development. This study reports that the drug has novel seleno-hormetic properties in both murine bone marrow and human liver cells. According with previous in vitro findings, mechanistic aspects of such properties were confirmed to include the activation of Nrf2 transcription factor and an increased expression of downstream stress response genes in the liver and in hematopoietic stem and progenitor cells of the myeloid lineage. These genes include glutathione S-transferase that is reported to represent a major player in the metabolism and pharmacological function of seleno-organic compounds. As a practical application, DSBA administration prevented bone marrow toxicities following acute exposure to sub-lethal doses of ionizing radiation in C57 BL/6 mice.In conclusion, this study demonstrates for the first time the pharmacological properties of DSBAin vivo. The findings suggest applications for this selenohormetine in radioprotection and prevention protocols.

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