Supercharged Protein Delivery via Cationic Lipids
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Solution Overview
Problem
Current methods for delivering functional effector proteins into mammalian cells are inefficient and often toxic, limiting their use for intracellular targeting and therapeutic applications, particularly for genome editing and gene modulation.
Innovation Solution
The use of supercharged proteins, either engineered or naturally occurring, to associate with functional effector proteins like nucleases or transcription factors, facilitating their delivery into cells through association with cationic lipids or polymers, which enhances cellular uptake and reduces cytotoxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional delivery methods (viral delivery, receptor-mediated delivery, cell-penetrating peptides) are used to deliver functional effector proteins into mammalian cells, then delivery can be achieved, but cytotoxicity and limited efficacy remain significant problems
Solution Approach 1:
The patent modifies the charge parameters of proteins by fusing them with supercharged protein domains (either positively charged or negatively charged). This parameter change enables the proteins to interact with cationic lipids or polymers, fundamentally altering their delivery characteristics and reducing cytotoxicity while maintaining delivery efficacy
Solution Approach 2:
The patent creates composite delivery systems by combining functional effector proteins with supercharged protein domains and cationic lipids/polymers. These composite structures leverage the complementary properties of each component: the functional protein provides the desired biological activity, the supercharged domain enables interaction with delivery vectors, and the cationic lipid/polymer facilitates cellular uptake
2Productivity
If higher concentrations of transduction agents are used to improve protein delivery efficiency, then more protein enters cells, but cytotoxicity increases significantly
Solution Approach 1:
The patent introduces cationic lipids and polymers as intermediary carriers that mediate the delivery of supercharged protein-fusion effector proteins. These intermediaries facilitate cellular uptake through a mechanism that is less toxic than direct use of high concentrations of conventional transduction agents like polyarginine or Tat peptides
3Ease of operation
If genetic fusion to cell-penetrating peptides (Tat, polyarginine) is used for protein delivery, then intracellular delivery can be achieved, but the process requires toxic concentrations and has low functional delivery efficiency
Solution Approach 1:
The patent inverts the conventional approach by using negatively charged supercharged protein domains instead of positively charged cell-penetrating peptides. This inversion allows the effector proteins to associate with cationic lipids/polymers for delivery, achieving similar intracellular delivery capability but with reduced toxicity requirements
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 efficient and specific delivery of proteins into cells with minimal toxicity, achieving high genome modification efficiency and specificity, as demonstrated by up to 80% modification of human cells with reduced off-target effects.
Implementation Method 1
fusing or associating functional effector proteins (e.g., nucleases, transcriptional activators/repressors, Cas9 proteins including variants and fusions thereof, etc.) with positively charged supercharged proteins allows for delivery of the proteins to the interior of cells
Implementation Method 2
fusing or associating functional effector proteins with negatively charged supercharged proteins allows for the proteins to assocoaite with cationic lipids or cationic polymers, which provides potent delivery of the proteins to the interior of a cell
Data Source
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AI summary
Compositions, methods, strategies, kits, and systems for the supercharged protein-mediated delivery of functional effector proteins into cells in vivo, ex vivo, or in vitro are provided. Compositions, methods, strategies, kits, and systems for delivery of functional effector proteins using cationic lipids and cationic polymers are also provided. Functional effector proteins include, without limitation, transcriptional modulators (e.g., repressors or activators), recombinases, nucleases (e.g., RNA-programmable nucleases, such as Cas9 proteins; TALE nuclease, and zinc finger nucleases), deaminases, and other gene modifying/editing enzymes. Functional effector proteins include TALE effector proteins, e.g., TALE transcriptional activators and repressors, as well as TALE nucleases. Compositions, methods, strategies, and systems for the delivery of functional effector proteins into cells is useful for therapeutic and research purposes, including, but not limited to, the targeted manipulation of a gene associated with disease, the modulation of the expression level of a gene associated with disease, and the programming of cell fate.