ABSTRACT
Therapeutic gene silencing by RNA interference relies on the safe and efficient in vivo delivery of small interfering RNAs (siRNAs). Polyethylenimines are among the most studied cationic polymers for gene delivery. For several reasons including superior tolerability, small linear PEIs would be preferable over branched PEIs, but they show poor siRNA complexation. Their chemical modification for siRNA formulation has not been extensively explored so far. We generated a set of small linear PEIs bearing tyrosine modifications (LPxY), leading to substantially enhanced siRNA delivery and knockdown efficacy in vitro in various cell lines, including hard-to-transfect cells. The tyrosine-modified linear 10 kDa PEI (LP10Y) is particularly powerful, associated with favorable physicochemical properties and very high biocompatibility. Systemically administered LP10Y/siRNA complexes reveal antitumor effects in mouse xenograft and patient-derived xenograft (PDX) models, and their direct application into the brain achieves therapeutic inhibition of orthotopic glioma xenografts. LP10Y is particularly interesting for therapeutic siRNA delivery.
Subject(s)
Genetic Therapy , Neoplasms, Experimental , Polyethyleneimine , RNA, Small Interfering , Transfection , Animals , Cell Line, Tumor , Humans , Mice , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Neoplasms, Experimental/genetics , Neoplasms, Experimental/therapy , Polyethyleneimine/chemistry , Polyethyleneimine/pharmacology , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Glioblastomas (GBMs) are the most malignant brain tumors and are essentially incurable even after extensive surgery, radiotherapy, and chemotherapy, mainly because of extensive infiltration of tumor cells into the adjacent normal tissue. Thus, the evaluation of novel drugs in malignant glioma treatment requires sophisticated ex vivo models that approach the authentic interplay between tumor and host environment while avoiding extensive in vivo studies in animals. This paper describes the standardized setup of an organotypic brain tissue slice tandem-culture system, comprising of normal brain tissue from adult mice and tumor tissue from human glioblastoma xenografts, and explore its utility for assessing inhibitory effects of test drugs. The microscopic analysis of vertical sections of the slice tandem-cultures allows for the simultaneous assessment of (i) the invasive potential of single cells or cell aggregates and (ii) the space occupying growth of the bulk tumor mass, both contributing to malignant tumor progression. The comparison of tissue slice co-cultures with spheroids vs. tissue slice tandem-cultures using tumor xenograft slices demonstrates advantages of the xenograft tandem approach. The direct and facile application of test drugs is shown to exert inhibitory effects on bulk tumor growth and/or tumor cell invasion, and allows their precise quantitation. In conclusion, we describe a straightforward ex vivo system mimicking the in vivo situation of the tumor mass and the normal brain in GBM patients. It reduces animal studies and allows for the direct and reproducible application of test drugs and the precise quantitation of their effects on the bulk tumor mass and on the tumor's invasive properties.
