Vector-free intracellular delivery by reversible permeabilization
Inventors
Maguire, Michael • O'Dea, Shirley
Assignees
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Publication Number
US-11981915-B2
Publication Date
2024-05-14
Expiration Date
Abstract
The invention provides a solution to the problem of transfecting non-adherent cells. Devices and delivery compositions containing ethanol and an isotonic salt solution are used for delivery of compounds and compositions to non-adherent cells.
Core Innovation
The invention provides a system and delivery approach for intracellular delivery into a population of cells in a well using a vector-free, reversible permeabilization. A differential pressure applicator applies a differential pressure to the well, and a delivery solution applicator contacts the cells with an atomized delivery solution formed by gas-propelling an aqueous isotonic solution containing a payload and an alcohol at greater than 5 percent (v/v) concentration.
The payload comprises a gene editing composition, and the system further includes a stop solution applicator configured to deliver a stop solution to the well and a culture medium applicator configured to deliver a culture medium to the well. The delivery is described as non-viral, vector-free, and gentle reversible permeabilization, with rapid membrane resealing and endocytosis-independent uptake.
The documented approach is presented as Soluporation for intracellular delivery into non-adherent mammalian cells, including human T cells and PBMC-derived T cells. The platform is described as providing mechanistic evidence of rapid cytoplasmic loading and membrane resealing.
Claims Coverage
The independent claim coverage centers on one system for atomized, vector-free intracellular delivery of a gene editing composition using an alcohol-containing isotonic aqueous spray, differential pressure applied to a well, and stop-solution plus culture-medium delivery.
Differential pressure applicator to the well
A differential pressure applicator configured to apply a differential pressure to the well, wherein the well comprises a population of cells.
Vector-free alcohol-containing isotonic gene editing spray delivery
A delivery solution applicator configured to deliver atomized delivery solution to the well, wherein the delivery solution is vector-free, includes an aqueous solution including a payload and an alcohol at greater than 5 percent (v/v) concentration, the aqueous solution is isotonic, and the payload comprises a gene editing composition.
Stop-solution and culture-medium delivery sequence
A stop solution applicator configured to deliver a stop solution to the well, and a culture medium applicator configured to deliver a culture medium to the well.
Overall, the claims require a differential-pressure-enabled, atomized delivery system that uses a vector-free, isotonic aqueous alcohol formulation containing a gene editing composition payload, plus stop-solution and culture-medium delivery to recover the cell population after delivery.
Stated Advantages
Improved cell viability/function compared with electroporation/nucleofection.
Reduced gene-expression perturbation compared with electroporation/nucleofection.
Rapid membrane resealing is described.
Documented Applications
Intracellular delivery of gene editing compositions into non-adherent mammalian cells, including human T cells and PBMC-derived T cells.
Gene editing outcomes in T cells using CRISPR/Cas9 RNP targeting PDCD1 (PD-1 knockdown).
CAR expression from delivered CAR mRNA (CD19 CAR).
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