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Vector-free delivery of gene editing proteins and compositions to cells and … — Avectas Limited (US20240279683A1)

Avectas Limited · Google Patents

Google Patents · Patents · License: Open Access

patent, google patents, intellectual property, US20240279683A1, Avectas Limited, Michael Maguire, en, 2024

ABSTRACT

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.

Description

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser. No. 16/067,431, filed Jun. 29, 2018, which is a National Stage Application, filed under 35 U.S.C. Â§ 371, of International Application No. PCT/IB2016/001895 filed Dec. 22, 2016, which claims benefit of priority to U.S. Provisional Application No. 62/273,284, filed Dec. 30, 2015, the entire contents of each of which are incorporated herein by reference.

TECHNICAL FIELD

The subject matter described herein relates to vector-free delivery of gene editing compounds and complexes to cells and tissues.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named â048831_506D01US_ST25.xmlâ, which was created on Apr. 11, 2024 and is 32.4 KB in size, is hereby incorporated by reference in its entirety.

BACKGROUND

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas genome engineering system (hereinafter the âCRISPR-Cas systemâ) enables researchers to modify genomic DNA inside cells. Three components are required for this system: Cas9, which may be derived from Streptococcus pyogenes, is an endonuclease that is complexed with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to form an RNA-guided endonuclease (RGEN). These RGENs cleave chromosomal DNA in cells, producing site-specific double-strand breaks (DSBs). Repair of these DSBs can occur via endogenous high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ) resulting in targeted mutagenesis and chromosomal rearrangements. The specificity of the RGEN is determined by Watson-Crick base pairing between the crRNA and the target DNA and by Cas9 recognition of the NGG-trinucleotide protospacer adjacent motif (PAM). crRNA and tracrRNA can be fused to form a single-guide RNA (gRNA or sgRNA). A major advantage of CRISPR-Cas9 over other genome editing nucleases is the ability to readily reprogram the specificity of Cas9 by replacing the crRNA, in contrast to zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) whose DNA-targeting specificities must be altered by protein engineering strategies.

The CRISPR-Cas system has been most commonly delivered into cells in the form of plasmid DNA, which encodes for the three components, either as separate or combined plasmids. But plasmids are associated with off-target edits. Plasmids can also integrate randomly in the genome or at Cas9-generated sites. While the latter event can at least be predicted and monitored, the former can be difficult to detect and so can be more problematic. Regardless of the site of integration, these foreign sequences can cause host immune responses, which creates challenges for the use of gene-edited stem cells or primary cells in clinical applications. Furthermore, cells transfected with plasmids for clinical applications are regarded as genetically modified by regulatory authorities and as such are subject to lengthy and costly regulatory procedures.

SUMMARY OF THE INVENTION

The devices, methods, and systems described below solve many of the problems and drawbacks associates with previous gene-editing approaches by providing a reliable and efficient system for delivering the building blocks, e.g., proteins or gene editing complexes, of a gene editing system thereby avoiding delivery of plasmid DNA or other vectors encoding such proteins. The present system also provides advantages over other approaches such as electroporation, magnetofection, liposome-mediated transfection, lentiviral-mediated transfection, or intracellular injection methods

Although several gene editing systems use a DNA plasmid form of the Cas9 and the guide RNAs, the invention described herein delivers the RNP [e.g., (gene editing protein(s) and guide RNA(s)] to achieve a more transient gene editing effect, thus leading to fewer off-target effects. Although electroporation can be used to deliver proteins, e.g., gene editing proteins, to cells, the methods, systems, and compositions described herein offer several advantages compared to electroporation. For example, the ethanol-mediated spray delivery system of the invention delivers gene editing proteins and complexes with greater efficiency, e.g., 10%, 20%, 50%, 75%, 2-fold or more, compared to electroporation and with comparable or better viability of the cells after delivery and comparable or better gene editing efficiency compared to electroporation. Moreover, cell function, e.g., differentiation of stem cells or activation of immune cells, is better preserved (e.g., 10%, 20%, 50%, 75%, 2-fold or more) using the ethanol spray methods described compared to electroporation. In addition unlike the system described herein, other methods such as liposome-mediated transfection or electroporation, the latter approaches require that adherent cells be detached from their substratum in order to transfect them using, e.g., a electroporator, and such an extra manipulation step compromises both the sterility and the biology/function of the cells. Moreover, multiple dosing with delivery cargo (gene delivery proteins and/or gene delivery complexes) is not be possible using other methods, e.g., electroporation, due to high levels of cell damage associated with such procedures. The compositions and methods described here

permit

2, 3, 4, 5, or more doses (delivery spray, followed by stop solution), a process that increases efficiency delivery of the cargo to cells and amount of those gene editing composition(s) into the target cells. Target cells include primary cells (e.g., those directly cultured from their source organ tissue or obtained from blood) as well as immortalized cells, e.g., cell lines, and adherent cells as well as suspension, e.g., free-floating, cells. Primary human cells are directly cultured from their source organ tissue or blood cells

Electroporation is a widely used vector-/carrier-free method but while it can be efficient for delivery of nucleic acids and/or proteins to some cell types, toxicity can be high, particularly in primary cells. Alternative membrane disrupting methods are therefore required. As discussed above, the devices, methods, and systems described herein are advantageous over such other techniques, and in particular advantageous over electroporation.

Aspects of the present subject matter provide methods for delivering a gene editing composition across a plasma membrane of a cell. In preferred embodiments, the method does not comprise a vector, e.g., a lentiviral vector, a liposome, or electroporation. In some embodiments, the method comprises contacting the cells or delivering to the cells plasmid DNA encoding a gene editing protein; in other embodiments, the method does not comprise contacting the cells or delivering to the cells plasmid DNA encoding a gene editing protein. In some embodiments, the method comprises contacting the cells or delivering to the cells RNA encoding a gene editing protein; in other embodiments, the method does not comprise contacting the cells or delivering to the cells RNA encoding a gene editing protein. The methods described here include providing a population of cells and contacting the population of cells with a volume of aqueous solution, e.g., containing gene editing proteins and or complexes. The aqueous solution may comprise the gene editing composition and an alcohol at a concentration of at least about 2%. In various embodiments, the volume is a function of: (i) exposed surface area of the population of cells; or (ii) a number of cells in the population of cells.

Preferably, the solution is delivered to the cells in the form of a spray or mist, e.g., a plurality of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more aqueous particles per cell. For example, the cells are coated with the spray but not soaked or submersed in the delivery compound-containing solution. A spray or mist comprises a liquid that is blown or driven through the air in the form of small drops.

An example of conditions to achieve a coating of a population of coated cells include delivery of a fine particle spray, e.g., the conditions exclude dropping or pipetting a bolus volume of solution on the cells such that a substantial population of the cells are soaked or submerged by the volume of fluid. Thus, the mist or spray comprises a ratio of volume of fluid to cell volume. Alternatively, the conditions comprise a ratio of volume of mist or spray to exposed cell area, e.g., area of cell membrane that is exposed when the cells exist as a confluent or substantially confluent layer on a substantially flat surface such as the bottom of a tissue culture vessel, e.g., a well of a tissue culture plate, e.g., a microtiter tissue culture plate.

Accordingly, there is a need to provide a vector-free e.g., viral vector-free, approach for delivering biologically relevant payloads, e.g., compounds or compositions, across a plasma membrane and into cells. âCargoâ or âpayloadâ are terms used to describe a compound, or composition that is delivered via an aqueous solution across a cell plasma membrane and into the interior of a cell.

In some examples, the aqueous solution comprises alcohol at a concentration between about 2% and about 50%; at least about 2% and less than about 20, 19, 18, 17, 16, or 15%; or at least about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7. 7.5, 8. 8.5, 9. 9.5, 10, 11, 12, 13, 14, or 15%. In some aspects, the alcohol is at a concentration less than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2%. In some aspects, the alcohol, e.g. ethanol, concentration does not exceed 50%. In some aspects, the alcohol, e.g, ethanol, concentration does not exceed 70%. In some examples, the alcohol concentration is about 25%. The alcohol concentration may vary depending on the cell type, for example the alcohol concentration may be 25%, 27% or 33%. For example, certain target cells, e.g., T cells such as primary T cells, are characterized by a higher tolerance for ethanol and maintain viability and functionality after treatment using delivery buffers with greater than 25% ethanol, e.g., 27%, 30%, 33%, 35% or more. NK cells, e.g., primary NK cells, may also have such a tolerance level for the presence/concentration of ethanol in the cargo delivery buffer.

One or more of the following features can be included in any feasible combination. The volume of solution to be delivered to the cells is a plurality of units, e.g., a spray, e.g., a plurality of droplets of aqueous particles. The volume is described relative to an individual cell or relative to the exposed surface area of a confluent or substantially confluent (e.g., at least 75%, at least 80% confluent, e.g., 85%, 90%, 95%, 97%, 98%, 100%) cell population. For example, the volume can be between 6.0Ã10 â7 microliter per cell and 7.4Ã10 â4 microliter per cell. The volume is between 4.9Ã10 â6 microliter per cell and 2.2Ã10 â3 microliter per cell. The volume can be between 9.3Ã10 â6 microliter per cell and 2.8Ã10 â5 microliter per cell. The volume can be about 1.9Ã10 â5 microliters per cell, and about is within 10 percent. The volume is between 6.0Ã10 â7 microliter per cell and 2.2Ã10 â3 microliter per cell. The volume can be between 2.6Ã10 9 microliter per square micrometer of exposed surface area and 1.1Ã10 â6 microliter per square micrometer of exposed surface area. The volume can be between 5.3Ã10 â8 microliter per square micrometer of exposed surface area and 1.6Ã10 â7 microliter per square micrometer of exposed surface area. The volume can be about 1.1Ã10 â7 microliter per square micrometer of exposed surface area. The term âaboutâ can be, e.g., within 10 percent, of a stated unit or amount.

Confluency of cells refers to cells in contact with one another on a surface. For example, it can be expressed as an estimated (or counted) percentage, e.g., 10% confluency means that 10% of the surface, e.g., of a tissue culture vessel, is covered with cells, 100% means that it is entirely covered. For example, adherent cells grow two dimensionally on the surface of a tissue culture well, plate or flask. Non-adherent cells can be spun down, pulled down by a vacuum, or tissue culture medium aspiration off the top of the cell population, or removed by aspiration or vacuum removal from the bottom of the vessel.

24 well=about 2.0 cm 2 ; deliver 20 Î¼l 48 well=about 0.8 cm 2 ; deliver 5 Î¼l 96 well=about 0.3 cm 2 ; deliver 2.5 Î¼l

In this example, the volume of solution that lands in each well size was measured. A ratio of âvolume to surface areaâ or âvolume to cell numberâ is generally used.

In another aspect, the culture plate can include sample wells selected from 1, 6, 9, 12, 24, 48, 96, 384, and 1536 wells, in particular examples the culture plate has 96 sample wells. The diameter of the sample well may range from 0.1 mm to 100 mm; for example, a 24-well culture plate may have a diameter of about 15.6 mm, a 48-well culture plate may have a diameter of about 11 mm and a 96-well culture plate may have a diameter of about 6.4 mm. Approximate well dimensions and spray delivery volumes are shown below.

Single Well

Approx.

Single Well

Delivery spray

Diameter

Approx. Growth

Volume per

Microtiter Plate

(Bottom-mm)

<td class="de

RELATED APPLICATIONS

TECHNICAL FIELD

The subject matter described herein relates to vector-free delivery of gene editing compounds and complexes to cells and tissues.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named â048831_506D01US_ST25.xmlâ, which was created on Apr. 11, 2024 and is 32.4 KB in size, is hereby incorporated by reference in its entirety.

BACKGROUND

SUMMARY OF THE INVENTION

permit

24 well=about 2.0 cm 2 ; deliver 20 Î¼l 48 well=about 0.8 cm 2 ; deliver 5 Î¼l 96 well=about 0.3 cm 2 ; deliver 2.5 Î¼l

In this example, the volume of solution that lands in each well size was measured. A ratio of âvolume to surface areaâ or âvolume to cell numberâ is generally used.

Single Well

Approx.

Single Well

Delivery spray

Diameter

Approx. Growth

Volume per

Microtiter Plate

(Bottom-mm)

Area (cm 2 )

single well

1536 Well Plateâ

1.63 Ã 1.63*

0.025

0.1-10

Î¼l

384 Well Plateâ

2.7 Ã 2.7*

0.056

0.1-10

Î¼l

96 Well Plate

6.4

0.32

Î¼l**

48 Well Plate

0.64

Î¼l

*square wells;

**2.5 Î¼l as determined by the volume of solution that lands in each well size (see above)

Typically, the aqueous solution includes a payload to be delivered across a cell membrane and into cell, and the second volume is a buffer or culture medium that does not contain the payload. Alternatively, the second volume (buffer or media) can also contain payload. In some embodiments, the aqueous solution includes a payload and an alcohol, and the second volume does not contain alcohol (and optionally does not contain payload). The population of cells can be in contact with said aqueous solution for 0.01-10 minutes, e.g., 0.1-10 minutes, prior to adding a second volume of buffer or culture medium to submerse or suspend said population of cells. The buffer or culture medium can be phosphate buffered saline (PBS). The population of cells can be in contact with the aqueous solution for 2 seconds to 5 minutes prior to adding a second volume of buffer or culture medium to submerse or suspend the population of cells. The population of cells can be in contact with the aqueous solution, e.g., containing the payload, for 30 seconds to 2 minutes prior to adding a second volume of buffer or culture medium, e.g., without the payload, to submerse or suspend the population of cells. The population of cells can be in contact with a sprayed solution for about 1-2 minutes prior to adding the second volume of buffer or culture medium to submerse or suspend the population of cells. In some examples, preparation of the aqueous solution is less than about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute before delivery.

In some embodiments, addition of the alcohol to the aqueous solution may be after 5, 10, 15, 20, 25, 30, 45 minutes of incubation (for example, at 20-30 minutes).

During the time between spraying of cells and addition of buffer or culture medium, the cells remain hydrated by the layer of moisture from the spray volume.

The gene editing composition may include a compound that edits genomic DNA. For example, the gene editing composition may include a compound or complex that cleaves, nicks, splices, rearranges, translocates, recombines, or otherwise alters genomic DNA. Alternatively or in addition, a gene editing composition may include a compound that (i) may be included a gene-editing complex that cleaves, nicks, splices, rearranges, translocates, recombines, or otherwise alters genomic DNA; or (ii) may be processed or altered to be a compound that is included in a gene-editing complex that cleaves, nicks, splices, rearranges, translocates, recombines, or otherwise alters genomic DNA. In various embodiments, the gene editing composition comprises one or more of (a) gene editing protein; (b) RNA molecule; and/or (c) ribonucleoprotein (RNP).

In some embodiments, the gene editing composition comprises a gene editing protein, and the gene editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, a Cre recombinase, a Hin recombinase, or a Flp recombinase. In additional embodiments, the gene editing protein may be a fusion proteins that combine homing endonucleases with the modular DNA binding domains of TALENs (megaTAL). For example, megaTAL may be delivered as a protein or alternatively, a mRNA encoding a megaTAL protein is delivered to the cells.

In various embodiments, the gene editing composition comprises a RNA molecule, and the RNA molecule comprises a sgRNA, a crRNA, and/or a tracrRNA.

In certain embodiments, the gene editing composition comprises a RNP, and the RNP comprises a Cas protein and a sgRNA or a crRNA and a tracrRNA.

Aspects of the present subject matter are particularly useful for controlling when and for how long a particular gene-editing compound is present in a cell.

In various implementations of the present subject matter, the gene editing composition is detectable in a population of cells, or the progeny thereof, for (a) about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12 or 0.5-72 hours after the population of cells is contacted with the aqueous solution, or (b) less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, 0.5-2, 0.5-6, 6-12 or 0.5-72 hours after the population of cells is contacted with the aqueous solution.

In some embodiments, the genome of cells in the population of cells, or the progeny thereof, comprises at least one site-specific recombination site for the Cre recombinase, Hin recombinase, or Flp recombinase.

Aspects of the present invention relate to cells that comprise one gene editing compound, and inserting another gene editing compound into the cells. For example, one component of an RNP could be introduced into cells that express or otherwise already contain another component of the RNP. For example, cells in a population of cells, or the progeny thereof, may comprise a sgRNA, a crRNA, and/or a tracrRNA. In some embodiments the population of cells, or the progeny thereof, expresses the sgRNA, crRNA, and/or tracrRNA. Alternatively or in addition, cells in a population of cells, or the progeny thereof, express a Cas protein.

Various implementations of the subject matter herein include a Cas protein. In some embodiments, the Cas protein is a Cas9 protein or a mutant thereof. Exemplary Cas proteins (including Cas9 and non-limiting examples of Cas9 mutants) are described herein.

In various aspects, the concentration of Cas9 protein may range from about 0.1 to about 25 Î¼g. For example, the concentration of Cas9 may be about 1 Î¼g, about 5 Î¼g, about 10 Î¼g, about 15 Î¼g, or about 20 Î¼g. Alternatively, the concentration of Cas9 may range from about 10 ng/Î¼L to about 300 ng/Î¼L; for example from about 10 ng/Î¼L to about 200 ng/Î¼l; or from about 10 ng/Î¼L to about 100 ng/Î¼l, or from about 10 ng/Î¼L to about 50 ng/Î¼l.

In certain embodiments, the gene editing composition comprises (a) a first sgRNA molecule and a second sgRNA molecule, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; (b) a first RNP comprising a first sgRNA and a second RNP comprising a second sgRNA, wherein the nucleic acid sequence of the first sgRNA molecule is different from the nucleic acid sequence of the second sgRNA molecule; (c) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule; (d) a first crRNA molecule and a second crRNA molecule, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule, and further comprising a tracrRNA molecule; or (e) a first RNP comprising a first crRNA and a tracrRNA and a second RNP comprising a second crRNA and a tracrRNA, wherein the nucleic acid sequence of the first crRNA molecule is different from the nucleic acid sequence of the second crRNA molecule.

In aspects, the ratio of the Cas9 protein to guide RNA may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

In embodiments, increasing the number of times that cells go through the delivery process (alternatively, increasing the number of doses), may increase the percentage edit; wherein, in some embodiments the number of doses may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

In various embodiments, the first and second sgRNA or first and second crRNA molecules together comprise nucleic acid sequences complementary to target sequences flanking a gene, an exon, an intron, an extrachromosomal sequence, or a genomic nucleic acid sequence, wherein the gene, an exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence is about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100, kilobases in length or is at least about 1, 2, 3, 4, 5, 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-100, kilobases in length. In some embodiments, the use of pairs of RNPs comprising the first and second sgRNA or first and second crRNA molecules may be used to create a polynucleotide molecule comprising the gene, exon, intron, extrachromosomal sequence, or genomic nucleic acid sequence.

In certain embodiments, the target sequence of a sgRNA or crRNA is about 12 to about 25, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 17-23, or 18-22, nucleotides long. In some embodiments, the target sequence is 20 nucleotides long or about 20 nucleotides long.

In various embodiments, the first and second sgRNA or first and second crRNA molecules are complementary to sequences flanking an extrachromosomal sequence that is within an expression vector.

Aspects of the present subject matter relate to the delivery of multiple components of a gene-editing complex, where the multiple components are not complexed together. In some embodiments, gene editing composition comprises at least one gene editing protein and at least one nucleic acid, wherein the gene editing protein and the nucleic acid are not bound to or complexed with each other.

The present subject matter allows for high gene editing efficiency while maintaining high cell viability. In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more of the population of cells, or the progeny thereof, become genetically modified after contact with the aqueous solution. In various embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99%, 1-99%, or more of the population of cells, or the progeny thereof, are viable after contact with the aqueous solution.

In certain embodiments, the gene editing composition induces single-strand or double-strand breaks in DNA within the cells. In some embodiments the gene editing composition further comprises a repair template polynucleotide. In various embodiments, the repair template comprises (a) a first flanking region comprising nucleotides in a sequence complementary to about 40 to about 90 base pairs on one side of the single or double strand break and a second flanking region comprising nucleotides in a sequence complementary to about 40 to about 90 base pairs on the other side of the single or double strand break; or (b) a first flanking region comprising nucleotides in a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single or double strand break and a second flanking region comprising nucleotides in a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on the other side of the single or double strand break. Non-limiting descriptions relating to gene editing (including repair templates) using the CRISPR-Cas system are discussed in Ran et al. (2013) Nat Protoc. 2013 November; 8(11): 2281-2308, the entire content of which is incorporated herein by reference. Embodiments involving repair templates are not limited to those comprising the CRISPR-Cas system.

In various implementations of the present subject matter, the volume of aqueous solution is delivered to the population of cells in the form of a spray. In some embodiments, the volume is between 6.0Ã10 â7 microliter per cell and 7.4Ã10 â4 microliter per cell. In certain embodiments, the spray comprises a colloidal or sub- particle comprising a diameter of 10 nm to 100 Î¼m. In various embodiments, the volume is between 2.6Ã10 â9 microliter per square micrometer of exposed surface area and 1.1Ã10 â6 microliter per square micrometer of exposed surface area.

In some embodiments, the RNP has a size of approximately 100 â«Ã100 â«Ã50 â« or 10 nmÃ10 nmÃ5 nm. In various embodiments, the size of spray particles is adjusted to accommodate at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more RNPs per spray particle.

For example, contacting the population of cells with the volume of aqueous solution may be performed by gas propelling the aqueous solution to form a spray. In certain embodiments, the population of cells is in contact with said aqueous solution for 0.01-10 minutes (e.g., 0.1-10 minutes) prior to adding a second volume of buffer or culture medium to submerse or suspend said population of cells.

In some embodiments, the buffer or culture medium comprises phosphate buffered saline (PBS). In various embodiments, the aqueous solution comprises an ethanol concentration of about 5 to about 50%. In non-limiting examples, said aqueous solution comprises one or more of (a) about 75 to about 98% H 2 O; (b) about 2 to 50% ethanol; (c) about 6 to about 91 mM sucrose; (d) about 2 to about 35 mM potassium chloride; (e) about 2 to about 35 mM ammonium acetate; (f) about 1 to about 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES); (g) about 0.1 to about 10 mM or a magnesium salt or about 0.5 to about 10 mM magnesium chloride; and/or (h) about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 mM of a magnesium salt or magnesium chloride. As shown in FIG. 8 , a delivery solution comprising 2.5 mM magnesium chloride showed a surprising increase in delivery efficiency compared to delivery solution not comprising magnesium chloride.

In various embodiments, the population of cells includes at least one of primary or immortalized cells. For example, the population of cells may include mesenchymal stem cells, lung cells, neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, and human embryonic kidney (HEK) cells, primary or immortalized hematopoietic stem cell (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human cord blood CD34+ cells, B cells. Non limiting examples of T cells may include CD8+ or CD4+ T cells. In some aspects, the CD8+ subpopulation of the CD3+ T cells are used. CD8+ T cells may be purified from the PBMC population by positive isolation using anti-CD8 beads. In some aspects primary NK cells are isolated from PBMCs and GFP mRNA may be delivered by platform delivery technology (i.e., 3% expression and 96% viability at 24 hours). In additional aspects, NK cell lines, e.g., NK92 may be used.

Cell types also include cells that have previously been modified for example T cells, NK cells and MSC to enhance their therapeutic efficacy. For example: T cells or NK cells that express chimeric antigen receptors (CAR T cells, CAR NK cells, respectively); T cells that express modified T cell receptor (TCR); MSC that are modified virally or non-virally to overexpress therapeutic proteins that complement their innate properties (eg. delivery of Epo using lentiviral vectors or BMP-2 using AAV-6) (reviewed in Park et al, Methods, 2015 August; 84-16.); MSC that are primed with non-peptidic drugs or magnetic nanoparticles for enhanced efficacy and externally regulated targeting respectively (Park et al., 2015); MSC that are functionalised with targeting moieties to augment their homing toward therapeutic sites using enzymatic modification (eg. Fucosyltransferase), chemical conjugation (eg. modification of SLe X on MSC by using N-hydroxy-succinimide (NHS) chemistry) or non-covalent interactions (eg. engineering the cell surface with palmitated proteins which act as hydrophobic anchors for subsequent conjugation of antibodies) (Park et al., 2015). For example, T cells, e.g., primary T cells or T cell lines, that have been modified to express chimeric antigen receptors (CAR T cells) may further be treated according to the invention with gene editing proteins and or complexes containing guide nucleic acids specific for the CAR encoding sequences for the purpose of editing the gene(s) encoding the CAR, thereby reducing or stopping the expression of the CAR in the modified T cells.

Aspects of the present subject matter also provide a composition for delivering a payload across a plasma membrane of a cell, the composition comprising an aqueous solution including the payload and an alcohol at a concentration of at least 2%. In some embodiments the composition further comprises a magnesium salt. In certain embodiments, the composition comprises about 0.1 to about 10 mM of a magnesium salt or about 0.1 to about 10 mM magnesium chloride. A magnesium salt comprises magnesium ions.

Aspects of the present subject matter further provide a composition for delivering a payload across a plasma membrane of a cell, the composition comprising an aqueous solution including the payload, an alcohol at a concentration of at least 2%, less than about 46 mM of a salt lacking magnesium ions, less than about 121 mM sugar, less than 19 mM buffering agent, and about 0.1 to about 10 mM of a magnesium salt.

In certain embodiments, the buffering agent is a weak acid or a weak base that is effective to adjust or maintain the pH of the aqueous solution at a pH of about 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, or 8.5. In various embodiments, the alcohol is methanol, ethanol, isopropyl alcohol, butanol, or benzyl alcohol. In some embodiments, the salt that lacks a magnesium ion comprises a Na or a K ion. In some embodiments, the salt is NaCl, KCl, Na 2 HPO 4 , C 2 H 3 O 2 NH 4 , or KH 2 PO 4 . In certain embodiments, the sugar comprises sucrose. The buffering agent comprise, e.g., 4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid, 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid, 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, or piperazine-N,Nâ²-bis(2-ethanesulfonic acid).

Aspects of the present invention provide an apparatus for delivering a gene editing composition across a plasma membrane of a cell. In various embodiments, the apparatus comprises: a pneumatic generator producing gas under a pressure; an atomizer operatively coupled to the pneumatic generator; a reservoir configured to contain a volume of aqueous solution, the aqueous solution including the gene editing composition and an alcohol at greater than 2 percent concentration; and a valve between the pneumatic generator and atomizer. In some embodiments, the valve is switchable between a closed position for preventing the gas under the pressure from activating the atomizer and an open position for allowing the gas under the pressure to activate the atomizer to produce colloidal droplets from the aqueous solution. In various embodiments, the valve has a switching speed less than about 250 milliseconds. In certain embodiments, the atomizer is oriented to contact a population of cells with the aqueous solution and wherein the volume is a function of: (i) exposed surface area of the population of cells; or (ii) a number of cells in the population of cells.

Additional apparatus, systems, methods, compositions, and articles described or illustrated herein are also provided.

In an aspect, delivering a payload across a plasma membrane of a cell includes providing a population of cells and contacting the population of cells with a volume of an aqueous solution. The aqueous solution includes the payload and an alcohol content greater than 5 percent concentration. The volume of the aqueous solution may be a function of exposed surface area of the population of cells, or may be a function of a number of cells in the population of cells.

In another aspect, a composition for delivering a payload across a plasma membrane of a cell includes an aqueous solution including the payload, an alcohol at greater than 5 percent concentration, less than 46 mM salt, less than 121 mM sugar, and less than 19 mM buffering agent. For example, the alcohol, e.g., ethanol, concentration does not exceed 50%.

One or more of the following features can be included in any feasible combination. The volume of solution to be delivered to the cells is a plurality of units, e.g., a spray, e.g., a plurality of droplets or aqueous particles. The volume is described relative to an individual cell or relative to the exposed surface area of a confluent or substantially confluent (e.g., at least 75%, at least 80% confluent, e.g., 85%, 90%, 95%, 97%, 98%, 100%) cell population. For example, the volume can be between 6.0Ã10 â7 microliter per cell and 7.4Ã10 â4 microliter per cell. The volume is between 4.9Ã10 â6 microliter per cell and 2.2Ã10 â3 microliter per cell. The volume can be between 9.3Ã10 â6 microliter per cell and 2.8Ã10 â5 microliter per cell. The volume can be about 1.9Ã10 â5 microliters per cell, and about is within 10 percent. The volume is between 6.0Ã10 â7 microliter per cell and 2.2Ã10 â3 microliter per cell. The volume can be between 2.6Ã10 â9 microliter per square micrometer of exposed surface area and 1.1Ã10 â6 microliter per square micrometer of exposed surface area. The volume can be between 5.3Ã10 â8 microliter per square micrometer of exposed surface area and 1.6Ã10 â7 microliter per square micrometer of exposed surface area. The volume can be about 1.1Ã10 â7 microliter per square micrometer of exposed surface area. About can be within 10 percent.

Contacting the population of cells with the volume of aqueous solution can be performed by gas propelling the aqueous solution to form a spray. The gas can include nitrogen, ambient air, or an inert gas. The spray can include discrete units of volume ranging in size from, 10 nm to 100 Î¼m, e.g., 30-100 Î¼m in diameter. The spray includes discrete units of volume with a diameter of about 30-50 Î¼m. For example, a total volume of aqueous solution of 20 Î¼l can be delivered in a spray to a cell-occupied area of about 1.9 cm 2 , e.g., one well of a 24-well culture plate (see also description above regarding volume and well size/area). A total volume of aqueous solution of 10 Î¼l is delivered to a cell-occupied area of about 0.95 cm 2 , e.g., one well of a 48-well culture plate. Typically, the aqueous solution includes a payload to be delivered across a cell membrane and into cell, and the second volume is a buffer or culture medium that does not contain the payload. Alternatively, the second volume (buffer or media) can also contain payload. In some embodiments, the aqueous solution includes a payload and an alcohol, and the second volume does not contain alcohol (and optionally does not contain payload). The population of cells can be in contact with said aqueous solution for 0.1-10 minutes prior to adding a second volume of buffer or culture medium to submerse or suspend said population of cells. The buffer or culture medium can be phosphate buffered saline (PBS). The population of cells can be in contact with the aqueous solution for 2 seconds to 5 minutes prior to adding a second volume of buffer or culture medium to submerse or suspend the population of cells. The population of cells can be in contact with the aqueous solution, e.g., containing the payload, for 30 seconds to 2 minutes prior to adding a second volume of buffer or culture medium, e.g., without the payload, to submerse or suspend the population of cells. The population of cells can be in contact with a spray for about 1-2 minutes prior to adding the second volume of buffer or culture medium to submerse or suspend the population of cells. During the time between spraying of cells and addition of buffer or culture medium, the cells remain hydrated by the layer of moisture from the spray volume.

The aqueous solution can include an ethanol concentration of 5 to 30%. The aqueous solution can include one or more of 75 to 98% H 2 O, 2 to 45% ethanol, 6 to 91 mM sucrose, 2 to 35 mM KCl, 2 to 35 mM ammonium acetate, and 1 to 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES).

The population of cells can include adherent cells or non-adherent cells. The adherent cells can include at least one of primary mesenchymal stem cells, fibroblasts, monocytes, macrophages, lung cells, neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells or immortalized cells, such as cell lines. The population of cells can include non-adherent cells. The non-adherent cells can include at least one of primary hematopoietic stem cell (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human cord blood CD34+ cells, B cells, or cell lines such as the Jurkat T cell line.

The population of cells can be substantially confluent, such as greater than 75 percent confluent. Confluency of cells refers to cells in contact with one another on a surface. For example, it can be expressed as an estimated (or counted) percentage, e.g., 10% confluency means that 10% of the surface, e.g., of a tissue culture vessel, is covered with cells, 100% means that it is entirely covered. For example, adherent cells grow two dimensionally on the surface of a tissue culture well, plate or flask. Non-adherent cells can be spun down, pulled down by a vacuum, or tissue culture medium aspiration off the top of the cell population, or removed by aspiration or vacuum removal from the bottom of the vessel. The population of cells can form a monolayer of cells.

In some embodiments, a composition or solution of the present subject matter comprises an alcohol; a salt; a sugar; a buffering agent; and/or ammonium acetate. The alcohol can be selected from, e.g., methanol, ethanol, isopropyl alcohol, butanol and benzyl alcohol. The salt can be selected from, e.g., NaCl, KCl, Na 2 HPO 4 , KH 2 PO 4 , and C 2 H 3 O 2 NH. The sugar can include, e.g., sucrose. The buffering agent can include, e.g., 4-2-(hydroxyethyl)-1-piperazineethanesulfonic acid, 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid, 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, or piperazine-N,Nâ²-bis(2-ethanesulfonic acid). In some examples, the solution or buffer comprises magnesium ions, e.g., magnesium ions are optionally provided in the form of a salt such as MgCl 2 .

The present subject matter relates to a method for delivering gene-editing compounds and complexes across a plasma membrane. The method of the present subject matter comprises introducing a compound or complex to an aqueous composition to form a matrix; atomizing the matrix into a spray; and contacting the matrix with a plasma membrane.

The example methods described herein include a payload, wherein the payload includes an alcohol. By the term âan alcoholâ is meant a polyatomic organic compound including a hydroxyl (âOH) functional group attached to at least one carbon atom. The alcohol may be a monohydric alcohol and may include at least one carbon atom, for example methanol. The alcohol may include at least two carbon atoms (e.g. ethanol). In other aspects, the alcohol comprises at least three carbons (e.g. isopropyl alcohol). The alcohol may include at least four carbon atoms (e.g., butanol), or at least seven carbon atoms (e.g., benzyl alcohol). The example payload may include no more than 50% (v/v) of the alcohol, more preferably, the payload comprises 2-45% (v/v) of the alcohol, 5-40% of the alcohol, and 10-40% of the alcohol. The payload may include 20-30% (v/v) of the alcohol.

Most preferably, the payload includes 25% (v/v) of the alcohol. Alternatively, the payload can include 2-8% (v/v) of the alcohol, or 2% of the alcohol. The alcohol may include ethanol and the payload comprises 5, 10, 20, 25, 30, 40, or 50% (v/v) of the ethanol. Example methods may include methanol as the alcohol, and the payload may include 5, 10, 20, 25, 30, 40, or 50% (v/v) of the methanol. The payload may include 2-45% (v/v) of methanol, 20-30% (v/v), or 25% (v/v) methanol. Preferably, the payload includes 20-30% (v/v) of methanol. Further alternatively, the alcohol is butanol and the payload comprises 2, 4, or 8% (v/v) of the butanol.

In some aspects of the present subject matter, the payload is in a hypotonic solution or buffer. The payload solution may have an osmotic concentration of 171 mOsm/L. According to example methods, the payload solution has an osmotic concentration of 171 mOsm/L at room temperature.

According to the present subject matter, the payload may include at least one salt. The salt may be selected from NaCl, KCl, Na 2 HPO 4 , C 2 H 3 O 2 NH 4 and KH 2 PO 4 . According to example methods, the payload includes each of NaCl, KCl, Na 2 HPO 4 , and KH 2 PO 4 . The payload may include less than 46 mM salt. Further, the payload includes 2-35 mM salt, or 10-15 mM salt (e.g., 12 mM salt). According to example methods, the salt is KCl and the payload includes 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mM KCl, and more preferably 12 mM KCl.

According to example methods of the present subject matter, the payload may include a sugar (e.g., a sucrose, or a disaccharide). According to example methods, the payload comprises less than 121 mM sugar, 6-91 mM, or 26-39 mM sugar. Still further, the payload includes 32 mM sugar (e.g., sucrose). Optionally, the sugar is sucrose and the payload comprises 6.4, 12.8, 19.2, 25.6, 32, 64, 76.8, or 89.6 mM sucrose.

According to example methods of the present subject matter, the payload may include a buffering agent (e.g. a weak acid or a weak base). The buffering agent may include a zwitterion. According to example methods, the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The payload may comprise less than 19 mM buffering agent (e.g., 1-15 mM, or 4-6 mM or 5 mM buffering agent). According to example methods, the buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and the payload comprises 1, 2, 3, 4, 5, 10, 12, 14 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Further preferably, the payload comprises 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

According to example methods of the present subject matter, the payload includes ammonium acetate. The payload may include less than 46 mM ammonium acetate (e.g., between 2-35 mM, 10-15 mM, ore 12 mM ammonium acetate). The payload may include 2.4, 4.8, 7.2, 9.6, 12, 24, 28.8, or 33.6 mM ammonium acetate.

The methods described herein include a second aspect of the present subject matter, where a second payload (e.g. an aqueous solution) including 68 mM NaCl, 1.4 mM KCl, 5 mM Na 2 HPO 4 , and 0.9 mM KH 2 PO 4 is provided. The pH of the second payload may be pH 7.4.

In various embodiments, the volume of aqueous solution performed by gas propelling the aqueous solution may include, e.g., compressed air (e.g. ambient air). These and other implementations may include inert gases, for example, helium, neon, and argon.

In certain aspects of the present subject matter, the population of cells may include adherent cells (e.g., lung, kidney, immune cells such as macrophages) or non-adherent cells (e.g., suspension cells).

In certain aspects of the present subject matter, the population of

CLAIMS

Claims ( 14 )

1 - 51 . (canceled)

52 . An apparatus for delivering a gene editing composition across a plasma membrane of a cell, the apparatus comprising:

a pneumatic generator producing gas under a pressure; an atomizer operatively coupled to the pneumatic generator; a reservoir configured to contain a volume of aqueous solution, the aqueous solution including the gene editing composition and an alcohol at greater than 2 percent concentration; and a valve between the pneumatic generator and atomizer, the valve switchable between a closed position for preventing the gas under the pressure from activating the atomizer and an open position for allowing the gas under the pressure to activate the atomizer to produce colloidal droplets from the aqueous solution, the valve having a switching speed less than 250 milliseconds; wherein the atomizer is oriented to spray an area configured to contain a population of cells with the aqueous solution and wherein the volume is a function of: (i) exposed surface area of the population of cells; or (ii) a number of cells in the population of cells.

53 - 61 . (canceled)

62 . The apparatus of claim 52 , further comprising, contacting the population of cells with the volume of aqueous solution by gas propelling the aqueous solution to form a spray.

63 . The apparatus of claim 62 , wherein the gas comprises nitrogen, ambient air, or an inert gas.

64 . The apparatus of claim 62 , wherein the spray includes discrete units of volume ranging in size from, 10 nm to 100 Î¼m.

65 . The apparatus of claim 52 , wherein the colloidal droplets have a diameter of 30-100 Î¼m.

66 . The apparatus of claim 52 , wherein the alcohol comprises methanol, ethanol, isopropyl alcohol, butanol, or benzyl alcohol.

67 . The apparatus of claim 66 , comprising about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% ethanol.

68 . The apparatus of claim 52 , wherein the aqueous solution comprises less than about 46 mM of a salt, less than about 121 mM sugar, and less than 19 mM buffering agent.

69 . The apparatus of claim 68 , wherein the salt is present in the aqueous solution and selected from a group consisting of NaCl, KCl, Na 2 HPO 4 , C 2 H 3 O 2 NH 4 , and KH 2 PO 4 .

70 . The apparatus of claim 52 , wherein the gene editing composition comprises one or more of (a) gene editing protein; (b) RNA molecule; and/or (c) ribonucleoprotein (RNP).

71 . The apparatus of claim 52 , wherein the gene editing composition includes a Cas9 protein and a gRNA complex.

72 . The apparatus of claim 52 , wherein the cell comprises a stem cell.

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US18/509,634

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US11827899B2

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2023-11-28

Vector-free delivery of gene editing proteins and compositions to cells and … — Avectas Limited (US20240279683A1)

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