Gene therapy of RAG-2−/− mice: sustained correction of the immunodeficiency

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 3942-3949 ◽  
Author(s):  
Frank Yates ◽  
Michèle Malassis-Séris ◽  
Daniel Stockholm ◽  
Cécile Bouneaud ◽  
Frédérique Larousserie ◽  
...  

Patients with mutations of either RAG-1 or RAG-2 genes suffer from severe combined immunodeficiency (SCID) characterized by the lack of T and B lymphocytes. The only curative treatment today consists of hematopoietic stem cell (HSC) transplantation, which is only partially successful in the absence of an HLA genoidentical donor, thus justifying research to find an alternative therapeutic approach. To this end, RAG-2–deficient mice were used to test whether retrovirally mediated ex vivo gene transfer into HSCs could provide long-term correction of the immunologic deficiency. Murine RAG-2−/−Sca-1+ selected bone marrow cells were transduced with a modified Moloney leukemia virus (MLV)–based MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) retroviral vector containing the RAG-2 cDNA and transplanted into RAG-2−/− sublethally irradiated mice (3Gy). Two months later, T- and B-cell development was achieved in all mice. Diverse repertoire of T cells as well as proliferative capacity in the presence of mitogens, allogeneic cells, and keyhole limpet hemocyanin (KLH) were shown. B-cell function as shown by serum Ig levels and antibody response to a challenge by KLH also developed. Lymphoid subsets and function were shown to be stable over a one-year period without evidence of any detectable toxicity. Noteworthy, a selective advantage for transduced lymphoid cells was evidenced by comparative provirus quantification in lymphoid and myeloid lineages. Altogether, this study demonstrates the efficiency of ex vivo RAG-2 gene transfer in HSCs to correct the immune deficiency of RAG-2−/− mice, constituting a significant step toward clinical application.

Blood ◽  
2002 ◽  
Vol 100 (1) ◽  
pp. 72-79 ◽  
Author(s):  
Emily J. Tsai ◽  
Harry L. Malech ◽  
Martha R. Kirby ◽  
Amy P. Hsu ◽  
Nancy E. Seidel ◽  
...  

Abstract X-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain of cytokine receptors, γc. Because bone marrow transplantation (BMT) for XSCID does not provide complete immune reconstitution for many patients and because of the natural selective advantage conferred on lymphoid progenitors by the expression of normal γc, XSCID is a good candidate disease for therapeutic retroviral gene transfer to hematopoietic stem cells. We studied XSCID patients who have persistent defects in B-cell and/or combined B- and T-cell function despite having received T cell–depleted haploidentical BMT. We compared transduction of autologous B-cell lines and granulocyte colony-stimulating factor–mobilized peripheral CD34+ cells from these patients using an MFGS retrovirus vector containing the γc gene IL2RG pseudotyped with amphotropic, gibbon ape leukemia virus, or RD114 envelopes. Transduced B-cell lines and peripheral CD34+ cells demonstrated provirus integration and new cell-surface γc expression. The chimeric sheep model was exploited to test development of XSCID CD34+ cells into mature myeloid and lymphoid lineages. Transduced and untransduced XSCID CD34+ cells injected into developing sheep fetuses gave rise to myeloid cells. However, only transduced γc+ progenitors from XSCID patients developed into T and B cells. These results suggest that gene transfer to autologous peripheral CD34+ cells using MFGS-gc retrovirus may benefit XSCID patients with persistent T- and B-cell deficits despite prior BMT.


Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 1002-1009 ◽  
Author(s):  
Masayoshi Kobune ◽  
Yoshinori Ito ◽  
Yutaka Kawano ◽  
Katsunori Sasaki ◽  
Hiroaki Uchida ◽  
...  

AbstractHematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However, the mechanisms involved in controlling the number and abilities of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in coculture with CD34+ cells and human stromal cells. Ihh mRNA was expressed in primary and telomerized human (hTERT) stromal cells, and its receptor molecules were detected in CD34+ cells. Ihh gene transfer into hTERT stromal cells enhanced their hematopoietic supporting potential, which was elevated compared with control stromal cells, as indicated by the colony-forming units in culture (CFU-Cs) (26-fold ± 2-fold versus 59-fold ± 3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's], 63-fold ± 37-fold versus 349-fold ± 116-fold). Engraftments of nonobese diabetic/severe combined immunodeficiency–ß2m–/– (NOD/SCID–ß2m–/–) repopulating cells (RCs) expanded on Ihh stromal cells were significantly higher compared with control coculture results, and engraftment was neutralized by addition of an antihedgehog antibody. Limiting dilution analysis indicated that NOD/SCID–ß2m–/– RCs proliferated efficiently on Ihh stromal cells, compared with control stromal cells. These results indicate that Ihh gene transfer could enhance the primitive hematopoietic support ability of human stromal cells.


Author(s):  
Shirly Frizinsky ◽  
Erez Rechavi ◽  
Ortal Barel ◽  
Yu-Nee Lee ◽  
Amos Simon ◽  
...  

Background: During the process of generating diverse T and B cell receptor (TCR and BCR, respectively) repertoires, double strand DNA breaks are produced. Subsequently, these breaks are corrected by a complexed system led mainly by the non-homologous end-joining (NHEJ). Mutations in proteins involved in this process, including the XLF/ Cernunnos gene, cause severe combined immunodeficiency syndrome (SCID) along with neurodevelopmental disease and susseptability to inoizing radiation. Objective: To provide new clinical and immunological insights on XLF/Cernunnos deficiency, arising from a newly diagnosed patient with severe immunodeficiency. Methods: A male infant, born to consanguineous parents, suspected of having primary immunodeficiency underwent immunological and genetic work up. This included a thorough assessment of T cell phenotyping and lymphocyte activation by mitogen stimulation tests, whole exome sequencing (WES), TCR repertoire Vβ repertoire via flow cytometry analysis and TCR and BCR via next generation sequencing (NGS). Results: Clinical findings included microcephaly, recurrent bacterial viral pneumonia and failure to thrive. Immune workup revealed lymphopenia, reduced T cell function and hypogammaglubolinemia. A skewed TCR Vβ repertoire, TCR gamma (TRG) repertoire and BCR repertoire were determined in the patient. Genetic analysis identified a novel autosomal recessive homozygous missense mutation in XLF/Cernunnos c. A580Ins.T; p. M194fs. The patient underwent a successful hematopoietic stem cell transplantation (HSCT). Conclusions: A novel XLF/Cernunnos mutation is reported in a patient presented with SCID phenotype that displayed clonally expanded T and B cells. An adjusted HSCT was safe to ensure full T cell immune reconstitution.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5246-5246
Author(s):  
Chantal Lagresle-Peyrou ◽  
Pierre Charneau ◽  
Christophe Hue ◽  
Karine Mollier ◽  
Isabelle Andre-Schmutz ◽  
...  

Abstract Patients lacking expression of either RAG-1 or RAG-2 suffer from a Severe Combined Immuno-Deficiency (SCID) disease characterized by an early block in T and B lymphocytes differentiation leading to the absence of both mature lymphocyte subsets. This disease accounts for about 20% of SCID and the only curative treatment is hematopoietic stem cell transplantation, usually successful when an HLA-genoidentical donor is available. In the absence of such a donor, the success rate decreases along with the degree of HLA disparity between donor and recipient. Ex-vivo gene therapy of hematopoietic stem cells can be considered as an alternative treatment as a selective advantage of transgene-expressing cells is expected. Moreover, constitutive expression of only one of the two RAG proteins should not be harmful as concomitant expression of both genes is required for the recombination activity. We used a lentivecteur containing the RAG-1 cDNA transgene as a therapeutic vector to transduce bone marrow CD34+ cells obtained from RAG-1 deficient patients. The transduced cells were injected into N0D-SCID mice previously irradiated (3Gy) and treated with an anti-TMb1 antibody. Ten weeks after transplantation, in all treated mice, 35±15% of the bone-marrow cells express the human CD45 marker. In this population, 24±2% co-express CD19 and IgM demonstrating that B cell differentiation capacity has been restored. We also detected some CD33+ cells attesting the presence of human myeloid progenitors cells. Altogether, these results suggest that both lymphoid and myeloid precursors have been transduced and demonstrate that gene transfer into hematopoietic cells can reconstitute B cell development in vivo. Our data support the hypothesis that gene therapy could represent a possible alternative to bone marrow transplantation in RAG-1 deficient SCID disease.


Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 460-460
Author(s):  
Chantal Lagresle-Peyrou ◽  
Frank Yates ◽  
Michele Malassis-Seris ◽  
Christophe Hue ◽  
Estelle Morillon ◽  
...  

Abstract Patients affected by severe combined immunodeficiency (SCID) have a profound defect in T cell development and the only curative treatment is hematopoietic stem cell transplantation (HSCT). When an HLA-identical family related donor exists, the success rate of HSCT is 90% while in all others cases, it ranges between 40 and 70%. In SCID patients also presenting B cell differentiation defects, such as in RAG-1 and RAG-2 deficiencies, the survival rate is significantly reduced. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG-1 cDNA. To this end, murine RAG-1 deficient hematopoietic stem cells were transduced with a retroviral modified Moloney leukemia virus vector containing the RAG-1cDNA and transplanted into RAG-1-deficient mice. B and T cell development was achieved in all the recipient mice and the reconstitution is stable over time, attesting for a selective advantage of transduced progenitors. The T cell compartment was found to be diverse and functional as shown by Va and Vb TCR immunoscope analysis as well as proliferation assays in the presence of mitogens. The mature circulating B lymphocytes were not abundant but functional as the serum immunoglobulin levels and specific antibody response against the T cell dependent KLH antigen were always within normal range. Noteworthy, a high transgene copy number was detected in all lymphoid organs and this was associated with a risk of insertional mutagenesis as observed in one mouse. Altogether, these results demonstrate that RAG-1 gene transfer can correct immunodeficiency associated with RAG-1 defect but that human application would require the use of lentivirus vectors with self inactivating long terminal repeats in order to decrease the risk of lymphoproliferative diseases.


Blood ◽  
2006 ◽  
Vol 107 (8) ◽  
pp. 3091-3097 ◽  
Author(s):  
Suk See Ting–De Ravin ◽  
Douglas R. Kennedy ◽  
Nora Naumann ◽  
Jeffrey S. Kennedy ◽  
Uimook Choi ◽  
...  

AbstractX-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency and early mortality, the only potential cure being hematopoietic stem cell (HSC) transplantation or gene therapy. Current clinical gene therapy protocols targeting HSCs are based upon ex vivo gene transfer, potentially limited by the adequacy of HSC harvest, transduction efficiencies of repopulating HSCs, and the potential loss of their engraftment potential during ex vivo culture. We demonstrate an important proof of principle by showing achievement of durable immune reconstitution in XSCID dogs following intravenous injection of concentrated RD114-pseudotyped retrovirus vector encoding the corrective gene, the interleukin-2 receptor γ chain (γc). In 3 of 4 dogs treated, normalization of numbers and function of T cells were observed. Two long-term–surviving animals (16 and 18 months) showed significant marking of B lymphocytes and myeloid cells, normalization of IgG levels, and protective humoral immune response to immunization. There were no adverse effects from in vivo gene therapy, and in one dog that reached sexual maturity, sparing of gonadal tissue from gene transfer was demonstrated. This is the first demonstration that in vivo gene therapy targeting HSCs can restore both cellular and humoral immunity in a large-animal model of a fatal immunodeficiency.


Blood ◽  
2003 ◽  
Vol 102 (3) ◽  
pp. 843-848 ◽  
Author(s):  
Christine S. McCauslin ◽  
John Wine ◽  
Linzhao Cheng ◽  
Kim D. Klarmann ◽  
Fabio Candotti ◽  
...  

Abstract Efficient retroviral gene transfer to pluripotential hematopoietic stem cells (PHSCs) requires ex vivo culture in multiple hematopoietic growth factors (HGFs) to promote cell division. While treatment of PHSCs with HGF can render stem cells viable targets for retroviral infection, HGFs can promote differentiation, loss of self-renewal potential, and affect the homing/engraftment capacity of PHSCs. To avoid the negative impacts observed with ex vivo transduction protocols, we developed a murine model for in vivo retroviral infection by direct intrafemoral injection (DII), thus abolishing the need for removal of cells from their native microenvironment and the signals necessary to maintain their unique physiology. Using this approach we have demonstrated in vivo retroviral gene transfer to colony-forming units–c (CFU-c), short-term reconstituting cells, and PHSCs. Moreover, direct intrafemoral injection of Jak3 knock-out mice with retroviral particles encoding the Jak3 gene resulted in reconstitution of normally deficient lymphocyte populations concomitant with improved immune function. In addition, DII can be used to target the delivery of other gene therapy vectors including adenoviral vectors to bone marrow cells in vivo. Taken together, these results demonstrate that in vivo retroviral gene transfer by direct intrafemoral injection may be a viable alternative to current ex vivo gene transfer approaches.


Blood ◽  
2021 ◽  
Author(s):  
Yuqing Yang ◽  
Andrew J Kueh ◽  
Zoe Grant ◽  
Waruni Abeysekera ◽  
Alexandra L Garnham ◽  
...  

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac) and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used two complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1 null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Hbo1 deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors (HPCs). The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1 and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.


Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 102-110 ◽  
Author(s):  
Craig Dorrell ◽  
Olga I. Gan ◽  
Daniel S. Pereira ◽  
Robert G. Hawley ◽  
John E. Dick

Abstract Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin− CD34+CD38−cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin−CD34+ CD38− cells and SRC, CD34+–enriched lineage–depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34+CD38− cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP+ human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34+CD38− phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34+CD38− phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34+CD38+ cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)


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