CRISPR-Cas9 Conjugates for Liver Targeting
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Solution Overview
Problem
Current methods for delivering gene editing agents face challenges such as insertional mutagenesis, hepatotoxicity, and transient pharmacological benefits due to the use of viral vectors, and existing agents struggle with cell membrane penetration and endosomal escape, leading to safety concerns and inefficiencies in targeted delivery.
Innovation Solution
Development of compounds comprising ribonucleoproteins, such as Cas9 or Cpf1, conjugated with endosomal escape agents and targeting moieties, which facilitate selective delivery and uptake by liver cells, enhancing pharmacokinetics and pharmacodynamics while minimizing toxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If viral vectors are used to deliver gene editing agents, then delivery efficiency is improved, but safety risks increase due to insertional mutagenesis and hepatotoxicity
Solution Approach 1:
The patent extracts the harmful viral vector component from the delivery system while retaining the beneficial gene editing function. By using non-viral delivery methods (lipid nanoparticles, polymers, or naked nucleic acids) combined with CRISPR-Cas9 components, the system achieves safe delivery without the insertional mutagenesis and hepatotoxicity associated with viral vectors, thus resolving the contradiction between delivery efficiency and safety
Solution Approach 2:
The patent employs composite delivery systems combining multiple components (e.g., lipid nanoparticles with ionizable lipids, polymer-nucleic acid conjugates, or protein-nucleic acid complexes) to achieve both efficient delivery and enhanced safety. These composite materials provide controlled release, improved cellular uptake, and reduced toxicity compared to traditional viral vectors, simultaneously addressing delivery efficiency and safety concerns
2Ease of operation
If cell permeable molecules are used for drug delivery, then cell penetration is improved, but tissue selectivity decreases leading to off-target effects
Solution Approach 1:
The patent introduces targeting ligands (antibodies, peptides, or small molecules) as intermediaries that mediate between the cell permeable delivery vehicle and the target tissue. These ligands bind to specific receptors on target cells (e.g., hepatocytes), enabling selective uptake through receptor-mediated endocytosis while maintaining the cell penetration capability of the permeable molecule, thus resolving the contradiction between penetration efficiency and tissue selectivity
Solution Approach 2:
The patent modifies the delivery system to have different properties at different locations: the core maintains cell permeability while the surface is functionalized with tissue-specific targeting moieties. This local differentiation allows the system to penetrate cells efficiently wherever it goes, but only triggers uptake in target tissues through the surface ligands, achieving both high penetration and high selectivity simultaneously
3Productivity
If endosomal uptake pathway is used for delivery, then cellular internalization is improved, but endosomal escape efficiency decreases due to lysosomal degradation
Solution Approach 1:
The patent employs endosomolytic agents (fusogenic peptides, pH-sensitive lipids, or cationic polymers) that rapidly disrupt the endosomal membrane upon pH change or membrane contact, causing the endosome to rupture and release its contents into the cytoplasm. This rushing through mechanism minimizes the time the cargo spends in the endosomal compartment, preventing lysosomal degradation while maintaining efficient internalization, thus resolving the contradiction between uptake efficiency and escape capability
Solution Approach 2:
The patent designs delivery systems with pH-sensitive or redox-sensitive properties that change their conformation or charge state in response to the endosomal environment. These parameter changes trigger membrane disruption or cargo release at the appropriate time and location, enabling efficient escape from endosomes after internalization without compromising uptake efficiency, thereby resolving the contradiction between internalization and escape
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 compounds enable efficient and safe delivery of gene editing agents to liver cells, overcoming previous delivery issues by promoting effective endosomal escape and targeted action, thereby improving therapeutic outcomes for liver diseases.
Implementation Method 1
Certain sugars, such as galactose, N-acetyl galactosamine, and other galactose derivatives including those described by M. G. Finn and V. Mascitti et al. in the Journal of the American Chemical Society, 134, 1978 (2012) have been used as targeting agents for hepatocytes due to the binding to asialoglycoprotein receptors (ASGPR) that are present on the surface of hepatocytes.
Implementation Method 2
Various reagents, such as chloroquine, polyethyleneimine [PEI], certain highly charged cationic compounds, fusogenic peptides, and inactivated adenoviruses, have been developed that are intended to quickly disrupt the endosome in order to minimize the amount of time that a delivered bioactive agent spends in the endosome-like environment.
Data Source
AI summary
The present application provides compounds, compositions, uses thereof for the treatment of diseases, conditions and/or disorders, and uses thereof as asialoglycoprotein receptor (ASGPR) targeting agents.