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2.
Mol Ther ; 32(6): 1658-1671, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38532630

RESUMO

Base editing of hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematologic diseases. However, the feasibility of using adenine-base-edited HSPCs for treating X-linked severe combined immunodeficiency (SCID-X1), the influence of dose-response relationships on immune cell generation, and the potential risks have not been demonstrated in vivo. Here, a humanized SCID-X1 mouse model was established, and 86.67% ± 2.52% (n = 3) of mouse hematopoietic stem cell (HSC) pathogenic mutations were corrected, with no single-guide-RNA (sgRNA)-dependent off-target effects detected. Analysis of peripheral blood over 16 weeks post-transplantation in mice with different immunodeficiency backgrounds revealed efficient immune cell generation following transplantation of different amounts of modified HSCs. Therefore, a large-scale infusion of gene-corrected HSCs within a safe range can achieve rapid, stable, and durable immune cell regeneration. Tissue-section staining further demonstrated the restoration of immune organ tissue structures, with no tumor formation in multiple organs. Collectively, these data suggest that base-edited HSCs are a potential therapeutic approach for SCID-X1 and that a threshold infusion dose of gene-corrected cells is required for immune cell regeneration. This study lays a theoretical foundation for the clinical application of base-edited HSCs in treating SCID-X1.


Assuntos
Adenina , Linfócitos B , Modelos Animais de Doenças , Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Linfócitos T , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Animais , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Humanos , Adenina/análogos & derivados , Linfócitos B/imunologia , Linfócitos B/metabolismo , Camundongos SCID , Terapia Genética/métodos , Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas
3.
Front Cell Infect Microbiol ; 14: 1341236, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38410723

RESUMO

Bacille Calmette-Guérin (BCG) is a live strain of Mycobacterium bovis (M.bovis) for use as an attenuated vaccine to prevent tuberculosis (TB) infection, while it could also lead to an infection in immunodeficient patients. M.bovis could infect patients with immunodeficiency via BCG vaccination. Disseminated BCG disease (BCGosis) is extremely rare and has a high mortality rate. This article presents a case of a 3-month-old patient with disseminated BCG infection who was initially diagnosed with hemophagocytic syndrome (HPS) and eventually found to have X-linked severe combined immunodeficiency (X-SCID). M.bovis and its drug resistance genes were identified by metagenomics next-generation sequencing (mNGS) combined with targeted next-generation sequencing (tNGS) in blood and cerebrospinal fluid. Whole exome sequencing (WES) revealed a pathogenic variant in the common γ-chain gene (IL2RG), confirming X-SCID. Finally, antituberculosis therapy and umbilical cord blood transplantation were given to the patient. He was successfully cured of BCGosis, and his immune function was restored. The mNGS combined with the tNGS provided effective methods for diagnosing rare BCG infections in children. Their combined application significantly improved the sensitivity and specificity of the detection of M.bovis.


Assuntos
Síndromes de Imunodeficiência , Tuberculose Latente , Mycobacterium bovis , Tuberculose , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Masculino , Lactente , Criança , Humanos , Mycobacterium bovis/genética , Vacina BCG/efeitos adversos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/diagnóstico , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/complicações , Tuberculose/microbiologia , Síndromes de Imunodeficiência/complicações , Sequenciamento de Nucleotídeos em Larga Escala
4.
Tissue Eng Part A ; 30(3-4): 144-153, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37950719

RESUMO

The airway epithelia (AE) play a role in the clearance of foreign substances through ciliary motility and mucus secreted. We developed an artificial trachea that is made of collagen sponges and polypropylene mesh for the regeneration of the tracheal defect, and it was used for a clinical study. Then, a model in which the luminal surface of an artificial trachea was covered with a human-induced pluripotent stem cell-derived AE (hiPSC-AE) was transplanted into the tracheal defect of nude rats to promote epithelialization. In the future, this model was expected to be applied to research on infectious diseases and drug discovery as a trachea-humanized rat model. However, at present, sufficient engraftment has not been achieved to evaluate functional recovery in transplanted cells. Therefore, this study focused on immunosuppression in recipient rats. Nude rats lack T cell function and are widely used for transplantation experiments; however, more severe immunosuppressed recipients are preferred for xenotransplantation. Several strains of immunodeficient rats were created as rats that exhibit more severe immunodeficiency until now. In this study, to establish a trachea-humanized rat model in which human AE function can be analyzed to improve engraftment efficiency, engraftment efficiency in nude rats and X-linked severe combined immunodeficiency (X-SCID) rats following hiPSC-AE transplantation was compared. In the analysis of the proportion of engrafted cells in total cells at the graft site, the engraftment efficiency of epithelial cells tended to be high in X-SCID rats, although no statistical difference was found between the two groups, whereas the engraftment efficiency of mesenchymal cells was higher in X-SCID rats. Furthermore, the number of immune cells that accumulated in the grafts showed that a pan T cell marker, that is, CD3-positive cells, did not differ between the two strains; however, CD45-positive cells and major histocompatibility complex (MHC) class II-positive cells significantly decreased in X-SCID rats. These results indicate that X-SCID rats are more useful for the transplantation of hiPSC-AE into the tracheae to generate trachea-humanized rat models.


Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Ratos , Animais , Camundongos , Ratos Nus , Linfócitos T , Traqueia , Camundongos SCID
5.
Sci Adv ; 9(40): eadg9959, 2023 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-37801507

RESUMO

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.


Assuntos
Vetores Genéticos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Vetores Genéticos/genética , Terapia Genética , Retroviridae/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Linfócitos T
6.
J Med Case Rep ; 17(1): 307, 2023 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-37461086

RESUMO

BACKGROUND: X-linked severe combined immunodeficiency is caused by IL2RG gene mutation. Several variations have been identified in the IL2RG gene, which potentially can prevent the production of nonfunctional proteins. Herein, a novel X-linked variant in the IL2RG gene is reported in twin brothers, associated with inflammatory bowel symptoms. CASE PRESENTATION: The patients were 26-month-old monozygotic twin middle-eastern males with failure to thrive and several inpatient admissions due to severe chronic nonbloody diarrhea that started at the age of 12 months. Pancolitis was revealed after performing upper and lower gastrointestinal endoscopies on the twin with more severe gastrointestinal symptoms. Flow cytometric evaluation of the peripheral blood cells showed low levels of CD4+ cells in both patients. Next generation sequencing-based gene panel test results of the two patients proved a novel heterozygous missense X-linked IL2RG mutation (70330011 A > G, p.Trp197Arg) in one of the patients, which was predicted to be deleterious (CADD score of 28), which soon after was confirmed by Sanger segregation in his twin brother. Both parents were wild types and had never experienced similar symptoms. The patients received an human leukocyte antigen (HLA)-matched cord blood transplant. The twin with more severe gastrointestinal symptoms died 1 month after transplantation. In his brother, watery diarrhea eventually subsided after transplantation. CONCLUSION: Intestinal involvement in X-linked severe combined immunodeficiency is a rare presentation that might be neglected. The increasing availability of genetic screening tests worldwide could be helpful for early detection of such lethal primary immunodeficiency diseases and in implementing effective interventions to handle the severe outcomes.


Assuntos
Doenças Inflamatórias Intestinais , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Masculino , Humanos , Lactente , Pré-Escolar , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Irmãos , Mutação , Doenças Inflamatórias Intestinais/genética , Diarreia/genética , Subunidade gama Comum de Receptores de Interleucina/genética
7.
Cell Transplant ; 32: 9636897231178460, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37278405

RESUMO

Previous studies transplanted human-induced pluripotent stem cells (hiPSCs)-derived mesenchymal stem cells (iMSCs) into thyroid cartilage defect of X-liked severe combined immunodeficiency (X-SCID) rats and confirmed transplanted cell survival and cartilage regeneration. Thus, this study aimed to investigate the contribution of iMSC transplantation to thyroid cartilage regeneration of nude rats. iMSCs were induced from hiPSCs via a neural crest cell lineage. Then, clumps formed from an iMSC/extracellular matrix complex were transplanted into thyroid cartilage defects in nude rats. The larynx was removed and histological and immunohistochemical analyses were performed 4 or 8 weeks after the transplantation. Human nuclear antigen (HNA)-positive cells were observed in 11 of 12 (91.7%) rats, which indicated that transplanted iMSCs survived in thyroid cartilage defects in nude rats. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells in 8 of 12 rats (66.7%), which indicated cartilage-like regeneration. Cartilage-like regeneration in nude rats in this study was comparable to the previous report on X-SCID rats (HNA-positive cells were observed in all 14 rats and cartilage-like regeneration was observed in 10 of 14 rats). This result suggests that nude rats could be an alternative to X-SCID rats in thyroid cartilage regeneration experiments using iMSCs, and this nude rat cartilage transplantation model may develop cartilage regeneration research concerning fewer problems such as infection due to immunosuppression.


Assuntos
Células-Tronco Pluripotentes Induzidas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Ratos , Animais , Células-Tronco Pluripotentes Induzidas/metabolismo , Ratos Nus , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/metabolismo , Diferenciação Celular , Cartilagens Laríngeas , Células-Tronco Mesenquimais/metabolismo
9.
Int J Mol Med ; 51(3)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36799159

RESUMO

Maternal engraftment is frequently present in X­linked severe combined immunodeficiency (X­SCID) patients caused by pathogenic mutations in IL2GR. However, the functional status of the engrafted cells remains unclear because of the difficulty in separately evaluating the function of the maternal and autologous cells. The present study reported an X­SCID patient with a de novo c.677C>T (p.R226H) variant in exon 5 of IL2RG, exhibiting recurrent and persistent infections from 3­months­old. After the male patient suffering recurrent pneumonia and acute hematogenous disseminated tuberculosis when 13­months­old, single­cell RNA sequencing was applied to characterize the transcriptome landscape of his bone marrow mononuclear cells (BMMNCs). A novel bioinformatic analysis strategy was designed to discriminate maternal and autologous cells at single­cell resolution. The maternal engrafted cells consisted primarily of T, NKT and NK cells and the patient presented with the coexistence of autologous cells of these cell types. When compared respectively with normal counterparts, both maternal and autologous T and NKT cells increased the transcription of some important cytokines (GZMB, PRF1 and NKG7) against infections, but decreased the expression of a number of key transcription factors (FOS, JUN, TCF7 and LEF1) related to lymphocyte activation, proliferation and differentiation. Notably, the expression of multiple inhibitory factors (LAG3, CTLA4 and HAVCR2) were substantially enhanced in the T and NKT cells of both origins. In conclusion, both maternal and autologous T and NKT cells exhibited exhaustion­like dysfunction in this X­SCID patient suffering recurrent and persistent infections.


Assuntos
Células T Matadoras Naturais , Imunodeficiência Combinada Severa , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Lactente , Masculino , Células T Matadoras Naturais/patologia , Infecção Persistente , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/patologia , Análise de Célula Única , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
10.
J Clin Immunol ; 43(2): 358-370, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36260239

RESUMO

Abnormally high γδ T cell numbers among individuals with atypical SCID have been reported but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG (NM_000206.3) c.172C > T;p.(Pro58Ser) variant. Here, we have further investigated the index patient's abnormally large γδ T cell population in terms of function and phenotype by studying IL2RG cell surface expression, STAT tyrosine phosphorylation and blast formation in response to interleukin stimulation, immunophenotyping, TCRvγ sequencing, and target cell killing. In contrast to his âºß T cells, the patient's γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2 + population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. His TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C > A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in γδ T cells, though initially present only in part of them. IL2RG-Pro58Ser/Phe178Leu variant showed higher cell surface expression compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline p.(Pro58Ser) variant. In conclusion, our report indicates that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells.


Assuntos
Linfócitos Intraepiteliais , Imunodeficiência Combinada Severa , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Linfócitos Intraepiteliais/patologia , Células HEK293 , Receptores de Antígenos de Linfócitos T gama-delta/genética , Subunidade gama Comum de Receptores de Interleucina/genética
11.
Genes (Basel) ; 13(12)2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36553615

RESUMO

X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the IL2RG gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the IL2RG c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.


Assuntos
Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Sistemas CRISPR-Cas/genética , Mosaicismo , Edição de Genes/métodos , Terapia Genética/métodos
12.
Front Immunol ; 13: 1075351, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36569925

RESUMO

A young man with X-linked severe combined immunodeficiency developed a persistent vaccine-derived rubella virus (VDRV) infection, with the emergence of cutaneous granulomas more than fifteen years after receipt of two doses of measles-mumps-rubella (MMR) vaccine. Following nasopharyngeal swab (NP) collection, VDRV was detected by real-time polymerase chain reaction (RT-qPCR) and sequencing, and live, replication-competent VDRV was isolated in cell culture. To assess duration and intensity of viral shedding, sequential respiratory samples, one cerebrospinal fluid sample, and two urine samples were collected over 15 months, and VDRV RNA was detected in all samples by RT-qPCR. Live VDRV was cultured from nine of the eleven respiratory specimens and from one urine specimen. To our knowledge, this was the first reported instance of VDRV cultured from respiratory specimens or from urine. To assess potential transmission to close contacts, NP specimens and sera were collected from all household contacts, all of whom were immunocompetent and previously vaccinated with MMR. VDRV RNA was not detected in any NP swabs from the contacts, nor did serologic investigations suggest VDRV transmission to any contacts. This report highlights the need to understand the prevalence and duration of VDRV shedding in granuloma patients and to estimate the risk of VDRV transmission to immune and non-immune contacts.


Assuntos
Imunodeficiência Combinada Severa , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Masculino , Humanos , Vírus da Rubéola , Vacina contra Sarampo-Caxumba-Rubéola/efeitos adversos , Granuloma/genética
13.
Curr Opin Pediatr ; 34(6): 580-588, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36165614

RESUMO

PURPOSE OF REVIEW: The current review highlights how inborn errors of immunity (IEI) due to IL-2 receptor (IL-2R) subunit defects may result in children presenting with a wide variety of infectious and inflammatory presentations beyond typical X-linked severe combined immune deficiency (X-SCID) associated with IL-2Rγ. RECENT FINDINGS: Newborn screening has made diagnosis of typical SCID presenting with severe infections less common. Instead, infants are typically diagnosed in the first days of life when they appear healthy. Although earlier diagnosis has improved clinical outcomes for X-SCID, atypical SCID or other IEI not detected on newborn screening may present with more limited infectious presentations and/or profound immune dysregulation. Early management to prevent/control infections and reduce inflammatory complications is important for optimal outcomes of definitive therapies. Hematopoietic stem cell transplant (HSCT) is curative for IL-2Rα, IL-2Rß, and IL-2Rγ defects, but gene therapy may yield comparable results for X-SCID. SUMMARY: Defects in IL-2R subunits present with infectious and inflammatory phenotypes that should raise clinician's concern for IEI. Immunophenotyping may support the suspicion for diagnosis, but ultimately genetic studies will confirm the diagnosis and enable family counseling. Management of infectious and inflammatory complications will determine the success of gene therapy or HSCT.


Assuntos
Receptores de Interleucina-2 , Imunodeficiência Combinada Severa , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Homeostase , Subunidade alfa de Receptor de Interleucina-2 , Receptores de Interleucina-2/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Recém-Nascido
14.
Front Immunol ; 13: 883446, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35874699

RESUMO

To address inborn errors of immunity (IEI) which were underdiagnosed in resource-limited regions, our centre developed and offered free genetic testing for the most common IEI by Sanger sequencing (SS) since 2001. With the establishment of The Asian Primary Immunodeficiency (APID) Network in 2009, the awareness and definitive diagnosis of IEI were further improved with collaboration among centres caring for IEI patients from East and Southeast Asia. We also started to use whole exome sequencing (WES) for undiagnosed cases and further extended our collaboration with centres from South Asia and Africa. With the increased use of Next Generation Sequencing (NGS), we have shifted our diagnostic practice from SS to WES. However, SS was still one of the key diagnostic tools for IEI for the past two decades. Our centre has performed 2,024 IEI SS genetic tests, with in-house protocol designed specifically for 84 genes, in 1,376 patients with 744 identified to have disease-causing mutations (54.1%). The high diagnostic rate after just one round of targeted gene SS for each of the 5 common IEI (X-linked agammaglobulinemia (XLA) 77.4%, Wiskott-Aldrich syndrome (WAS) 69.2%, X-linked chronic granulomatous disease (XCGD) 59.5%, X-linked severe combined immunodeficiency (XSCID) 51.1%, and X-linked hyper-IgM syndrome (HIGM1) 58.1%) demonstrated targeted gene SS should remain the first-tier genetic test for the 5 common X-linked IEI.


Assuntos
Agamaglobulinemia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/genética , Criança , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sequenciamento do Exoma , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
15.
Nat Commun ; 13(1): 3710, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35764638

RESUMO

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where 'emergency' life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken ß-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety.


Assuntos
Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Antígenos CD34/genética , Células Clonais , Terapia Genética , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia
16.
Iran J Allergy Asthma Immunol ; 21(1): 92-97, 2022 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-35524383

RESUMO

Loss-of-function mutations in magnesium transporter 1 (MAGT1) gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasm (X-MEN), a disease with quite diverse clinical and immunological consequences. The phenotypic characteristics of the initially described patients included CD4+ T cell lymphopenia, immune deficiency, EBV viremia, and EBV-related lymphoproliferative disease. To date, a total of 25 patients have been reported. The spectrum of the MAGT1 defect ranges from other viral infections (HSV, VZV, CMV, MCV) and sinopulmonary bacterial infections, autoimmune diseases, non-EBV driven lymphoproliferative disease, Castleman disease, HHV8+ Kaposi's sarcoma, vasculitis (Kawasaki) to glycosylation defects in new patients. Here, we report 2 patients from two different families with novel MAGT1 mutations and different clinical features. The first patient presented with B cell lymphoma and low IgM level without recurrent infections. The second patient presented with recurrent upper respiratory tract infections, Kawasaki-like disease, hypogammaglobulinemia, and T cell lymphopenia. X-MEN disease is the first phenotype identified due to MAGT1 mutation. The identification of new mutations and atypical presentations will clarify whether there is a relationship between the genotype and the phenotype and the characteristics of the disease.


Assuntos
Proteínas de Transporte de Cátions , Infecções por Vírus Epstein-Barr , Linfopenia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Proteínas de Transporte de Cátions/genética , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4 , Humanos , Mutação , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
17.
Genes Immun ; 23(2): 66-72, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35264785

RESUMO

XMEN (X-linked immunodeficiency with magnesium defect) is caused by loss-of-function mutations in MAGT1 which is encoded on the X chromosome. The disorder is characterised by CD4 lymphopenia, severe chronic viral infections and defective T-lymphocyte activation. XMEN patients are susceptible to Epstein-Barr virus infections and persistently low levels of intracellular Mg2+. Here we describe a patient that presented with multiple recurrent infections and a subsequent diffuse B-cell lymphoma. Molecular genetic analysis by exome sequencing identified a novel hemizygous MAGT1 nonsense mutation c.1005T>A (NM_032121.5) p.(Cys335*), confirming a diagnosis of XMEN deficiency. Follow-up immunophenotyping was performed by antibody staining and flow cytometry; proliferation was determined by 3H-thymidine uptake after activation by PHA and anti-CD3. Cytotoxic natural killer cell activity was assessed with K562 target cells using the NKTESTTM assay. While lymphocyte populations were superficially intact, B cells were largely naive with a reduced memory cell compartment. Translated NKG2D was absent on both NK and T cells in the proband, and normally expressed in the carrier mother. In vitro NK cell activity was intact in both the proband and his mother. This report adds to the growing number of identified XMEN cases, raising awareness of a, still rare, X-linked immunodeficiency.


Assuntos
Proteínas de Transporte de Cátions , Infecções por Vírus Epstein-Barr , Neoplasias , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Proteínas de Transporte de Cátions/genética , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4 , Humanos , Mutação , Neoplasias/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/diagnóstico , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
18.
Hum Genet ; 141(7): 1279-1286, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35182234

RESUMO

Mutations in the X-linked gene MAGT1 cause a Congenital Disorder of Glycosylation (CDG), with two distinct clinical phenotypes: a primary immunodeficiency (XMEN disorder) versus intellectual and developmental disability. It was previously established that MAGT1 deficiency abolishes steady-state expression of the immune response protein NKG2D (encoded by KLRK1) in lymphocytes. Here, we show that the reduced steady-state levels of NKG2D are caused by hypoglycosylation of the protein and we pinpoint the exact site that is underglycosylated in MAGT1-deficient patients. Furthermore, we challenge the possibility that supplementation with magnesium restores NKG2D levels and show that the addition of this ion does not significantly improve NKG2D steady-state expression nor does it rescue the hypoglycosylation defect in CRISPR-engineered human cell lines. Moreover, magnesium supplementation of an XMEN patient did not result in restoration of NKG2D expression on the cell surface of lymphocytes. In summary, we demonstrate that in MAGT1-deficient patients, the lack of NKG2D is caused by hypoglycosylation, further elucidating the pathophysiology of XMEN/MAGT1-CDG.


Assuntos
Proteínas de Transporte de Cátions , Síndromes de Imunodeficiência , Transtornos Linfoproliferativos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Magnésio/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
19.
J Clin Immunol ; 42(1): 108-118, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34655400

RESUMO

X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus (EBV) infection and N-linked glycosylation defect (XMEN) disease is an inborn error of immunity caused by loss-of-function mutations in the magnesium transporter 1 (MAGT1) gene. The original studies of XMEN patients focused on impaired magnesium regulation, leading to decreased EBV-cytotoxicity and the loss of surface expression of the activating receptor "natural killer group 2D" (NKG2D) on CD8+ T cells and NK cells. In vitro studies showed that supraphysiological supplementation of magnesium rescued these defects. Observational studies in 2 patients suggested oral magnesium supplementation could decrease EBV viremia. Hence, we performed a randomized, double-blind, placebo-controlled, crossover study in 2 parts. In part 1, patients received either oral magnesium L-threonate (MLT) or placebo for 12 weeks followed by 12 weeks of the other treatment. Part 2 began with 3 days of high-dose intravenous (IV) magnesium sulfate (MgSO4) followed by open-label MLT for 24 weeks. One EBV-infected and 3 EBV-naïve patients completed part 1. One EBV-naïve patient was removed from part 2 of the study due to asymptomatic elevation of liver enzymes during IV MgSO4. No change in EBV or NKG2D status was observed. In vitro magnesium supplementation experiments in cells from 14 XMEN patients failed to significantly rescue NKG2D expression and the clinical trial was stopped. Although small, this study indicates magnesium supplementation is unlikely to be an effective therapeutic option in XMEN disease.


Assuntos
Proteínas de Transporte de Cátions , Infecções por Vírus Epstein-Barr , Neoplasias , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Linfócitos T CD8-Positivos , Proteínas de Transporte de Cátions/genética , Estudos Cross-Over , Suplementos Nutricionais , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/fisiologia , Humanos , Magnésio/metabolismo , Magnésio/uso terapêutico , Neoplasias/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
20.
Front Immunol ; 13: 1067417, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36685559

RESUMO

Introduction: Ex vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs. Methods: We compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)-IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)-IL2RG donor following double-strand DNA break at the endogenous IL2RG locus. Results and discussion: In vitro differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI-IL2RG HSPCs with efficient T cell development following TI-IL2RG in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before (in vitro) or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.


Assuntos
Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Animais , Camundongos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Dependovirus , Sistemas CRISPR-Cas , Camundongos SCID , Células-Tronco Hematopoéticas
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