Sortase-Mediated Red Blood Cell Surface Modification
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Solution Overview
Problem
Current methods for engineering red blood cells face limitations in producing enucleated red blood cells that can carry therapeutic or diagnostic agents effectively, as existing systems either disrupt plasma membrane integrity or have short in vivo survival rates, and lack suitable in vitro systems for synchronized erythroid expansion and differentiation.
Innovation Solution
An in vitro multi-phase culturing process for differentiating human CD34+ peripheral blood cells into enucleated red blood cells, combined with a sortagging system for surface modification, allowing for the conjugation of agents to the cell surface using sortase-mediated transpeptidation reactions, enabling efficient labeling and prolonged in vivo survival of modified red blood cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If genetic engineering or chemical modifications are used to modify cell surfaces, then surface function can be modulated or new functions can be conferred, but many surface proteins do not tolerate insertions above a certain size without suffering impairments in their function or expression, and many chemical modifications require non-physiological reaction conditions
Solution Approach 1:
The patent uses sortase A as an intermediary enzyme that mediates the conjugation of agents to cell surface proteins. The sortase recognizes a specific peptide sequence (LPXTG) and catalyzes the formation of an amide bond between the threonine residue and the amino group of the agent, enabling modification without directly interacting with the protein structure itself. This intermediary mechanism allows for versatile surface functionalization while preserving protein integrity and function.
2Adaptability or versatility
If existing systems are used to engineer red blood cells, then cell surface modification can be achieved, but plasma membrane integrity is disrupted or in vivo survival rates are short
Solution Approach 1:
The sortase-mediated conjugation system serves as an intermediary that enables surface modification without disrupting plasma membrane integrity. The enzymatic reaction occurs on the cell surface under physiological conditions, attaching therapeutic or diagnostic agents to surface proteins while maintaining membrane structure and cell viability, thereby extending in vivo survival rates.
Solution Approach 2:
The patent changes the parameters of the modification process by using enzymatic catalysis under physiological conditions rather than harsh chemical conditions. This parameter change (from chemical to enzymatic, from non-physiological to physiological conditions) preserves cell integrity and extends survival while achieving the desired surface modification.
3Productivity
If conventional culturing methods are used for red blood cell production, then cell expansion can be achieved, but synchronized erythroid expansion and terminal differentiation cannot be achieved
Solution Approach 1:
The culturing process is segmented into distinct phases: expansion phase and differentiation phase. During the expansion phase, cells are cultured under conditions that promote proliferation. During the differentiation phase, conditions are changed to induce terminal differentiation and enucleation. This temporal segmentation allows for synchronized production of enucleated red blood cells while maintaining high productivity.
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 process synchronizes erythroid expansion and terminal differentiation, producing enucleated red blood cells that can be effectively labeled and modified for therapeutic or diagnostic purposes, with extended in vivo survival and retention of cell viability, overcoming previous limitations in red blood cell engineering.
Implementation Method 1
a sortagging system for surface modification of red blood cells, in which an agent of interest is conjugated to the surface of the enucleated red blood cells... using sortase-mediated transpeptidation reactions
Data Source
AI summary
Methods for the in vitro production of enucleated red blood cells and the enucleated red blood cells thus prepared are provided. Such enucleated red blood cells may express a sortaggable surface protein, which allows for surface modification in the presence of a sortase. Also described herein are surface modified enucleated red blood cells, e.g., conjugated with an agent of interest such as a peptide, a detectable label, or a chemotherapeutic agent, and uses thereof in delivering the agent to a subject.