Generation of new LGMDR1 models with CRISPR/Cas9 and studies to expand insight into the disease.

254 p.La LGMDR1 es la forma más común de las distrofias musculares de cinturas, y está causada por mutaciones en el gen CAPN3. Este gen codifica la proteína calpaína 3, una proteasa no lisosomal que se expresa principalmente en el músculo esquelético. La enfermedad, que actualmente no tiene cura ni tratamiento disponible, se caracteriza clínicamente por una debilidad muscular progresiva que afecta tanto a las cinturas pélvica y escapular como a los músculos proximales de las extremidades. Actualmente, la investigación en LGMDR1 requiere por un lado de mejores modelos animales y celulares para su investigación, ya que los existentes presentan limitaciones por la ausencia de un proceso distrófico claro, y por otro lado de una mayor comprensión del impacto de la ausencia de calpaína 3 en el músculo. Con esto en mente, en esta tesis se fijaron como objetivos principales 1) Desarrollarun modelo in vitro basado en líneas isogénicas de iPSCs KO para calpaína 3 mediante el uso de CRISPR/Cas9.2) Evaluar las consecuencias de la ausencia de calpaína 3 en procesos musculares mediante el uso del modelo generado y el modelo en ratón existente (C3KO). 3) Generar un nuevo modelo in vivo KO de calpaína 3 en la especie porcina mediante el uso de CRISPR/Cas9. Tras el trabajo realizado, se obtuvo el modelo in vitro que seplanteó y se caracterizó, el cual diferenciaba adecuadamente a células musculares. Los estudios con el modelo generado y el ratón C3KO concluyeron que la miogénesis está afectada en ausencia de calpaína 3, se estudió la respuesta al daño muscular agudo y se identificaron proteínas y funciones desreguladas. En cuanto al modelo porcino, tras optimizar el protocolo para la obtención de embriones KO de calpaína 3, se realizaron varias transferencias embrionarias, pero ninguna de las gestaciones dieron como resultado un lechón KO por lo que no se llegó a conseguir el modelo

Mechanisms of E2F2-mediated transcriptional repression

In this work we wanted to study the mechanism of E2F2-mediated repression. Our hypothesis is that E2F2 activates the expression of one or more E2F members of the “repressor” subset of the family through the E2F motifs present in their promoters, and those repressor E2F(s) would subsequently repress the target promoters. To address this hypothesis, we focused on E2F7. E2F7 is a repressor that lacks the Rb binding domain, and associates with DNA through E2F binding sites (de Bruin et al., 2003). Furthermore, E2F7 itself is also regulated by E2F motifs on its own promoter, and it has been shown to repress DNA metabolism and replication genes in late S-phase (de Bruin et al., 2003; Westendorp et al., 2012). E2F7, together with E2F8 has been found to form heterodimers, being critical on cell proliferation and development, and both seem to have similar functions (Li et al., 2008). Preliminary results from Zubiaga’s group have indicated that E2F2 activates E2F7 transcription in U2OS cells, suggesting that E2F2’s repressor function could be mediated by E2F7. For this purpose, we focused on studying E2F7’s role on the target genes previously known to be repressed by E2F2: Chk1 and Mcm5. The specific aims for this work were the following: - Confirm that E2F2 induces E2F7 in HEK-293T cells - Assess whether E2F7 acts as a transcriptional repressor on E2F sites - Evaluate the role of E2F7 on E2F2-mediated transcriptional repression of Chk1 and Mcm5

Patient-Specific iPSC-Derived Cellular Models of LGMDR1

Limb-girdle muscular dystrophy recessive 1 (LGMDR1) represents one of the most common types of LGMD in the population, where patients develop a progressive muscle degeneration. The disease is caused by mutations in calpain 3 gene, with over 500 mutations reported to date. However, the molecular events that lead to muscle wasting are not clear, nor the reasons for the great clinical variability among patients, and this has so far hindered the development of effective therapies. Here we generate human induced pluripotent stem cells (iPSCs) from skin fibroblasts of 2 healthy controls and 4 LGMDR1 patients with different mutations. The generated lines were able to differentiate into myogenic progenitors and myotubes in vitro and in vivo, upon a transient PAX7 overexpressing protocol. Thus, we have generated myogenic cellular models of LGMDR1 that harbor different CAPN3 mutations within a human genetic background, and which do not derive from muscular biopsies. These models will allow us to investigate disease mechanisms and test therapies. Despite the variability found among iPSC lines that was unrelated to CAPN3 mutations, we found that patient-derived myogenic progenitors and myotubes express lower levels of DMD, which codes a key protein in satellite cell regulation and myotube maturation.This work has been funded by grants from Ilundain Foundation, Isabel Gemio Foundation, Fundació La Caixa, Basque Government (2015111038), Catalan Government (2017-SGR-899 and CERCA Programme), Provincial Council of Gipuzkoa (A.LdM 114/17), and Instituto de Salud Carlos III (PI14/00436, PS09/00660 and RD16/0011/0024). A.M.-A and N.N.-G. received a studentship from the Department of Education, University and Research of the Basque Government (BFI-2012-19, PRE2013-1-1168

Mechanisms of E2F2-mediated transcriptional repression

In this work we wanted to study the mechanism of E2F2-mediated repression. Our hypothesis is that E2F2 activates the expression of one or more E2F members of the “repressor” subset of the family through the E2F motifs present in their promoters, and those repressor E2F(s) would subsequently repress the target promoters. To address this hypothesis, we focused on E2F7. E2F7 is a repressor that lacks the Rb binding domain, and associates with DNA through E2F binding sites (de Bruin et al., 2003). Furthermore, E2F7 itself is also regulated by E2F motifs on its own promoter, and it has been shown to repress DNA metabolism and replication genes in late S-phase (de Bruin et al., 2003; Westendorp et al., 2012). E2F7, together with E2F8 has been found to form heterodimers, being critical on cell proliferation and development, and both seem to have similar functions (Li et al., 2008). Preliminary results from Zubiaga’s group have indicated that E2F2 activates E2F7 transcription in U2OS cells, suggesting that E2F2’s repressor function could be mediated by E2F7. For this purpose, we focused on studying E2F7’s role on the target genes previously known to be repressed by E2F2: Chk1 and Mcm5. The specific aims for this work were the following: - Confirm that E2F2 induces E2F7 in HEK-293T cells - Assess whether E2F7 acts as a transcriptional repressor on E2F sites - Evaluate the role of E2F7 on E2F2-mediated transcriptional repression of Chk1 and Mcm5