Codon-Optimized IL-15 Sequences for Mammalian Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for expressing interleukin-15 (IL-15) in mammalian cells are inefficient due to negative signals within the RNA sequence, such as potential splice sites and low stability determinants, leading to suboptimal production of biologically active IL-15 protein.
Innovation Solution
Modifying the IL-15 coding sequence to minimize splice sites and stability issues, and using heterologous signal peptides and propeptides, such as those from tissue plasminogen activator, to enhance mRNA stability and protein expression, resulting in increased IL-15 production levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native IL-15 coding sequences are used for expression in mammalian cells, then the sequence maintains natural biological fidelity, but expression efficiency is low due to negative signals within the RNA sequence
Solution Approach 1:
The patent applies codon optimization to change the nucleotide sequence parameters of IL-15 coding regions while maintaining the amino acid sequence. This involves substituting codons with synonymous alternatives that are more efficiently translated in mammalian cells, removing cryptic splice sites, and optimizing GC content to eliminate negative RNA signals, thereby dramatically improving expression levels without altering the protein product
Solution Approach 2:
The patent introduces heterologous signal peptides and propeptides as intermediary elements to facilitate proper protein folding, secretion, and stability. These intermediary sequences act as chaperones that guide the IL-15 protein through the secretory pathway and enhance its bioactivity, allowing the optimized coding sequence to produce functional protein at high levels
2Productivity
If codon optimization is applied to IL-15 coding sequence, then mRNA stability and translation efficiency improve, but the sequence diverges from native mammalian sequences
Solution Approach 1:
The patent systematically modifies nucleotide parameters including codon usage frequency, GC content distribution, and absence of cryptic regulatory elements. These parameter changes optimize the sequence for mammalian transcription and translation machinery while using computational algorithms to maintain reading frame integrity and avoid introducing frameshifts or premature stop codons, ensuring the protein product remains identical to native IL-15
3Productivity
If heterologous signal peptides and propeptides are used, then protein expression and stability increase, but the expression system becomes more complex
Solution Approach 1:
The patent divides the IL-15 expression construct into distinct functional segments: a heterologous signal peptide segment for secretion, a propeptide segment for folding and stability, and the mature IL-15 coding segment. This segmentation allows each element to be independently optimized and facilitates modular assembly of the expression cassette, making the complex system more manageable and adaptable to different expression vectors
Data Source
AI summary
The present invention provides for nucleic acids improved for the expression of interleukin-15 (IL-15) in mammalian cells. The invention further provides for methods of expressing IL-15 in mammalian cells by transfecting the cell with a nucleic acid sequence encoding an improved IL-15 sequence. The present invention further provides expression vectors, and IL-15 and IL 15 receptor alpha combinations (nucleic acid and protein) that increase IL-15 stability and potency in vitro and in vivo. The present methods are useful for the increased bioavailability and biological effects of IL-15 after DNA, RNA or protein administration in a subject (e.g. a mammal, a human).