RdCVF Expression Vector Signal Peptide Optimization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The challenge lies in achieving efficient and effective expression and secretion of rod-derived cone viability factor (RdCVF) for treating ocular diseases, particularly retinitis pigmentosa, as existing methods face difficulties in large-scale recombinant expression and delivery due to post-translational events affecting protein levels and secretion pathways.
Innovation Solution
The use of an expression vector comprising a RdCVF-encoding nucleotide sequence with an insulin growth factor-1 signal peptide and a furin cleavage consensus sequence, along with an exogenous signal peptide such as human growth hormone or brain-derived neurotrophic factor, to enhance secretion and therapeutic delivery of RdCVF, utilizing mammalian cells and viral vectors like AAV for intraocular administration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RdCVF is expressed using native signal peptide sequences, then the protein structure is preserved, but secretion efficiency is insufficient
Solution Approach 1:
The patent uses heterologous signal peptide sequences from IGF-1, hGH, BDNF, or GUSB as intermediary elements to replace the native RdCVF signal peptide. These intermediary signal peptides act as mediators that redirect the RdCVF protein through more efficient secretory pathways, thereby improving secretion efficiency while maintaining the functional integrity of the mature RdCVF protein after signal peptide cleavage.
2Quantity of substance
If large-scale recombinant expression is attempted, then production volume increases, but post-translational events reduce protein levels
Solution Approach 1:
The patent changes the critical parameter of signal peptide sequence to optimize protein secretion at large scale. By substituting the native signal peptide with heterologous signal peptides from IGF-1, hGH, BDNF, or GUSB, the system achieves improved recognition by the secretory pathway machinery, thereby maintaining high protein expression levels even during large-scale recombinant production.
3Measurement precision
If gene therapy vector delivery is used, then targeted delivery to retina is achieved, but therapeutic protein levels are insufficient
Solution Approach 1:
The patent employs heterologous signal peptides as intermediary elements within the gene therapy vector construct. These intermediary signal peptides enhance the secretion efficiency of RdCVF from transduced retinal cells, thereby increasing the quantity of therapeutic protein delivered to the target tissue while maintaining the precise targeting capability of the AAV vector system.
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 improves the secretion and therapeutic efficacy of RdCVF, enabling effective treatment of ocular diseases by ensuring higher levels of RdCVF protein expression and secretion, thereby supporting cone photoreceptor survival and visual function.
Implementation Method 1
Secreted proteins comprise a signal peptide, which is recognized during translation by the signal recognition pathway
Implementation Method 2
Subsequently, the signal peptide is cleaved by a membrane -bound signal peptidase and the mature secreted protein enters a secretory vesicle
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The invention is related to methods of producing rod-derived cone viability factor (RdCVF). This invention also relates to the treatment of an ocular disease in a mammal using RdCVF. Also provided are expression vectors for high secreted expression of RdCVF of using nucleotide sequences encoding heterologous signal proteins and optionally markers for furin cleavage.