RESUMEN
Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.
Asunto(s)
Péptidos de Penetración Celular/farmacología , Sistemas de Liberación de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/métodos , Animales , Bacteriófago T7 , Biotinilación , Células CHO , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/toxicidad , Dicroismo Circular , Cricetulus , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Microscopía Fluorescente , Distrofia Muscular de Duchenne/tratamiento farmacológico , Biblioteca de Péptidos , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Phylomers are peptides derived from biodiverse protein fragments. Genetically encoded Phylomer libraries have been constructed, containing hundreds of billions of peptides derived from virtually all of the few thousand fold families found in the protein universe. They offer a rich source of high quality hits against diverse target sequences and have been used for three main purposes: firstly, to identify and validate targets in phenotypic screens; secondly, to block protein interactions with nanomolar potency binding affinities; thirdly as a source of more efficient cell penetrating peptides for the delivery of a wide range of biologics. Phylomer libraries are being increasingly used in applications such as phenotypic screening where the numbers of peptides which can be feasibly screened is limited. Phylomers also offer access to the intracellular target landscape, which remains largely undruggable by conventional means.