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The 117-nucleotide (nt) RNA, called the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor, has been shown to be an efficient vector for the construction of RNA nanoparticles for the delivery of small interfering RNA (siRNA) into specific cancer or viral-infected cells. Currently, chemical synthesis of 117-nt RNA is not feasible commercially. In addition, labeling at specific locations on pRNA requires the understanding of its modular organization. Here, we report multiple approaches for the construction of a functional 117-base pRNA using two synthetic RNA fragments with variable modifications. The resulting bipartite pRNA was fully competent in associating with other interacting pRNAs to form dimers, as demonstrated by the packaging of DNA via the nanomotor and the assembly of phi29 viruses in vitro. The pRNA subunit assembled from bipartite fragments harboring siRNA or receptor-binding ligands were equally competent in assembling into dimers. The subunits carrying different functionalities were able to bind cancer cells specifically, enter the cell, and silence specific genes of interest. The pRNA nanoparticles were subsequently processed by Dicer to release the siRNA embedded within the nanoparticles. The results will pave the way toward the treatment of diseases using synthetic pRNA/siRNA chimeric nanoparticles. [ABSTRACT FROM AUTHOR]

A new strategy of peptide half-life extension has been evaluated. We investigated libraries of a small and very stable protein scaffold called Nanofitin, capable of high affinity for protein targets. We have identified Nanofitins targeting Human and mouse Serum Albumin, which could significantly improve the pharmacokinetics of an active associated peptide, mobilizing the patient's own albumin without external source. To demonstrate the impact of this approach on half-life extension, a genetic fusion of an Exenatide peptide with an Albumin Binding Nanofitin (ABNF) was performed. Specific activity of Exenatide-ABNF was measured and unaffected by the fusion. In vivo mice results provided convincing data (t½ of 8 min for Exenatide peptide compared to 20 h for Exenatide-ABNF) with sustained pharmacological activity over 3 days. This study constitutes a proof-of-concept of in vivo half-life extension of a biologic using an ABNF. Besides, the absence of cysteine in the Nanofitin scaffold, which is therefore devoid of structuring disulfide bonds, allows manufacturing in microbial cost effective systems. [Display omitted] • A serum albumin binding Nanofitin (ABNF) with high affinity for human and mouse serum albumin was developed. • Exenatide showed increased stability, extended plasma half-life and sustained therapeutic efficacy when fused to the ABNF. • ABNF can be effectively used for the stabilization and plasma half-life extension of therapeutic peptides or proteins. [ABSTRACT FROM AUTHOR]

Introduction Nanofitins are low molecular weight, single chain and cysteine-free protein scaffolds able to selectively bind a defined biological target. They derive from Sac7d bacterial protein family and are highly stable over a wide range of pH (0–13) and temperature (Tm ~80 °C). Their extreme stability, low cost of production and high tolerability for chemical coupling make Nanofitins a very interesting alternative to antibodies and their fragments. Here, a hexahistidine tagged model Nanofitin (H4) directed against hen egg white lysozyme was radiolabelled and injected in mice to provide a baseline biodistribution and pharmacokinetic profiles to support future Nanofitin development programs. Method A single cysteine residue has been genetically inserted in a model Nanofitin and its regioselective radiolabelling has been performed with 4-[ 18 F]fluorobenzamido-N-ethylamino-maleimide ([ 18 F]FBEM). The synthesis of [ 18 F]FBEM has been completely implemented on a radiosynthesis unit (FastLab) including HPLC purification and formulation. Coupling with the [ 18 F]FBEM has been achieved on a solid support (Ni magnetic beads) allowing rapid purification at room temperature without organic solvent. PET-MRI studies on C57BL/6 mice were conducted after injection of [ 18 F]FBEM-Cys-H4 in order to access the biodistribution of this Nanofitin model. Results Radiochemical yield (decay corrected) of 54 ± 7% (n = 4) was obtained after optimization for coupling the [ 18 F]FBEM to Nanofitin. Pharmacokinetics results of [ 18 F]FBEM-Cys-H4 revealed a fast clearance through the liver and the kidneys. Conclusion An efficient new method on Ni magnetic beads was developed to radiolabelled his-tagged biomolecules with [ 18 F]FBEM. This procedure was applied on a Nanofitin model Cys-H4 and biodistribution kinetic studies were achieved to evaluate the potential use of Nanofitin for diagnostic imaging. Fast clearance indicates that Nanofitins represent very interesting tools for diagnostic imaging. Image 1 [ABSTRACT FROM AUTHOR]