Immortalized Myoblast Cell Lines for Hypoxic Protein Delivery
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Solution Overview
Problem
Current methods for generating immortalized human myoblast cell lines for therapeutic protein secretion face challenges such as immunogenicity, limited scalability, and instability under hypoxic conditions, which hinder their effectiveness in encapsulation technologies for chronic protein delivery.
Innovation Solution
A method involving the transduction of human myoblasts with lentiviral vectors encoding for CDK4 and hTERT, followed by selection and cloning to achieve immortalized cell lines that can differentiate and maintain high protein secretion levels, even under hypoxic conditions, without the use of antibiotics, and can be genetically engineered to produce therapeutic proteins like GM-CSF.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If human primary myoblast cells are immortalized by retrovirus transduction of CD4 and hTERT with antibiotic selection, then the cells achieve immortalization and prolonged survival, but the cells become immunogenic and unsuitable for therapeutic use
Solution Approach 1:
The patent removes the antibiotic selection step from the immortalization process, extracting the harmful element (antibiotic resistance genes that cause immunogenicity) while maintaining the beneficial immortalization effect through lentiviral transduction of CDK4 and hTERT without selective pressure
Solution Approach 2:
The patent uses transient lentiviral transduction instead of stable integrating vectors with selection markers, allowing the cells to be immortalized without carrying permanent genetic markers that would cause immunogenicity
2Duration of action of stationary object
If primary human myoblasts are immortalized with hTERT, CDK4R24C mutant and cyclin dl, then the cells achieve immortalization, but they lose myogenic potential and scalability is limited
Solution Approach 1:
The patent changes the genetic parameters by using wild-type CDK4 instead of CDK4R24C mutant and adding CD4 to the transduction combination, which restores myogenic potential while maintaining immortalization
Solution Approach 2:
The patent segments the immortalization process into two separate lentiviral transductions (one for CDK4 and one for hTERT) rather than using a single complex vector system, allowing for better control and maintenance of cell properties
3Reliability
If cells are selected with antibiotics during immortalization, then the cells achieve stable proliferation, but the cells develop immunogenicity against the antibiotic resistance genes
Solution Approach 1:
The patent extracts and removes the antibiotic selection step entirely from the protocol, achieving stable proliferation through lentiviral transduction without introducing foreign genetic elements that would trigger immune responses
4Duration of action of stationary object
If encapsulated cells are used for chronic protein delivery, then the cells provide prolonged therapeutic effect, but the cells must survive under difficult metabolic conditions including hypoxia
Solution Approach 1:
The patent changes the cellular metabolic parameters by selecting for cells with enhanced hypoxia response and using lentiviral vectors with promoters active under low oxygen conditions, enabling survival and protein secretion in the hypoxic encapsulation environment
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
The resulting immortalized human myoblast cell lines exhibit prolonged survival, stability, and enhanced protein secretion capabilities, making them suitable for encapsulation and long-term therapeutic protein delivery, including in hypoxic environments, and can be used for various diseases like cancer and neurodegenerative disorders.
Implementation Method 1
transduction of human myoblasts isolated from a muscle tissue from a donor with at least one lentiviral vector encoding for CDK4 protein and at least one lentiviral vector encoding for hTERT protein
Data Source
AI summary
The present invention relates to immortalized human cells particularly useful for cell encapsulation therapy and methods of preparation and use thereof.