Inverted BGH 3' UTR for Prolonged hCFTR Expression
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Solution Overview
Problem
Current gene therapy approaches face challenges in achieving and maintaining high levels of transgene expression in the lungs, particularly for cystic fibrosis, due to factors like thick mucus secretions and immunogenic responses, which reduce gene transfer efficiency and lead to transient expression.
Innovation Solution
The development of nucleic acid constructs that incorporate a 3' portion of the bovine growth hormone (BGH) gene in inverted orientation, combined with codon optimization and CpG depletion, to enhance the persistence and duration of hCFTR mRNA expression when delivered as compacted DNA nanoparticles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional gene transfer methods are used in CF lungs, then gene transfer can be achieved, but thick mucus secretions reduce transfer efficiency and expression is transient
Solution Approach 1:
The patent optimizes multiple parameters of the expression plasmid including codon utilization, CpG content, and 3' untranslated region sequences to enhance both transfer efficiency and expression persistence. The inverted BGH 3' UTR is a specific parameter change that prevents silencing and maintains high-level expression for months
Solution Approach 2:
The patent uses an intermediary element - the inverted 3' UTR of the BGH gene - that mediates between the transgene and host cell machinery to achieve prolonged expression. This intermediary prevents silencing mechanisms while maintaining high-level transcription
2Quantity of substance
If standard expression plasmids are used, then initial transgene expression can be achieved, but promoter down-regulation and CpG methylation cause expression to shut off
Solution Approach 1:
The patent ensures continuity of useful action by designing an expression plasmid that maintains high-level transcription over time. The inverted BGH 3' UTR prevents silencing, allowing continuous mRNA production for months without promoter down-regulation or CpG methylation effects
Solution Approach 2:
The patent applies inversion by placing the BGH 3' UTR in reverse orientation. This inverted configuration prevents formation of silencing structures and heterochromatin that would otherwise shut off transcription, thereby maintaining continuous expression
3Ease of operation
If CMV promoter is used for initial expression, then transient expression is achieved for toxicity evaluation, but expression rapidly shuts off in the lung
Solution Approach 1:
The patent segments the expression plasmid into functional modules: a promoter for initial transcription and an inverted BGH 3' UTR for sustained expression. This segmentation allows the CMV promoter to provide initial transient expression for toxicity evaluation while the inverted 3' UTR maintains long-term expression
Data Source
AI summary
Efficient and prolonged hCFTR expression is one of the major obstacles for cystic fibrosis lung therapy. hCFTR mRNA expression levels depend on eukaryotic expression cassette components, prokaryotic backbone elements, and the gene transfer method may also influence transcriptional silencing mechanisms. A codon-optimized and CpG-reduced human CFTR gene (CO-CFTR) was made. Various vector modifications were tested to facilitate extended duration of CO-CFTR expression. Insertion of an extended 3′BGH transcribed sequence (712 bp) in an inverted orientation produced prolonged expression of CO-CFTR expression at biologically relevant levels. Further studies revealed that prolonged CO-CFTR expression is dependant on the orientation of the extended BGH 3′ BGH transcribed sequence and its transcription, is not specific to the UbC promoter, and is less dependent on other vector backbone elements.