RNA protein granules modulate tau isoform expression and induce neuronal sprouting.

The neuronal microtubule-associated protein Tau is expressed in different variants, and changes in Tau isoform composition occur during development and disease. Here, we investigate a potential role of the multivalent tau mRNA-binding proteins G3BP1 and IMP1 in regulating neuronal tau expression. We demonstrate that G3BP1 and IMP1 expression induces the formation of structures, which qualify as neuronal ribonucleoprotein (RNP) granules and concentrate multivalent proteins and mRNA. We show that RNP granule formation leads to a >30-fold increase in the ratio of high molecular weight to low molecular weight tau mRNA and an ∼12-fold increase in high molecular weight to low molecular weight Tau protein. We report that RNP granule formation is associated with increased neurite formation and enhanced process growth. G3BP1 deletion constructs that do not induce granule formation are also deficient in inducing neuronal sprouting or changing the expression pattern of tau. The data indicate that granule formation driven by multivalent proteins modulates tau isoform expression and suggest a morphoregulatory function of RNP granules during health and disease.

Downregulation of PIK3CA via antibody-esiRNA-complexes suppresses human xenograft tumor growth.

Precision cancer therapy requires on the one hand detailed knowledge about a tumor's driver oncogenes and on the other hand an effective targeted therapy that specifically inhibits these oncogenes. While the determination of genomic landscape of a tumor has reached a very precise level, the respective therapy options are scarce. The application of small inhibitory (si) RNAs is a promising field of investigation. Here, we present the effective in vivo-treatment of colorectal cancer (CRC) xenograft tumors with antibody-complexed, endoribonuclease-prepared small inhibitory (esi)RNAs. We chose heterogeneous endoribonuclease-prepared siRNA pools (esiRNAs) against the frequently mutated genes PIK3CA and KRAS and coupled them to the anti-EGFR antibody cetuximab, which was internalized specifically into the tumor cells. esiRNA pools have been shown to exhibit superior specificity in target gene knockdown compared to classic siRNAs. We identified a significant decrease in tumor growth upon this treatment due to decreased tumor cell proliferation. The ex vivo-analysis of the xenograft CRC tumors revealed the expected downregulation of the intended direct targets PIK3CA and KRAS on protein level. Moreover, known downstream targets for EGFR signaling such as p-ERK, p-AKT, and c-MYC were decreased as well. We therefore propose the use of antibody-esiRNA complexes as a novel experimental treatment option against key components of the EGFR signaling cascade.

Antibody-coupled siRNA as an efficient method for in vivo mRNA knockdown.

Knockdown of genes by RNA interference (RNAi) in vitro requires methods of transfection or transduction, both of which have limited impact in vivo. As a virus-free approach, we chemically coupled cell surface receptors internalizing antibodies to the short interfering RNA (siRNA) carrier peptide protamine using the bispecific cross-linker sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). First, protamine was conjugated amino-terminally to sulfo-SMCC, and then this conjugate was coupled via cysteine residues to the IgG backbone to carry siRNA. This complex can efficiently find, bind and internalize into receptor-positive cells in vitro and in vivo, which can be checked by flow cytometry, fluorescence microscopy and western blotting. This method obtains results similar to those of siRNA targeting molecules engineered by genetic fusions between receptor-binding and siRNA carrier units, with the advantage of using readily available purified proteins without the need for engineering, expression and purification of respective constructs. The procedure for coupling the complex takes ∼ 2 d, and the functional assays take ∼ 2 weeks.

Antibody-mediated delivery of anti-KRAS-siRNA in vivo overcomes therapy resistance in colon cancer.

PURPOSE: KRAS mutations are frequent driver mutations in multiple cancers. KRAS mutations also induce anti-EGFR antibody resistance in adenocarcinoma such as colon cancer. The aim of this study was to overcome anti-EGFR antibody resistance by coupling the antibody to KRAS-specific siRNA. EXPERIMENTAL DESIGN: The anti-EGFR antibody was chemically coupled to siRNA. The resulting complex was tested for antibody binding efficiency, serum stability and ability to deliver siRNA to EGFR-expressing cells. Western blotting, viability, apoptosis, and colony formation assays were performed for efficacy evaluation in vitro. Furthermore, therapeutic activity of the antibody-KRAS-siRNA complexes was examined in in vivo xenograft mouse tumor models. RESULTS: Antibody-siRNA complexes were targeted and internalized via the EGFR receptor. Upon internalization, target gene expression was strongly and specifically repressed, followed by a reduced proliferation and viability, and induced apoptosis of the cells in vitro. Clonogenic growth of mutant KRAS-bearing cells was suppressed by KRAS-siRNA-anti-EGFR antibody complexes. In xenograft mouse models, anti-EGFR antibody-KRAS-siRNA complexes significantly slowed tumor growth in anti-EGFR-resistant cells. CONCLUSIONS: The coupling of siRNA against KRAS to anti-EGFR antibodies provides a novel therapy approach for KRAS-mutated EGFR-positive cancer cells in vitro and in vivo. These findings provide an innovative approach for cancer-specific siRNA application and for enhanced therapeutic potential of monoclonal antibody therapy and personalized treatment of cancer entities.