IL-12 Expression via Modified Nucleic Acid Sequences

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Solution Overview

Problem

Current methods for expressing interleukin-12 (IL-12) in mammalian cells are inefficient due to issues such as potential splice sites and low stability sequences in native IL-12 sequences, leading to suboptimal production of the IL-12 heterodimer.

Innovation Solution

The development of modified nucleic acid sequences encoding IL-12p35 and IL-12p40 with increased GC content and altered codons to minimize splice sites and stability issues, resulting in high-level expression of IL-12 using dual promoter plasmids and expression vectors in mammalian cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If native IL-12 nucleic acid sequences are used for expression, then the sequence maintains natural biological properties, but expression efficiency is low due to splice sites and stability issues

Engineering Contradiction:

ImproveIL-12 expression levelVSAvoidsequence stability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies parameter changes by modifying the nucleic acid sequence parameters including increasing GC content from approximately 40% to over 60%, altering codon usage patterns, and changing sequence composition while maintaining the amino acid sequence. These parameter modifications eliminate splice site sequences and instability elements, resulting in enhanced expression levels up to 5.6-fold compared to native sequences.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If codon optimization is performed to increase GC content, then expression efficiency improves, but sequence identity to native IL-12 decreases

Engineering Contradiction:

Improveexpression efficiencyVSAvoidsequence identity

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The patent applies local quality by making targeted modifications to specific regions of the nucleic acid sequence. Codon optimization is applied locally to enhance expression in mammalian cells while maintaining the overall protein sequence integrity. The modifications are concentrated in regions affecting stability and expression rather than uniformly altering the entire sequence, preserving essential biological properties.

Inventive Principle:

Principle #3Local quality

3Reliability

If splice site sequences are eliminated through modification, then expression stability improves, but potential loss of natural regulation occurs

Engineering Contradiction:

Improveexpression stabilityVSAvoidnatural regulation

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent applies the extraction principle by removing specific problematic elements from the native sequence including splice site sequences and other instability elements. By extracting these problematic sequences while retaining the functional coding regions, the invention achieves stable expression in mammalian cells without the negative effects of premature splicing or degradation, while still maintaining immunological activity.

Inventive Principle:

Principle #2Taking out (Extraction)

Data Source

PatentUS7833754B2IL-12 for expression in mammalian cell

Publication Date: 2010.11.16 THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY DEPARTMENT OF HEALTH & HUMAN SERVICES

AI summary

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.