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Supplemental Digital Content is available in the text., Background: Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder characterized by premature aging and death mainly because of myocardial infarction, stroke, or heart failure. The disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells. Patients look healthy at birth, and symptoms typically emerge in the first or second year of life. Assessing the reversibility of progerin-induced damage and the relative contribution of specific cell types is critical to determining the potential benefits of late treatment and to developing new therapies. Methods: We used CRISPR-Cas9 technology to generate LmnaHGPSrev/HGPSrev (HGPSrev) mice engineered to ubiquitously express progerin while lacking lamin A and allowing progerin suppression and lamin A restoration in a time- and cell type–specific manner on Cre recombinase activation. We characterized the phenotype of HGPSrev mice and crossed them with Cre transgenic lines to assess the effects of suppressing progerin and restoring lamin A ubiquitously at different disease stages as well as specifically in vascular smooth muscle cells and cardiomyocytes. Results: Like patients with HGPS, HGPSrev mice appear healthy at birth and progressively develop HGPS symptoms, including failure to thrive, lipodystrophy, vascular smooth muscle cell loss, vascular fibrosis, electrocardiographic anomalies, and precocious death (median lifespan of 15 months versus 26 months in wild-type controls, P

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 24-01-2020, Esta tesis tiene embargado el acceso al texto completo hasta el 24-07-2021, El Síndrome de Progeria de Hutchinson-Gilford (HGPS, por sus siglas en inglés) es un trastorno genético fatal extremadamente raro caracterizado por envejecimiento acelerado y muerte prematura a una edad promedio de 14,6 años. El HGPS "clásico" está causado por una mutación de novo c.1824 C>T en el exón 11 del gen LMNA, que codifica las laminas A y C. Se trata de una mutación que, a pesar de ser sinónima, promueve la expresión de progerina, una forma anómala de prelamina A que no puede madurar correctamente, permaneciendo farnesilada y unida a la membrana nuclear interna, afectando muchos procesos celulares de manera dominante-negativa. Los pacientes con HGPS tienen un aspecto normal al nacer, pero desarrollan síntomas de la enfermedad típicamente durante el primer y segundo año de vida. Teniendo en cuenta que no existe una cura definitiva para HGPS y que los pacientes son diagnosticados cuando los síntomas están ya presentes, es crítico determinar si el daño causado por la progerina es reversible o si la progresión de HGPS puede disminuir o detenerse mediante la supresión de la expresión de esta proteína. También es necesario investigar la contribución relativa de factores sistémicos y específicos de tejido al desarrollo de HGPS, para evaluar la efectividad de posibles terapias futuras diseñadas para suprimir la expresión de progerina en tejidos específicos, lo que sería más sencillo que la supresión de progerina en todo el cuerpo. Para abordar estas preguntas, en esta Tesis Doctoral utilizamos el sistema CRISPR-Cas9 para generar ratones LmnaHGPSrev, el primer modelo de progeria “reversible” con expresión ubicua de progerina que puede suprimirse de manera controlada espacio-temporalmente tras la activación de la recombinasa Cre, que además permite el restablecimiento de la expresión de la lamina A. Hemos demostrado que los ratones LmnaHGPSrev/HGPSrev recapitulan las características principales de HGPS en humanos, incluyendo alteraciones en el desarrollo, problemas cardiovasculares y muerte prematura. Además, mostramos que el tratamiento de estos ratones progeroides mediante la activación de la Cre con tamoxifeno en una etapa avanzada de la enfermedad aumenta su supervivencia. Finalmente, la terapia génica con virus adeno-asociados que sobreexpresan Cre mejora el crecimiento postnatal de ratones progéricos adultos con síntomas iniciales de HGPS. Estos y futuros estudios con ratones LmnaHGPSrev/HGPSrev más allá del alcance de la presente Tesis Doctoral, arrojarán información importante sobre los mecanismos celulares y moleculares de HGPS y facilitarán el camino para desarrollar terapias más eficientes., Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder characterized by accelerated aging and premature death at an average age of 14.6 years. “Classical” HGPS is caused by a heterozygous de novo c.1824 C>T dominant synonymous point mutation in the LMNA gene, which encodes for lamin A and C. This mutation promotes the expression of a mutant protein called progerin, an aberrant form of prelamin A that cannot undergo complete maturation. Progerin remains permanently farnesylated and firmly anchored to the inner nuclear membrane, affecting many cellular processes in a dominant-negative manner. HGPS patients appear normal at birth but develop symptoms of the disease typically during the first and second year of life. Taking into account that there is still no definitive cure for HGPS and that patients are diagnosed when symptoms are already present, it is critically important to ascertain whether the damage caused by progerin expression is reversible or if disease progression can be slowed down or halted upon progerin suppression. It is also necessary to investigate the relative contribution of systemic and tissue-specific factors to the development of HGPS to assess the effectiveness of potential future therapies designed to suppress progerin expression in specific tissues, which, if proven effective, would likely be less challenging than whole-body progerin suppression. In order to address these questions, in this Doctoral Thesis we use the CRISPR-Cas9 system to generate LmnaHGPSrev mice, the first “reversible” mouse model of progeria which expresses progerin ubiquitously and allows a controlled spatio-temporal suppression of progerin expression with concomitant restoration of lamin A expression upon activation of the Cre recombinase. We demonstrate that LmnaHGPSrev/HGPSrev mice recapitulate the main features of human HGPS, including failure to thrive, cardiovascular alterations and premature death. Moreover, we show that Cre activation upon tamoxifen treatment starting at an advanced stage of the disease in LmnaHGPSrev/HGPSrev Ubc-CreERTtg/+ progeroid mice prolongs their life span. Finally, we show that gene therapy with adeno-associated virus overexpressing Cre ameliorates postnatal growth of adult LmnaHGPSrev/HGPSrev mice with initial symptoms of HGPS at the initiation of the therapy. These studies with the LmnaHGPSrev mouse model, and future studies beyond the scope of the present Doctoral Thesis, will shed significant light on the cellular and molecular mechanisms of HGPS and pave the way to developing more efficient therapies.

Vascular stiffness is a major cause of cardiovascular disease during normal aging and in Hutchinson–Gilford progeria syndrome (HGPS), a rare genetic disorder caused by ubiquitous progerin expression. This mutant form of lamin A causes premature aging associated with cardiovascular alterations that lead to death at an average age of 14.6 years. We investigated the mechanisms underlying vessel stiffness in LmnaG609G/G609G mice with ubiquitous progerin expression, and tested the effect of treatment with nitrites. We also bred LmnaLCS/LCSTie2Cre+/tgand LmnaLCS/LCSSM22αCre+/tg mice, which express progerin specifically in endothelial cells (ECs) and in vascular smooth muscle cells (VSMCs), respectively, to determine the specific contribution of each cell type to vascular pathology. We found vessel stiffness and inward remodeling in arteries of LmnaG609G/G609G and LmnaLCS/LCSSM22αCre+/tg, but not in those from LmnaLCS/LCSTie2Cre+/tgmice. Structural alterations in aortas of progeroid mice were associated with decreased smooth muscle tissue content, increased collagen deposition, and decreased transverse waving of elastin layers in the media. Functional studies identified collagen (unlike elastin and the cytoskeleton) as an underlying cause of aortic stiffness in progeroid mice. Consistent with this, we found increased deposition of collagens III, IV, V, and XII in the media of progeroid aortas. Vessel stiffness and inward remodeling in progeroid mice were prevented by adding sodium nitrite in drinking water. In conclusion, LmnaG609G/G609G arteries exhibit stiffness and inward remodeling, mainly due to progerin-induced damage to VSMCs, which causes increased deposition of medial collagen and a secondary alteration in elastin structure. Treatment with nitrites prevents vascular stiffness in progeria, This study was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU, grants SAF2016‐79490‐R and SAF2016‐80305‐P), with co‐funding from the European Regional Development Fund (ERDF, “Una manera de hacer Europa”). RvK and RKA are supported by NIH grants AG047373, T32‐GM008076, F31HL142160 and NSF grant CMMI 1548571. L.d.C. was supported by a Jordi Soler postdoctoral grant from the Red de Investigación Cardiovascular (RETIC Program, Instituto de Salud Carlos III), and A.S.‐L. was supported by a Severo Ochoa predoctoral grant from the MCIU (SVP‐2014‐068334) and by a grant from Asociación Apadrina la Ciencia‐Ford España‐Ford Motor Company Fund. The CNIC is supported by the MCIU and the Pro‐CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV‐2015‐0505)