TRAP Binding Site for Viral Vector Production
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Solution Overview
Problem
The production of viral vectors encoding therapeutic nucleotides often results in reduced titers due to factors like genome size, instability, nucleotide bias, and protein expression in production cells, which affects packaging, infectivity, and immune response, necessitating effective repression of nucleotide expression during vector production without impairing vector functionality.
Innovation Solution
The use of modified nucleic acid sequences incorporating tryptophan RNA-binding attenuation protein (TRAP) binding sites and Kozak sequences to repress transgene expression in viral vector production cells, including improved 5'UTR leader sequences, spacer sequences, and overlapping multi-cloning sites, ensures low transgene expression during production while maintaining vector titers and functionality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the nucleotide of interest is expressed in viral vector production cells, then the vector genome can be packaged, but vector titers are substantially reduced
Solution Approach 1:
The TRAP binding site is incorporated into the nucleotide of interest sequence before transfection into production cells. This preliminary design enables TRAP protein to bind and repress translation of the NOI during vector production, preventing harmful protein expression while maintaining genome packaging. The repression mechanism is built into the vector construct itself, allowing production cells to package the genome without expressing the therapeutic protein that would reduce titers.
Solution Approach 2:
TRAP protein serves as an intermediary molecule that binds to the TRAP binding site in the NOI sequence. This intermediary mechanism allows control over NOI translation without directly modifying the vector genome structure or packaging signals. The TRAP-TRAP binding site interaction mediates repression of harmful protein expression while permitting genome packaging, thus resolving the contradiction between packaging efficiency and vector titer.
2Reliability
If the nucleotide of interest is expressed in production cells, then therapeutic effect is achieved, but immune response is triggered
Solution Approach 1:
The TRAP binding site is designed into the NOI sequence in advance, enabling conditional translation control. In production cells, TRAP binds to repress translation and prevent immune response. In target cells lacking TRAP, the NOI is translated to produce therapeutic protein. This preliminary design ensures therapeutic effect while avoiding immune response during production.
Solution Approach 2:
The translation repression mechanism is applied locally to the NOI sequence through the TRAP binding site, while leaving the rest of the vector genome and its functions unchanged. This local control allows differential expression: repression in production cells (preventing immune response) and activation in target cells (achieving therapeutic effect).
3Quantity of substance
If the nucleotide of interest is large, then therapeutic genome capacity is met, but reverse transcription and integration efficiency decrease
Solution Approach 1:
The harmful effect of large genome size on integration efficiency is extracted and addressed separately through translation repression. By using TRAP to repress NOI translation during production, the patent isolates the problem of large genome size from its negative consequences, allowing efficient packaging and delivery of large therapeutic genomes without the expected reduction in integration efficiency.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach achieves consistent and robust repression of transgene expression in production cells, enhancing vector titers and reducing immune response, while ensuring effective transduction and long-term expression in target cells.
Implementation Method 1
tryptophan RNA-binding attenuation protein (TRAP) binding sites
Data Source
AI summary
The present invention relates to a nucleic acid sequence comprising a nucleotide of interest and a tryptophan RNA-binding attenuation protein (TRAP) binding site, and optionally a Kozak sequence, wherein said TRAP binding site overlaps the Kozak sequence and/or the ATG start codon of the nucleotide of interest. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest and a Kozak sequence, wherein said Kozak sequence comprises a portion of a tryptophan RNA-binding attenuation protein (TRAP) binding site. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest and TRAP binding site wherein the TRAP binding site comprises a portion of the start codon ATG of said nucleotide of interest or wherein the ATG start codon comprises a portion of the TRAP binding site. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest, a binding site for tryptophan RNA-binding attenuation protein (TRAP), a multiple cloning site and a Kozak sequence, wherein said multiple cloning site is overlapping with or located downstream to the 3′ KAGN2-3 repeat of the TRAP binding site and upstream of the Kozak sequence.