RESUMEN
The EWS/FLI1 fusion gene is well characterized as a driver of Ewing's sarcoma. Bi-shRNA EWS/FLI1 is a functional plasmid DNA construct that transcribes both siRNA and miRNA-like effectors each of which targets the identical type 1 translocation junction region of the EWS/FLI1 transcribed mRNA sequence. Previous preclinical and clinical studies confirm the safety of this RNA interference platform technology and consistently demonstrate designated mRNA and protein target knockdown at greater than 90% efficiency. We initiated development of pbi-shRNA EWS/FLI1 lipoplex (LPX) for the treatment of type 1 Ewing's sarcoma. Clinical-grade plasmid was manufactured and both sequence and activity verified. Target protein and RNA knockdown of 85-92% was demonstrated in vitro in type 1 human Ewing's sarcoma tumor cell lines with the optimal bi-shRNA EWS/FLI1 plasmid. This functional plasmid was placed in a clinically tested, liposomal (LP) delivery vehicle followed by in vivo verification of activity. Type 1 Ewing's sarcoma xenograft modeling confirmed dose related safety and tumor response to pbi-shRNA EWS/FLI1 LPX. Toxicology studies in mini-pigs with doses comparable to the demonstrated in vivo efficacy dose resulted in transient fever, occasional limited hypertension at low- and high-dose assessment and transient liver enzyme elevation at high dose. These results provide the justification to initiate clinical testing.
Asunto(s)
Liposomas , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , ARN Interferente Pequeño/genética , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/genética , Animales , Sitios de Unión , Línea Celular Tumoral , Citocinas/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Técnicas de Silenciamiento del Gen , Orden Génico , Terapia Genética , Humanos , Mediadores de Inflamación , Masculino , Proteínas de Fusión Oncogénica/administración & dosificación , Proteínas de Fusión Oncogénica/química , Plásmidos/administración & dosificación , Plásmidos/genética , Proteína Proto-Oncogénica c-fli-1/administración & dosificación , Proteína Proto-Oncogénica c-fli-1/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , Proteína EWS de Unión a ARN/administración & dosificación , Proteína EWS de Unión a ARN/química , Sarcoma de Ewing/mortalidad , Sarcoma de Ewing/patología , Sarcoma de Ewing/terapia , Reparación del Gen Blanco , Transfección , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The KRAS oncogene, present in over 90% of pancreatic ductal adenocarcinomas, is most frequently the result of one of three gain-of-function substitution mutations of codon 12 glycine. Thus far, RAS mutations have been clinically refractory to both direct and selective inhibition by systemic therapeutics. This report presents the results of pre-clinical assessment of a lipoplex comprising a plasmid-encoded, modular bi-functional shRNA (bi-shRNA), which executes selective and multi-mutant allelic KRASG12mut gene silencing, encased within a fusogenic liposome systemic delivery vehicle. Using both a dual luciferase reporter system and a Restriction Fragment Length Polymorphism (RFLP) assay, selective discrimination of KRASG12mut from KRASwt was confirmed in vitro in PANC1 cells. Subsequently, systemic administration of the bi-shRNAKRAS fusogenic lipoplex into female athymic Nu/Nu mice bearing PANC1 xenografts demonstrated intratumoral plasmid delivery, KRASG12mut knockdown, and inhibition of tumor growth, without adverse effect. Clinical trials with the bi-shRNA lipoplex have been implemented.