RESUMO
Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2(-/-) mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2(-/-) mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.
Assuntos
Proteínas de Ligação a DNA/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Animais , Linfócitos B/metabolismo , Proliferação de Células , Quimerismo , Cromatina , Códon/genética , Feminino , Dosagem de Genes , Rearranjo Gênico , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Fenótipo , Plasmídeos , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Baço/citologia , Baço/metabolismo , Vírus Formadores de Foco no Baço/genética , Linfócitos T/metabolismo , Transdução Genética , TransgenesRESUMO
Persistence of malignant clones is a major determinant of adverse outcome in patients with hematologic malignancies. Despite the fact that the majority of patients with acute myeloid leukemia (AML) achieve complete remission after chemotherapy, a large proportion of them relapse as a result of residual malignant cells. These persistent clones have a competitive advantage and can re-establish disease. Therefore, targeting strategies that specifically diminish cell competition of malignant cells while leaving normal cells unaffected are clearly warranted. Recently, our group identified YBX1 as a mediator of disease persistence in JAK2-mutated myeloproliferative neoplasms. The role of YBX1 in AML, however, remained so far elusive. Here, inactivation of YBX1 confirms its role as an essential driver of leukemia development and maintenance. We identify its ability to amplify the translation of oncogenic transcripts, including MYC, by recruitment to polysomal chains. Genetic inactivation of YBX1 disrupts this regulatory circuit and displaces oncogenic drivers from polysomes, with subsequent depletion of protein levels. As a consequence, leukemia cells show reduced proliferation and are out-competed in vitro and in vivo, while normal cells remain largely unaffected. Collectively, these data establish YBX1 as a specific dependency and therapeutic target in AML that is essential for oncogenic protein expression.