RESUMO
Glial cell-line-derived neurotrophic factor (GDNF) is a potent neuroprotective agent in cellular and animal models of Parkinson's disease (PD). However, CNS delivery of GDNF in clinical trials has proven challenging due to blood-brain barrier (BBB) impermeability, poor diffusion within brain tissue, and large brain size. We report that using non-toxic mobilization-enabled preconditioning, hematopoietic stem cell (HSC) transplantation-based macrophage-mediated gene delivery may provide a solution to overcome these obstacles. Syngeneic bone marrow HSCs were transduced ex vivo with a lentiviral vector expressing macrophage promoter-driven GDNF and transplanted into 14-week-old MitoPark mice exhibiting PD-like impairments. Transplant preconditioning with granulocyte colony-stimulating factor (G-CSF) and AMD3100 was used to vacate bone marrow stem cell niches. Chimerism reached â¼80% after seven transplantation cycles. Transgene-expressing macrophages infiltrated degenerating CNS regions of MitoPark mice (not wild-type littermate controls), resulting in increased GDNF levels in the midbrain. Macrophage GDNF delivery not only markedly improved motor and non-motor dysfunction, but also dramatically mitigated the loss of dopaminergic neurons in both substantia nigra and the ventral tegmental area and preserved axonal terminals in the striatum. Striatal dopamine levels were almost completely restored. Our data support further development of mobilization-enabled HSC transplantation (HSCT)-based macrophage-mediated GDNF gene delivery as a disease-modifying therapy for PD.
RESUMO
Mammalian aging is associated with reduced tissue regeneration and loss of physiological integrity. With age, stem cells diminish in their ability to regenerate adult tissues, likely contributing to age-related morbidity. Thus, we replaced aged hematopoietic stem cells (HSCs) with young-donor HSCs using a novel mobilization-enabled hematopoietic stem cell transplantation (HSCT) technology as an alternative to the highly toxic conditioning regimens used in conventional HSCT. Using this approach, we are the first to report an increase in median lifespan (12%) and a decrease in overall mortality hazard (HR: 0.42, CI: 0.273-0.638) in aged mice following transplantation of young-donor HSCs. The increase in longevity was accompanied by reductions of frailty measures and increases in food intake and body weight of aged recipients. Young-donor HSCs not only preserved youthful function within the aged bone marrow stroma, but also at least partially ameliorated dysfunctional hematopoietic phenotypes of aged recipients. This compelling evidence that mammalian health and lifespan can be extended through stem cell therapy adds a new category to the very limited list of successful anti-aging/life-extending interventions. Our findings have implications for further development of stem cell therapies for increasing health and lifespan.
Assuntos
Senescência Celular , Fragilidade/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Longevidade , Doadores de Tecidos , Transplantados , Fatores Etários , Animais , Peso Corporal , Medula Óssea/fisiologia , Ingestão de Alimentos , Feminino , Fragilidade/sangue , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , FenótipoRESUMO
Lentiviral vectors are increasingly used as efficient gene transfer tools in the experimental and clinical gene therapy treatment of acquired and inherited genetic diseases. Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal, as well as multi-lineage differentiation and maintenance of the lymphohematopoietic system throughout life. As such, HSC transplantation (HSCT) has proven to be a powerful therapeutic modality for the treatment of both malignant and nonmalignant disorders. Transduction of lentiviral vectors into HSCs may offer long-term stable expression of a therapeutic gene in both preclinical and clinical settings. The purpose of this chapter is to describe an optimized procedure for lentiviral transduction of mouse HSCs followed by HSCT.
Assuntos
Células da Medula Óssea/metabolismo , Vetores Genéticos/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Transdução Genética , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , CamundongosRESUMO
We identified 29 G-quadruplex binding proteins by affinity purification and quantitative LC-MS/MS. We demonstrated that the DEAD-box RNA helicases Dbp2, Ded1 and Mss116 preferentially bind to G-quadruplex nucleic acids in vitro and destabilize RNA quadruplexes, suggesting new potential roles for these helicases in disruption of quadruplex structures in RNA.
Assuntos
RNA Helicases DEAD-box/química , Quadruplex G , Proteínas de Saccharomyces cerevisiae/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Cromatografia Líquida de Alta Pressão , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Cinética , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em TandemRESUMO
Glial cell line-derived neurotrophic factor (GDNF) exhibits potent neuroprotective properties in preclinical models of Parkinson's disease (PD), but challenges in GDNF delivery have been reported from clinical trials. To address this barrier, we developed a hematopoietic stem cell transplantation-based macrophage-mediated GDNF therapy platform. Here, we introduced a regulatable lentiviral vector (LV-MSP-Tet-Off-hGDNF) to allow the expression of human GDNF (hGDNF) to be adjusted or stopped by oral administration of doxycycline (Dox). C57BL/6J mice were lethally irradiated with head protection and then transplanted with syngeneic bone marrow cells transduced with either the hGDNF-expressing vector or a corresponding GFP-expressing vector, LV-MSP-Tet-Off-GFP. Suppression of vector gene expression was achieved through administration of Dox in drinking water. To create a toxin-induced Parkinsonian model, mice were injected in two cycles with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to yield nigral cell/striatal dopamine loss and behavioral deficits. During the presence of Dox in the drinking water, plasma GDNF was at a basal level, whereas during the absence of Dox, plasma GDNF was significantly elevated, indicating reliable regulation of therapeutic gene expression. Midbrain GDNF levels were altered in parallel, although these did not return completely to basal levels during the periods of Dox withdrawal. Motor activities of the MPTP-Tet-off-hGDNF group were comparable to those of the Tet-off-GFP (subject to no MPTP treatment) group, but substantially better than those of the MPTP-Tet-off-GFP group. Interestingly, the improvement in motor activities was sustained during the Dox-withdrawn periods in MPTP-Tet-off-hGDNF animals. Neuroprotection by therapeutic GDNF expression was further evidenced by significant amelioration of nigral tyrosine hydroxylase loss after both the first and second MPTP treatment cycles. These data suggest that neurotrophic factor expression can be upregulated to achieve efficacy or downregulated in case of off-target effects or adverse events, a feature that may eventually increase the acceptance of this potentially neuroprotective/disease-modifying PD therapy.