nucleic acids improved for the expression of interleukin-12 (IL-12); improved cytokine expression in mammalian cells by optimizing all steps of gene expression of the cytokine
Inventors
Felber, Barbara • Pavlakis, George N. • Rosati, Margherita
Assignees
National Institutes of Health NIH • US Department of Health and Human Services
Publication Number
US-7833754-B2
Publication Date
2010-11-16
Expiration Date
2027-01-12
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Abstract
The present invention provides for nucleic acids improved for the expression of interleukin-12 (IL-12) in mammalian cells. The invention further provides for methods of expressing IL-12 in mammalian cells by transfecting the cell with a nucleic acid sequence encoding an improved IL-12 sequence.
Core Innovation
The invention provides nucleic acid sequences, expression vectors, and mammalian cells that enable high-level expression of interleukin-12 (IL-12) in mammalian cells. Specifically, it offers nucleic acid pairs encoding IL-12 protein heterodimers composed of IL-12p35 and IL-12p40 subunits, where each nucleic acid sequence has optimized codons differing significantly from native sequences, resulting in improved expression of IL-12 protein.
The problem solved addresses the inefficient expression of native mammalian IL-12 sequences. Native IL-12 nucleic acid sequences contain signals such as potential splice sites and low stability determinants, including A/T rich sequences, which negatively affect mRNA stability and processing, limiting IL-12 protein production. By optimizing codon usage, eliminating these negative signals, and increasing GC content, the invention achieves substantially increased expression levels of biologically active IL-12 heterodimers in mammalian cells.
Claims Coverage
The claims include two primary independent claims covering nucleic acid sequence pairs encoding IL-12 heterodimers and methods of expressing these nucleic acids in mammalian cells, along with related dependent claims covering expression vectors, cells, and compositions.
Nucleic acid sequence pair encoding optimized IL-12 heterodimer
A nucleic acid sequence pair encoding an IL-12 protein heterodimer comprising an IL-12p35 nucleic acid sequence and an IL-12p40 nucleic acid sequence, wherein the IL-12p35 nucleic acid has at least 95% sequence identity to SEQ ID NO:3, and the IL-12p40 nucleic acid has at least 95% sequence identity to SEQ ID NO:6.
Expression vectors containing optimized IL-12 nucleic acid sequences
Expression vectors comprising the nucleic acid sequence pair as defined, designed for high-level expression of IL-12 subunits in mammalian cells.
Mammalian cells containing optimized IL-12 nucleic acid sequences or expression vectors
Isolated mammalian cells that comprise the nucleic acid sequence pair or expression vectors encoding optimized IL-12p35 and IL-12p40 sequences.
Composition comprising optimized IL-12 nucleic acid sequences and pharmaceutical excipients
Pharmaceutical compositions including the nucleic acid sequence pair and a pharmaceutically acceptable excipient suitable for use as vaccines or adjuvants.
Method of expressing IL-12 in mammalian cells
Methods of introducing recombinant vectors encoding the nucleic acid sequence pair into mammalian cells to express the IL-12 heterodimer with at least 95% sequence identity to SEQ ID NOs:3 and 6.
Nucleic acid sequence pairs with at least 80% sequence identity showing increased expression
Nucleic acid sequence pairs encoding IL-12p35 and IL-12p40 having at least 80% sequence identity to SEQ ID NOs:3 and 6, respectively, that provide at least a 2-fold increase in IL-12 expression compared to native sequences, with elevated GC content and removal of all potential splice sites.
Codon optimization at defined positions in IL-12 sequences
The nucleic acid sequences include non-native codons at specific codon positions identified in FIGS. 6 and 8 that contribute to improved expression.
The claims comprehensively cover optimized IL-12 nucleic acid sequences with specific codon modifications, expression vectors incorporating these sequences, mammalian cells expressing IL-12 heterodimers, compositions for pharmaceutical use, and methods for expressing IL-12 in mammalian cells. The inventive focus is on codon optimization and nucleic acid sequence modifications that enhance IL-12 expression.
Stated Advantages
Significant increase in expression levels of IL-12 heterodimer in mammalian cells, achieving up to 5-6 fold or more compared to native sequences.
Improved mRNA stability and processing by removal of potential splice sites and reduction of low stability A/T-rich sequences.
Use of optimized codons increases GC content, contributing to enhanced transcription and translation efficiency.
Enables high-level expression of biologically active IL-12 suitable for use as molecular adjuvants in vaccines.
Documented Applications
Use of optimized IL-12 nucleic acid sequences as molecular adjuvants co-administered with vaccine antigens to enhance vaccine-induced immune responses.
Delivery of IL-12 nucleic acid sequences as naked DNA via injections or other delivery methods (e.g., liposome-based, electroporation, gene gun) for DNA vaccination.
Expression of IL-12 in cultured mammalian cells for research or therapeutic protein production applications.
Potential use in antimicrobial therapy, anticancer therapy, and stimulation of mucosal immunity as adjuvants.
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