Patent ID: 6458560
Filing Date: 2002-10-01
Classification: A61K,C07K,C12N

Abstract:
A method for making a selected polypeptide comprising:(a) introducing into a cell a eukaryotic layered vector initiation system, comprising a 5â€² promoter which directs synthesis of alphavirus RNA in vivo from cDNA, a 5â€² sequence which directs transcription of alphavirus RNA, a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins, a heterologous nucleic acid sequence encoding for said selected polypeptide and an alphavirus RNA polymerase recognition sequence, wherein said nucleic acid sequence which operably encodes all four alphaviral nonstructural proteins further comprises an altered nucleic acid sequence encoding for nonstructural protein 2 (nsP2) such that when said altered nucleic acid sequence is operably incorporated into an RNA vector replicon, the time required to reach 50% inhibition of host-cell directed macromolecular synthesis following expression in mammalian cells is increased, as compared to an RNA vector replicon having a wild-type alphavirus nsP2; (b) maintaining said cell under conditions and for a time sufficient to permit expression of said selected polypeptide; and (c) expressing said selected polypeptide.