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1.
Zhonghua Er Ke Za Zhi ; 57(6): 429-433, 2019 Jun 02.
Artigo em Chinês | MEDLINE | ID: mdl-31216799

RESUMO

Objective: To explore the clinical value of genetic screening for early identification of WAS gene-related disorders in newborns. Methods: This was a retrospective study. Neonatal Genome Project from Children's Hospital of Fudan University collected 5 800 high-risk newborns in the neonatal intensive care unit to study the patients' genetic causes using high-throughput sequencing from January 2016 to December 2017. Eleven newborns (all were boys) with pathogenic or likely pathogenic variants in WAS gene were enrolled. Data of clinical characteristics,gene variants and genotype-phenotype correlation were collected and summarized. Results: Eleven patients included 5 cases with Wiskott-Aldrich syndrome (WAS) and 6 cases with X-linked thrombocytopenia (XLT).Two patients with WAS developed clinical manifestations in the early neonatal period,and 3 patients in 5-8 weeks after birth. Three neonates with XLT were hospitalized for other diseases in the first place.Their platelet count was found to be reduced after admission to hospital, and diagnosis was made after genetic testing. Eleven pathogenic or likely pathogenic variants in WAS gene were identified. Among them, 7 were first reported in this study, including 2 frame shift variants c.138delG and c.388_390del, 4 splicing variants c.1453+1G>A,c.734+1G>C,c.135G>A and c.1453+3G>C, and 1 missense variant c.1118C>T. The other 4 reported variants were c.777+1G>A,c.107_108delTT, c.436delC and c.1509_*3delAGTG. Conclusions: The clinical features of WAS gene-related disorders in neonatal period lack specificity. Genetic screening in newborns plays an important role in the early diagnosis of diseases and provides providing evidence for the early intervention.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Testes Genéticos/métodos , Trombocitopenia/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/diagnóstico , Criança , Análise Mutacional de DNA , Diagnóstico Precoce , Humanos , Recém-Nascido , Masculino , Mutação , Estudos Retrospectivos , Trombocitopenia/genética , Síndrome de Wiskott-Aldrich/genética
3.
Lancet Haematol ; 6(5): e239-e253, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30981783

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy. METHODS: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32). FINDINGS: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 109 per L in one patient, 50-100 × 109 per L in five patients, and more than 100 × 109 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy. INTERPRETATION: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available. FUNDING: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Pré-Escolar , Feminino , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Itália , Masculino , Mutação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Condicionamento Pré-Transplante/métodos , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética
5.
Clin Lab ; 64(11)2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30549999

RESUMO

BACKGROUND: X-linked thrombocytopenia (XLT) is a milder form of Wiskott-Aldrich syndrome (WAS), characterized predominantly by thrombocytopenia with small-sized platelets. Mutations in the WAS gene are responsible for the disease. We herein detected a new mutation in the WAS gene responsible for XLT in a 3-generation Chinese pedigree. METHODS: Peripheral blood samples were collected from 7 members in the family. WAS gene was amplified from genomic DNA isolated from leucocytes, and then direct sequencing was performed. RESULTS: Three male members of this family (the proband, his younger brother and maternal uncle) had thrombocytopenia and decreased mean platelet volume. A homozygous mutation (T>C) was found at nucleotide position 319 in exon 3, causing the amino acid Tyr (T) to be abnormally changed to His (H) at position 107. Two female members (the proband's mother and grandmother) were carriers of the mutation. CONCLUSIONS: XLT is easy to misdiagnose as immune thrombocytopenic purpura (ITP). The diagnosis of XLT should be considered in any male with congenital microthrombocytopenia or early onset of microthrombocytope-nia (< 7 fL). This article adds to the growing number of known mutations associated with XLT.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação de Sentido Incorreto , Trombocitopenia/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Adolescente , Grupo com Ancestrais do Continente Asiático/genética , Sequência de Bases , China , Análise Mutacional de DNA , Éxons/genética , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Doenças Genéticas Ligadas ao Cromossomo X/etnologia , Humanos , Masculino , Volume Plaquetário Médio , Linhagem , Trombocitopenia/sangue , Trombocitopenia/etnologia , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/etnologia
6.
Front Immunol ; 9: 2554, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30450104

RESUMO

Early diagnosis of primary immunodeficiency disorders (PID) is vital and allows directed treatment, especially in syndromes with severe or profound combined immunodeficiency. In PID patients with perinatal CMV or other opportunistic, invasive infections (e.g., Pneumocystis or Aspergillus), multi-organ morbidity may already arise within the first months of life, before hematopoietic stem cell transplantation (HSCT) or gene therapy can be undertaken, compromising the definitive treatment and outcome. Deficiency of Wiskott-Aldrich syndrome (WAS) protein-interacting protein (WIP deficiency) causes an autosomal recessive, WAS-like syndrome with early-onset combined immunodeficiency that has been described in three pedigrees to date. While WAS typically includes combined immunodeficiency, microthrombocytopenia, and eczema, the clinical and laboratory phenotypes of WIP-deficient patients-including lymphocyte subsets, platelets, lymphocyte proliferation in vitro, and IgE-varied widely and did not entirely recapitulate WAS, impeding early diagnosis in the reported patients. To elucidate the phenotype of WIP deficiency, we provide a comprehensive synopsis of clinical and laboratory features of all hitherto-described patients (n = 6) and WIP negative mice. Furthermore, we summarize the treatment modalities and outcomes of these patients and review in detail the course of one of them who was successfully treated with serial, unconditioned, maternal, HLA-identical donor lymphocyte infusions (DLI) against life-threatening, invasive CMV infection, followed by a TCRαß/CD19-depleted, treosulfan/melphalan-conditioned, peripheral blood HSCT and repetitive, secondary-prophylactic, CMV-specific DLI with 1-year post-HSCT follow-up. This strategy could be useful in other patients with substantial premorbidity, considered "too bad to transplant," who have an HLA-identical family donor, to eliminate infections and bridge until definitive treatment.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Proteínas do Citoesqueleto/genética , Síndromes de Imunodeficiência/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transfusão de Linfócitos , Síndrome de Wiskott-Aldrich/genética , Animais , Proteínas de Transporte/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/terapia , Feminino , Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas , Histocompatibilidade , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/terapia , Lactente , Camundongos , Camundongos Knockout , Linhagem , Fenótipo , Doadores de Tecidos , Condicionamento Pré-Transplante
7.
Curr Opin Allergy Clin Immunol ; 18(6): 453-458, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30299399

RESUMO

PURPOSE OF REVIEW: Conventional gene therapy has been a successful, curative treatment modality for many primary immune deficiencies with significant improvements in the last decade. However, the risk of leukemic transformation with viral-mediated gene addition still remains, and unregulated gene addition is not an option for certain diseases in which the target gene is closely controlled. The recent bloom in genome modification platforms has created the opportunity to site-specifically correct mutated DNA base pairs or insert a corrective cDNA minigene while maintaining gene expression under control of endogenous regulatory elements. RECENT FINDINGS: There is an abundance of ongoing research utilizing programmable nucleases to facilitate site-specific gene correction of many primary immune deficiencies including X-linked severe combined immune deficiency, X-linked chronic granulomatous disease, Wiskott-Aldrich syndrome, X-linked hyper-IgM syndrome, X-linked agammaglobulinemia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked. In all, these studies have demonstrated the ability to integrate corrective DNA sequences at a precise location in the genome at rates likely to either cure or ameliorate disease. SUMMARY: Gene editing for primary immune deficiency (PID) has advanced to the point to that translation to clinical trials is likely to occur in the next several years. At the current pace of research in DNA repair mechanisms, stem cell biology, and genome-editing technology, targeted genome modification represents the next chapter of gene therapy for PID.


Assuntos
Agamaglobulinemia , Edição de Genes/métodos , Doenças Genéticas Ligadas ao Cromossomo X , Terapia Genética/métodos , Síndrome de Wiskott-Aldrich , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Agamaglobulinemia/terapia , Animais , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Humanos , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/terapia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia
8.
J Biol Chem ; 293(39): 15136-15151, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30104412

RESUMO

Wiskott-Aldrich syndrome protein (WASP) activates the actin-related protein 2/3 homolog (Arp2/3) complex and regulates actin polymerization in a physiological setting. Cell division cycle 42 (Cdc42) is a key activator of WASP, which binds Cdc42 through a Cdc42/Rac-interactive binding (CRIB)-containing region that defines a subset of Cdc42 effectors. Here, using site-directed mutagenesis and binding affinity determination and kinetic assays, we report the results of an investigation into the energetic contributions of individual WASP residues to both the Cdc42-WASP binding interface and the kinetics of complex formation. Our results support the previously proposed dock-and-coalesce binding mechanism, initiated by electrostatic steering driven by WASP's basic region and followed by a coalescence phase likely driven by the conserved CRIB motif. The WASP basic region, however, appears also to play a role in the final complex, as its mutation affected both on- and off-rates, suggesting a more comprehensive physiological role for this region centered on the C-terminal triad of positive residues. These results highlight the expanding roles of the basic region in WASP and other CRIB-containing effector proteins in regulating complex cellular processes and coordinating multiple input signals. The data presented improve our understanding of the Cdc42-WASP interface and also add to the body of information available for Cdc42-effector complex formation, therapeutic targeting of which has promise for Ras-driven cancers. Our findings suggest that combining high-affinity peptide-binding sequences with short electrostatic steering sequences could increase the efficacy of peptidomimetic candidates designed to interfere with Cdc42 signaling in cancer.


Assuntos
Neoplasias/genética , Proteína da Síndrome de Wiskott-Aldrich/química , Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/química , Actinas/química , Actinas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Cinética , Neoplasias/química , Neoplasias/patologia , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas ras/química , Proteínas ras/genética
9.
BMJ Case Rep ; 20182018 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-29991546

RESUMO

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder, described as a clinical triad of microthrombocytopenia, eczema and recurrent infections. Different mutations in WAS gene have been identified, resulting in various phenotypes and a broad range of disease severity, ranging from classic WAS to X-linked thrombocytopenia and X-linked neutropenia. WAS in some cases can be fatal without haematopoietic stem cell transplantation early in life. In this particular case, we present a novel mutation with a unique presentation. An 18-year-old man incidentally found to have macrothrombocytopenia and neutropenia at 16 years of age later found to be hemizygous for c. 869T>C (p.Ile290Thr) mutation in WAS gene. The late presentation, absence of other manifestations of WAS and presence of macrothrombocytopenia, rather than microthrombocytopenia, which is usually a characteristic finding in WAS, misled the initial diagnosis. On review of literature, this mutation has not been reported as causing WAS.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação , Neutropenia/genética , Trombocitopenia/genética , Síndrome de Wiskott-Aldrich/genética , Adolescente , Humanos , Masculino , Síndrome de Wiskott-Aldrich/complicações , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética
11.
Cell Stem Cell ; 23(1): 132-146.e9, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29979988

RESUMO

Genes that regulate hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation are tightly controlled by regulatory regions. However, mapping such regions relies on surface markers and immunophenotypic definition of HSCs. Here, we use γ-retroviral integration sites (γRV ISs) from a gene therapy trial for 10 patients with Wiskott-Aldrich syndrome to mark active enhancers and promoters in functionally defined long-term repopulating HSCs. Integration site clusters showed the highest ATAC-seq signals at HSC-specific peaks and strongly correlated with hematopoietic risk variants. Tagged genes were significantly enriched for HSC gene sets. We were able to map over 3,000 HSC regulatory regions in late-contributing HSCs, and we used these data to identify miR-10a and miR-335 as two miRNAs regulating early hematopoiesis. In this study, we show that viral insertion sites can be used as molecular tags to assess chromatin conformation on functionally defined cell populations, thereby providing a genome-wide resource for regulatory regions in human repopulating long-term HSCs.


Assuntos
Cromatina/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Diferenciação Celular , Proliferação de Células , Terapia Genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia , Síndrome de Wiskott-Aldrich/terapia
12.
BMC Med Genet ; 19(1): 123, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-30029636

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome is an X-linked recessive immunodeficiency due to mutations in Wiskott-Aldrich syndrome (WAS) gene. WAS gene is encoded for a multifunctional protein with key roles in actin polymerization, signaling pathways, and cytoskeletal rearrangement. Therefore, the impaired protein or its absence cause phenotypic spectrum of the disease. Since identification of novel mutations in WAS gene can help uncover the exact pathogenesis of Wiskott-Aldrich syndrome, the purpose of this study was to investigate disease causing-mutation in an Iranian male infant suspicious of this disorder. CASE PRESENTATION: The patient had persistent thrombocytopenia from birth, sepsis, and recurrent gastrointestinal bleeding suggestive of both Wiskott-Aldrich syndrome and chronic colitis in favor of inflammatory bowel disease (IBD). To find mutated gene in the proband, whole exome sequencing was performed for the patient and its data showed a novel, private, hemizygous splice site mutation in WAS gene (c.360 + 1G > C). CONCLUSIONS: Our study found a novel, splice-site mutation in WAS gene and help consider the genetic counselling more precisely for families with clinical phenotypes of both Wiskott-Aldrich syndrome and inflammatory bowel disease and may suggest linked pathways between these two diseases.


Assuntos
Colite/genética , Mutação/genética , Sítios de Splice de RNA/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Éxons/genética , Humanos , Lactente , Doenças Inflamatórias Intestinais/genética , Irã (Geográfico) , Masculino , Proteínas/genética , Trombocitopenia/genética
16.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 35(2): 207-209, 2018 Apr 10.
Artigo em Chinês | MEDLINE | ID: mdl-29652993

RESUMO

OBJECTIVE: To detect potential mutation of the WAS gene in a Chinese family affected with Wiskott-Aldrich syndrome. METHODS: Peripheral blood samples were collected from the proband and his family members. All exons and flanking regions of the WAS gene were subjected to PCR amplification - Sanger sequencing as well as restriction endonuclease analysis. Plasma level of B-cell activating factor (BAFF) was also determined for all family members. RESULTS: A hemizygous mutation (c.257G>A) of the WAS gene was identified in all patients from the family, for which the patient's mother was heterozygous. The same mutation was not found among healthy members of the family. Compared with unaffected members, all patients had a higher level of BAFF. CONCLUSION: The c.257G>A mutation of the WAS gene probably underlies the Wiskott-Aldrich syndrome in this family.


Assuntos
Mutação , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Fator Ativador de Células B/sangue , Pré-Escolar , Heterozigoto , Humanos , Masculino
18.
J Allergy Clin Immunol ; 142(5): 1605-1617.e4, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29447842

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency caused by mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in hematopoietic cells. A high proportion of patients experience autoimmunity caused by a breakdown in T- and B-cell tolerance. Moreover, excessive production of type I interferon (IFN-I) by plasmacytoid dendritic cells (pDCs) contributes to autoimmune signs; however, the factors that trigger excessive innate activation have not been defined. OBJECTIVE: Neutrophil extracellular traps (NETs) emerged as major initiating factors in patients with diseases such as systemic lupus erythematosus and rheumatoid arthritis. In this study we explored the possible involvement of aberrant neutrophil functions in patients with WAS. METHODS: We evaluated the expression of a set of granulocyte genes associated with NETs in a cohort of patients with WAS and the presence of NET inducers in sera. Using a mouse model of WAS, we analyzed NET release by WASp-null neutrophils and evaluated the composition and homeostasis of neutrophils in vivo. By using depletion experiments, we assessed the effect of neutrophils in promoting inflammation and reactivity against autoantigens. RESULTS: Transcripts of genes encoding neutrophil enzymes and antimicrobial peptides were increased in granulocytes of patients with WAS, and serum-soluble factors triggered NET release. WASp-null neutrophils showed increased spontaneous NETosis, induced IFN-I production by pDCs, and activated B cells through B-cell activating factor. Consistently, their depletion abolished constitutive pDC activation, normalized circulating IFN-I levels, and, importantly, abolished production of autoantibodies directed against double-stranded DNA, nucleosomes, and myeloperoxidase. CONCLUSIONS: These findings reveal that neutrophils are involved in the pathogenic loop that causes excessive activation of innate cells and autoreactive B cells, thus identifying novel mechanisms that contribute to the autoimmunity of WAS.


Assuntos
Interferon Tipo I/imunologia , Neutrófilos/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Adolescente , Adulto , Animais , Autoanticorpos/imunologia , Linfócitos B/imunologia , Pré-Escolar , Células Dendríticas/imunologia , Armadilhas Extracelulares , Feminino , Expressão Gênica , Humanos , Lactente , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome de Wiskott-Aldrich/genética , Adulto Jovem
19.
J Clin Immunol ; 38(1): 13-27, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29086100

RESUMO

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder originally described by Dr. Alfred Wiskott in 1937 and Dr. Robert Aldrich in 1954 as a familial disease characterized by infections, bleeding tendency, and eczema. Today, it is well recognized that the syndrome has a wide clinical spectrum ranging from mild, isolated thrombocytopenia to full-blown presentation that can be complicated by life-threatening hemorrhages, immunodeficiency, atopy, autoimmunity, and cancer. The pathophysiology of classic and emerging features is being elucidated by clinical studies, but remains incompletely defined, which hinders the application of targeted therapies. At the same time, progress of hematopoietic stem cell transplantation and gene therapy offer optimistic prospects for treatment options aimed at the replacement of the defective lymphohematopoietic system that have the potential to provide a cure for this rare and polymorphic disease.


Assuntos
Genes Ligados ao Cromossomo X/genética , Transplante de Células-Tronco Hematopoéticas , Mutação/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/fisiopatologia , Autoimunidade , Eczema , Terapia Genética , Humanos , Neoplasias , Trombocitopenia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia
20.
Hum Gene Ther ; 29(3): 366-380, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28922955

RESUMO

Primary immunodeficiencies, including Wiskott-Aldrich syndrome (WAS), are a main target for genome-editing strategies using specific nucleases (SNs) because a small number of corrected hematopoietic stem cells could cure patients. In this work, we have designed various WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific zinc finger nucleases (ZFNs), using K-562 cells as a cellular model and plasmid nucleofection or integration-deficient lentiviral vectors (IDLVs) for delivery. The various CRISPR/Cas9 and ZFN SNs showed similar efficiency when using plasmid nucleofection for delivery. However, dual IDLVs expressing ZFNs were more efficient than dual IDLVs expressing Cas9 and guide RNA or all-in-one IDLVs, expressing Cas9 and guide RNA in the same vector. The specificity of heterodimeric ZFNs and CRISPR/Cas9, measured by increments in γ-H2AX focus formation in WAS-edited cells, was similar for both, and both outperformed homodimeric ZFNs independently of the delivery system used. Interestingly, we show that delivery of SNs, using IDLVs, is more efficient and less genotoxic than plasmid nucleofection. We also show the similar behavior of heterodimeric ZFNs and CRISPR/Cas9 for homology-directed gene knock-in strategies, with 88 and 83% of the donors inserted in the WAS locus, respectively, whereas when using homodimeric ZFNs only 45% of the insertions were on target. In summary, our data indicate that CRISPR/Cas9 and heterodimeric ZFNs are both good alternatives to further develop SN-based gene therapy strategies for WAS. However, IDLV delivery of WAS-specific heterodimeric ZFNs was the best option of all systems compared in this study.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Loci Gênicos , Lentivirus , Transdução Genética , Síndrome de Wiskott-Aldrich/genética , Nucleases de Dedos de Zinco , Humanos , Células K562 , Síndrome de Wiskott-Aldrich/metabolismo , Nucleases de Dedos de Zinco/biossíntese , Nucleases de Dedos de Zinco/genética
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