Spy-C Mouse Model for Rapid cMyBP-C Protein Exchange
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Solution Overview
Problem
Current methods are inadequate for rapidly replacing cardiac myosin binding protein-C (cMyBP-C) in sarcomeres, hindering the study of its function, phosphorylation sites, and the effects of mutations, due to complex dynamic interactions and difficulties in manipulating large muscle filaments.
Innovation Solution
A gene-edited mouse model platform using a Spy-C mouse model with a protease recognition site and SpyTag peptide allows for the rapid exchange of modified or mutant cMyBP-C proteins, enabling their introduction into sarcomeres with precise modifications and functional studies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional AAV or adenoviral methods are used to manipulate cMyBP-C, then some level of protein delivery is achieved, but the replacement efficiency is limited and manipulation of large thick filaments remains difficult
Solution Approach 1:
The cMyBP-C protein is divided into two functional segments: the N-terminal domain (C0-C7) that can be rapidly exchanged and the C-terminal domain (C8-C10) that anchors to the thick filament. This segmentation allows the stable C-terminal portion to remain while the N-terminal portion is replaced using the SpyCatcher-SpyTag system, achieving efficient manipulation without requiring replacement of the entire protein.
Solution Approach 2:
The SpyCatcher-SpyTag peptide pair serves as an intermediary mechanism enabling specific covalent bonding between the introduced recombinant cMyBP-C and the endogenous cMyBP-C scaffold. This intermediary system facilitates precise protein replacement at the molecular level, overcoming the limitations of traditional viral delivery methods.
2Ease of operation
If in vitro assays are used to study cMyBP-C, then some experimental control is achieved, but the unique localization of cMyBP-C within the sarcomere is not preserved and partial fragments are used in excess
Solution Approach 1:
The Spy-C mouse model is pre-engineered with the SpyTag peptide inserted into the cMyBP-C gene at the C7-C8 domain junction. This preliminary genetic modification allows for rapid and specific replacement of the N-terminal domain in vivo, preserving the protein's native sarcomeric localization and physiological context while enabling experimental manipulation.
3Loss of information
If cMyBP-C is studied using current methods, then some functional analysis is possible, but the complex dynamic interactions with binding partners make the study extremely complex and time-consuming
Solution Approach 1:
The invention applies modifications at specific local regions of the cMyBP-C protein (N-terminal domains C0-C7) while leaving the C-terminal anchoring domains (C8-C10) unchanged. This localized approach allows functional studies of specific domains without disrupting the overall protein structure or its interactions with thick filament binding partners, thereby reducing experimental complexity.
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 enables the rapid introduction of genetic modifications to cMyBP-C in its native position, facilitating the study of its function and disease-related mutations, and provides a platform for modifying other sarcomeric proteins, enhancing research and drug screening capabilities.
Implementation Method 1
The protease recognition site allows the cMyBP-C protein to be cleaved to eliminate the N-Terminus
Implementation Method 2
the SpyTag peptide allows a different peptide to be attached to the remaining portion of cMyBP-C
Data Source
AI summary
Methods and compositions for rapidly replacing cMyBP-C in sarcomeres featuring the creation of Spy-C mice, which are mice genetically engineered to express cMyBP-C with a protease recognition site and SpyTag peptide introduced into the cMyBP-C gene. In permeabilized myocytes from the Spy-C mice, the cMyBP-C protein can be cleaved at the protease recognition site, and the N-Terminus of cMyBP-C can be removed while the C-terminus remains anchored to the thick filament. A new peptide featuring the SpyCatcher sequence can be covalently bonded to the remaining portion of cMyBP-C, thereby creating a modified cMyBP-C protein. The methods and compositions of the present invention allow for the reconstitution of full-length cMyBP-C at the precise position of native cMyBP-C in the sarcomere and allow for a variety of modifications to be introduced to cMyBP-C in situ.