CRISPR Base Editing of Hepatocytes for MHC Disruption
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Solution Overview
Problem
The limitations of hepatocyte transplantation for liver diseases include the scarcity of high-quality hepatocytes and insufficient engraftment and long-term acceptance of allografts, primarily due to the immunogenic nature of human hepatocytes and the challenges in procurement and expansion.
Innovation Solution
Genetically modifying human hepatocytes by disrupting major histocompatibility complex (MHC) Class I or Class II genes using a base editor and guide RNAs to reduce alloreactivity, thereby enhancing engraftment and long-term acceptance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If human hepatocytes are transplanted to treat liver disease, then therapeutic effect is achieved, but alloreactivity causes insufficient engraftment and long-term acceptance
Solution Approach 1:
The patent extracts and removes the harmful immunogenic components (MHC Class I and Class II genes) from the hepatocytes through CRISPR-Cas9 gene editing. By specifically targeting and disrupting these genes, the patent eliminates the primary cause of alloreactivity while preserving the therapeutic function of the hepatocytes, thereby resolving the contradiction between therapeutic effect and engraftment success
Solution Approach 2:
The patent converts the harmful immunogenicity of hepatocytes into a benefit by using the same MHC genes that cause rejection as targets for CRISPR-Cas9 editing. The guide RNAs are designed to specifically bind to MHC Class I and Class II gene sequences, directing the Cas9 enzyme to precisely disrupt these harmful genes, thereby transforming the source of rejection into the mechanism for improving engraftment
2Quantity of substance
If hepatocytes are procured from donor livers, then hepatocyte supply is obtained, but limited donor liver availability restricts expansion
Solution Approach 1:
The patent applies preliminary genetic modification to hepatocytes before transplantation, using CRISPR-Cas9 to pre-disrupt MHC genes in vitro. This preliminary action reduces alloreactivity in advance, allowing hepatocytes to be expanded and prepared beforehand without risking rejection, thereby increasing both supply quantity and procurement productivity
Solution Approach 2:
The patent uses CRISPR-Cas9 system as an intermediary tool to modify hepatocytes. The guide RNAs and Cas9 enzyme serve as mediators that enable precise genetic disruption of MHC genes, facilitating the transformation of hepatocytes from immunogenic to immunoevasive without requiring donor liver transplantation, thus expanding supply capacity
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 genetic modification of hepatocytes reduces alloreactivity, addressing the scarcity and engraftment issues, allowing for improved transplantation outcomes and expanded hepatocyte supply through humanized animal models for therapeutic use.
Implementation Method 1
disrupting one or more major histocompatibility complex (MHC) Class I or Class II genes in isolated human hepatocytes or in a hepatocyte progenitor cell by introducing a base editor and one or more gRNAs that hybridize with a target sequence in the one or more Class I or Class II genes
Data Source
AI summary
The present invention provides methods of producing genetically modified human hepatocytes suitable for hepatocyte transplantation comprising: disrupting one or more major histocompatibility complex (MHC) Class I or Class II genes in isolated human hepatocytes or in a hepatocyte progenitor cell by introducing a base editor and one or more gRNAs that hybridize with a target sequence in the one or more Class I or Class II genes, thereby producing genetically modified human hepatocytes.