Vector-free delivery of gene editing proteins and compositions to cells and tissues
Inventors
Maguire, Michael • O'Dea, Shirley
Assignees
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Publication Number
US-11827899-B2
Publication Date
2023-11-28
Expiration Date
Abstract
The present subject matter provides a method for delivering a gene editing composition across a plasma membrane of a cell. Related apparatus, system, techniques, compositions, and articles are also described.
Core Innovation
The invention relates to a method for delivering a gene editing composition across a plasma membrane of a cell. The method contacts a population of mesenchymal stem cells (MSCs), U2OS, Jurkat or T cells with a volume of a hypotonic aqueous solution that comprises a gene editing composition, including a Cas9 protein and a gRNA complex, enabling formation of DNA single-strand breaks and/or DNA double-strand breaks and/or inclusion of a repair template polynucleotide.
The hypotonic aqueous solution is formulated to promote delivery and Cas9 activity while preventing precipitation/aggregation, using ethanol at about 25% (v/v) together with sucrose, potassium chloride, ammonium acetate, HEPES, and MgCl2. The contacting is performed by gas propelling the hypotonic aqueous solution to form a spray, thereby delivering the composition across the plasma membrane through spray contact with cells.
The invention further defines the contacting in relation to exposed surface area or a number of cells, where the sprayed volume is a function of exposed surface area of the population of cells or a number of cells in the population of cells. In supporting embodiments, the solution may include additives comprising a heat shock protein or a sugar alcohol while excluding glycerol, including embodiments with α-crystallin or sorbitol to protect Cas9 activity.
Claims Coverage
The document includes one independent claim (clm-00001) directed to a plasma-membrane delivery method using a hypotonic, ethanol-containing aqueous spray with Cas9 protein/gRNA. Dependent claims refine the gene-editing functional payload, delivery contact timing/volume normalization, spray particle characteristics, and formulation additives/exclusions.
Gas-propelled spray delivery across plasma membrane using a hypotonic aqueous solution
Contacting a population of cells with a volume of a hypotonic aqueous solution comprising a gene editing composition, wherein contacting is performed by gas propelling the hypotonic aqueous solution to form a spray for delivering the gene editing composition across a plasma membrane.
Cas9 protein and gRNA complex in a hypotonic ethanol-containing formulation
The gene editing composition includes a Cas9 protein and a gRNA complex, and the hypotonic aqueous solution comprises ethanol at a concentration of about 25% (v/v) and defined solutes including about 32 mM sucrose, about 12 mM potassium chloride, about 12 mM ammonium acetate, about 5 mM HEPES, and about 2.5 mM MgCl2.
Volume defined as a function of exposed surface area or number of cells
Defining the contacting volume as a function of either exposed surface area of the population of cells or a number of cells in the population of cells.
Gene editing composition that induces DNA breaks and/or includes a repair template polynucleotide
A gene editing composition that includes inducing single-strand breaks or double-strand breaks in DNA within cells and/or includes a repair template polynucleotide.
Repair template flanking regions complementary to DNA breaks
Using a repair template polynucleotide containing first and second flanking regions complementary to a single or double strand break, with specified ranges/thresholds of base-pair complementarity on each side.
Additives comprising a heat shock protein or a sugar alcohol and excluding glycerol
Using an aqueous solution that may include an additive comprising a heat shock protein or a sugar alcohol, excluding glycerol, including embodiments where the heat shock protein is α-crystallin at about 1 μM to about 500 μM or the sugar alcohol is sorbitol at about 1% to about 10% weight/volume.
Overall, the claim coverage centers on delivering a Cas9 protein/gRNA gene editing composition across a cell plasma membrane using a hypotonic aqueous, ethanol-containing formulation that is gas propelled to form a spray, with the delivery volume normalized to exposed surface area or cell number. Dependent claims further constrain the DNA-break and repair template features and refine formulation additives with glycerol exclusion.
Stated Advantages
Protecting Cas9 activity by reducing ethanol-induced precipitation/aggregation while avoiding deleterious additives like glycerol.
Preserving viability/functionality of clinically relevant cells during delivery.
Improved gene editing efficiency relative to electroporation in MSCs, with comparable viability.
Successful gene editing in adherent and suspension primary cells and cell lines.
Documented Applications
Gene editing in mesenchymal stem cells (MSCs), including bone marrow-derived MSCs, and in other cell populations including U2OS, Jurkat T cells, primary T cells, NK cells, PBMC, and HSC.
Delivery of Cas9 protein/gRNA to enable gene editing outcomes assessed using ddPCR and drop-off probe assays, including in cells such as A549, Jurkat, primary T cells, and MSCs.
Transient Cas9 protein expression after spray exposure.
Delivery and assessment using reporter proteins (ovalbumin-FITC/OVA-FITC reporter delivery) and related functionality/activation markers (CD69/CD25) in relevant cell types.
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