Ectopic synthesis of high-Mr calcitonin by the BEN lung carcinoma cell line reflects aberrant proteolytic processing

AbstractCloning and nucleotide sequence analysis of the human calcitonin mRNA from the BEN lung carcinoma cell line, a cell line known to secrete high-Mr forms of calcitonin, showed no difference in the Coding region at the nucleotide level compared with calcitonin mRNA isolated from medullary thyroid carcinoma which secretes calcitonin monomer. Therefore, the secretion of high-Mr forms of calcitonin reflects the absence or limited activity of proteolytic processing enzymes within the secretory pathway of this cell line. In all other respects, as judged by RNA blotting and S1 mapping, calcitonin/α-CGRP expression was identical to that found in medullary thyroid carcinoma, including the differential use of an alternative splice donor site within intron 1. The BEN cell line also produces low levels of α-CGRP mRNA and secretes CGRP antigenic determinants. Analysis of plasma CGRP levels in 12 patients with anaplastic lung carcinoma showed elevated levels in 11 of these, suggesting that CGRP may be an important diagnostic marker for this disease

Lipid-peptide nanocomplexes for mRNA delivery in vitro and in vivo

Despite recent advances in the field of mRNA therapy, the lack of safe and efficacious delivery vehicles with pharmaceutically developable properties remains a major limitation. Here, we describe the systematic optimisation of lipid-peptide nanocomplexes for the delivery of mRNA in two murine cancer cell types, B16-F10 melanoma and CT26 colon carcinoma as well as NCI-H358 human lung bronchoalveolar cells. Different combinations of lipids and peptides were screened from an original lipid-peptide nanocomplex formulation for improved luciferase mRNA transfection in vitro by a multi-factorial screening approach. This led to the identification of key structural elements within the nanocomplex associated with substantial improvements in mRNA transfection efficiency included alkyl tail length of the cationic lipid, the fusogenic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. The peptide component (K16GACYGLPHKFCG) was further improved by the inclusion of a linker, RVRR, that is cleavable by the endosomal enzymes cathepsin B and furin, and a hydrophobic motif (X-S-X) between the mRNA packaging (K16) and receptor targeting domains (CYGLPHKFCG). Nanocomplex transfections of a murine B16-F10 melanoma tumour supported the inclusion of cholesterol for optimal transfection in vivo as well as in vitro. In vitro transfections were also performed with mRNA encoding interleukin-15 as a potential immunotherapy agent and again, the optimised formulation with the key structural elements demonstrated significantly higher expression than the original formulation. Physicochemical characterisation of the nanocomplexes over time indicated that the optimal formulation retained biophysical properties such as size, charge and mRNA complexation efficiency for 14 days upon storage at 4 °C without the need for additional stabilising agents. In summary, we have developed an efficacious lipid-peptide nanocomplex with promising pharmaceutical development properties for the delivery of therapeutic mRNA