RESUMO
The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.
Assuntos
Gangliosidoses GM2 , Doença de Tay-Sachs , Desoxirribonuclease I/metabolismo , Fibroblastos/metabolismo , Proteína Ativadora de G(M2) , Gangliosídeo G(M2)/genética , Gangliosídeo G(M2)/metabolismo , Gangliosidoses GM2/genética , Gangliosidoses GM2/metabolismo , Gangliosidoses GM2/terapia , Edição de Genes , Globosídeos/metabolismo , Glicosaminoglicanos/metabolismo , Hexosaminidase A/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Lipossomos/metabolismo , Doença de Tay-Sachs/genética , Doença de Tay-Sachs/metabolismo , Doença de Tay-Sachs/terapia , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
P3 mAb is an IgM monoclonal antibody specific for N-glycolyl-containing gangliosides. The immunogenicity of the P3 idiotype has been previously described by immunizing syngeneic BALB/c mice with the purified murine IgM or the mouse-human chimeric IgG antibody. In the present work we study the antibody response against the idiotype of P3 mAb through immunization with DNA. We used small immune proteins (SIP) consisting on the idiotype in the scFv format, covalently linked to gamma1CH3, the self-dimerizing domain of murine IgG1. SIPs were previously shown to be appropriate to induce specific anti-idiotypic responses. By gene gun immunization, a polyspecific response was occasionally generated, particularly with the P3 idiotype. A single shot of DNA was sufficient to induce a strong and long-lasting anti-P3 idiotype response. In addition, by delivery of the same DNA construct with a recombinant adeno-associated virus the unique immunogenicity of the P3 idiotype was demonstrated. The requirement of T cells in the anti-P3 idiotype response was indicated by the lack of P3-specific anti-idiotypic antibodies following immunization of both, allogeneic C57BL/6 and athymic BALB/c mice.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Gangliosídeo G(M2)/imunologia , Gangliosídeo G(M3)/imunologia , Idiótipos de Imunoglobulinas/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Anti-Idiotípicos/genética , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Biolística , DNA/imunologia , Feminino , Gangliosídeo G(M2)/genética , Gangliosídeo G(M3)/genética , Humanos , Imunização , Idiótipos de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
A point mutation within exon 5 of beta-hexosaminidase alpha chain gene was identified earlier in a Puerto Rican patient with GM2-gangliosidosis B1 variant (the DN-allele) [K. Ohno and K. Suzuki: J. Neurochem. 50:316-318, 1988]. Oligonucleotide probes designed to detect either the normal or the DN-allele showed that four additional patients carried the same mutation. These patients were of Italian, French, Spanish, and English/Italian/Hungarian origin. Three of them, as well as our original patient, were compound heterozygotes with positive signals for both the mutant and normal probes, while the Spanish patient was positive only for the DN-allele. A patient from Czechoslovakia was negative for the DN-allele. Thus, the specific mutation originally found in the Puerto Rican patient has a surprisingly wide geographic and ethnic distribution. This mutation can account for the B1 variant phenotype in five of the six B1 variant patients so far examined.