Innovative Therapeutic and Delivery Approaches Using Nanotechnology to Correct Splicing Defects Underlying Disease

Alternative splicing of pre-mRNA contributes strongly to the diversity of cell- and tissue-specific protein expression patterns. Global transcriptome analyses have suggested that >90% of human multiexon genes are alternatively spliced. Alterations in the splicing process cause missplicing events that lead to genetic diseases and pathologies, including various neurological disorders, cancers, and muscular dystrophies. In recent decades, research has helped to elucidate the mechanisms regulating alternative splicing and, in some cases, to reveal how dysregulation of these mechanisms leads to disease. The resulting knowledge has enabled the design of novel therapeutic strategies for correction of splicing-derived pathologies. In this review, we focus primarily on therapeutic approaches targeting splicing, and we highlight nanotechnology-based gene delivery applications that address the challenges and barriers facing nucleic acid-based therapeutics

Improved synthesis and characterization of cholesteryl oleate-loaded cationic solid lipid nanoparticles with high transfection efficiency for gene therapy applications

The development of new nanoparticle formulations that are capable of high transfection efficiency without toxicity is essential to provide new tools for gene therapy. However, the issues of complex, poorly reproducible manufacturing methods, and low efficiencies during in vivo testing have prevented translation to the clinic. We have previously reported the use of cholesteryl oleate as a novel excipient for solid lipid nanoparticles (SLNs) for the development of highly efficient and nontoxic nucleic acid delivery carriers. Here, we performed an extensive characterization of this novel formulation to make the scale up under Good Manufacturing Practice (GMP) possible. We also describe the complete physicochemical and biological characterization of cholesteryl oleate-loaded SLNs to ensure the reproducibility of this formula and the preservation of its characteristics before and after the lyophilization process. We defined the best manufacturing method and studied the influence of some parameters on the obtained nanoparticles using the Quality by Design (ICH Q8) guideline to obtain cholesteryl oleate-loaded SLNs that remain stable during storage and guarantee in vitro nucleic acid delivery efficacy. Our results indicate that this improved formulation is suitable for gene therapy with the possibility of scale-up the manufacturing of nanoparticles under GMP conditions

Predictores de riesgo en una cohorte española con cardiolaminopatías. Registro REDLAMINA

[Abstract] Introduction and objectives. According to sudden cardiac death guidelines, an implantable cardioverter-defibrillator (ICD) should be considered in patients with LMNA-related dilated cardiomyopathy (DCM) and ≥ 2 risk factors: male sex, left ventricular ejection fraction (LVEF) < 45%, nonsustained ventricular tachycardia (NSVT), and nonmissense genetic variants. In this study we aimed to describe the clinical characteristics of carriers of LMNA genetic variants among individuals from a Spanish cardiac-laminopathies cohort (REDLAMINA registry) and to assess previously reported risk criteria. Methods. The relationship between risk factors and cardiovascular events was evaluated in a cohort of 140 carriers (age ≥ 16 years) of pathogenic LMNA variants (54 probands, 86 relatives). We considered: a) major arrhythmic events (MAE) if there was appropriate ICD discharge or sudden cardiac death; b) heart failure death if there was heart transplant or death due to heart failure. Results. We identified 11 novel and 21 previously reported LMNA-related DCM variants. LVEF < 45% (P = .001) and NSVT (P < .001) were related to MAE, but not sex or type of genetic variant. The only factor independently related to heart failure death was LVEF < 45% (P < .001). Conclusions. In the REDLAMINA registry cohort, the only predictors independently associated with MAE were NSVT and LVEF < 45%. Therefore, female carriers of missense variants with either NSVT or LVEF < 45% should not be considered a low-risk group. It is important to individualize risk stratification in carriers of LMNA missense variants, because not all have the same prognosis.[Resumen] Introducción y objetivos. Según las guías de muerte súbita, se debe considerar un desfibrilador automático implantable (DAI) para los pacientes con miocardiopatía dilatada debida a variantes en el gen de la lamina (LMNA) con al menos 2 factores: varones, fracción de eyección del ventrículo izquierdo (FEVI) < 45%, taquicardia ventricular no sostenida (TVNS) y variantes no missense. Nuestro objetivo es describir las características clínicas de una cohorte española de pacientes con cardiolaminopatías (registro REDLAMINA) y evaluar los criterios de riesgo vigentes. Métodos. Se evaluó la relación entre factores de riesgo y eventos cardiovasculares en una cohorte de 140 portadores de variantes en LMNA (54 probandos, 86 familiares, edad ≥ 16 años). Se consideró: a) evento arrítmico mayor (EAM) si hubo descarga apropiada del DAI o muerte súbita, y b) muerte por insuficiencia cardiaca, incluidos los trasplantes. Resultados. Se identificaron 11 variantes nuevas y 21 previamente publicadas. La FEVI < 45% (p = 0,001) y la TVNS (p < 0,001) se relacionaron con los EAM, pero no el sexo o el tipo de variante (missense frente a no missense). La FEVI < 45% (p < 0,001) fue el único factor relacionado con la muerte por insuficiencia cardiaca. Conclusiones. En el registro REDLAMINA, los únicos 2 predictores asociados con EAM fueron la TVNS y la FEVI < 45%. No se debería considerar grupo de bajo riesgo a las portadoras de variantes missense con TVNS o FEVI < 45%. Es importante individualizar la estratificación del riesgo de los portadores de variantes missense en LMNA, porque no todas tienen el mismo pronóstico.This study received a grant from the Proyecto de investigación de la Sección de Insuficiencia Cardiaca 2017 from the Spanish Society of Cardiology and grants from the Instituto de Salud Carlos III (ISCIII) [PI14/0967, PI15/01551, AC16/0014] and ERA-CVD Joint Transnational Call 2016 (Genprovic). Grants from the ISCIII and the Ministerio de Economía y Competitividad de España (Spanish Department of Economy and Competitiveness) are supported by the Plan Estatal de I+D+i 2013-2016: Fondo Europeo de Desarrollo Regional (FEDER) “Una forma de hacer Europa”