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    OPTIMIZED POLYNUCLEOTIDES FOR PROTEIN EXPRESSION
    4.
    发明申请
    OPTIMIZED POLYNUCLEOTIDES FOR PROTEIN EXPRESSION 有权

    公开(公告)号：US20250064986A1

    公开(公告)日：2025-02-27

    申请号：US18726955

    申请日：2023-01-06

    Applicant: Precision BioSciences, Inc.

    Inventor： Jason Richard Harris ,  Armin Hekele

    IPC: A61K48/00 ,  C12N9/22 ,  C12N15/86 ,  C12N15/88

    Abstract: A method for expressing and delivering a polynucleotide encoding a protein of interest is provided herein. Specifically, the protein of interest can be a nuclease associated with a gene-editing system with increased half-life of the mRNA encoding an engineered nuclease, such that the protein level and the gene editing efficiency of the engineered nuclease is increased. In particular, the mRNA comprises a specific combination of 5′ UTR sequence, Kozak sequence, and 3′ UTR sequence. Further provided herein are pharmaceutical compositions comprising the polynucleotides, and methods of modifying the genome of a eukaryotic cells using the polynucleotides disclosed herein.

    Method for screening a signal peptide for efficient expression and secretion of a heterologous polypeptide in mammalian cells
    10.
    发明申请
    Method for screening a signal peptide for efficient expression and secretion of a heterologous polypeptide in mammalian cells 有权

    公开(公告)号：US20250059561A1

    公开(公告)日：2025-02-20

    申请号：US18721961

    申请日：2022-12-23

    Applicant: Helsingin yliopisto

    Inventor： Ville Paavilainen ,  Juho Kellosalo ,  Paul Carlson ,  Katja Rosti ,  Maryna Green ,  Rahul Nanekar

    IPC: C12N15/86 ,  C12N15/10

    Abstract: The present disclosure is directed to a method for screening a signal peptide for efficient expression and secretion of a heterologous polypeptide in mammalian cells, the method comprising the steps of: providing a pool of viral expression vectors encoding a polypeptide of interest with various candidate signal peptides, wherein each viral expression vector of said pool comprises at least a polynucleotide encoding a fusion protein, said polynucleotide comprising: i) a promoter, ii) a sequence encoding a signal peptide, iii) a sequence encoding the polypeptide of interest, iv) optionally a sequence encoding an epitope tag, and v) a sequence encoding a C-terminal signal peptide for glycosylphosphatidylinositol, GPI, attachment or a sequence encoding a transmembrane domain; transforming host cells with said pool of viral vectors so that each host cell is preferably transformed on average by only one viral vector from said pool; expressing said fusion protein in said host cells in order to produce fusion proteins which are GPI-anchored to the host cell surface or alternatively which are anchored to the host cell surface by a transmembrane domain of said fusion protein; contacting said host cells with a first binding reagent, preferably an antibody, specifically binding to said polypeptide of interest or alternatively if said epitope tag is present in the fusion protein with a first binding reagent specifically binding to said epitope tag, wherein said binding reagent is optionally labelled with a fluorescent label or other means of detection; dividing the transformed host cells into at least two groups based on the fluorescence characteristics of each host cell; and performing next generation sequencing to the group of host cells showing the most efficient fusion protein expression in order to identify an optimal signal peptide for the polypeptide of interest in the host cell. The present disclosure is also directed to a vector, a host cell or a DNA library for use in said method.

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