Reprogramming of cells using self-amplifying RNA

Abstract

The present disclosure provides a method for reprogramming a cell, the method comprising passing a cell suspension containing the cell and self-amplifying RNA encoding a reprogramming factor through a constriction, the constriction deforming the cell, thereby causing perturbation of the cell and allowing the reprogramming factor to enter the cell.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 345,339, filed May 24, 2022, which is hereby incorporated by reference in its entirety.

[0002] The present disclosure generally relates to methods of reprogramming cells to differentiate into different types of cells (e.g., neurons) by using one or more constrictions and self - amplifying RNA encoding reprogramming factors.

Background Art

[0003] Cell - replacement - based regenerative medicine is at the forefront of having a major impact on health. However, significant challenges remain, particularly in the production of functional transplantable cells that are consistent, efficient, and cost - effective. Although it is possible to produce cells for various purposes by induced pluripotent stem cell (iPSC) differentiation and somatic cell differentiation conversion (i.e., direct reprogramming), current methods are often complex and inefficient. For example, with currently available iPSC differentiation methods, it typically takes up to several weeks, and in some cases, months, to reach the desired final cell type. Furthermore, the resulting cell products are inevitably heterogeneous cell populations in which the desired cells are present over a wide frequency range. Similarly, in somatic cell differentiation conversion, reprogramming with lentiviral vectors has been useful, but this method has a risk of insertional mutagenesis. Also, methods such as electroporation and lipofection can have an adverse effect on cell health and the recovery of sensitive primary cells and can also cause other cytotoxicity problems. Therefore, there is still a need for more efficient and cost - effective approaches for delivering differentiation or reprogramming factors to generate cells that can be used for various therapeutic applications such as regenerative medicine.

Summary of the Invention

[0004] Disclosed herein is a method of inducing the expression of a reprogramming factor within a cell, the method comprising passing a cell suspension containing the cells through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in a perturbation within the membrane of the cell and thereby allows a self-amplifying RNA encoding the reprogramming factor to enter the cell through the perturbation when the self-amplifying RNA contacts the cell, such that the reprogramming factor is expressed within the cell.

[0005] In some embodiments, the reprogramming factor is expressed within the cell for at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, or at least about 25 days after the self-amplifying RNA has entered the cell through the perturbation. In some embodiments, the expression of the reprogramming factor within the cell is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to the corresponding expression in a reference cell after the self-amplifying RNA has entered the cell through the perturbation.

[0006] Also disclosed herein is a method of enhancing the expression of a reprogramming factor within a cell, the method comprising passing a cell suspension containing the cells through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in a perturbation within the membrane of the cell and thereby allows a self-amplifying RNA encoding the reprogramming factor to enter the cell through the perturbation when the self-amplifying RNA contacts the cell, such that the expression of the reprogramming factor is enhanced compared to the corresponding expression in a reference cell.

[0007] In some embodiments, the reference cells include corresponding cells that passed through the constriction under a set of parameters, where the reference cells contacted a non-self-amplifying RNA encoding a reprogramming factor. In some embodiments, the reference cells include corresponding cells that were not passed through the constriction under a set of parameters, where the reference cells contacted a self-amplifying RNA encoding a reprogramming factor.

[0008] In some embodiments, enhanced expression includes (i) an increase in the level of expression, (ii) an extension of the period of expression, or (iii) both (i) and (ii). In some embodiments, the level of expression of the reprogramming factor in the cell is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold increased compared to the level of expression of the reprogramming factor in the reference cells. In some embodiments, the period of expression of the reprogramming factor in the cell is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold increased compared to the period of expression of the reprogramming factor in the reference cells.

[0009] The present disclosure further provides a method of reprogramming a cell, comprising passing a cell suspension containing the cell through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters causes perturbations in the cell membrane, and thus self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbations when contacting the cell, thereby reprogramming the cell.

[0010] In some embodiments, the reprogramming factor is expressed in the cell for at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, or at least about 25 days after the self-amplifying RNA enters the cell through the perturbations. In some embodiments, the expression of the reprogramming factor in the cell increases by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold after the self-amplifying RNA enters the cell through the perturbations.

[0011] Some embodiments of the present disclosure are methods of enhancing the reprogramming of a cell, comprising passing a cell suspension containing the cell through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters causes perturbations in the cell membrane, and thus self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbations when contacting the cell, resulting in enhanced reprogramming of the cell as compared to a reference method.

[0012] In some embodiments, the reference method includes passing a cell suspension through a constriction under a set of parameters and contacting the cell suspension with a non-self-amplifying RNA encoding a reprogramming factor. In some embodiments, the reference method does not include passing a cell suspension through a constriction under a set of parameters, where the cell suspension contacts a self-amplifying RNA.

[0013] In some embodiments, enhancement of cell reprogramming includes (i) an increase in the number of reprogrammed cells, (ii) a reduction in the duration required for cell reprogramming, or (iii) both (i) and (ii). In some embodiments, the number of reprogrammed cells is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold compared to the reference method. In some embodiments, the duration required to reprogram a cell is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the reference method.

[0014] In some embodiments, cell reprogramming includes inducing the cell to differentiate into a neuron. In some embodiments, cell reprogramming includes inducing the expression of other reprogramming factors within the cell.

[0015] Also provided herein is a method of producing neurons, comprising passing a cell suspension through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in perturbations within the membranes of the cells present in the cell suspension, such that self-amplifying RNA encoding a reprogramming factor can enter the cells through the perturbations when contacting the cells, and as a result, the cells differentiate into neurons. Also provided herein is a method of enhancing the production of neurons, comprising passing a cell suspension through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in perturbations within the membranes of the cells present in the cell suspension, such that self-amplifying RNA encoding a reprogramming factor can enter the cells through the perturbations when contacting the cells, and as a result, the production of neurons is enhanced as compared to a reference method.

[0016] In some embodiments, the reference method comprises passing a cell suspension through a constriction under a set of parameters and contacting the cell suspension with non-self-amplifying RNA encoding a reprogramming factor. In some embodiments, the reference method does not comprise passing a cell suspension through a constriction under a set of parameters, wherein the cell suspension contacts self-amplifying RNA.

[0017] For any of the methods provided herein (e.g., the methods described above), in some embodiments, the method further comprises contacting the cell suspension with self-amplifying RNA prior to passing the cell suspension through the constriction. In some embodiments, the cells contact self-amplifying RNA prior to passing the cell suspension through the constriction. In some embodiments, the method further comprises contacting the cell suspension with self-amplifying RNA during passing the cell suspension through the constriction. In some embodiments, the cells contact self-amplifying RNA during passing the cell suspension through the constriction. In some embodiments, the method further comprises contacting the cell suspension with self-amplifying RNA after passing the cell suspension through the constriction. In some embodiments, the cells contact self-amplifying RNA after passing the cell suspension through the constriction.

[0018] For any of the methods provided herein (e.g., the methods described above), in some embodiments, the reprogramming factors include transcription factors. In some embodiments, the reprogramming factors include Neurogenin 2 (Ngn2, Neurog2), Atonal BHLH Transcription Factor 1 (Atoh1), Achaete-Scute Family BHLH Transcription Factor 1 (Ascl1), Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), LIM Homeobox Transcription Factor 1 Alpha (Lmx1a), Engrailed Homeobox 1 (EN1), POU Class 3 Homeobox 2 (POU3F2; Brn2), Myelin Transcription Factor 1-Like (Myt1l), Forkhead Box A2 (Foxa2), Paired-Like Homeodomain 3 (Pitx3), SRY-Box Transcription Factor 2 (SOX2), MicroRNA 124 (mir124), or combinations thereof.

[0019] For any of the methods provided herein (e.g., the methods described above), in some embodiments, the method further includes contacting a cell suspension with a payload, whereby when the payload contacts the cell, the payload can also enter the cell through perturbation. In some embodiments, the method includes contacting the cell suspension with the payload before passing through a constriction of the cell suspension. In some embodiments, the cell contacts the payload before passing through a constriction of the cell suspension. In some embodiments, the method further includes contacting the cell suspension with the payload during passing through a constriction of the cell suspension. In some embodiments, the cell contacts the payload during passing through a constriction of the cell suspension. In some embodiments, the method includes contacting the cell suspension with the payload after passing through a constriction of the cell suspension. In some embodiments, the cell contacts the payload after passing through a constriction of the cell suspension.

[0020] When the cell contacts both the payload and the saRNA, in some embodiments, the self-amplifying RNA and the payload contact the cell simultaneously. In some embodiments, the self-amplifying RNA and the payload contact the cell sequentially.

[0021] In some embodiments, the payload includes nucleic acids, polypeptides, lipids, carbohydrates, small molecules, metal-containing compounds, antibodies, transcription factors, nanoparticles, liposomes, fluorescently labeled molecules, or combinations thereof. In some embodiments, the nucleic acids include DNA, RNA, or both. In some embodiments, the DNA includes recombinant DNA, cDNA, genomic DNA, or combinations thereof. In some embodiments, the RNA includes siRNA, non-self-amplifying mRNA, microRNA (miRNA), lncRNA, tRNA, shRNA, self-amplifying mRNA (saRNA), or combinations thereof. In some embodiments, the small molecules include impermeable small molecules.

[0022] In some embodiments, the payload includes additional reprogramming factors. In some embodiments, the additional reprogramming factors include Neurogenin 2 (Ngn2, Neurog2), Atonal BHLH Transcription Factor 1 (Atoh1), Achaete-Scute Family BHLH Transcription Factor 1 (Ascl1), Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), LIM Homeobox Transcription Factor 1 Alpha (Lmx1a), Engrailed Homeobox 1 (EN1), POU Class 3 Homeobox 2 (POU3F2; Brn2), Myelin Transcription Factor 1-Like (Myt1l), Forkhead Box A2 (Foxa2), Paired-Like Homeodomain 3 (Pitx3), SRY-Box Transcription Factor 2 (SOX2), MicroRNA 124 (mir124), or combinations thereof.

[0023] For any of the methods provided herein (e.g., the methods described above), in some embodiments, the cells include stem cells, somatic cells, or both. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells, tissue-specific stem cells, mesenchymal stem cells, or combinations thereof. In some embodiments, the stem cells are iPSCs. In some embodiments, the somatic cells include blood cells. In some embodiments, the blood cells are PBMCs. In some embodiments, the PBMCs include immune cells. In some embodiments, the immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells, or combinations thereof.

[0024] For any of the methods provided herein (e.g., the methods described above), in some embodiments, the set of parameters is selected from cell density; pressure; length, width, and / or depth of the constriction; diameter of the constriction; diameter of the cells; temperature; inlet angle of the constriction; outlet angle of the constriction; length, width, and / or width of the approach region; surface characteristics of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity); operating flow rate; payload concentration, viscosity of the cell suspension, osmotic pressure, salt concentration, serum content, and / or pH; time in the constriction; shear rate in the constriction; type of payload, or combinations thereof. In some embodiments, the cell density is at least about 6×10 7 cells / mL, at least about 7×10 7 cells / mL, at least about 8×10 7 cells / mL, at least about 9×10 7 cells / mL, at least about 1×10 8 cells / mL, at least about 1.1×10 8 cells / mL, at least about 1.2×10 8 cells / mL, at least about 1.3×10 8 cells / mL, at least about 1.4×10 8 cells / mL, at least about 1.5×10 8 cells / mL, at least about 2.0×10 8 cells / mL, at least about 3.0×108 cells / mL, at least about 4.0×10 8 cells / mL, at least about 5.0×10 8 cells / mL, at least about 6.0×10 8 cells / mL, at least about 7.0×10 8 cells / mL, at least about 8.0×10 8 cells / mL, at least about 9.0×10 8 cells / mL, or at least about 1.0×10 9 cells / mL or more. In some embodiments, the pressure is at least about 30 psi, at least about 35 psi, at least about 40 psi, at least about 45 psi, at least about 50 psi, at least about 55 psi, at least about 60 psi, at least about 65 psi, at least about 70 psi, at least about 75 psi, at least about 80 psi, at least about 85 psi, at least about 90 psi, at least about 95 psi, at least about 100 psi, at least about 110 psi, at least about 120 psi, at least about 130 psi, at least about 140 psi, or at least about 150 psi.

[0025] For any of the methods provided herein (e.g., the methods described above), in some embodiments, the constriction is contained within the microfluidic chip. In some embodiments, the diameter of the constriction is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the diameter of one or more cells of the cell population.

[0026] In some embodiments, the length of the constriction is between about 0 μm and about 100 μm. In some embodiments, the length of the constriction is less than about 1 μm. In some embodiments, the length of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2.5 μm, less than about 5 μm, less than about 7.5 μm, less than about 10 μm, less than about 12.5 μm, less than about 15 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, or less than about 100 μm. In some embodiments, the length of the constriction is about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 2.5 μm, about 5 μm, about 7.5 μm, about 10 μm, about 12.5 μm, about 15 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. In some embodiments, the length of the constriction is about 10 μm.

[0027] In some embodiments, the width of the constriction is between about 0 μm and about 10 μm. In some embodiments, the width of the constriction is less than about 1 μm, less than about 2 μm, less than about 3 μm, less than about 4 μm, less than about 5 μm, less than about 6 μm, less than about 7 μm, less than about 8 μm, less than about 9 μm, or less than about 10 μm. In some embodiments, the width of the constriction is between about 3 μm and about 10 μm. In some embodiments, the width of the constriction is about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In some embodiments, the width of the constriction is about 6 μm.

[0028] In some embodiments, the depth of the constriction is at least about 1 μm. In some embodiments, the depth of the constriction is at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 110 μm, or at least about 120 μm. In some embodiments, the depth of the constriction is from about 5 μm to about 90 μm. In some embodiments, the depth of the constriction is about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or about 90 μm. In some embodiments, the depth of the constriction is about 70 μm.

[0029] In some embodiments, the length of the constriction is about 10 μm, the width of the constriction is about 6 μm, and the depth of the constriction is about 70 μm.

[0030] In some embodiments, the methods provided herein include contacting a cell suspension with a plurality of self-amplifying RNAs, a plurality of payloads, or both, (i) before passage of the cell suspension through a constriction, (ii) during passage of the cell suspension through the constriction, (iii) after passage of the cell suspension through the constriction, or (iv) any combination of (i)-(iii). In some embodiments, the cell is in contact with a plurality of self-amplifying RNAs, wherein at least two or more of the plurality of self-amplifying RNAs can enter the cell through perturbation. In some embodiments, the cell is in contact with a plurality of payloads, wherein at least two or more of the plurality of payloads can enter the cell through perturbation. In some embodiments, the plurality of self-amplifying RNAs includes at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 or more self-amplifying RNAs. In some embodiments, each of the plurality of self-amplifying RNAs is different. In some embodiments, at least two of the plurality of self-amplifying RNAs are the same.

[0031] In some embodiments, the plurality of payloads includes at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 or more payloads. In some embodiments, each of the plurality of payloads is different. In some embodiments, at least two of the plurality of payloads are the same.

[0032] In some embodiments, the plurality of self-amplifying RNAs contact the cell simultaneously. In some embodiments, the plurality of self-amplifying RNAs contact the cell sequentially. In some embodiments, the plurality of payloads contact the cell simultaneously. In some embodiments, the plurality of payloads contact the cell sequentially. In some embodiments, the plurality of self-amplifying RNAs and the plurality of payloads contact the cell simultaneously. In some embodiments, the plurality of self-amplifying RNAs and the plurality of payloads contact the cell sequentially.

[0033] For any of the methods provided herein (e.g., the methods described above), in some embodiments, the method includes passing a cell suspension through a plurality of constrictions. In some embodiments, the plurality of constrictions includes at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1,000 or more distinct constrictions. In some embodiments, each constriction of the plurality of constrictions is the same. In some embodiments, one or more of the plurality of constrictions are different. In some embodiments, one or more of the constrictions differ in their length, depth, width, or combinations thereof.

[0034] In some embodiments, each constriction of the plurality of constrictions is associated with the same self - amplifying RNA. In some embodiments, one or more of the plurality of constrictions are associated with different self - amplifying RNAs. In some embodiments, the plurality of constrictions includes a first constriction associated with a first self - amplifying RNA and a second constriction associated with a second amplifying RNA, where the cell suspension passes through the first constriction, whereby the first self - amplifying RNA can enter the cells, and then the cell suspension passes through the second constriction, whereby the second self - amplifying RNA can enter the cells. In some embodiments, the cell suspension passes through the second constriction at least about 1 minute, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, or at least about 1 day after the cell suspension has passed through the first constriction.

[0035] For any of the methods provided herein (e.g., the methods described above), in some embodiments, the method further comprises contacting the cell suspension with an additional compound. In some embodiments, the additional compound comprises a nucleic acid, polypeptide, lipid, carbohydrate, small molecule, metal-containing compound, antibody, transcription factor, nanoparticle, liposome, fluorescently labeled molecule, or a combination thereof. In some embodiments, the additional compound is a nucleic acid encoding an enzyme that confers resistance to an antibiotic. In some embodiments, the contacting of the additional compound with the cell suspension occurs simultaneously with the contacting of the cell suspension with the self-amplifying RNA. In some embodiments, the contacting of the additional compound with the cell suspension occurs before or after the contacting of the cell suspension with the self-amplifying RNA.

[0036] In some embodiments, the method further comprises collecting the cell suspension that has passed through the constriction and treating the cell suspension with an antibiotic. In some embodiments, the enzyme comprises puromycin-N-acetyltransferase and the antibiotic is puromycin. In some embodiments, after treatment with the antibiotic, the percentage of cells containing the self-amplifying RNA present in the cell suspension is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold.

[0037] Some embodiments of the present disclosure relate to a population of cells comprising reprogramming factors, where the population of cells is produced using any of the methods provided herein. Also provided herein is a population of reprogrammed cells produced using any of the methods of the present disclosure. The present disclosure further provides a population of neurons produced using any of the methods provided herein.

[0038] This specification provides a composition comprising any one of a population of cells, a population of reprogrammed cells, and / or a population of neurons described herein. In some embodiments, such a composition further comprises a pharmaceutically acceptable excipient.

[0039] Also provided herein is a kit comprising any one of a population of cells, a population of reprogrammed cells, and / or a population of neurons described herein and instructions for use.

[0040] The present disclosure provides a composition comprising a population of cells and a self-amplifying RNA encoding a reprogramming factor under a set of parameters for deforming one or more cells of the population of cells, wherein the one or more cells comprise a perturbation of the cell membrane sufficient for the self-amplifying RNA to enter the one or more cells. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0041] This specification provides a cell comprising a perturbation of the cell membrane by a set of parameters for deforming the cell, wherein the perturbation of the cell membrane of the cell is caused by the set of parameters for deforming the cell, and thus a self-amplifying RNA encoding a reprogramming factor can enter the cell. Also provided herein is a cell comprising a reprogramming factor, wherein the reprogramming factor enters the cell through a perturbation of the cell membrane by a set of parameters for deforming the cell, the perturbation of the cell membrane of the cell is caused by the set of parameters for deforming the cell, and thus a self-amplifying RNA encoding the reprogramming factor can enter the cell through the perturbation. This specification provides a composition comprising such cells. In some embodiments, such a composition further comprises a pharmaceutically acceptable excipient.

[0042] Some aspects of the present disclosure provide a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject any one of the compositions provided herein. In some aspects, the disease or disorder includes neuropathy. In some aspects, the neuropathy includes Parkinson's disease.

Brief Description of the Drawings

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BEST MODE FOR CARRYING OUT THE INVENTION

[0044] The present disclosure generally relates to methods of producing neurons. More specifically, the methods provided herein involve intracellular delivery of self-amplifying RNA encoding a payload (e.g., a reprogramming factor) to a cell (e.g., an iPSC), thereby inducing the cell to differentiate into a neuron. In some embodiments, intracellular delivery of the self-amplifying RNA to the cell involves passing a cell suspension containing a population of cells through a constriction under one or more parameters, such that the cells are temporarily deformed, and as a result, the cell membrane of the cells is perturbed. Such perturbation allows the self-amplifying RNA to enter the cell when the cell contacts the self-amplifying RNA.

[0045] As further described herein, the delivery method of the present disclosure (squeeze delivery) has certain distinct characteristics that are not shared by other delivery methods known in the art. For example, in addition to an improved ability to deliver various types of payloads into cells, the squeeze processing method described herein minimizes the lasting effects on cells. Compared to conventional delivery methods such as electroporation, the squeeze processing method of the present disclosure maintains both the structural and functional integrity of the squeezed cells. In contrast to the delivery methods provided herein, electroporation may induce extensive and lasting changes in gene expression, which can lead to non-specific activation of cells (e.g., human T cells) and delayed proliferation upon antigen stimulation. In this method, any changes to the cells (e.g., perturbation of the cell membrane) are transient and are rapidly repaired when the cells are removed from the constriction. Non-limiting examples of various aspects are shown in the present disclosure.

[0046] I. General Techniques Some of the techniques and procedures described or referred to herein are generally well understood and are by those skilled in the art in conventional methodologies, e.g., Molecular Cloning: A Laboratory Manual (Sambrook et al., 4 thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R.I. Freshney, 6 thed., J.Wiley and Sons, 2010); Oligonucleotide Synthesis (M.J.Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E.Cellis, ed., Academic Press, 1998); Introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A.Doyle, J.B.Griffiths, and D.G.Newell, eds., J.Wiley and Sons, 1993 - 8); Handbook of Experimental Immunology (D.M.Weir and C.C.Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E.Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J.Wiley and Sons, 2002); Immunobiology (C.A.Janeway et al., 2004); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D.Catty., ed., IRL Press, 1988 - 1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C.Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 2011), etc. are generally used by using widely used methodologies described therein.

[0047] II. Definitions For the purpose of interpreting this specification, the following definitions apply, and whenever appropriate, terms used in the singular form also include the plural form, and vice versa. If any of the definitions described below conflicts with any document incorporated herein by reference, the definitions described in this specification shall prevail. Further definitions are described throughout the detailed description.

[0048] As used herein, the singular terms "a", "an", and "the" refer to one or more of that entity unless otherwise indicated. Thus, the terms "a" (or "an" or "the"), "one or more", and "at least one" can be used interchangeably herein.

[0049] The aspects and aspects of the present disclosure described herein are understood to include "comprising", "consisting of", and "consisting essentially of" aspects and aspects. Whenever aspects or aspects are described herein using the term "comprising", it is also understood that alternative similar aspects or aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0050] Furthermore, as used herein, "and / or" can be interpreted as a specific disclosure of each of two recited features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" is intended to encompass each of the following aspects when used in phrases such as "A, B, and / or C": A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0051] For all of the compositions described herein and all of the methods of using the compositions described herein, the compositions can be either inclusive of the recited components or steps or "consisting essentially of" the recited components or steps. When a composition is described as "consisting essentially of" the recited components, the composition contains the recited components and can further contain other components that do not substantially affect the disclosed methods, but does not contain any other components that substantially affect the disclosed methods other than those explicitly recited; or, if the composition contains additional components that substantially affect the disclosed methods other than the recited components, the composition does not contain the additional components in a concentration or amount sufficient to substantially affect the disclosed methods. When a method is described as "consisting essentially of" the recited steps, the method contains the recited steps and can further contain other steps that do not substantially affect the disclosed methods, but does not contain any other steps that substantially affect the disclosed methods other than those explicitly recited. By way of non-limiting specific example, when a composition is described as "consisting essentially of" components, the composition can further contain any amount of a pharmaceutically acceptable carrier, vehicle, or diluent, and other such components that do not substantially affect the disclosed methods.

[0052] Units, prefixes, and symbols are shown in the form approved by the International System of Units (SI). Numerical ranges are to be inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure that may have by reference to the entire specification. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0053] The term "about" is used herein to mean approximately, roughly, generally, or within the region thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical value. Generally, the term "about" can modify a numerical value above and below the stated value by a variation of, for example, up to or down 10 percent (higher or lower).

[0054] As used herein, the term "constriction" refers to a narrowed passageway. In some embodiments, the constriction is a microfluidic channel such as may be contained within a microfluidic device. In some embodiments, the constriction is a pore or is contained within a pore. Where the constriction is a pore, in some embodiments, the pore is contained on a surface. Unless otherwise indicated, the term "constriction" refers to both microfluidic channels and pores, as well as other suitable constrictions available in the art. Accordingly, where applicable, disclosures regarding microfluidic channels can also apply to pores and / or other suitable constrictions available in the art. Similarly, where applicable, disclosures regarding pores can also apply equally to microfluidic channels and / or other suitable constrictions available in the art.

[0055] As used herein, the term "pore" refers to an opening including, but not limited to, a hole, fissure, cavity, aperture, break, gap, or perforation in a material. In some embodiments, (where shown) the term refers to pores within the surface of a microfluidic device as described in this disclosure. In some embodiments, (where shown) the pore can refer to pores in a cell wall and / or cell membrane.

[0056] As used herein, the term "membrane" refers to a selective barrier or sheet containing pores. The term includes, but is not limited to, flexible sheet-like structures that function as a boundary or lining. In some embodiments, the term refers to a surface or filter containing pores. This term is different from the "cell membrane" that refers to the semipermeable membrane surrounding the cytoplasm of a cell.

[0057] As used herein, the term "filter" refers to a porous article that can selectively pass through pores. In some embodiments, the term refers to a surface or membrane containing pores.

[0058] As used herein, the terms "deform" and "deformation" (including their derivatives) refer to physical changes within a cell. As described herein, when a cell passes through a constriction (such as those of the present disclosure), it is subjected to various forces by a restrictive physical environment, including but not limited to mechanical deformation forces and / or shear forces that cause perturbations in the cell membrane. As used herein, "perturbation" within the cell membrane refers to any opening in the cell membrane that does not exist under normal steady-state conditions (e.g., when no deformation force is applied to the cell). Perturbations can include holes, tears, cavities, apertures, pores, breaks, gaps, perforations, or combinations thereof.

[0059] As used herein, the term "heterogeneous" refers to something whose structure or composition is mixed or not uniform. In some embodiments, the term refers to pores having various sizes, shapes, or distributions within a given surface.

[0060] As used herein, the term "homogeneous" refers to something whose structure or composition is consistent or uniform throughout. In some embodiments, the term refers to pores having a consistent size, shape, or distribution within a given surface.

[0061] As used herein, the terms "polynucleotide" or "nucleic acid" refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases, but is not limited to these. The backbone of a polynucleotide can include sugars and phosphate groups (typically found in RNA or DNA), or modified or substituted sugars or phosphate groups. Alternatively, the backbone of a polynucleotide can include polymers of synthetic subunits such as phosphoramidates, and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products either by synthesizing the complementary strand and annealing the strands under appropriate conditions or by de novo synthesizing the complementary strand using DNA polymerase with an appropriate primer. As described herein, nucleic acids that can be delivered to cells using the squeeze processing methods provided herein include RNA (e.g., mRNA). As used herein, "RNA" includes both self-amplifying RNA (e.g., self-amplifying mRNA) and non-self-amplifying RNA (e.g., non-self-amplifying mRNA). As used herein, the term "self-amplifying RNA" refers to an RNA molecule that can replicate within a host and as a result can lead to an increase in the amount of RNA and the protein encoded by the RNA (e.g., a reprogramming factor). As used herein, the term "mRNA" refers to any polynucleotide (either self-amplifying or non-self-amplifying) that encodes at least one polypeptide.

[0062] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Further, for the purposes of the present disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally substantially conservative) to the native sequence, so long as the protein maintains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or accidental, such as due to mutations in the host producing the protein or errors resulting from PCR amplification.

[0063] III. Methods of the Present Disclosure In some embodiments, the present disclosure relates to a method of delivering self-amplifying RNA (saRNA) into a cell by passing the cell through a constriction (such as those described herein). As demonstrated herein, when a cell passes through a constriction, the cell is temporarily deformed, thereby perturbing the cell membrane. Perturbation within the cell membrane can enable various payloads (including self-amplifying RNA) to enter the cell or be carried intracellularly (e.g., via diffusion). The specific process by which the cell passes through the constriction and is temporarily deformed is referred to herein as "squeeze treatment," "squeeze delivery," or "squeezing."

[0064] As demonstrated herein, the methods provided herein can modify different aspects of cells. In some aspects, different cells of interest can be produced using the delivery methods provided herein by using saRNA encoding molecules (i.e., “reprogramming factors”) that can reprogram cells. As further described herein, in some aspects, reprogramming factors useful in the present disclosure include differentiation factors. As used herein, the term “differentiation factor” refers to any agent that can induce the differentiation of cells into different types of cells. Unless otherwise indicated, the terms “reprogramming factor” and “differentiation factor” can be used interchangeably in the present disclosure. Thus, as used herein, the terms “neuronal reprogramming factor” or “neuronal differentiation factor” can be used interchangeably and refer to an agent that can reprogram and / or induce cells to differentiate into neurons. As used herein, a neuronal reprogramming factor is not particularly limited as long as the agent can induce cells (e.g., stem cells or PBMCs) to differentiate into neurons. Such neuronal reprogramming factors are known in the art. Non-limiting examples of such factors include Neurogenin 2 (Ngn2, Neurog2), Atonal BHLH Transcription Factor 1 (Atoh1), Achaete-Scute Family BHLH Transcription Factor 1 (Ascl1), Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) (also called Nuclear Receptor Related 1 Protein (Nurr1)), LIM Homeobox Transcription Factor 1 Alpha (Lmx1a), POU Class 3 Homeobox 2 (POU3F2; Brn2), Myelin Transcription Factor 1-Like (Myt1l), Forkhead Box A2 (Foxa2), Paired-Like Homeodomain 3 (Pitx3), Engrailed Homeobox 1 (EN1), SRY-Box Transcription Factor 2 (SOX2), MicroRNA 124 (mir124), or combinations thereof. In some aspects, the neuronal reprogramming factor is Ngn2.In some embodiments, the neuronal reprogramming factor is Atoh1. In some embodiments, the neuronal reprogramming factor is Ascl1. In some embodiments, the neuronal reprogramming factor is NR4A2. In some embodiments, the neuronal reprogramming factor is Nurr1. In some embodiments, the neuronal reprogramming factor is Lmx1a. In some embodiments, the neuronal reprogramming factor is POU3F2. In some embodiments, the neuronal reprogramming factor is Myt1l. In some embodiments, the neuronal reprogramming factor is Foxa2. In some embodiments, the neuronal reprogramming factor is Pitx3. In some embodiments, the neuronal reprogramming factor is SOX2.

[0065] The above transcription factors generally activate signal transduction pathways that induce neuronal differentiation, but in some embodiments, the neuronal reprogramming factors useful in the present disclosure can also inhibit signal transduction pathways, such as pathways that interfere with neuronal differentiation. Thus, in some embodiments, the neuronal reprogramming factor comprises siRNA capable of inhibiting p53 signaling. In some embodiments, the neuronal reprogramming factor comprises miRNA (e.g., miR-214).

[0066] As further described and demonstrated herein, the above reprogramming factors can be delivered to cells (e.g., stem cells or PBMCs) alone or in combination. For example, such transcription factors can be encoded by a single saRNA (e.g., a polycistronic saRNA), and the single saRNA can be delivered to cells using squeeze delivery. In some embodiments, multiple transcription factors can be delivered to cells separately. For example, as demonstrated herein, the delivery methods provided herein (e.g., squeeze treatment) can be used to deliver an saRNA encoding a first transcription factor, a first mRNA encoding a transcription factor, a second mRNA encoding a transcription factor, a third mRNA encoding a transcription factor, a fourth mRNA encoding a transcription factor, and a fifth mRNA encoding a transcription factor. In some embodiments, the delivery methods provided herein (e.g., squeeze treatment) can be used to deliver an saRNA encoding a first transcription factor in combination with at least one mRNA, at least two mRNAs, at least three mRNAs, at least four mRNAs, at least five mRNAs, or at least six mRNAs, wherein each of the mRNAs encodes a payload (e.g., a reprogramming factor).

[0067] In some embodiments, when combinations of reprogramming factors (or any other payloads described herein) are involved, they can be delivered to cells using a single squeeze process. For example, a cell suspension contains multiple payloads, and they are delivered to the cells in combination ("co-delivery"). In some embodiments, multiple payloads (e.g., saRNAs encoding reprogramming factors in combination with one or more additional payloads) can be sequentially delivered to cells. As used herein, the term "sequential delivery" refers to delivering multiple payloads to cells, where a first payload (e.g., an saRNA encoding a reprogramming factor) is delivered to the cells, and then a second (or subsequent) payload (e.g., a non-self-amplifying RNA encoding a reprogramming factor) is delivered to the cells. In some embodiments, the first payload, the second payload, or both the first and second payloads can be delivered to cells using a squeeze process. For example, in some embodiments, the first payload can be delivered to cells using a squeeze process, and the second payload can be delivered to cells using a non-squeeze process (e.g., transfection). In some embodiments, the first payload can be delivered to cells using a non-squeeze process (e.g., transfection), and the second payload can be delivered to cells using a squeeze process. In some embodiments, the first payload can be delivered to cells using a first squeeze, and then the second payload can be delivered to cells using a second squeeze (also referred to herein as "sequential squeeze" or "sequential squeeze process"). Thus, sequential delivery useful in the present disclosure can include multiple squeeze processes. In some embodiments, each of the multiple squeeze processes delivers a distinct payload to the cells. In some embodiments, one or more of the multiple squeeze processes are without payload delivery.For example, in some embodiments, the sequential delivery method described herein includes a first squeeze, a second squeeze, and a third squeeze, where the first squeeze includes passing a cell without a payload through a first constriction, the second squeeze includes passing the cell from the first squeeze through a second constriction to deliver a first payload (e.g., saRNA encoding a reprogramming factor) to the cell, and the third squeeze includes passing the cell from the second squeeze through a third constriction to deliver a second payload to the cell. Without being bound by any theory, in some embodiments, passing a cell without a payload through a first constriction (i.e., the first squeeze) helps prepare the cell for subsequent payload delivery, e.g., it can improve the delivery efficiency of the first payload and / or the second payload.

[0068] In some embodiments, combinations of payloads can be repeatedly delivered to a cell (e.g., a stem cell or PBMC). For example, in some embodiments, a combination of transcription factors is delivered to a cell in a first squeeze treatment and then the combination of transcription factors is delivered to the cell again in a second squeeze treatment. In some embodiments, the first squeeze treatment includes a microfluidic device (e.g., a chip) having multiple rows of constrictions, and thus the squeeze treatment is performed on a single microfluidic device (e.g., a chip). As further described herein, in some embodiments, the second squeeze treatment can be performed immediately after the cell has undergone the first squeeze treatment (e.g., immediately after the cell has passed through the constriction of the first squeeze treatment). In some embodiments, the second squeeze treatment can be performed some time after the first squeeze treatment (e.g., at least about 1 minute, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, or at least about 1 day after the cell has passed through the constriction of the first squeeze treatment).

[0069] The present disclosure generally discloses the use of saRNAs encoding reprogramming factors, but it will be apparent to those skilled in the art that the disclosure regarding such saRNAs is equally applicable to other types of payloads. Thus, as is apparent from the present disclosure, the squeezing process or squeezing method provided herein can also be used to deliver additional cargo (also referred to herein as "payload") into cells. Non-limiting examples of such payloads are provided elsewhere in the present disclosure. Unless otherwise indicated, the terms "payload" and "reprogramming factor" are used interchangeably.

[0070] Accordingly, in some aspects, provided herein is a method of inducing the expression of a payload (e.g., a reprogramming factor) in a cell, the method comprising passing a cell suspension containing the cells through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in perturbations within the membranes of the cells, such that self-amplifying RNA encoding the payload can enter the cells through the perturbations when contacting the cells, and as a result, the payload is expressed in the cells. As is apparent from the present disclosure, in some aspects, the payload is a reprogramming factor.

[0071] In some embodiments, the above method enables long-term expression of the encoded payload (e.g., reprogramming factors) when expressed intracellularly. For example, in some embodiments, after squeezing and delivering saRNA to cells (e.g., iPSCs), the encoded payload is then expressed intracellularly for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year.

[0072] As demonstrated herein, Applicant has confirmed that a specific combination of squeeze delivery and saRNA (also referred to herein as "the delivery method provided herein" or variations thereof) is far more effective at inducing the expression of payloads (e.g., reprogramming factors) intracellularly compared to other approaches in the art. Thus, in some embodiments, provided herein is a method of enhancing the expression of a payload (e.g., a reprogramming factor) intracellularly, the method comprising passing a cell suspension containing cells through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in perturbations within the cell membrane such that when a payload encoding a reprogramming factor contacts the cell, the payload can enter the cell through the perturbations, resulting in enhanced expression of the payload as compared to the corresponding expression in reference cells. As used herein, the term "reference cells" refers to any cells (e.g., iPSCs) that have not been modified to contain saRNA using squeeze delivery. For example, in some embodiments, the reference cells include corresponding cells that have passed through the constriction under a set of parameters but have not contacted saRNA. In some embodiments, such reference cells contact a non-self-amplifying RNA (e.g., mRNA) encoding the payload. Thus, in some embodiments, the reference cells include corresponding cells (e.g., iPSCs) that have been subjected to squeeze treatment but have contacted an mRNA encoding the payload. In some embodiments, the reference cells include corresponding cells that have contacted a self-amplifying RNA encoding the payload and the reference cells have not been squeeze-treated. For example, in some embodiments, the reference cells contact saRNA using other delivery methods in the art (e.g., electroporation or lipofection).

[0073] In some embodiments, the expression of the encoded payload (e.g., a reprogramming factor) within the cell is increased (e.g., higher expression levels) compared to a reference cell (e.g., a cell that has been squeezed and contacted with non-self-amplifying mRNA). In some embodiments, the encoded payload is expressed for a much longer period of time within the cell compared to a reference cell (e.g., a cell that has been squeezed and contacted with non-self-amplifying mRNA). In some embodiments, both the expression and the expression period of the encoded payload are increased compared to a reference cell (e.g., a cell that has been squeezed and contacted with non-self-amplifying mRNA).

[0074] In some embodiments, the expression level of the encoded payload (e.g., a reprogramming factor) within the cell is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold compared to the expression level of the encoded payload in a reference cell. In some embodiments, the period of expression of the encoded payload within the cell is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold compared to the period of expression of the encoded payload in a reference cell.

[0075] As is apparent from the present disclosure, the methods provided herein can be particularly effective in delivering reprogramming factors to cells (e.g., iPSCs), thereby inducing reprogramming of the cells. Accordingly, in some aspects, the present disclosure is directed to a method of reprogramming a cell, the method comprising passing a cell suspension comprising the cell through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in perturbations within the membrane of the cell, such that self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbations when the self-amplifying RNA contacts the cell, thereby reprogramming the cell. In some aspects, compared to other delivery methods in the art, the methods provided herein can significantly enhance reprogramming of the cell. Accordingly, in some aspects, provided herein is a method of enhancing reprogramming of a cell, the method comprising passing a cell suspension comprising the cell through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in perturbations within the membrane of the cell, such that self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbations when the self-amplifying RNA contacts the cell, resulting in enhanced reprogramming of the cell as compared to a reference method.

[0076] As used herein, the term "reference method" refers to a delivery method that does not include delivering saRNA (e.g., encoding a reprogramming factor) to a cell (e.g., iPSC) using a squeezing process. For example, in some embodiments, the reference method includes passing a cell suspension through a constriction under a set of parameters, but the cells do not contact the saRNA described herein. In some embodiments, such a method includes contacting the cells with a non-self-amplifying RNA (e.g., mRNA) encoding a payload (e.g., a reprogramming factor). Thus, in some embodiments, the reference method includes passing a cell suspension through a constriction under a set of parameters and contacting the cell suspension with a non-self-amplifying RNA encoding a reprogramming factor. In some embodiments, the reference method includes contacting a cell (e.g., iPSC) with saRNA, but the cell has not undergone a squeezing process. For example, in some embodiments, the reference method includes contacting the cell with saRNA using other delivery methods in the art (e.g., electroporation or lipofection).

[0077] In some embodiments, compared to the reference method, the delivery method provided herein (e.g., squeezing process combined with saRNA) results in a much larger number of reprogrammed cells. In some embodiments, compared to the reference method, the delivery method provided herein has a much shorter duration required to reprogram the cells. In some embodiments, compared to the reference method, the delivery method provided herein enables both an increase in the number of reprogrammed cells and a decrease in the duration required to reprogram the cells.

[0078] In some embodiments, by using a squeeze process to deliver saRNA encoding a reprogramming factor into cells, the number of reprogrammed cells is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold compared to a reference method. In some embodiments, by using a squeeze process to deliver saRNA encoding a reprogramming factor into cells, the duration required to reprogram the cells is shortened by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference method.

[0079] As will be apparent from the present disclosure, the methods provided herein can be useful for generating many different types of cells. In some embodiments, as demonstrated herein, the reprogramming factors described herein can induce the differentiation of cells (e.g., iPSCs) into neurons. Thus, in some embodiments, provided herein is a method of generating neurons, comprising passing a cell suspension through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in perturbations within the membranes of the cells present in the cell suspension, such that self-amplifying RNA encoding a reprogramming factor can enter the cells through the perturbations when contacting the cells, and as a result, the cells differentiate into neurons. Non-limiting

[0080] Also provided herein is a method of enhancing neuron production, the method comprising passing a cell suspension through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters results in perturbation within the membranes of the cells present in the cell suspension, such that self-amplifying RNA encoding a reprogramming factor can enter the cells through the perturbation when the self-amplifying RNA contacts the cells, and as a result, neuron production is enhanced as compared to a reference method.

[0081] In some embodiments, use of the methods provided herein (e.g., a squeezing process using saRNA encoding a reprogramming factor) can increase the number of neurons produced, e.g., as compared to a reference method. In some embodiments, the methods provided herein can decrease the duration required to produce neurons, e.g., as compared to a reference method. In some embodiments, the methods provided herein can decrease both the number of neurons that can be produced and the duration required to produce neurons.

[0082] In some embodiments, using the methods provided herein (e.g., squeeze treatment with saRNA encoding reprogramming factors), the number of neurons produced is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold compared to a reference method. In some embodiments, using the methods provided herein (e.g., squeeze treatment with saRNA encoding reprogramming factors), the duration required to produce neurons is shortened by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference method.

[0083] The production of neurons (or any other cell of interest) can be evaluated using any suitable method known in the art. For example, in some embodiments, whether a cell (e.g., an iPSC) has been induced to differentiate into a neuron can be evaluated by detecting the expression of certain markers associated with neurons (a "neuronal marker"). Non-limiting examples of such markers include NeuroD1, FoxA2, NR4A2, tyrosine hydroxylase (TH), Lmx1a, Pitx3, Tuj1, Map2, or combinations thereof. In some embodiments, the marker includes a general neuronal marker (e.g., NeuroD1). In some embodiments, the marker includes a floor plate progenitor marker (e.g., FoxA2). In some embodiments, the marker includes a dopamine lineage marker (e.g., NR4A2, Pitx3, Lmx1a, and tyrosine hydroxylase). In some embodiments, the marker includes an early neuronal marker (e.g., Tuj1). In some embodiments, the marker includes a mature neuronal marker (e.g., Map2). Thus, unless otherwise indicated, the term "neuron" as used herein includes any cell that expresses one or more of the above neuronal markers.

[0084] Thus, in some embodiments, the delivery methods provided herein can increase the expression of any of the above markers intracellularly (e.g., by using squeeze processing to deliver saRNA encoding reprogramming factors that can induce cells to differentiate into neurons). In some embodiments, compared to a reference method, the expression of a neuronal marker is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold.

[0085] As will be apparent from the present disclosure, for any of the delivery methods provided herein (e.g., delivering saRNA to cells using squeeze delivery), in some embodiments, the method further comprises contacting a cell (e.g., a cell to be reprogrammed) with an saRNA encoding a payload (e.g., a reprogramming factor) prior to passing the cell suspension through the constriction. For example, in some embodiments, prior to passing the cell suspension through the constriction, the method comprises contacting the cells with an saRNA described herein (e.g., encoding a reprogramming factor) to generate a cell suspension (e.g., the cell suspension comprises both the cells to be reprogrammed and the reprogramming factor prior to passing the cell suspension through the constriction). In some embodiments, any of the delivery methods provided herein (e.g., delivering saRNA to cells using squeeze delivery) further comprises contacting the cells with an saRNA encoding a payload as the cell suspension (comprising the cells) passes through the constriction. In some embodiments, during passage of the cell suspension through the constriction, the cells first contact an saRNA described herein. In some embodiments, the cells contact the saRNA both before and during the passage step (i.e., the cell suspension passes through the constriction). In some embodiments, the method further comprises contacting the cells with an saRNA encoding a payload (e.g., a reprogramming factor) after passage of the cell suspension through the constriction. In some embodiments, after passage of the cell suspension through the constriction, the cells first contact an saRNA described herein. In some embodiments, the cells contact the saRNA before, during, and / or after the passage step. As further described elsewhere in the present disclosure, when the cells contact an saRNA encoding a payload (e.g., a reprogramming factor) after the passage step, the contact occurs immediately after the cells have passed through the constriction and there is still perturbation within the cell membrane.

[0086] As used herein, "contacting" between a cell and an saRNA encoding a payload (e.g., a reprogramming factor) does not require physical contact between the cell and the saRNA. As is apparent from the present disclosure, contact between a cell and an saRNA occurs as long as the saRNA can enter the cell when there is perturbation within the cell membrane of the cell. For convenience of explanation, in some embodiments, whether or not the cell and the saRNA are in physical contact, the cell and the saRNA are in contact when both the cell and the saRNA are present in the same cell suspension.

[0087] III.A. Cell Suspension In some embodiments, the cell suspensions described herein include any suitable cells known in the art that can be modified (e.g., by introducing an saRNA encoding a reprogramming factor using the squeezing method described herein).

[0088] In some embodiments, the cells are stem cells. As used herein, the term "stem cell" refers to a cell that has not only the ability to self-renew but also the ability to differentiate into other types of cells (e.g., neurons). In some embodiments, stem cells useful in the present disclosure include induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), tissue-specific stem cells (e.g., liver stem cells, cardiac stem cells, or neural stem cells), mesenchymal stem cells, hematopoietic stem cells (HSCs), or combinations thereof. In some embodiments, the stem cells are iPSCs.

[0089] In some embodiments, the cells are somatic cells. As used herein, the term "somatic cell" refers to any cell in the body that is not a gamete (sperm or egg), germ cell (cell that becomes a gamete), or stem cell. Non-limiting examples of somatic cells include blood cells, bone cells, muscle cells, nerve cells, or combinations thereof. In some embodiments, the somatic cells useful in the present disclosure include blood cells. In some embodiments, the blood cells are peripheral blood mononuclear cells (PBMCs). As used herein, "PBMC" refers to any peripheral blood cell having a round nucleus. In some embodiments, the PBMCs include immune cells. As used herein, the term "immune cell" refers to any cell involved in immune function. In some embodiments, the immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells, or combinations thereof. In some embodiments, the blood cells are red blood cells. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are cells of a cancer cell line such as HeLa cells. In some embodiments, the cancer cells are tumor cells. In some embodiments, the cancer cells are circulating tumor cells (CTCs). In some embodiments, the cells are fibroblasts such as primary fibroblasts or neonatal foreskin fibroblasts (Nuff cells). In some embodiments, the cells are cells of an immortalized cell line such as HEK293 cells or CHO cells. In some embodiments, the cells are skin cells. In some embodiments, the cells are germ cells, e.g., oocytes, egg cells, or zygotes. In some embodiments, the cells are cell clusters, such as embryos, provided that the cell clusters are not disrupted when passing through pores.

[0090] In some embodiments, the cell suspensions useful in the present disclosure include a population of mixed or purified cells. In some embodiments, the cell suspension is a mixed cell population such as whole blood, lymph, PBMCs, or combinations thereof. In some embodiments, the cell suspension is a purified cell population. In some embodiments, the cells are primary cells or cells of a cell line.

[0091] As demonstrated herein, the intracellular delivery of saRNA encoding a payload (e.g., a reprogramming factor) can be controlled by one or more parameters of the process by which the cell suspension passes through the constriction. In some embodiments, specific characteristics of the cell suspension may affect the intracellular delivery of the saRNA. Such characteristics include, but are not limited to, osmotic pressure, salt concentration, serum content, cell concentration, pH, temperature, or combinations thereof. Additional disclosure regarding such parameters is provided throughout this disclosure.

[0092] In some embodiments, the cell suspension comprises a homogeneous population of cells. In some embodiments, the cell suspension comprises a heterogeneous population of cells (e.g., whole blood, or a mixture of cells in a physiological medium other than saline or blood). In some embodiments, the cell suspension comprises an aqueous solution. In some embodiments, the aqueous solution comprises a cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, bulking agents, surfactants, lubricants, vitamins, polypeptides, agents that affect actin polymerization, or combinations thereof. In some embodiments, the cell culture medium comprises DMEM, OptiMEM, EVIDM, RPMI, or combinations thereof. Additionally, the solution buffer can include one or more lubricants (pluronics or other surfactants) designed to reduce or eliminate surface clogging and improve cell viability. Exemplary surfactants include, but are not limited to, poloxamers, polysorbates, sugars such as mannitol, animal-derived serum, and albumin proteins.

[0093] When the suspension contains a particular type of cell, in some embodiments, the cells can be treated with a solution that helps deliver a payload (e.g., a reprogramming factor) into the interior of the cells. In some embodiments, the solution contains an agent that affects actin polymerization. In some embodiments, the agent that affects actin polymerization includes latrunculin A, cytochalasin, colchicine, or a combination thereof. For example, in some embodiments, after incubating the cells in a depolymerization solution such as latrunculin A for about 1 hour, the cells can be passed through the constriction to depolymerize the actin cytoskeleton. In some embodiments, after incubating the cells in colchicine (Sigma) for about 2 hours, the cells can be passed through the constriction to depolymerize the microtubule network.

[0094] In some embodiments, a characteristic of the cell suspension that can affect the intracellular delivery of saRNA encoding a payload (e.g., a reprogramming factor) is the viscosity of the cell suspension. As used herein, the term "viscosity" refers to the internal resistance to flow exhibited by a fluid. In some embodiments, the viscosity of the cell suspension is between about 8.9×10 -4 Pa·s and about 4.0×10 -3 Pa·s, between about 8.9×10 -4 Pa·s and about 3.0×10 -3 Pa·s, between about 8.9×10 -4 Pa·s and about 2.0×10 -3 Pa·s, or between about 8.9×10 -4 Pa·s and about 1.0×10 -3It is between Pa·s. In some embodiments, the viscosity is between about 0.89 cP and about 4.0 cP, between about 0.89 cP and about 3.0 cP, between about 0.89 cP and about 2.0 cP, or between about 0.89 cP and about 1.0 cP. In some embodiments, a shear thinning effect is observed in which the viscosity of the cell suspension decreases under shear strain conditions. The viscosity can be measured by any suitable method known in the art, including but not limited to viscometers such as glass capillary viscometers or rheometers. A viscometer measures the viscosity under one flow condition, while a rheometer is used to measure the viscosity that changes with flow conditions. In some embodiments, the viscosity of a shear thinning solution such as blood is measured. In some embodiments, the viscosity is measured between about 0 °C and about 45 °C. For example, the viscosity of the cell suspension can be measured at room temperature (e.g., about 20 °C), physiological temperature (e.g., about 37 °C), a temperature higher than physiological temperature (e.g., above about 37 °C to about 45 °C or higher), low temperature (e.g., about 0 °C to about 4 °C), or a temperature between these exemplary temperatures.

[0095] III.B. Payload As described and demonstrated herein, the delivery methods provided by the present disclosure are particularly useful for delivering saRNAs encoding reprogramming factors capable of inducing the differentiation of cells into neurons intracellularly. In some embodiments, the delivery methods provided herein can further include contacting the cells with one or more additional payloads such that, as a result, the plurality of payloads can enter the cells through perturbation when contacting the cells. In some embodiments, the additional payloads can be delivered to the cells simultaneously with the saRNAs (e.g., encoding reprogramming factors) described herein. For example, in some embodiments, the additional payloads can be present in the cell suspension with the saRNAs described herein before, during, and / or after the passage step in which the cell suspension passes through the constriction. In some embodiments, the cell suspension can include at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 or more additional payloads. Where multiple payloads are involved, in some embodiments, the multiple payloads can be delivered to the cells as a single unit (e.g., a single saRNA encoding the multiple payloads). In some embodiments, the multiple payloads can be delivered to the cells as multiple units. For example, using the delivery methods provided herein, an saRNA encoding a first payload (e.g., a reprogramming factor) and a non-self-amplifying RNA encoding a second payload (e.g., a reprogramming factor) can be delivered.

[0096] As further described elsewhere in this disclosure, in some embodiments, the cell suspension can pass through a plurality of constrictions. In such embodiments, the payload can be carried into the cell when the cell passes through one or more of the plurality of constrictions. In some embodiments, when multiple payloads are involved where a payload is carried into the cell each time the cell passes through one or more of the plurality of constrictions, each of the payloads may be the same. In some embodiments, one or more of the payloads are different. Additional disclosure regarding the delivery of saRNA described herein (also referred to herein as "multiple delivery") in combination with one or more additional payloads is provided elsewhere in this disclosure.

[0097] As will be apparent from the present disclosure, any suitable payload known in the art can be delivered to cells using the methods described herein (e.g., in combination with the saRNAs described herein). Non-limiting examples of such payloads include nucleic acids, polypeptides, lipids, carbohydrates, small molecules, metal-containing compounds, antibodies, transcription factors, nanoparticles, liposomes, fluorescently labeled molecules, or combinations thereof. In some embodiments, the small molecules include impermeable small molecules. As used herein, "impermeable small molecule" refers to a small molecule that does not naturally cross the cell membrane of a cell. In some embodiments, additional payloads that can be delivered to cells using the delivery methods provided herein include nucleic acids. In some embodiments, the nucleic acids include DNA, RNA, or both. In some embodiments, the DNA includes recombinant DNA, cDNA, genomic DNA, or combinations thereof. In some embodiments, the RNA includes siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, self-amplifying mRNA (saRNA), or combinations thereof. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is shRNA. In some embodiments, the RNA is miRNA. In some embodiments, the RNA is saRNA. For example, in some embodiments, the delivery methods described herein can include delivering a first saRNA encoding a first payload (e.g., a reprogramming factor) and a second saRNA encoding a second payload (e.g., a reprogramming factor) to a cell (e.g., an iPSC), where the first and second saRNAs are both delivered to the cell using a squeeze treatment. As further described herein, in some embodiments, the first and second saRNAs can be delivered to the cell simultaneously. In some embodiments, the first and second saRNAs can be delivered to the cell sequentially.In some embodiments, the delivery methods provided herein include delivering a first saRNA encoding a first payload (e.g., a reprogramming factor) and a non-self-amplifying RNA encoding a second payload (e.g., a reprogramming factor).

[0098] In some embodiments, the additional compound is a nucleic acid encoding an enzyme that confers resistance to an antibiotic. Without being bound by any theory, adding these compounds to a cell suspension containing multiple cells and reprogramming factors can increase the purity of the mixture containing modified cells (i.e., reprogrammed / differentiated cells). By adding a specific antibiotic related to the additional compound, the purity of the mixture containing modified cells (e.g., including differentiated neurons) can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold. Non-limiting examples of such additional compounds include puromycin-N-acetyltransferase, and the antibiotic is puromycin.

[0099] III.C. Stenosis III.C.1. Microfluidic Channel As described herein, constrictions are used to effect physical deformation within a cell, thereby creating perturbations within the cell's cell membrane, which enables delivery of a payload (e.g., a reprogramming factor) into the cell. In some embodiments, the constrictions are within channels (referred to herein as "microfluidic channels" or "channels") contained within a microfluidic device. When multiple channels are involved, in some embodiments, the multiple channels can be arranged in parallel and / or in series within the microfluidic device. In some embodiments, the cells described herein can pass through at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1,000 or more distinct constrictions. In some embodiments, the cells described herein pass through more than about 1,000 distinct constrictions. In some embodiments, multiple constrictions can be part of a single microfluidic device (e.g., a multi-row constriction chip). In some embodiments, one or more of the multiple constrictions can be part of different microfluidic devices. For example, in some embodiments, the cells described herein (e.g., stem cells or PBMCs) undergo a first squeezing process where the cells pass through a first constriction of a first microfluidic device (e.g., a chip). Next, after the cells have undergone the first squeezing process (e.g., passed through the first constriction), the cells undergo a second squeezing process where the cells pass through a second constriction within a second microfluidic device (e.g., a chip). In some embodiments, each of the constrictions is the same (e.g., having the same length, width, and / or depth).In some embodiments, one or more of the constrictions are different. When multiple constrictions are used, the multiple constrictions can include a first constriction associated with a first reprogramming factor and a second constriction associated with a second reprogramming factor, where the cell suspension passes through the first constriction such that the first reprogramming factor is delivered to one or more of the plurality of cells, and then the cell suspension passes through the second constriction such that the second reprogramming factor is delivered to one or more of the plurality of cells. In some embodiments, the cell suspension passes through the second constriction at least about 1 minute, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, or at least about 1 day after the cell suspension has passed through the first constriction.

[0100] In some embodiments, when a cell suspension passes through a plurality of constrictions (e.g., multiple squeezing processes), the cells remain viable after passing through each constriction. As is apparent from the present disclosure, in some embodiments, the plurality of constrictions can include two or more constrictions present within a single microfluidic device (e.g., a multi-row constriction chip), and thus the cells sequentially pass through the plurality of constrictions. In some embodiments, the plurality of constrictions are part of separate microfluidic devices, and thus, a first constriction is associated with a first microfluidic device and a second constriction is associated with a second microfluidic device. For example, as demonstrated herein (e.g., Example 1), in some embodiments, the cells pass through a first constriction associated with a first microfluidic device (e.g., a chip) (i.e., a first squeezing process). After the cells pass through the first constriction, the cells pass through a second constriction associated with a second microfluidic device (e.g., a chip) (i.e., a second squeezing process). In some embodiments, the cells are cultured in a medium after passing through the first constriction and before the cells pass through the second constriction. In some embodiments, the cells are cultured for at least about 1 minute, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, or at least about 1 day before passing through the second constriction. As is apparent from the present disclosure, in some embodiments, the first and second constrictions have the same length, depth, and / or width. In some embodiments, the first and second constrictions can have different lengths, depths, and / or widths.

[0101] In some embodiments, after passing through the constriction, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cells remain viable. When cells pass through multiple constrictions (e.g., part of a single microfluidic device or separate microfluidic devices), after passing through each of the multiple constrictions, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cells remain viable. The viability of the cells can be measured using any suitable method known in the art. In some embodiments, the viability of the cells can be measured using a Nucleocounter NC-200, an Orflo Moxi Go II cell counter, or both.

[0102] Exemplary microfluidic channels containing cell-deforming constrictions for use in the methods disclosed herein are described in U.S. Patent Application Publication No. 2020 / 0277566A1, U.S. Patent Application Publication No. 2020 / 0332243A1, U.S. Patent Application Publication No. 2020 / 0316604A1, U.S. Provisional Application No. 63 / 131,423, and U.S. Provisional Application No. 63 / 131,430, each of which is incorporated herein by reference in its entirety.

[0103] In some embodiments, the microfluidic channels (i.e., including constrictions) described herein include a lumen and are configured such that cells suspended in a buffer (e.g., cell suspension) can pass through the channels. Microfluidic channels useful in the present disclosure can be fabricated using any suitable material available in the art, including but not limited to silicon, metal (e.g., stainless steel), plastic (e.g., polystyrene), ceramic, glass, crystalline substrate, amorphous substrate, polymer (e.g., polymethyl methacrylate (PMMA), PDMS, cyclic olefin copolymer (COC)), or combinations thereof. In some embodiments, the material is silicon. The manufacture of the microfluidic channels can be performed by any method known in the art, including but not limited to dry etching, e.g., deep reactive ion etching, wet etching, photolithography, injection molding, laser ablation, SU-8 masks, or combinations thereof. In some embodiments, the manufacture is performed using dry etching.

[0104] In some embodiments, the microfluidic channels useful in the present disclosure include an inlet portion, a central point, and an outlet portion. In some embodiments, the cross-section of one or more of the inlet portion, the central point, and / or the outlet portion can vary. For example, the cross-section can be circular, elliptical, an elongated slit, square, hexagonal, or triangular in shape.

[0105] The inlet portion defines a constriction angle. In some embodiments, by adjusting the constriction angle (e.g., increasing or decreasing it), clogging of the constriction can be reduced or prevented. In some embodiments, the angle of the outlet portion can also be adjusted. For example, in some embodiments, the angle of the outlet portion can be configured to reduce the likelihood of turbulent flow, which can result in non-laminar flow. In some embodiments, the walls of the inlet portion and / or the outlet portion are straight. In some embodiments, the walls of the inlet portion and / or the outlet portion are curved.

[0106] In some embodiments, the length, depth, and / or width of the constriction can vary. In some embodiments, the delivery efficiency of the payload can be controlled by adjusting (e.g., increasing or decreasing) the length, depth, and / or width of the constriction. As used herein, the term "delivery efficiency" refers to the amount of payload delivered into the cell. For example, an increase in delivery efficiency can occur when the total amount of payload delivered increases.

[0107] In some embodiments, the constriction has a length of less than about 1 μm. In some embodiments, the constriction has a length from about 0 μm to about 100 μm. In some embodiments, the length of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2.5 μm, less than about 5 μm, less than about 7.5 μm, less than about 10 μm, less than about 12.5 μm, less than about 15 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, or less than about 100 μm. In some embodiments, the length of the constriction is about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 2.5 μm, about 5 μm, about 7.5 μm, about 10 μm, about 12.5 μm, about 15 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. In some embodiments, the length of the constriction is about 10 μm. In some embodiments, the constriction has a length of about 0 μm. For example, in some embodiments, a microfluidic device (e.g., a chip) useful in the present disclosure comprises a constriction that resembles two diamond points joined together such that the length of the constriction is about 0 μm.

[0108] In some embodiments, the width of the constriction is between about 0 μm and about 10 μm. In some embodiments, the width of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2 μm, less than about 3 μm, less than about 4 μm, less than about 5 μm, less than about 6 μm, less than about 7 μm, less than about 8 μm, less than about 9 μm, or less than about 10 μm. In some embodiments, the width of the constriction is about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In some embodiments, the width of the constriction is between about 3 μm and about 10 μm. In some embodiments, the width of the constriction is about 6 μm.

[0109] In some embodiments, the depth of the constriction is at least about 1 μm. In some embodiments, the depth of the constriction is at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 110 μm, or at least about 120 μm. In some embodiments, the depth of the constriction is between about 5 μm and about 90 μm. In some embodiments, the depth of the constriction is about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or about 90 μm. In some embodiments, the depth of the constriction is about 70 μm.

[0110] In some embodiments, the length of the constriction is about 10 μm, the width of the constriction is about 6 μm, and the depth of the constriction is about 70 μm. In some embodiments, the length of the constriction is 10 μm, the width of the constriction is 6 μm, and the depth of the constriction is 70 μm.

[0111] In some embodiments, the diameter of the constriction (e.g., contained within a microfluidic channel) is a function of the diameter of one or more cells passing through the constriction. Without being bound by any theory, in some embodiments, the diameter of the constriction is smaller than the diameter of the cell, and thus, when the cell passes through the constriction, a deforming force is applied to the cell, resulting in a temporary physical deformation of the cell.

[0112] Accordingly, in some embodiments, the diameter of the constriction (also referred to herein as the "constriction diameter") is from about 20% to about 99% of the diameter of the cell. In some embodiments, the constriction diameter is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. As will be apparent from the present disclosure, by adjusting (e.g., increasing or decreasing) the diameter of the constriction, the delivery efficiency of the payload into the cell can also be controlled.

[0113] III.C.2. Surfaces with Pores In some embodiments, the constrictions described herein include pores contained on a surface. Non-limiting examples of pores contained on a surface that can be used in the present disclosure are described, for example, in U.S. Patent Application Publication No. 2019 / 0382796A1, which is hereby incorporated by reference in its entirety.

[0114] In some embodiments, a surface useful in the present disclosure (i.e., including one or more pores that may cause physical deformation of a cell when passing through the pores) can be made using any suitable material available in the art and / or can adopt any one of a plurality of forms. Non-limiting examples of such materials include synthetic or natural polymers, polycarbonate, silicon, glass, metal, alloy, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose ester, porcelain, ceramic, or combinations thereof.

[0115] In some embodiments, the surface includes a filter. In some embodiments, the filter is a tangential flow filter. In some embodiments, the surface includes a membrane. In some embodiments, the surface includes a sponge or sponge-like matrix. In some embodiments, the surface includes a matrix. In some embodiments, the surface includes a tortuous path surface. In some embodiments, the tortuous path surface includes cellulose acetate.

[0116] The surfaces disclosed herein (i.e., those including one or more pores) can have any suitable shape known in the art. When the surface has a two-dimensional shape, the surface can be, but is not limited to, circular, oval, round, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal. In some embodiments, the surface is round. When the surface has a three-dimensional shape, in some embodiments, the surface can be, but is not limited to, cylindrical, conical, or cube-like.

[0117] As will be apparent from the present disclosure, the surfaces useful in the present disclosure (e.g., those including one or more pores) can have various cross-sectional widths and thicknesses. In some embodiments, the cross-sectional width of the surface is between about 1 mm and about 1 m. In some embodiments, the surface has a defined thickness. In some embodiments, the surface thickness is uniform. In some embodiments, the surface thickness is variable. For example, in some embodiments, a particular portion of the surface is thicker or thinner than other portions of the surface. In such embodiments, the thickness of different portions of the surface can vary by about 1% to about 90%. In some embodiments, the thickness of the surface is between about 0.01 μm and about 5 mm.

[0118] The cross-sectional width of the pore can depend on the type of cell targeted by the payload. In some embodiments, the pore diameter is a function of the diameter of the cell or cluster of cells to be targeted. In some embodiments, the pore diameter is sized such that the cell is perturbed (i.e., physically deformed) as it passes through the pore. In some embodiments, the pore diameter is smaller than the diameter of the cell. In some embodiments, the pore diameter is from about 20% to about 99% of the diameter of the cell. In some embodiments, the pore diameter is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the diameter of the cell. In some embodiments, the pore diameter is about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, or about 15 μm or greater.

[0119] The inlet and outlet of the pores can have various angles. In some embodiments, by adjusting the pore angle (e.g., increasing or decreasing), pore clogging can be reduced or prevented. In some embodiments, the flow rate (i.e., the rate at which cells or a suspension containing cells pass through the pores) is between about 0.001 mL / cm / second and about 100 L / cm / second. For example, the angle of the inlet portion or the outlet portion can be between about 0 degrees and about 90 degrees. In some embodiments, the pores have the same inlet angle and outlet angle. In some embodiments, the pores have different inlet angles and outlet angles. In some embodiments, the edges of the pores are smooth, for example, rounded or curved. As used herein, a "smooth" pore edge has a continuous flat and uniform surface without protrusions, ridges, or non-uniform portions. In some embodiments, the pore edges are sharp. As used herein, a "sharp" pore edge has a thin edge that is pointed or at an acute angle. In some embodiments, the pore passage is linear. As used herein, a "linear" pore passage does not include curves, bends, angles, or other irregularities. In some embodiments, the pore passage is curved. As used herein, a "curved" pore passage is curved or deviates from a straight line. In some embodiments, the pore passage has a plurality of curves, for example, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more curves.

[0120] The pores can have any shape known in the art, including two-dimensional or three-dimensional shapes. The pore shape (e.g., cross-sectional shape) can be, but is not limited to, circular, elliptical, round, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. In some embodiments, the cross-section of the pore is a round shape. In some embodiments, the three-dimensional shape of the pore is cylindrical or conical. In some embodiments, the pores have grooved inlet and outlet shapes. In some embodiments, the pore shape is homogeneous (i.e., consistent or regular) among the pores within a given surface. In some embodiments, the pore shape is heterogeneous (i.e., mixed or diverse) among the pores within a given surface.

[0121] Surfaces useful in the present disclosure can have a single pore. In some embodiments, surfaces useful in the present disclosure include a plurality of pores. In some embodiments, the pores occupy from about 10% to about 80% of the total surface area of the surface. In some embodiments, the surface contains from about 1.0×10 5 to about 1.0×10 30 total pores. In some embodiments, the surface contains pores between about 10 and about 1.0×10 2 per square millimeter of surface area. 15

[0122] The pores can be distributed in various ways within a given surface. In some embodiments, the pores are distributed parallel within a given surface. In some embodiments, the pores are arranged and distributed in the same direction within a given surface and are spaced the same distance apart. In some embodiments, the pore distribution is ordered or homogeneous. In such embodiments, the pores may be distributed in a regular and systematic pattern within a given surface or may be spaced the same distance apart. In some embodiments, the pore distribution is random or heterogeneous. For example, in some embodiments, the pores are distributed in an irregular and disorderly pattern within a given surface or are spaced different distances apart.

[0123] In some embodiments, multiple surfaces are used such that cells pass through multiple pores, where the pores are on different surfaces. In some embodiments, the multiple surfaces are continuously distributed. The multiple surfaces can have homogeneous or heterogeneous surface sizes, shapes, and / or roughnesses. The multiple surfaces can further contain pores having homogeneous or heterogeneous pore diameters, shapes, and / or numbers, thereby enabling simultaneous delivery of different payloads to different cell types.

[0124] In some embodiments, individual pores, such as the pores of the surfaces that can be used in the present disclosure, have a uniform width dimension (i.e., a constant width along the length of the pore passage). In some embodiments, individual pores have a variable width (i.e., a width that increases or decreases along the length of the pore passage). In some embodiments, the pores within a given surface have the same individual pore depth. In some embodiments, the pores within a given surface have different individual pore depths. In some embodiments, the pores are directly adjacent to each other. In some embodiments, the pores are separated from each other by a certain distance. In some embodiments, the pores are separated from each other by a distance of about 0.001 μm to about 30 mm.

[0125] In some embodiments, the surface is coated with a material. The material can be selected from any material known in the art, including but not limited to Teflon, adhesion coatings, surfactants, proteins, adhesion molecules, antibodies, anticoagulants, factors that regulate cell function, nucleic acids, lipids, carbohydrates, transmembrane proteins, or combinations thereof. In some embodiments, the surface is coated with polyvinylpyrrolidone. In some embodiments, the material is covalently bonded to the surface. In some embodiments, the material is non-covalently bonded to the surface. In some embodiments, surface molecules are released when the cells pass through the pores.

[0126] In some embodiments, the surface has modified chemical properties. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged. In some embodiments, the surface is positively and / or negatively charged. In some embodiments, the surface can be positively charged in some regions and negatively charged in other regions. In some embodiments, the surface has an overall positive charge or an overall negative charge. In some embodiments, the surface can be any one of a smooth surface, an electropolished surface, a rough surface, or a plasma-treated surface. In some embodiments, the surface contains zwitterions or bipolar compounds. In some embodiments, the surface is plasma-treated.

[0127] In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe, such as a plastic or glass syringe. In some embodiments, the surface is contained within a plastic filter holder. In some embodiments, the surface is contained within a pipette tip.

[0128] III.D. Cell Perturbation As described herein, when a cell passes through a constriction, the cell physically deforms, and thus perturbations (e.g., holes, tears, cavities, apertures, pores, breaks, gaps, perforations) occur in the cell membrane of the cell. Such perturbations in the cell membrane are transient and are sufficient for any of the payloads (e.g., reprogramming factors) described herein to be delivered into the cell. The cell has a self-healing mechanism by which the cell can repair the disruption of the cell membrane. See Blazek et al., Physiology (Bethesda) 30(6):438-48 (Nov. 2015), which is hereby incorporated by reference in its entirety. Thus, in some embodiments, when a cell passes through a constriction (e.g., a microfluidic channel or pore), the perturbation of the cell membrane can be reduced or eliminated so that the payload delivered into the cell cannot exit the cell.

[0129] In some embodiments, the perturbation of the cell membrane persists for about 1.0×10 -9 seconds to about 2 hours after the pressure is removed (e.g., after the cell has passed through the constriction). In some embodiments, the cell perturbation persists for about 1.0×10 -9 seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. In some embodiments, the cell perturbation persists for about 1.0×10 -9 seconds to about 1.0×10 -1 seconds, about 1.0×10 -9 seconds to about 1.0×10 -2 seconds, about 1.0×10 -9 seconds to about 1.0×10 -3 seconds, about 1.0×10 -9 seconds to about 1.0×10 -4 seconds, about 1.0×10 -9 seconds to about 1.0×10 -5 seconds, about 1.0×10 -9 seconds to about 1.0×10 -6 seconds, about 1.0×10 -9 seconds to about 1.0×10 -7 seconds, or about 1.0×10 -9 seconds to about 1.0×10 -8 seconds. In some embodiments, the cell perturbation persists for about 1.0×10 -8 seconds to about 1.0×10 -1 seconds, about 1.0×10 -7 seconds to about 1.0×10 -1 seconds, about 1.0×10 -6 seconds to about 1.0×10 -1 seconds, about 1.0×10 -5 seconds to about 1.0×10 -1 seconds, about 1.0×10 -4 seconds to about 1.0×10 -1 seconds, about 1.0×10 -3 seconds to about 1.0×10 -1 seconds, or about 1.0×10 -2 seconds to about 1.0×10 -1 seconds. The cell perturbations (e.g., pores or holes) created by the methods described herein are not formed as a result of the organization of polypeptide subunits to form multimeric pore structures such as those created by complement or bacteriolysin.

[0130] In some embodiments, as the cell passes through the constriction, the pressure applied to the cell temporarily damages the cell membrane, resulting in passive diffusion of materials through perturbation. In some embodiments, the cell is deformed or perturbed only for a short period of time, for example, on the order of 100 μs or less, to minimize the possibility that the apoptosis pathway is activated via the cell signaling mechanism, although other periods are possible (e.g., nanoseconds to hours). In some embodiments, the cell deforms in less than about 1.0×10 -9 seconds to less than about 2 hours. In some embodiments, the cell deforms in less than about 1.0×10 -9 seconds to less than about 1 second, less than about 1 second to less than about 1 minute, or less than about 1 minute to less than about 1 hour. In some embodiments, the cell deforms in about 1.0×10 -9 seconds to about 2 hours. In some embodiments, the cell deforms in about 1.0×10 -9 seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. In some embodiments, the cell deforms in about 1.0×10 -9 seconds to about 1.0×10 -1 seconds, about 1.0×10 -9 seconds to about 1.0×10 -2 seconds, about 1.0×10 -9 seconds to about 1.0×10 -3 seconds, about 1.0×10 -9 seconds to about 1.0×10 -4 seconds, about 1.0×10 -9 seconds to about 1.0×10 -5 seconds, about 1.0×10 -9 seconds to about 1.0×10 -6 seconds, about 1.0×10 -9 seconds to about 1.0×10 -7 seconds, or about 1.0×10 -9 seconds to about 1.0×10 -8 seconds. In some embodiments, the cell deforms between about 1.0×10 -8 seconds to about 1.0×10 -1 seconds, about 1.0×10 -7 seconds to about 1.0×10 -1 seconds, about 1.0×10 -6 seconds to about 1.0×10 -1 seconds.-5 seconds to about 1.0×10 -1 seconds, about 1.0×10 -4 seconds to about 1.0×10 -1 seconds, about 1.0×10 -3 seconds to about 1.0×10 -1 seconds, or about 1.0×10 -2 seconds to about 1.0×10 -1 seconds and deform. In some embodiments, deforming the cell occurs, but is not limited to, for a time in the range of about 1 μsecond to at least about 750 μseconds, such as at least about 1 μsecond, at least about 10 μseconds, at least about 50 μseconds, at least about 100 μseconds, at least about 500 μseconds, or at least about 750 μseconds.

[0131] In some embodiments, delivery of the saRNA encoding the payload (e.g., reprogramming factor) into the cell occurs simultaneously with the cell passing through the constriction. In some embodiments, delivery of the saRNA encoding the payload into the cell can occur after the cell has passed through the constriction (i.e., when perturbation of the cell membrane still exists and before the cell membrane of the cell has recovered). In some embodiments, delivery of the saRNA encoding the payload into the cell occurs within about several minutes after the cell has passed through the constriction. In some embodiments, the intracellular perturbation after the cell has passed through the constriction is corrected within about 5 minutes after the cell has passed through the constriction.

[0132] In some embodiments, the viability of the cells (e.g., stem cells or PBMCs) after passing through the constriction is from about 5% to about 100%. In some embodiments, the cell viability after passing through the constriction is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, the cell viability is about 1.0×10 after the cell has passed through the constriction -2It is measured at seconds to at least about 10 days later. For example, the cell viability can be measured about 1.0×10 -2 seconds to about 1 second later, about 1 second to about 1 minute later, about 1 minute to about 30 minutes later, or about 30 minutes to about 2 hours later. In some embodiments, the cell viability is about 1.0×10 -2 seconds to about 2 hours later, about 1.0×10 -2 seconds to about 1 hour later, about 1.0×10 -2 seconds to about 30 minutes later, about 11.0×10 -2 seconds to about 1 minute later, about 1.0×10 -2 seconds to about 30 seconds later, about 1.0×10 -2 seconds to about 1 second later, or about 1.0×10 -2 seconds to about 0.1 second later. In some embodiments, the cell viability is measured about 1.5 hours to about 2 hours later, about 1 hour to about 2 hours later, about 30 minutes to about 2 hours later, about 15 minutes to about 2 hours later, about 1 minute to about 2 hours later, about 30 seconds to about 2 hours later, or about 1 second to about 2 hours later after the cells pass through the constriction. In some embodiments, the cell viability is measured about 2 hours to about 5 hours later, about 5 hours to about 12 hours later, about 12 hours to about 24 hours later, or about 24 hours to about 10 days later after the cells pass through the constriction.

[0133] III.E. Delivery Parameters As is apparent from the present disclosure, using the squeeze processing method provided herein, multiple parameters can affect the intracellular delivery efficiency of saRNAs (e.g., encoding reprogramming factors) described herein. Thus, by adjusting one or more of the delivery parameters (e.g., increasing or decreasing), the delivery of the payload into the cell can be improved. Accordingly, in some aspects, the present disclosure relates to a method of increasing the delivery of a payload (e.g., a reprogramming factor) into a cell, where the method includes adjusting one or more parameters through which a cell suspension passes through a constriction, where the cell suspension includes a population of cells, and where the one or more parameters increase the delivery of the payload to one or more cells of the population of cells as compared to a reference parameter. As described elsewhere in the present disclosure, the payload can contact the population of cells before, during, or after the squeeze step.

[0134] In some aspects, by adjusting one or more of the delivery parameters, the delivery of the payload (e.g., a reprogramming factor) to one or more cells is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold increased compared to delivering the payload agent to the corresponding cells using the reference parameter.

[0135] In some aspects, one or more delivery parameters that can be adjusted to enhance the delivery efficiency of the parameter include cell density (i.e., the concentration of cells present in the cell suspension, for example), pressure, or both. Additional examples of delivery parameters that can be adjusted are described elsewhere in the present disclosure.

[0136] In some aspects, the cell density is about 1×10 7cells / mL, approximately 2×10 7 cells / mL, approximately 3×10 7 cells / mL, approximately 4×10 7 cells / mL, approximately 5×10 7 cells / mL, approximately 6×10 7 cells / mL, approximately 7×10 7 cells / mL, approximately 8×10 7 cells / mL, approximately 9×10 7 cells / mL, approximately 1×10 8 cells / mL, approximately 1.1×10 8 cells / mL, approximately 1.2×10 8 cells / mL, approximately 1.3×10 8 cells / mL, approximately 1.4×10 8 cells / mL, approximately 1.5×10 8 cells / mL, approximately 2.0×10 8 cells / mL, approximately 3.0×10 8 cells / mL, approximately 4.0×10 8 cells / mL, approximately 5.0×10 8 cells / mL, approximately 6.0×10 8 cells / mL, approximately 7.0×10 8 cells / mL, approximately 8.0×10 8 cells / mL, approximately 9.0×10 8 cells / mL, or approximately 1.0×10 9 cells / mL or more. In some embodiments, the cell density is approximately 6×10 7 cells / mL to approximately 1.2×10 8 cells / mL.

[0137] In some embodiments, the pressure is approximately 20 psi, approximately 25 psi, approximately 30 psi, approximately 35 psi, approximately 40 psi, approximately 50 psi, approximately 55 psi, approximately 60 psi, approximately 65 psi, approximately 70 psi, approximately 75 psi, approximately 80 psi, approximately 85 psi, approximately 90 psi, approximately 95 psi, approximately 100 psi, approximately 110 psi, approximately 120 psi, approximately 130 psi, approximately 140 psi, approximately 150 psi, approximately 160 psi, approximately 170 psi, approximately 180 psi, approximately 190 psi, or approximately 200 psi or more. In some embodiments, the pressure is approximately 30 psi to approximately 90 psi.

[0138] In some embodiments, certain types of devices (e.g., microfluidic chips) may also affect the delivery efficiency of the payloads (e.g., reprogramming factors) described herein. In the case of microfluidic chips, different chips may have different constriction parameters, such as the length, depth, and width of the constriction; the inlet angle, outlet angle, length, depth, and width of the approach region, and the like. As described herein, such variables may affect the delivery of payloads into cells using the squeezing methods of the present disclosure. In some embodiments, the length of the constriction is at most 100 μm. For example, in some embodiments, the length is about 1 μm, about 5 μm, 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. In some embodiments, the length of the constriction is less than 1 μm. In some embodiments, the length of the constriction is less than about 1 μm, less than about 5 μm, less than about 10 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, or less than about 100 μm. In some embodiments, the constriction has a length of about 10 μm.

[0139] In some embodiments, the width of the constriction is at most 10 μm. In some embodiments, the width of the constriction is less than about 1 μm, less than about 2 μm, less than about 3 μm, less than about 4 μm, less than about 5 μm, less than about 6 μm, less than about 7 μm, less than about 8 μm, less than about 9 μm, or less than about 10 μm. In some embodiments, the width is between about 3 μm and about 10 μm. In some embodiments, the width is about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In some embodiments, the width of the constriction is about 6 μm.

[0140] In some embodiments, the depth of the constriction is at least about 1 μm. In some embodiments, the depth of the constriction is at least about 1 μm, at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 110 μm, or at least about 120 μm. In some embodiments, the depth is from about 5 μm to about 90 μm. In some embodiments, the depth is about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or about 90 μm. In some embodiments, the depth of the constriction is about 70 μm.

[0141] In some embodiments, the length is about 10 μm, the width is about 6 μm, and the depth is about 70 μm.

[0142] Further examples of parameters that may affect payload delivery to cells include, but are not limited to, the dimensions of the constriction (e.g., length, width, and / or depth), the inlet angle of the constriction, the surface properties of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity), the operating flow rate, the payload concentration, the time for the cells to recover, or combinations thereof. Further parameters that may affect the delivery efficiency of the payload (e.g., reprogramming factors) include the velocity of the cells in the constriction, the shear rate in the constriction, the viscosity of the cell suspension, the velocity component perpendicular to the flow rate, and the time in the constriction. Such parameters can be designed to control payload delivery.

[0143] In some embodiments, the temperature used in the methods of the present disclosure may also affect the delivery efficiency of the payload to the cells, as well as the viability of the cells. In some embodiments, the squeezing process method is performed between about -5°C and about 45°C. For example, the method can be performed at room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), a temperature higher than physiological temperature (e.g., greater than about 37°C to 45°C or higher), or a low temperature (e.g., about -5°C to about 4°C), or a temperature between these exemplary temperatures.

[0144] A variety of methods can be utilized to drive the cells to pass through the constriction. For example, pressure can be applied with an inlet-side pump (e.g., a gas cylinder, a compressor), a vacuum can be applied with an outlet-side vacuum pump, capillary action can be applied via a tube, and / or gravity can be supplied to the system. Displacement-based flow systems (e.g., syringe pumps, peristaltic pumps, manual syringes or pipettes, pistons, etc.) can also be used. In some embodiments, the cells pass through the constriction by positive pressure. In some embodiments, the cells pass through the constriction by a constant pressure or a variable pressure. In some embodiments, the pressure is applied using a syringe. In some embodiments, the pressure is applied using a pump. In some embodiments, the pump is a peristaltic pump or a diaphragm pump. In some embodiments, the pressure is applied using a vacuum. In some embodiments, the cells pass through the constriction by g-force. In some embodiments, the cells pass through the constriction by capillary pressure.

[0145] In some embodiments, cells pass through a constriction by a fluid flow. In some embodiments, the fluid flow is a turbulent flow before the cells pass through the constriction. Turbulent flow is a fluid flow in which the magnitude and direction of the velocity at a given point change irregularly. In some embodiments, the fluid flow passing through the constriction is a laminar flow. Laminar flow is accompanied by a continuous flow in the fluid near a solid boundary, and the direction of the flow remains constant at any point. In some embodiments, the fluid flow is a turbulent flow after the cells pass through the constriction. The speed at which the cells pass through the constriction can be varied. In some embodiments, the cells pass through the constriction at a uniform cell speed. In some embodiments, the cells pass through the constriction at a varying cell speed.

[0146] In some embodiments, a combination treatment such as exposing to an electric field downstream of the constriction, for example, after the methods described herein, is used to deliver a payload. In some embodiments, the cells pass through an electric field generated by at least one electrode after passing through the constriction. In some embodiments, the electric field aids in the delivery of the payload to a second location within the cell, such as the cell nucleus. In some embodiments, one or more electrodes are proximate to the cell deformation constriction to generate the electric field. In some embodiments, the electric field is between about 0.1 kV / m and about 100 MV / m. In some embodiments, an integrated circuit is used to provide an electrical signal for driving the electrodes. In some embodiments, the cells are exposed to the electric field with a pulse width between about 1 nsec and about 1 sec and a period between about 100 ns and about 10 s.

[0147] III.F. Therapeutic Uses In some aspects, the present disclosure relates to the use of cells produced using the squeezing method described herein for treating various diseases or disorders. As will be apparent from the present disclosure, the methods and compositions provided herein may be useful for diseases and disorders for which cell replacement therapy can be used as a treatment. By replacing damaged cells with cells produced using the methods provided herein, in some aspects, one or more functions associated with the damaged cells are restored, thereby treating the disease or disorder. For example, in some aspects, neurons produced using the squeezing method provided herein can be administered to a subject suffering from neuropathy. Administration of such neurons may be useful for improving one or more symptoms associated with neuropathy.

[0148] As used herein, the terms "neuropathy" and "neuroimmune disorder" can be used interchangeably and refer to any disease and disorder of the central or peripheral nervous system. Unless otherwise specified, the terms "neuropathy" and "neuroimmune disorder" include all diseases or disorders of the nervous system, including autoimmune disorders. Non-limiting examples of neuropathies that can be treated by the present disclosure include brain tumors, neoplastic meningitis, leptomeningeal carcinomatosis (LMD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease (PD), Huntington's disease (HD), Alzheimer's disease (AD), or combinations thereof. In some embodiments, the neuropathy is Parkinson's disease.

[0149] IV. Compositions of the Present Disclosure In some aspects, the present disclosure provides a system for delivering a payload (e.g., a reprogramming factor) into a cell, the system comprising a microfluidic channel described herein, a cell suspension comprising a plurality of cells and the payload; wherein the constriction is configured such that the plurality of cells can pass through the microfluidic channel, and wherein passage of the plurality of cells causes deformation and disruption of the cell membranes of the cells, thereby enabling the payload to enter the cells.

[0150] In some aspects, the present disclosure provides a system for delivering a payload, comprising a surface having pores, a plurality of cells, and a cell suspension containing the payload; wherein the surface having pores is configured such that the plurality of cells can pass through the pores, and wherein passage of the plurality of cells causes deformation and disruption of the cell membranes of the cells, thereby enabling the payload to enter the cells. In some aspects, the surface is a filter or a membrane. In some aspects of the above aspects, the system further comprises at least one electrode for generating an electric field. In some aspects, the system is used to deliver a payload into cells by any of the methods described herein. The system can include any of the aspects described above for the methods disclosed above, including cell deformation constrictions, cell suspensions, cell perturbations, microfluidic channels, or surfaces having pores for providing delivery parameters. In some aspects, delivery parameters such as operating flow rate, cell and compound concentrations, velocity of cells within the constriction, and composition of the cell suspension (e.g., osmotic pressure, salt concentration, serum content, cell concentration, pH, etc.) are optimized for delivering a payload (e.g., a reprogramming factor) into cells.

[0151] In some aspects, the disclosure provides cells (e.g., neurons) produced using any of the methods provided herein. In some aspects, provided herein are cells that include a perturbation in the cell membrane, where the perturbation is by one or more parameters that deform the cell (e.g., the delivery parameters described herein), whereby a perturbation occurs in the cell membrane of the cell, and thus a payload (e.g., a reprogramming factor) can enter the cell. In some aspects, provided herein are cells that include a payload (e.g., a reprogramming factor), where the payload enters the cell through a perturbation of the cell membrane, and the perturbation is by one or more parameters that deform the cell (e.g., the delivery parameters described herein), whereby a perturbation occurs in the cell membrane of the cell and the payload enters the cell. In some aspects, such cells can include any of the cells described herein (e.g., stem cells or PBMCs).

[0152] In some aspects, the disclosure provides a composition comprising a plurality of cells, the plurality of cells produced by any of the methods described herein. Also provided herein is a composition comprising a population of cells and a payload (e.g., a reprogramming factor) under one or more parameters, such that one or more cells of the population of cells are deformed, whereby a perturbation occurs in the cell membrane of the one or more cells, where the perturbation of the cell membrane enables the payload to enter the one or more cells.

[0153] Also provided are kits or articles of manufacture for use in delivering a payload (e.g., a reprogramming factor) described herein into a cell. In some embodiments, the kit comprises a composition (e.g., a pore, a cell suspension, and / or a microfluidic channel or surface containing the payload) described herein in a suitable package. Suitable packaging materials are known in the art and include, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packages (such as sealed Mylar or plastic bags), and the like. These articles of manufacture can be further sterilized and / or sealed.

[0154] The present disclosure also provides kits that include the components of the methods described herein, and can further include instructions (s) for performing the methods for delivering a payload (e.g., a reprogramming factor) into a cell. The kits described herein can further include other buffers, diluents, filters, needles, syringes, and articles of manufacture with instructions for performing any of the methods described herein; for example, instructions for delivering a payload into a cell, and other materials.

[0155] The following examples are presented by way of illustration and not by way of limitation.

Example

[0156] Example 1: Analysis of GFP-Puro SaRNA Expression and Duration in iPSCs To determine the GFP expression and duration of GFP-Puro SaRNA, iPSCs were treated with Accutase and dissociated into single cells. The iPSCs were seeded at 1×10 8It was prepared at a density of cells / mL. Next, the prepared iPSCs were combined with GFP-Puro SaRNA (1.5 mg / ml or 0.3 mg / ml). The cell suspension was added to a constricted channel with the following dimensions: length 10 μm, width 6 μm, and depth 70 μm at room temperature in mTeSR™ Plus basal medium. Cells squeezed with GFP mRNA (non-self-amplifying) (0.125 mg / ml) were used as a positive control, and cells squeezed without cargo were used as a negative control. The concentrations of GFP-Puro SaRNA and GFP mRNA were adjusted based on their sizes so that the molecular concentrations were equal. After squeezing, the squeeze-loaded iPSCs were transferred to mTeSR™ Plus basal medium containing supplements and incubated at 37°C. A subset of iPSCs delivered with GFP-Puro SaRNA was treated with 5 μg / ml puromycin from day 1 to day 9. (See Figure 1A). Cells were collected on days 1, 3, 9, 15, and 21 for analysis of the green fluorescence signal by flow cytometry.

[0157] As shown in Figures 1B - 1D, GFP mRNA and GFP SaRNA had similar squeeze delivery efficiencies. However, the GFP MFI was more than 20-fold higher for saRNA compared to mRNA on day 1. Furthermore, puromycin selection was highly effective (95%+ GFP positive), which extended GFP expression beyond 9 days of culture. After removal of puromycin, the GFP% decreased from 95% (day 9) to 6% (day 21), suggesting that cells lost GFP-Puro saRNA in the absence of puromycin selection. The ratio of GFP+ saRNA cells to non-delivered cells decreased from days 1 - 3, but the MFI of GFP+ mRNA cells remained the same, suggesting a growth disadvantage for saRNA-delivered iPSCs. The MFI of GFP-positive cells remained high for GFP-Puro SaRNA on day 21, comparable to GFP mRNA on day 1.

[0158] Overall, these results indicate that GFP SaRNA was expressed at higher levels and for a longer period compared to GFP mRNA.

[0159] Example 2: Analysis of cells derived from Ascl1 saRNA after squeeze treatment To evaluate whether the differentiation of dopamine neurons can be induced by delivering Ascl1 saRNA into cells using the squeeze treatment method described herein, iPSCs were treated with Accutase and dissociated into single cells. Next, the iPSCs were prepared at a density of 1×10 8 cells / mL and combined in a cell suspension under any of the following conditions: (1) GFP saRNA; (2) Ascl1 saRNA; (3) Ascl1 saRNA + 5TF (FoxA2, Lmx1a, NR4A2, Pitx3, and EN1) mRNA (non-self-amplifying). The cell suspension was applied to a constriction of the same dimensions as described in Example 1 at a pressure of 60 psi at room temperature. After the cells passed through the constriction, the cells were collected and transferred to StemFlex basal medium containing supplements and incubated at 37°C for 6 hours. After 6 hours, the cells were washed with PBS to remove unwanted debris. A 1:1 mixture of StemFlex basal medium and N2B medium + B27 (100X) supplemented with 5 μg / ml puromycin was added. The mixture was incubated for 18 hours. Next, the medium was removed, and a mixture of NBM medium + B27 (50×) + BDNF + GDNF (1:1000) was added. Total RNA was collected from the cells on day 1 and day 4, and RT-qPCR was used to analyze the expression of general neuron markers (NeuroD1) and dopaminergic markers (FoxA2, Pitx3, Lmx1a, NR4A2, and TH).

[0160] As shown in Figures 2 and 3, all markers were upregulated compared to GFP SaRNA. Most markers had higher expression on day 4 than on day 1. All dopamine markers tested were higher in co-delivery of Ascl1 saRNA with 5TF (FoxA2, Lmx1a, NR4A2, Pitx3, and EN1) mRNA compared to Ascl1 saRNA alone, suggesting an additional effect from the 5TF mRNA.

[0161] Example 3: Analysis of Ascl1 saRNA-derived Dopamine Neurons after Squeezing Treatment To evaluate whether dopamine neurons can be generated by delivering Ascl1 saRNA into cells using the squeezing treatment method described herein, iPSCs were treated with Accutase and dissociated into single cells. Next, the iPSCs were prepared at a density of 1×108 cells / mL and combined with Ascl1 saRNA + 5TF (FoxA2, Lmx1a, NR4A2, Pitx3, and EN1) mRNA (non-self-amplifying) in the cell suspension. The cell suspension was added to a constriction of the same dimensions as described in Example 1 at room temperature with a pressure of 60 psi. After the cells passed through the constriction, the cells were collected and transferred to StemFlex basal medium containing supplements and incubated at 37°C for 6 hours. After 6 hours, the cells were washed with PBS to remove unwanted debris. A 1:1 mixture of StemFlex basal medium and N2B medium + B27 (100X) supplemented with 5 μg / ml puromycin was added. The mixture was incubated for 18 hours. Next, the medium was removed and a mixture of NBM medium + B27 (50X) + BDNF and GDNF (1:1000) was added. Half of the medium was removed every 2 days and NBM medium + B27 (50X) + BDNF and GDNF (1:1000) were added. On day 14, the cells were fixed and immunofluorescent staining was performed for the dopamine neuron marker tyrosine hydroxylase TH, the early neuron marker TUJ1, and the mature neuron marker MAP2.

[0162] As shown in Fig. 4, all neuron-like cells expressed the early neuron marker TUJ1. Some cells had high expression of the dopamine neuron marker tyrosine hydroxylase (green arrow), while some cells had low expression (red arrow). All neuron-like cells expressed the mature neuron marker MAP2, but some cells had high expression of the dopamine neuron marker tyrosine hydroxylase TH (green arrow), while some cells had low expression of TH (red arrow). These results further supported previous data and demonstrated that delivery of saRNA using squeeze treatment could effectively induce differentiation of iPSCs into neurons. Example 4: Analysis of kinetic differences between mRNA (non-self-amplifying) and saRNA after squeeze treatment

[0163] To evaluate the kinetic differences between mRNA and saRNA, iPSCs were treated with Accutase and dissociated into single cells. Next, the iPSCs were adjusted to 1×10 8Prepared at a density of cells / mL and combined in a cell suspension under any of the following conditions: (1) GFP saRNA; (2) 6TF (Ascl1, FoxA2, Lmx1a, NR4A2, Pitx3, and EN1) + PAC mRNA (non-self-amplifying) (3) Ascl1 saRNA; (4) Ascl1 SaRNA + 6TF (Ascl1, FoxA2, Lmx1a, NR4A2, Pitx3, and EN1) mRNA (non-self-amplifying). The cell suspension was added to a constriction of the same dimensions as described in Example 1 at room temperature at a pressure of 60 psi. After the cells passed through the constriction, the cells were collected and transferred to StemFlex basal medium containing supplements and incubated at 37 °C for 6 hours. After 6 hours, the cells were washed with PBS to remove unwanted debris. A 1:1 mixture of StemFlex basal medium and N2B medium + B27 (100X) supplemented with 5 μg / ml puromycin was added. The mixture was incubated for 18 hours. Next, the medium was removed, and it was a mixture of NBM medium + B27 (50X) + BDNF + GDNF (1:1000). Total RNA was collected from the cells on day 1 and day 4, and RT-qPCR was used to analyze the expression of a general neuron marker (NeuroD1) and dopaminergic markers (FoxA2, Pitx3, Lmx1a, NR4A2, and TH).

[0164] As shown in Figures 5 and 6, most markers had higher expression of the target gene on day 1 compared to day 4 for 6TF mRNA, indicating the transient nature of the mRNA. In samples containing saRNA, most target genes increased from day 1 to day 4, indicating the effect of long-term expression from saRNA.

[0165] In summary, the above results demonstrate that saRNA encoding reprogramming factors enables long-term expression of the encoded reprogramming factors and effective differentiation of iPSCs into neurons, especially when delivered using the squeeze treatment method.

[0166] Incorporation by reference All of the published documents, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual published document, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0167] equivalent Although various specific embodiments have been illustrated and described, the above specification is not limiting. It will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s). Many variations will be apparent to those skilled in the art in light of this specification.

Claims

1. A method for reprogramming cells, comprising passing a cell suspension containing the cells through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters causes a perturbation within the cell membrane, so that self-amplified RNA encoding a reprogramming factor can enter the cells through the perturbation when it comes into contact with the cells, thereby reprogramming the cells.

2. A method for enhancing cell reprogramming, comprising passing a cell suspension containing the cells through a constriction under a set of parameters, wherein passing the cell suspension through the constriction under the set of parameters causes perturbations within the cell membrane, so that self-amplified RNA encoding reprogramming factors can enter the cells through the perturbations when it comes into contact with the cells, thereby enhancing the cell reprogramming compared to a reference method. The reference method includes passing the cell suspension through the constriction under the set of parameters, and contacting the cell suspension with non-self-amplified RNA encoding the reprogramming factor, or The reference method, wherein the reference method does not include passing the cell suspension through the constriction under the set of parameters, where the cell suspension comes into contact with the self-amplified RNA.

3. The method according to claim 2, wherein the enhancement of the reprogramming of the cells includes (i) an increase in the number of reprogrammed cells, (ii) a reduction in the duration required for cell reprogramming, or (iii) both (i) and (ii).

4. The method according to claim 1, wherein the reprogramming of the cells includes inducing the cells to differentiate into neurons.

5. The method according to claim 1, further comprising contacting the cell suspension with the self-amplified RNA before, during, or after its passage through the constricted portion of the cell suspension.

6. The method according to claim 1, wherein the reprogramming factor includes a transcription factor.

7. The method according to claim 6, wherein the reprogramming factor comprises neurogenin 2 (Ngn2, Neurog2), Atonal BHLH transcription factor 1 (Atoh1), Achaete-Scute family BHLH transcription factor 1 (Ascl1), nuclear receptor subfamily 4 group A member 2 (NR4A2), LIM homeobox transcription factor 1 alpha (Lmx1a), Engraved homeobox (homobox) 1 (EN1), POU class 3 homeobox 2 (POU3F2; Brn2), myelin transcription factor 1-like (Myt1l), forkhead box A2 (Foxa2), paired-like homeodomain 3 (Pitx3), SRY box transcription factor 2 (SOX2), microRNA 124 (mir124), or a combination thereof.

8. The method according to claim 1, further comprising bringing the cell suspension into contact with a payload, thereby allowing the payload to enter the cells through the perturbation when the payload comes into contact with the cells.

9. The method according to claim 8, comprising bringing the cell suspension into contact with the payload before, during, or after its passage through the constricted portion of the cell suspension.

10. The method according to claim 8, wherein the self-amplified RNA and the payload come into contact with the cell sequentially or simultaneously.

11. The aforementioned payload, (i) nucleic acids, polypeptides, lipids, carbohydrates, small molecules, metal-containing compounds, antibodies, transcription factors, nanoparticles, liposomes, fluorescently labeled molecules, or combinations thereof, (ii) Additional reprogramming factors The method according to claim 8, including the method described in claim 8.

12. The method according to claim 11, wherein the additional reprogramming factor includes neurogenin 2 (Ngn2, Neurog2), Atonal BHLH transcription factor 1 (Atoh1), Achaete-Scute family BHLH transcription factor 1 (Ascl1), nuclear receptor subfamily 4 group A member 2 (NR4A2), LIM homeobox transcription factor 1 alpha (Lmx1a), Engraved homeobox (homobox) 1 (EN1), POU class 3 homeobox 2 (POU3F2; Brn2), myelin transcription factor 1-like (Myt1l), forkhead box A2 (Foxa2), paired-like homeodomain 3 (Pitx3), SRY box transcription factor 2 (SOX2), microRNA 124 (mir124), or a combination thereof.

13. The method according to any one of claims 1 to 12, wherein the cells include stem cells, somatic cells, or both.

14. (i) The stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells, tissue-specific stem cells, mesenchymal stem cells, or a combination thereof, and / or (ii) The somatic cells include blood cells, The method according to claim 13.

15. The method of claim 8, comprising (i) before the cell suspension passes through the constriction, (ii) during the cell suspension passes through the constriction, (iii) after the cell suspension passes through the constriction, or (iv) any combination of (i) to (iii).

16. (i) Each of the plurality of self-amplified RNAs is different, or (ii) At least two of the plurality of self-amplified RNAs are the same, (iii) Each of the above payloads is different, (iv) At least two of the above multiple payloads are the same, The method according to claim 15.

17. The method further includes passing the cell suspension through a plurality of constricted areas. (i) Each of the multiple constrictions is the same, (ii) One or more of the aforementioned constricted portions are different, (iii) Each of the multiple constrictions is associated with the same self-amplified RNA, or (iv) One or more of the constricted portions are associated with different self-amplified RNAs, The method according to claim 1.

18. The method according to claim 1, further comprising contacting the cell suspension with an additional compound, wherein the additional compound is a nucleic acid encoding an enzyme that confers resistance to an antibiotic.

19. The method according to claim 18, further comprising collecting the cell suspension that has passed through the constricted portion and treating the cell suspension with the antibiotic.

20. (i) The reprogramming of the cells includes inducing the cells to differentiate into neurons, (ii) The reprogramming factor includes and / or a transcription factor. (iii) The reprogramming factor includes neurogenin 2 (Ngn2, Neurog2), Atonal BHLH transcription factor 1 (Atoh1), Achaete-Scute family BHLH transcription factor 1 (Ascl1), nuclear receptor subfamily 4 group A member 2 (NR4A2), LIM homeobox transcription factor 1 alpha (Lmx1a), Engraved homeobox (homobox) 1 (EN1), POU class 3 homeobox 2 (POU3F2; Brn2), myelin transcription factor 1-like (Myt1l), forkhead box A2 (Foxa2), paired-like homeodomain 3 (Pitx3), SRY box transcription factor 2 (SOX2), microRNA 124 (mir124), or a combination thereof. The method according to claim 15.

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