RESUMO
Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1-/- mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vß gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1-/- mice using lentiviral SIN vectors.
Assuntos
Terapia Genética , Vetores Genéticos/administração & dosagem , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Lentivirus/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Animais , Linfócitos B/fisiologia , Western Blotting , Medula Óssea/metabolismo , Medula Óssea/patologia , Transplante de Medula Óssea , Proliferação de Células , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Rearranjo Gênico , Técnicas de Transferência de Genes , Humanos , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/imunologia , Baço/metabolismo , Linfócitos T/fisiologia , Transgenes/fisiologiaRESUMO
X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. Btk-/- mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK >100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary Btk-/- recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in Btk-/- mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels.
