Lentivirus Packaging System TAR RNA Binding Protein Yield
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Solution Overview
Problem
The third-generation lentivirus packaging system compromises lentivirus production efficiency while aiming to enhance safety, leading to a need for improved yield without compromising safety.
Innovation Solution
Incorporating a plasmid with a nucleotide sequence encoding TAR RNA binding protein and a TAR-reserved chimeric 5′ long terminal repeat (LTR) into the packaging system, along with specific packaging and envelope plasmids, to increase lentivirus yield and transduction efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the third-generation lentivirus packaging system is used to improve safety by removing tat and rev genes, then safety is improved, but lentivirus production efficiency deteriorates
Solution Approach 1:
The patent extracts only the essential TAR RNA binding domain from the tat gene and incorporates it into a modified packaging system. This selective extraction allows the system to retain the critical function of TAR binding for transcription activation while eliminating the harmful replication-competent risks associated with the complete tat and rev genes, thus resolving the contradiction between safety and productivity
Solution Approach 2:
The patent applies local quality by modifying specific regions of the LTR (Long Terminal Repeat) to create TAR-reserved chimeric 5'-LTR and 3'-LTR sequences. These localized modifications preserve the necessary transcriptional activation functions while maintaining the safety improvements of the third-generation system, enabling high-titer lentivirus production without compromising safety
2Reliability
If the third-generation lentivirus packaging system removes accessory genes to enhance safety, then safety is improved, but transcriptional activation capability deteriorates
Solution Approach 1:
The patent introduces an intermediary element - a plasmid expressing TAR RNA binding protein - that mediates the transcriptional activation function. This intermediary provides the necessary TAR binding capability to drive viral genome transcription while being separate from the complete tat gene, thus maintaining safety while restoring transcriptional activation capability for high-titer production
Data Source
AI summary
Provided is a lentivirus packaging system, which comprises: a transfer plasmid comprising a nucleotide sequence of TAR-reserved-chimeric 5′ long terminal repeat (LTR); at least one packaging plasmid comprising a nucleotide sequence encoding TAR RNA binding protein, a nucleotide sequence of rev gene, a nucleotide sequence of gag gene, and a nucleotide sequence of pol gene; and an envelope plasmid. Due to the expression of gene of TAR RNA binding protein by the packaging plasmids, the produced lentivirus has higher virus titer and can improve the transduction rate and the gene delivery efficiency during cell transduction. The present invention further provides a method of improving lentivirus production in a host cell, which comprises using the lentivirus packaging system to transfect the host cell. The present invention further provides a cell transduced by the lentivirus and a method of using the cell for treating cancer.