Reprogramming cells using self-amplifying RNA

Abstract

The present disclosure provides a method for reprogramming a cell wherein the method comprises passing a cell suspension comprising the cell and a self-expanding RNA encoding a reprogramming factor through a stenosis wherein the stenosis deforms the cell, resulting in perturbation of the cell such that the reprogramming factor enters the cell.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 345,339, filed on May 24, 2022, which is incorporated herein by reference in its entirety. Field of the Invention

[0003] The present disclosure generally relates to methods of reprogramming cells to differentiate into different cell types (eg, neurons) by using one or more constrictions and self-amplifying RNA encoding reprogramming factors. Background Art

[0004] Regenerative medicine based on cell replacement is about to have a significant health impact. However, there are still major challenges, especially in manufacturing consistent, efficient and cost-effective functional transplantable cells. Although induced pluripotent stem cell (iPSC) differentiation and somatic cell transdifferentiation (i.e., direct reprogramming) can produce various cells of interest, current methods are generally cumbersome and inefficient. For example, currently available iPSC differentiation methods usually take up to several weeks and even take several months in some cases to obtain the desired final cell type. In addition, the resulting cell product is inevitably a heterogeneous cell population with the desired cells present at a wide range of frequencies. Similarly, with somatic cell transdifferentiation, reprogramming by lentiviral vectors is useful, but this method has the risk of insertion mutagenesis. And, by methods such as electroporation and lipid transfection, it can adversely affect the recovery of cell health and sensitive primary cells, and cause other cytotoxicity problems. Therefore, there is still a need to deliver differentiation or reprogramming factors to generate more effective and cost-effective methods for cells that can be used for various therapeutic applications (such as in regenerative medicine). Summary of the invention

[0005] A method for inducing expression of reprogramming factors in cells is provided, comprising passing a cell suspension comprising cells through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes perturbations within the cell membrane, such that self-amplifying RNA encoding the reprogramming factors can enter the cells through the perturbations when in contact with the cells, and thereby cause the reprogramming factors to be expressed in the cells.

[0006] In some aspects, the reprogramming factors are 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 by perturbation. In some aspects, after the self-amplifying RNA enters the cell through perturbation, the expression of the reprogramming factor in the cell increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times.

[0007] Also provided is a method for enhancing the expression of reprogramming factors in cells, comprising passing a cell suspension comprising cells through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes perturbations within the cell membrane, such that self-amplifying RNA encoding the reprogramming factor can enter the cell through the perturbation when in contact with the cell, and thereby result in enhanced expression of the reprogramming factor compared to the corresponding expression in a reference cell.

[0008] In some aspects, the reference cell includes a corresponding cell that passes through the stenosis under the set of parameters, and wherein the reference cell contacts a non-self-amplifying RNA encoding a reprogramming factor. In some aspects, the reference cell includes a corresponding cell that does not pass through the stenosis under the set of parameters, and wherein the reference cell contacts a self-amplifying RNA encoding a reprogramming factor.

[0009] In some aspects, expression enhancement includes (i) expression level increase, (ii) expression duration is longer, or (iii) (i) and (ii) both. In some aspects, the expression level of reprogramming factor in cell increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times or at least about 500 times compared with the expression in reference cell. In some aspects, the duration of expression of the reprogramming factor in the cell is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, or at least about 500 times compared to the duration of expression in a reference cell.

[0010] The present disclosure also provides a method for reprogramming cells, comprising passing a cell suspension containing cells through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes perturbations within the cell membrane, such that self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbation when in contact with the cell, and thereby reprogram the cell.

[0011] In some aspects, the reprogramming factors are 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 by perturbation. In some aspects, after the self-amplifying RNA enters the cell through perturbation, the expression of the reprogramming factor in the cell increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times.

[0012] Some aspects of the present disclosure also provide a method for enhancing cell reprogramming, comprising passing a cell suspension comprising cells through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes a perturbation within the cell membrane, such that self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbation when in contact with the cell, and thereby resulting in enhanced reprogramming of the cell compared to a reference method.

[0013] In some aspects, the reference method includes passing a cell suspension through a stenosis under the set of parameters, and contacting the cell suspension with a non-self-amplifying RNA encoding a reprogramming factor. In some aspects, the reference method does not include passing a cell suspension through a stenosis under the set of parameters, and wherein contacting the cell suspension with a self-amplifying RNA.

[0014] In some aspects, cell reprogramming enhancement includes (i) a greater number of cells that have been reprogrammed, (ii) a shorter duration required to reprogram cells, or (iii) both (i) and (ii). In some aspects, the number of cells that have been reprogrammed is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, or at least about 500 times compared to a reference method. In some aspects, 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 a reference method.

[0015] In some aspects, the reprogramming of cells comprises inducing the cells to differentiate into neurons. In some aspects, the reprogramming of cells comprises inducing the expression of other reprogramming factors in the cells.

[0016] Also provided is a method for producing neurons, comprising passing a cell suspension through a stenosis under a set of parameters, wherein passing a cell suspension through a stenosis under the set of parameters causes a perturbation in a cell membrane present in the cell suspension, such that a self-amplifying RNA encoding a reprogramming factor can enter the cell by the perturbation when in contact with the cell, and thereby causing the cell to differentiate into a neuron. Provided herein is a method for enhancing the production of neurons, comprising passing a cell suspension through a stenosis under a set of parameters, wherein passing a cell suspension through a stenosis under the set of parameters causes a perturbation in a cell membrane present in the cell suspension, such that a self-amplifying RNA encoding a reprogramming factor can enter the cell by the perturbation when in contact with the cell, and thereby causing enhanced neuron production compared to a reference method.

[0017] In some aspects, the reference method includes passing a cell suspension through a stenosis under the set of parameters, and contacting the cell suspension with a non-self-amplifying RNA encoding a reprogramming factor. In some aspects, the reference method does not include passing a cell suspension through a stenosis under the set of parameters, and wherein contacting the cell suspension with a self-amplifying RNA.

[0018] For any method provided herein (e.g., such as those described above), in some aspects, the method also includes contacting the cell suspension with a self-amplification RNA before passing the cell suspension through the stenosis. In some aspects, the cell is contacted with a self-amplification RNA before passing the cell suspension through the stenosis. In some aspects, the method also includes contacting the cell suspension with a self-amplification RNA during passing the cell suspension through the stenosis. In some aspects, the cell is contacted with a self-amplification RNA during passing the cell suspension through the stenosis. In some aspects, the method also includes contacting the cell suspension with a self-amplification RNA after passing the cell suspension through the stenosis. In some aspects, the cell is contacted with a self-amplification RNA after passing the cell suspension through the stenosis.

[0019] For any method provided herein (for example, such as those described above), in some aspects, reprogramming factors include transcription factors. In some aspects, 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 1α (Lmx1a), Engrailed homeobox 1 (EN1), POU category 3 homeobox 2 (POU3F2; Brn2), myelin transcription factor 1 sample (Myt1l), forkhead box (Forkhead Box) A2 (Foxa2), paired-like homeodomain 3 (Pitx3), SRY box transcription factor 2 (SOX2), microRNA 124 (mir124) or a combination thereof.

[0020] For any method provided herein (e.g., such as those described above), in some aspects, the method further comprises contacting a cell suspension with a payload so that the payload can also enter the cell by disturbance when in contact with the cell. In some aspects, the method comprises contacting the cell suspension with a payload before passing the cell suspension through the stenosis. In some aspects, the cell is contacted with a payload before passing the cell suspension through the stenosis. In some aspects, the method further comprises contacting the cell suspension with a payload during passing the cell suspension through the stenosis. In some aspects, the cell is contacted with a payload during passing the cell suspension through the stenosis. In some aspects, the method comprises contacting the cell suspension with a payload after passing the cell suspension through the stenosis. In some aspects, the cell is contacted with a payload after passing the cell suspension through the stenosis.

[0021] When cells are contacted with a payload and saRNA simultaneously, in some aspects, the self-amplifying RNA and the payload are contacted with the cells simultaneously. In some aspects, the self-amplifying RNA and the payload are contacted with the cells sequentially.

[0022] In some aspects, the payload includes nucleic acid, polypeptide, lipid, carbohydrate, small molecule, metal-containing compound, antibody, transcription factor, nanoparticle, liposome, fluorescent marker molecule or a combination thereof. In some aspects, nucleic acid includes DNA, RNA or both. In some aspects, DNA includes recombinant DNA, cDNA, genomic DNA or a combination thereof. In some aspects, RNA includes siRNA, non-self-amplifying mRNA, microRNA (miRNA), lncRNA, tRNA, shRNA, self-amplifying mRNA (saRNA) or a combination thereof. In some aspects, small molecules include impermeable small molecules.

[0023] In some aspects, the payload includes additional reprogramming factors. In some aspects, 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 category 3 homeobox 2 (POU3F2; Brn2), myelin transcription factor 1-like (Myt1l), forkhead box (Forkhead Box) A2 (Foxa2), paired-like homeodomain 3 (Pitx3), SRY box transcription factor 2 (SOX2), microRNA 124 (mir124) or a combination thereof.

[0024] For any method provided herein (e.g., such as those described above), in some aspects, cells include stem cells, somatic cells, or both. In some aspects, stem cells are induced pluripotent stem cells (iPSC), embryonic stem cells, tissue-specific stem cells, mesenchymal stem cells, or a combination thereof. In some aspects, stem cells are iPSC. In some aspects, somatic cells include blood cells. In some aspects, blood cells are PBMCs. In some aspects, PBMCs include immune cells. In some aspects, immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells (DC), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells, or a combination thereof.

[0025] For any of the methods provided herein (e.g., such as those described above), in some aspects, the set of parameters is selected from cell density; pressure; length, width, and / or depth of the stenosis; diameter of the stenosis; diameter of the cells; temperature; entrance angle of the stenosis; exit angle of the stenosis; length, width, and / or width of the approach zone; surface properties of the stenosis (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity); operating flow rate; payload concentration; viscosity, osmotic pressure, salt concentration, serum content, and / or pH of the cell suspension; time in the stenosis; shear rate in the stenosis; type of payload, or a combination thereof. In some aspects, the cell density is at least about 6 x 10 7 cells / mL, at least about 7 x 10 7 cells / mL, at least about 8x10 7 cells / mL, at least about 9 x 10 7 cells / mL, at least about 1x 10 8 cells / mL, at least about 1.1 x 10 8 cells / mL, at least about 1.2 x 10 8 cells / mL, at least about 1.3 x 10 8 cells / mL, at least about 1.4 x 10 8 cells / mL, at least about 1.5x10 8 cells / mL, at least about 2.0 x 10 8 cells / mL, at least about 3.0 x 10 8 cells / mL, at least about 4.0 x 10 8 cells / mL, at least about 5.0 x 10 8 cells / mL, at least about 6.0 x 10 8 cells / mL, at least about 7.0 x 10 8 cells / mL, at least about 8.0 x 10 8cells / mL, at least about 9.0 x 10 8 cells / mL or at least about 1.0 x 10 9 In some aspects, 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.

[0026] For any method provided herein (e.g., such as those described above), in some aspects, the stenosis is contained in the microfluidic chip. In some aspects, the stenosis has a diameter of 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 in the cell population.

[0027] In some aspects, the length of the narrow is between about 0 μm and about 100 μm. In some aspects, the length of the narrow is less than about 1 μm. In some aspects, the length of the narrow 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 aspects, the length of the stenosis 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 aspects, the length of the stenosis is about 10 μm.

[0028] In some aspects, the narrow width is between about 0 μm and about 10 μm. In some aspects, the narrow width 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 aspects, the narrow width is between about 3 μm and about 10 μm. In some aspects, the narrow 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 aspects, the narrow width is about 6 μm.

[0029] In some aspects, the depth of the narrow is at least about 1 μm. In some aspects, the depth of the narrow 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 aspects, the depth of the narrow is about 5 μm to about 90 μm. In some aspects, the depth of the narrow 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 aspects, the depth of the narrow is about 70 μm.

[0030] In some aspects, the length of the stenosis is about 10 μm, the width of the stenosis is about 6 μm, and the depth of the stenosis is about 70 μm.

[0031] In some aspects, the method provided herein includes contacting a cell suspension with multiple self-amplification RNAs, multiple payloads, or both at the following time: (i) before the cell suspension is passed through stenosis, (ii) during the cell suspension is passed through stenosis, (iii) after the cell suspension is passed through stenosis, or (iv) (i) to any combination of (iii). In some aspects, cells are contacted with multiple self-amplification RNAs, and at least two or more of the multiple self-amplification RNAs can enter cells by disturbance. In some aspects, cells are contacted with multiple payloads, and at least two or more of the multiple payloads can enter cells by disturbance. In some aspects, multiple self-amplification RNAs include at least 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-amplification RNAs. In some aspects, each of the multiple self-amplification RNAs is different. In some aspects, at least two of the multiple self-amplification RNAs are identical.

[0032] In some aspects, the plurality of payloads comprises 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 aspects, each of the plurality of payloads is different. In some aspects, at least two of the plurality of payloads are the same.

[0033] In some aspects, multiple self-amplification RNAs are contacted with cells simultaneously. In some aspects, multiple self-amplification RNAs are contacted with cells in sequence. In some aspects, multiple payloads are contacted with cells simultaneously. In some aspects, multiple payloads are contacted with cells in sequence. In some aspects, multiple self-amplification RNAs and multiple payloads are contacted with cells simultaneously. In some aspects, multiple self-amplification RNAs and multiple payloads are contacted with cells in sequence.

[0034] For any of the methods provided herein (eg, such as those described above), in some aspects, the method comprises passing the cell suspension through a plurality of constrictions. In some aspects, a plurality of stenoses 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, 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 individual stenoses. In some aspects, each of the plurality of stenoses is the same. In some aspects, one or more of the plurality of stenoses is different. In some aspects, one or more stenoses vary in length, depth, width, or a combination thereof.

[0035] In some aspects, each of the plurality of narrows is associated with the same self-amplifying RNA. In some aspects, one or more of the plurality of narrows are associated with different self-amplifying RNAs. In some aspects, a plurality of narrows include a first narrow associated with a first self-amplifying RNA and a second narrow associated with a second amplified RNA, wherein a cell suspension is passed through the first narrow so that the first self-amplifying RNA can enter the cell, and then the cell suspension is passed through the second narrow so that the second self-amplifying RNA can enter the cell. In some aspects, 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 is passed through the first narrow, the cell suspension is passed through the second narrow.

[0036] For any method provided herein (for example, such as those described above), in some aspects, method also includes contacting cell suspension with additional compounds. In some aspects, additional compounds include nucleic acids, polypeptides, lipids, carbohydrates, small molecules, metal-containing compounds, antibodies, transcription factors, nanoparticles, liposomes, fluorescently labeled molecules or combinations thereof. In some aspects, additional compounds are nucleic acids encoding enzymes that confer antibiotic resistance. In some aspects, the contact of additional compounds and cell suspension occurs simultaneously with the contact of cell suspension and self-amplification RNA. In some aspects, the contact of additional compounds with cell suspension occurs before or after cell suspension contacts with self-amplification RNA.

[0037] In some respects, method also comprises collecting and passing narrow cell suspension and treating cell suspension with antibiotics.In some respects, enzyme comprises puromycin-N-acetyltransferase and antibiotic is puromycin.In some respects, after treating with antibiotics, the ratio of the cell that comprises self-amplification RNA that is present in cell suspension increases at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times or at least about 50 times.

[0038] Some aspects of the present disclosure relate to cell populations comprising reprogramming factors, wherein the cell population is produced using any method provided herein. Also provided herein is a reprogrammed cell population produced using any method of the present disclosure. The present disclosure also provides a neuron population produced using any method provided herein.

[0039] Provided herein are compositions comprising any of the cell populations, reprogrammed cell populations, and / or neuronal populations described herein. In some aspects, such compositions further comprise a pharmaceutically acceptable excipient.

[0040] Also provided herein is a kit comprising any of the cell populations, reprogrammed cell populations and / or neuronal populations described herein and instructions for use.

[0041] The present disclosure also provides a composition comprising a cell population and a self-amplifying RNA encoding a reprogramming factor that causes one or more cells in the cell population to deform under a set of parameters, wherein the one or more cells comprise a perturbation sufficient to allow the self-amplifying RNA to enter the cell membrane of the one or more cells. In some aspects, the composition further comprises a pharmaceutically acceptable excipient.

[0042] A cell is provided herein, comprising a disturbance in the cell membrane due to a set of parameters that deform the cell, thereby causing a disturbance in the cell membrane of the cell, so that the self-amplification RNA encoding the reprogramming factor can enter the cell. A cell is also provided herein, comprising a reprogramming factor, wherein the reprogramming factor enters the cell by a disturbance in the cell membrane due to a set of parameters that deform the cell, thereby causing a disturbance in the cell membrane of the cell, and allowing the self-amplification RNA encoding the reprogramming factor to enter the cell through the disturbance. A composition is provided herein, comprising such cells. In some aspects, this composition also comprises a pharmaceutically acceptable excipient.

[0043] Some aspects of the present disclosure provide a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject any composition provided herein. In some aspects, the disease or condition comprises a nervous system disorder. In some aspects, the nervous system disorder comprises Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figures 1A-1D The GFP expression dynamics in iPSC after squeezing delivery of GFP encoding saRNA or GFP encoding mRNA (i.e., non-self-amplification) are shown. In particular, the following groups of iPSCs are shown: (1) GFP encoding saRNA is received at a high dose (1.5 mg / mL) and then treated with puromycin (inverted triangle); (2) GFP encoding saRNA is received at a low dose (0.3 mg / mL) and then treated with puromycin (diamond); (3) only GFP encoding saRNA (i.e., no puromycin treatment) is received at a high dose (1.5 mg / mL) (square); (4) only GFP encoding saRNA (dark circles) is received at a low dose (0.3 mg / mL); and (5) only modified iPSCs (triangles) containing GFP encoding mRNA (i.e., non-self-amplification) are included. iPSCs squeezed without any payload are used as controls ("extrusion delivery only"; light circles). Flow cytometry is used to observe GFP expression on days 1, 3, 9, 15, and 21 after squeezing delivery. Figure 1A Provide a schematic diagram of the experimental design. Figure 1B The percentage of GFP expression in total iPSCs is shown. Figure 1C Shown in GFP + Mean fluorescence intensity (MFI) of GFP expression on iPSCs. Figure 1D Provided are flow cytometry histograms comparing GFP expression between different iPSCs.

[0045] Figure 2The expression of NeuroD1 (left), FoxA2 (middle), and NR4A2 (right) in iPSCs after squeeze delivery of one of the following: (1) Ascl1 encoding saRNA + five different mRNAs encoding FoxA2, ​​Lmx1A, NR4A2, Pitx3, and EN1 (triangles); (2) Ascl1 encoding saRNA alone (squares); and (3) GFP encoding saRNA alone (green). Expression was measured on days 1 and 4 after squeeze delivery and normalized to the fold increase of GFP encoding saRNA.

[0046] Figure 3 Shown is the expression of tyrosine hydroxylase (TH) (left), Lmx1A (middle), and Pitx3 (right) in iPSCs after squeeze delivery of one of the following: (1) Ascl1 encoding saRNA + five different mRNAs encoding FoxA2, ​​Lmx1A, NR4A2, Pitx3, and EN1 (triangles); (2) Ascl1 encoding saRNA alone (squares); and (3) GFP encoding saRNA alone (green). Expression was measured at days 1 and 4 after squeeze delivery and normalized to GFP encoding saRNA fold increase.

[0047] Figure 4 The expression of TUJ1 (early neuron marker), tyrosine hydroxylase (TH) (dopamine neuron marker) and MAP2 (mature neuron marker) in iPSCs after extrusion delivery of Ascl1 encoding saRNA + five different mRNAs encoding FoxA2, ​​Lmx1A, NR4A2, Pitx3 and EN1 is shown using immunofluorescence microscopy. In the first column, green and red arrows represent those iPSCs with high TH expression and low TH expression, respectively. In the second column, green arrows represent those iPSCs with high TH expression and further express TUJ1 (top frame) or MAP2 (bottom frame).

[0048] Figure 5Shown is the expression of NeuroD1 (left) and FoxA2 (right) in iPSCs after extrusion delivery of one of the following: (1) Ascl1 encoding saRNA + six different mRNAs encoding Ascl1, FoxA2, ​​Lmx1a, NR4A2, Pitx3 and EN1 (inverted triangles); (2) Ascl1 encoding saRNA alone (diamonds); (3) six different mRNAs encoding Ascl1, FoxA2, ​​Lmx1a, NR4A2, Pitx3 and EN1 + mRNA encoding puromycin N-acetyltransferase (PAC mRNA) (triangles); and (4) GFP encoding saRNA alone (circles). Expression was measured on days 1 and 4 after extrusion delivery and normalized to GFP encoding saRNA fold increase.

[0049] Figure 6 Shown is the expression of NR4A2 (left side 1st figure), Pitx3 (2nd figure), tyrosine hydroxylase (TH) (3rd figure) and Lmx1a (4th figure) in iPSC after squeeze delivery of one of the following: (1) Ascl1 encoding saRNA + six different mRNAs encoding Ascl1, FoxA2, ​​Lmx1a, NR4A2, Pitx3 and EN1 (inverted triangles); (2) Ascl1 encoding saRNA alone (diamonds); (3) six different mRNAs encoding Ascl1, FoxA2, ​​Lmx1a, NR4A2, Pitx3 and EN1 + mRNA encoding puromycin N-acetyltransferase (PAC mRNA) (triangles); and (4) GFP encoding saRNA alone (circles). Expression was measured on days 1 and 4 after squeeze delivery and normalized to GFP encoding saRNA fold increase. DETAILED DESCRIPTION

[0050] The present disclosure relates generally to methods for producing neurons.More specifically, methods provided herein include delivering the self-amplifying RNA encoding payload (e.g., reprogramming factor) intracellularly to cells (e.g., iPSC) so that cells are induced to differentiate into neurons. In some aspects, delivering the self-amplifying RNA intracellularly to cells includes passing a cell suspension comprising a cell colony through narrowing under one or more parameters so that the cell is temporarily deformed, thereby causing a disturbance of the cell membrane of the cell. This disturbance allows the self-amplifying RNA to enter the cell when contacting with the self-amplifying RNA.

[0051] As further described herein, the delivery method (extrusion delivery) of the present disclosure has certain unique characteristics that other delivery methods known in the art do not share. For example, in addition to improving the ability to deliver various types of payloads to cells, the extrusion treatment method described herein produces minimal lasting effects on cells. Compared with traditional delivery methods such as electroporation, the extrusion treatment method of the present disclosure retains the structural and functional integrity of extruded cells. In contrast to the delivery method provided herein, electroporation can induce extensive and lasting changes in gene expression, which can lead to delayed proliferation after nonspecific activation and antigen stimulation of cells (e.g., human T cells). Using the inventive method, any changes to cells (e.g., disturbances in the cell membrane) are temporary, and once cells are removed from stenosis, they will be quickly repaired. The present disclosure shows non-limiting examples of various aspects.

[0052] I. General Technology

[0053] Some of the techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly used using routine methods, such as, for example, the widely used methods described in the following literature: Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6th edition, J. Wiley and Sons, 2010); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods inMolecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (JPMather and PERoberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, J.Wiley and Sons, 1993-8); Handbook of Experimental Immunology (DMWeir and CC Blackwell, editors, 1996); GeneTransfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (J. E. Coligan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, ed., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., ed., JB Lippincott Company, 2011). .

[0054] II. Definitions

[0055] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth herein will prevail. Additional definitions are set forth throughout the detailed description.

[0056] As used herein, unless otherwise specified, the singular terms "a", "an", and "the" entity refer to one or more of the entity. Therefore, the terms "a" (or "an" or "the"), "one or more", and "at least one" can be used interchangeably herein.

[0057] It should be understood that the various aspects and aspects of the present disclosure described herein include various aspects of "comprising", "consisting of" and "consisting essentially of". It should also be understood that whenever various aspects are described herein with the wording "comprising", other similar various aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0058] In addition, when used herein, "and / or" should be regarded as a specific disclosure of each of two specific features or components with or without the other. Therefore, 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" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: 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).

[0059] For all compositions described herein and all methods using the compositions described herein, the composition may include the listed components or steps, or may "consist essentially of the listed components or steps". When a composition is described as "consisting essentially of the listed components", the composition contains the listed components and may also contain other components that do not have a substantial effect on the disclosed method, but does not contain any other components that have a substantial effect on the disclosed method other than those components explicitly listed; or if the composition contains additional components that have a substantial effect on the disclosed method other than the listed components, the composition does not contain the additional components in concentrations or amounts sufficient to have a substantial effect on the disclosed method. When a method is described as "consisting essentially of the listed steps", the method contains the listed steps and may also contain other steps that do not have a substantial effect on the disclosed method, but does not contain any other steps that have a substantial effect on the disclosed method other than the steps explicitly listed. As a non-limiting specific example, when a composition is described as "consisting essentially of a certain component", the composition may additionally contain any amount of pharmaceutically acceptable carriers, vehicles or diluents and other such components that do not have a substantial effect on the disclosed method.

[0060] Units, prefixes and symbols are expressed in a form acceptable to the Système International de Unites (SI). Numerical ranges include the number of defined ranges. The headings provided herein are not limitations on the various aspects of the present disclosure and can be obtained by reference to the entire specification. Therefore, the terms defined immediately below are more fully defined by reference to the entire specification.

[0061] As used herein, the term "about" means approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above or below the numerical values ​​recited. In general, the term "about" can modify a numerical value above or below the stated value, with a variation of, for example, plus or minus 10% (higher or lower).

[0062] As used herein, the term "stenosis" refers to a narrowed channel. In some aspects, the stenosis is a microfluidic channel, such as a microfluidic channel contained in a microfluidic device. In some aspects, the stenosis is a hole or is contained in a hole. When the stenosis is a hole, in some aspects, the hole is contained in the surface. Unless otherwise stated, the term stenosis refers to microfluidic channels and holes, as well as other suitable stenoses available in the art. Therefore, where appropriate, disclosures relating to microfluidic channels may also apply to holes and / or other suitable stenoses available in the art. Similarly, where appropriate, disclosures relating to holes may also apply to microfluidic channels and / or other suitable stenoses available in the art.

[0063] As used herein, the term "pore" refers to an opening, including but not limited to a hole, a crack, a cavity, a pore, a break, a gap, or a perforation in a material. In some aspects, (where indicated) the term refers to a pore in a microfluidic device surface, such as those described in the present disclosure. In some aspects, (where indicated) a pore may refer to a pore in a cell wall and / or a cell membrane.

[0064] As used herein, the term "membrane" refers to a selective barrier or sheet containing pores. The term includes, but is not limited to, a flexible sheet-like structure that acts as a border or lining. In some aspects, the term refers to a surface or filter containing pores. This term is different from the term "cell membrane," which refers to a semipermeable membrane that surrounds the cytoplasm of a cell.

[0065] As used herein, the term "filter" refers to a porous article that allows selective passage through the pores. In some aspects, the term refers to a surface or membrane containing pores.

[0066] As used herein, the terms "deform" and "deformity" (including derivatives thereof) refer to physical changes in cells. As described herein, when a cell passes through a narrow structure (such as those disclosed herein), it is subjected to various forces due to the restrictive physical environment, including but not limited to mechanical deformation forces and / or shear forces that cause disturbances in the cell membrane. As used herein, a "disturbance" in a cell membrane refers to any opening in the cell membrane that does not exist under normal steady state conditions (e.g., no deforming forces are applied to the cell). A disturbance may include a hole, a cleft, a cavity, a pore, a hole, a fracture, a gap, a perforation, or a combination thereof.

[0067] As used herein, the term "heterogeneous" refers to something that is mixed or non-uniform in structure or composition. In some aspects, the term refers to pores of different sizes, shapes, or distributions within a given surface.

[0068] As used herein, the term "homogeneous" refers to something that is consistent or uniform throughout a structure or composition. In some aspects, the term refers to pores of uniform size, shape, or distribution within a given surface.

[0069] As used herein, the term "polynucleotide" or "nucleic acid" refers to the polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, this term includes but is not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or polymers comprising purine bases and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The backbone of polynucleotides can include sugar and phosphate groups (as common in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of polynucleotides can include the polymer of synthetic subunits (such as phosphoramidates), and therefore can be oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. In addition, can be by synthesizing complementary chains and annealing chains under appropriate conditions, or by using DNA polymerase to synthesize complementary chains from the beginning with appropriate primers, double-stranded polynucleotides are obtained from the single-stranded polynucleotide products of chemical synthesis. As described herein, nucleic acids that can be delivered to cells using the extrusion treatment methods provided herein include RNA (e.g., mRNA). As used herein, "RNA" includes 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 be replicated in a host, thereby causing an increase in the amount of RNA and proteins encoded by RNA (e.g., reprogramming factors). As used herein, the term "mRNA" refers to any polynucleotide (self-amplifying or non-self-amplifying) encoding at least one polypeptide.

[0070] The terms "polypeptide" and "protein" can be used interchangeably to refer to amino acid residue polymers and are not limited to a minimum length. Such amino acid residue polymers may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. The definition encompasses full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of this disclosure, "polypeptide" refers to a protein comprising modifications to the native sequence, such as deletions, additions and substitutions (usually conservative properties), as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the protein or errors due to PCR amplification.

[0071] III. Methods of the Disclosure

[0072] In some aspects, the present disclosure relates to methods of delivering self-amplifying RNA (saRNA) to cells by passing the cells through a stenosis, such as those described herein. As demonstrated herein, when a cell passes through a stenosis, the cell temporarily deforms, causing the cell membrane of the cell to be disturbed. The disturbance within the cell membrane can allow various payloads, including self-amplifying RNA, to enter or be loaded into the cell (e.g., by diffusion). The specific process by which a cell passes through a stenosis and temporarily deforms is referred to herein as a "squeeze process" 、 "Extrusion delivery" or "extrusion".

[0073] As demonstrated herein, by the methods provided herein, different aspects of cells can be modified. In some aspects, by using saRNA encoding molecules (i.e., "reprogramming factors") that can reprogram cells, the delivery methods provided herein can be used to produce different cells of interest. As further described herein, in some aspects, the reprogramming factors that can be used for the present disclosure include differentiation factors. As used herein, the term "differentiation factor" refers to any agent that can induce cell differentiation into different types of cells. Unless otherwise indicated, the terms "reprogramming factor" and "differentiation factor" can be used interchangeably in the present disclosure. Therefore, as used herein, the terms "neuronal reprogramming factor" or "neuronal differentiation factor" can be used interchangeably and refer to agents that can reprogram and / or induce cell differentiation into neurons. As used herein, neuronal reprogramming factors are 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 known as nuclear receptor-related 1 protein (Nurr1)), LIM homeobox transcription factor 1 alpha (Lmx1a), POU category 3 homeobox 2 (POU3F2; Brn2), myelin transcription factor 1-like (Myt1l), forkhead box (Forkhead Box) A2 (Foxa2), paired-like homeodomain 3 (Pitx3), Engrailed homeobox 1 (EN1), SRY box transcription factor 2 (SOX2), microRNA 124 (mir124) or a combination thereof. In some aspects, the neuronal reprogramming factor is Ngn2. In some aspects, the neuronal reprogramming factor is Atoh1. In some aspects, the neuronal reprogramming factor is Ascl1. In some aspects, the neuronal reprogramming factor is NR4A2. In some aspects, the neuronal reprogramming factor is Nurr1. In some aspects, the neuronal reprogramming factor is Lmx1a. In some aspects, the neuronal reprogramming factor is POU3F2. In some aspects, the neuronal reprogramming factor is Myt11. In some aspects, the neuronal reprogramming factor is Foxa2. In some aspects, the neuronal reprogramming factor is Pitx3. In some aspects, the neuronal reprogramming factor is SOX2.

[0074] Although the above-mentioned transcription factors generally activate signal transduction pathways that induce neuronal differentiation, in some aspects, the neuronal reprogramming factors useful in the present disclosure may also inhibit signal transduction pathways, such as those that interfere with neuronal differentiation. Thus, in some aspects, the neuronal reprogramming factors include siRNA, such as siRNA that can inhibit p53 signaling. In some aspects, the neuronal reprogramming factors include miRNA (e.g., miR-214)

[0075] As further described and demonstrated herein, the reprogramming factors described above 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., polycistronic saRNA), and a single saRNA can be delivered to cells using extrusion delivery. In some aspects, multiple transcription factors can be delivered to cells respectively. For example, as demonstrated herein, the delivery method provided herein (e.g., extrusion treatment) can be used to deliver the first mRNA encoding the first transcription factor, the first mRNA encoding the transcription factor, the second mRNA encoding the transcription factor, the third mRNA encoding the transcription factor, the fourth mRNA encoding the transcription factor, and the fifth mRNA encoding the transcription factor. In some aspects, the delivery method provided herein (e.g., extrusion treatment) can be used to deliver the first transcription factor encoding saRNA and 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).

[0076] In some aspects, when it comes to a combination of reprogramming factors (or any other payload described herein), it can be delivered to cells using a single extrusion process, for example, a cell suspension comprises multiple payloads, which are delivered to cells in combination; "simultaneous delivery"). In some aspects, multiple payloads (e.g., a combination of saRNA encoding reprogramming factors and one or more additional payloads) can be delivered to cells in sequence. As used herein, the term "sequential delivery" refers to multiple payloads being delivered to cells, wherein a first payload (e.g., saRNA encoding a reprogramming factor) is delivered to cells, and then a second (or subsequent) payload (e.g., a non-self-amplifying RNA encoding a reprogramming factor) is delivered to cells. In some aspects, the first payload, the second payload, or the first and second payloads can be delivered to cells using an extrusion process. For example, in some aspects, the first payload can be delivered to cells using an extrusion process, and the second payload can be delivered to cells using a non-extrusion process (e.g., transfection). In some aspects, the first payload can be delivered to cells using a non-extrusion process (e.g., transfection), and the second payload can be delivered to cells using an extrusion process. In some aspects, a first payload can be delivered to a cell using a first extrusion, and then a second payload can be delivered to a cell using a second extrusion (also referred to herein as "sequential extrusion" or "sequential extrusion process"). Therefore, the sequential delivery that can be used for the present disclosure may include multiple extrusion processes. In some aspects, each of the multiple extrusion processes delivers a separate payload to a cell. In some aspects, one or more of the multiple extrusion processes do not involve the delivery of a payload. For example, in some aspects, the sequential delivery method described herein includes a first extrusion, a second extrusion, and a third extrusion, wherein the first extrusion includes passing a cell without any payload through a first stenosis, the second extrusion includes passing a cell from the first extrusion through a second stenosis to deliver a first payload (e.g., saRNA encoding a reprogramming factor) to the cell, and the third extrusion includes passing a cell from the second extrusion through a third stenosis to deliver a second payload to the cell. Without being bound by any one theory, in some aspects, passing a cell through a first stenosis (i.e., the first extrusion) without any payload can help prepare cells for subsequent payload delivery, for example, the delivery efficiency of the first payload and / or the second payload can be improved.

[0077] In some aspects, the combination of payloads can be repeatedly delivered to cells (e.g., stem cells or PBMCs). For example, in some aspects, a combination of transcription factors is delivered to cells using a first extrusion process; then, a second extrusion process is used to deliver the combination of transcription factors to cells again. In some aspects, the first extrusion process includes a microfluidic device (e.g., a chip) having multiple rows of stenosis, so that the extrusion process occurs on a single microfluidic device (e.g., a chip). As further described herein, in some aspects, the second extrusion process may occur immediately after the cell has been processed by the first extrusion process (e.g., immediately after the cell passes through the stenosis of the first extrusion process). In some aspects, the second extrusion process may occur after a certain time after the first extrusion process (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 passes through the stenosis of the first extrusion process).

[0078] Although the present disclosure generally discloses the use of saRNA encoding reprogramming factors, it is clear to those skilled in the art that the disclosures relating to such saRNAs may be equally applicable to other types of payloads. Therefore, as is clear from the present disclosure, the extrusion treatment or extrusion method provided herein may also be used to deliver additional cargo (also referred to herein as "payload") to cells. The present disclosure provides non-limiting examples of such payloads elsewhere. Unless otherwise indicated, the terms "payload" and "reprogramming factor" may be used interchangeably.

[0079] Therefore, in some aspects, a method for inducing expression of a payload (e.g., a reprogramming factor) in a cell is provided, comprising passing a cell suspension comprising cells through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes a disturbance in the cell membrane, such that the self-amplifying RNA encoding the payload can enter the cell by the disturbance when in contact with the cell, and thereby causing the payload to be expressed in the cell. As is clear from the present disclosure, in some aspects, the payload is a reprogramming factor.

[0080] In some aspects, the above methods allow for long-term expression of the encoded payload (e.g., reprogramming factors) when expressed in the cell. For example, in some aspects, after extrusion delivery of the saRNA to the cell (e.g., iPSC), the encoded payload is subsequently expressed in the cell 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 30 days, at least about 31 days, at least about 32 days, at least about 33 days, at least about 34 days, at least about 35 days, at least about 36 days, at least about 37 days, at least about 38 days, at least about 39 days, at least about 40 days, at least about 41 days, at least about 42 days, at least about 43 days, at least about 44 days, at least about 45 days, at least about 46 days, at least about 47 days, at least about 48 days, at least about 49 days, at least about 50 days, at least about 51 days, at least about At least about 8 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.

[0081] As demonstrated herein, the applicant has identified that, compared with other methods in the art, a specific combination of extrusion delivery and saRNA (also referred to herein as "delivery method provided herein" or its variants) more effectively induces the expression of payload (e.g., reprogramming factor) in cells. Therefore, in some aspects, a method for enhancing the expression of payload (e.g., reprogramming factor) in cells is provided herein, which includes passing a cell suspension containing cells through a narrowing under a set of parameters, wherein the cell suspension is passed through a narrowing under the set of parameters to cause a disturbance in the cell membrane, so that the self-amplification RNA encoding the payload can enter the cell by perturbation when contacting the cell, and thereby causing the expression of the payload to be enhanced compared to the corresponding expression in the reference cell. As used herein, the term "reference cell" refers to any cell (e.g., iPSC) that is not modified to include saRNA using extrusion delivery. For example, in some aspects, the reference cell includes a corresponding cell that passes through a narrowing under the set of parameters but is not contacted with saRNA. In some aspects, this reference cell is contacted with a non-self-amplification RNA (e.g., mRNA) encoding a payload. Thus, in some aspects, the reference cell comprises a corresponding cell (e.g., iPSC) that has undergone extrusion but is in contact with mRNA encoding a payload. In some aspects, the reference cell comprises a corresponding cell in contact with a self-amplifying RNA encoding a payload, wherein the reference cell has not been extruded. For example, in some aspects, the reference cell is contacted with saRNA using other delivery methods in the art (e.g., electroporation or lipofection).

[0082] In some aspects, expression of the encoded payload is increased (e.g., higher expression level) compared to a reference cell (e.g., a reference cell that has been extruded and contacted with a non-self-amplifying mRNA). In some aspects, the encoded payload is expressed in the cell for a longer duration compared to a reference cell (e.g., a reference cell that has been extruded and contacted with a non-self-amplifying mRNA). In some aspects, expression and duration of expression of the encoded payload is increased compared to a reference cell (e.g., a reference cell that has been extruded and contacted with a non-self-amplifying mRNA).

[0083] In some aspects, the expression level of the encoded payload (e.g., reprogramming factor) in 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 in a reference cell. In some aspects, the duration of expression of the encoded payload in the cell is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, or at least about 500 times compared to the duration of expression in a reference cell.

[0084] As is clear from the present disclosure, the method provided herein can be particularly effective in delivering reprogramming factors to cells (e.g., iPSC), and thus induce cell reprogramming.Therefore, in some respects, the present disclosure relates to a method for reprogramming cells, including making a cell suspension comprising cells pass through stenosis under a set of parameters, wherein making a cell suspension pass through stenosis under the set of parameters to cause disturbances in the cell membrane, so that the self-amplification RNA encoding reprogramming factors can enter cells by disturbances when contacting with cells, and thus reprogramming cells.In some respects, compared with other delivery methods in the art, the method provided herein allows for greatly enhancing the reprogramming of cells.Therefore, in some respects, a method for enhancing cell reprogramming is provided herein, including making a cell suspension comprising cells pass through stenosis under a set of parameters, wherein making a cell suspension pass through stenosis under the set of parameters to cause disturbances in the cell membrane, so that the self-amplification RNA encoding reprogramming factors can enter cells by disturbances when contacting with cells, and thus causing the reprogramming of cells to be enhanced compared with a reference method.

[0085] As used herein, the term "reference method" refers to a delivery method that does not include using extrusion treatment to deliver saRNA (e.g., encoding reprogramming factors) to cells (e.g., iPSC). For example, in some aspects, the reference method includes passing a cell suspension through a narrow under a set of parameters, but does not contact the cell with saRNA as described herein. In some aspects, this method includes contacting the cell with a non-self-amplification RNA (e.g., mRNA) encoding a payload (e.g., reprogramming factor). Therefore, in some aspects, the reference method includes passing a cell suspension through a narrow under the set of parameters, and contacting the cell suspension with a non-self-amplification RNA encoding a reprogramming factor. In some aspects, the reference method includes contacting cells (e.g., iPSC) with saRNA, but wherein the cell has not yet undergone extrusion treatment. For example, in some aspects, the reference method includes using other delivery methods (e.g., electroporation or lipofection) in the art to contact cells with saRNA.

[0086] In some aspects, compared with a reference method, the delivery method provided herein (e.g., a combination of extrusion treatment and saRNA) results in a larger number of reprogrammed cells. In some aspects, compared with a reference method, the delivery method provided herein requires a shorter duration to reprogram cells. In some aspects, compared with a reference method, the delivery method provided herein allows a larger number of reprogrammed cells and a shorter duration for reprogramming cells.

[0087] In some aspects, by delivering saRNA encoding a reprogramming factor to cells using an extrusion process, the number of cells that have been reprogrammed is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, or at least about 500 times, as compared to a reference method. In some aspects, by delivering saRNA encoding reprogramming factors to cells using an extrusion process, the duration required to reprogram cells 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 a reference method.

[0088] As is clear from the present disclosure, the methods provided herein can be used to generate many different types of cells. In some aspects, as demonstrated herein, the reprogramming factors described herein can induce cells (e.g., iPSCs) to differentiate into neurons. Therefore, in some aspects, a method for producing neurons is provided, comprising passing a cell suspension through a stenosis under a set of parameters, wherein passing a cell suspension through a stenosis under the set of parameters causes a disturbance in the cell membrane present in the cell suspension, so that the self-amplifying RNA encoding the reprogramming factor can enter the cell through the disturbance when in contact with the cell, and thereby causing the cell to differentiate into a neuron. Non-limiting

[0089] Also provided herein is a method for enhancing the generation of neurons, comprising passing a cell suspension through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes perturbations within cell membranes present in the cell suspension, such that self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbation when in contact with the cell, and thereby resulting in enhanced neuron generation compared to a reference method.

[0090] In some aspects, for example, compared to a reference method, using the methods provided herein (e.g., extrusion treatment using saRNA encoding reprogramming factors), the number of neurons produced can be increased. In some aspects, for example, compared to a reference method, using the methods provided herein, the time required to produce neurons can be reduced. In some aspects, using the methods provided herein, the number of neurons that can be produced and the duration required to produce neurons can be reduced.

[0091] In some aspects, the number of neurons generated using the methods provided herein (e.g., extrusion treatment using saRNA encoding a reprogramming factor) is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, or at least about 500 times, as compared to a reference method. In some aspects, the duration required to generate neurons using the methods provided herein (e.g., extrusion treatment using saRNA encoding a reprogramming factor) 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 a reference method.

[0092] Any suitable method known in the art can be used to assess the generation of neurons (or any other cells of interest). For example, in some aspects, it is possible to assess whether cells (e.g., iPSC) have been induced to differentiate into neurons by detecting the expression of certain markers ("neuronal markers") associated with neurons. Non-limiting examples of such markers include NeuroD1, FoxA2, ​​NR4A2, tyrosine hydroxylase (TH), Lmx1a, Pitx3, Tuj1, Map2, or a combination thereof. In some aspects, markers include general neuron markers (e.g., NeuroD1). In some aspects, markers include floor plate progenitor markers (e.g., FoxA2). In some aspects, markers include dopamine lineage markers (e.g., NR4A2, Pitx3, Lmx1a, and tyrosine hydroxylase). In some aspects, markers include early neuron markers (e.g., Tuj1). In some aspects, markers include mature neuron markers (e.g., Map2). Thus, unless otherwise indicated, as used herein, the term "neuron" includes any cell that expresses one or more of the neuronal markers described above.

[0093] Therefore, in some aspects, using the delivery method provided herein (e.g., using extrusion treatment to deliver saRNA encoding a reprogramming factor capable of inducing cell differentiation into neurons), the expression of any marker described above in the cell can be increased. In some aspects, compared with the reference method, the expression of neuronal markers is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, or at least about 500 times.

[0094] As is clear from the present disclosure, for any delivery method provided herein (e.g., using extrusion delivery to deliver saRNA to cells), in some aspects, the method also includes contacting cells (e.g., cells to be reprogrammed) with saRNA encoding payload (e.g., reprogramming factors) before passing the cell suspension through the stenosis. For example, in some aspects, before passing the cell suspension through the stenosis, the method includes contacting cells with saRNA as described herein (e.g., saRNA encoding reprogramming factors) to produce a cell suspension (e.g., before passing the cell suspension through the stenosis, the cell suspension includes cells to be reprogrammed and reprogramming factors). In some aspects, any delivery method provided herein (e.g., using extrusion delivery to deliver saRNA to cells) also includes contacting cells with saRNA encoding payload when the cell suspension (including cells) passes through the stenosis. In some aspects, during passing the cell suspension through the stenosis, first contacting the cell with saRNA as described herein. In some aspects, before passing through the step (i.e., passing the cell suspension through the stenosis) and during the passing step, the cell is contacted with saRNA. In some aspects, the method also includes contacting the cell with a saRNA encoding a payload (e.g., a reprogramming factor) after the cell suspension is passed through the stenosis. In some aspects, after the cell suspension is passed through the stenosis, the cell is first contacted with a saRNA as described herein. In some aspects, before, during, and / or after the step of passing through, the cell is contacted with a saRNA. As further described elsewhere in the disclosure, when the cell is contacted with a saRNA encoding a payload (e.g., a reprogramming factor) after the step of passing through, contact occurs soon after the cell passes through the stenosis, so that there is still disturbance in the cell membrane.

[0095] As used herein, "contact" between a cell and a saRNA encoding a payload (e.g., a reprogramming factor) does not require that the cell be in physical contact with the saRNA. As is clear from the present disclosure, once there is a perturbation in the cell membrane of the cell, contact between the cell and the saRNA will occur as long as the saRNA is able to enter the cell. To help illustrate, in some aspects, if both the cell and the saRNA are present in the same cell suspension, the cell and the saRNA are in contact regardless of whether the cell is in physical contact with the saRNA.

[0096] III.A. Cell suspension

[0097] In some aspects, the cell suspensions described herein comprise any suitable cells known in the art that can be modified (e.g., by introducing saRNA encoding reprogramming factors using the extrusion treatment methods provided herein.

[0098] In some aspects, the cell is a stem cell. As used herein, the term "stem cell" refers to a cell that not only has the ability to self-replicate but also has the ability to differentiate into other types of cells (e.g., neurons). In some aspects, stem cells that can be used for the present disclosure include induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), tissue-specific stem cells (e.g., liver stem cells, cardiac stem cells or neural stem cells), mesenchymal stem cells, hematopoietic stem cells (HSC) or a combination thereof. In some aspects, stem cells are iPSC.

[0099] In some aspects, the cell is a somatic cell. As used herein, the term "somatic cell" refers to any cell, germ cell (cell that continues to become a gamete) or stem cell of a non-gamete (sperm or egg) in the body. Non-limiting examples of somatic cells include blood cells, bone cells, muscle cells, nerve cells or a combination thereof. In some aspects, the somatic cells that can be used for the present disclosure include blood cells. In some aspects, blood cells are peripheral blood mononuclear cells (PBMC). As used herein, "PBMC" refers to any peripheral blood cell with a round nucleus. In some aspects, PBMC includes immune cells. As used herein, the term "immune cell" refers to any cell that plays a role in immune function. In some aspects, immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells (DC), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells or a combination thereof. In some aspects, blood cells are red blood cells. In some aspects, cells are cancer cells. In some aspects, the cancer cell is a cancer cell line cell, such as a HeLa cell. In some aspects, the cancer cell is a tumor cell. In some aspects, the cancer cell is a circulating tumor cell (CTC). In some aspects, the cell is a fibroblast, such as a primary fibroblast or a new human foreskin fibroblast (Nuff cell). In some aspects, the cell is an immortalized cell line cell, such as a HEK293 cell or a CHO cell. In some aspects, the cell is a skin cell. In some aspects, the cell is a germ cell, such as an oocyte, an egg or a fertilized egg. In some aspects, the cell is a cell cluster, such as an embryo, provided that the cell cluster is not destroyed when passing through the hole.

[0100] In some aspects, the cell suspensions that can be used for the present disclosure include mixed or purified cell populations. In some aspects, the cell suspensions are mixed cell populations, such as whole blood, lymph, PBMC or a combination thereof. In some aspects, the cell suspensions are purified cell populations. In some aspects, the cells are primary cells or cell line cells.

[0101] As demonstrated herein, delivery of saRNA encoding a payload (e.g., a reprogramming factor) to cells can be regulated by one or more parameters of a process that passes a cell suspension through a narrow region. In some aspects, specific features of a cell suspension can affect the delivery of saRNA to cells. Such features include, but are not limited to, osmotic pressure, salt concentration, serum content, cell concentration, pH, temperature, or a combination thereof. Additional disclosures relating to such parameters are provided throughout this disclosure.

[0102] In some aspects, cell suspension comprises homogenous cell group. In some aspects, cell suspension comprises heterogeneous cell group (for example, physiological saline solution or cell mixture in physiological medium beyond whole blood or blood). In some aspects, cell suspension comprises aqueous solution. In some aspects, aqueous solution comprises cell culture medium, PBS, salt, sugar, somatomedin, product of animal origin, bulking material, surfactant, lubricant, vitamin, polypeptide, agent affecting actin polymerization or its combination. In some aspects, cell culture medium comprises DMEM, OptiMEM, EVIDM, RPMI or its combination. In addition, solution buffer may comprise one or more lubricants (Pluronic (pluronic) or other surfactants), which may be designed to reduce or eliminate surface obstruction and improve cell viability. Exemplary surfactants include but are not limited to poloxamer (poloxamer), polysorbate, sugar (such as mannitol), serum and albumin of animal origin.

[0103] When the suspension includes certain types of cells, in some aspects, it is possible to use a solution treatment cell that helps to deliver a payload (e.g., a reprogramming factor) to the interior of the cell. In some aspects, the solution includes an agent that affects actin polymerization. In some aspects, the agent that affects actin polymerization includes Latrunculin A, cytochalasin, colchicine, or a combination thereof. For example, in some aspects, cells can be incubated in a depolymerization solution (such as Latrunculin A) for about 1 hour, and then the cells are passed through to depolymerize the narrow actin cytoskeleton. In some aspects, cells can be incubated in colchicine (Sigma) for about 2 hours, and then the cells are passed through a narrow microtubule network to depolymerize.

[0104] In some aspects, a characteristic of a cell suspension that can affect the delivery of saRNA encoding a payload (e.g., a reprogramming factor) to a cell 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 aspects, the viscosity of the cell suspension is about 8.9 x 10 -4 Pa·s to about 4.0x 10 -3 Pa·s, about 8.9x 10 -4 Pa·s to about 3.0x 10 -3 Pa·s, about 8.9x 10 -4 Pa·s is about 2.0x10 -3 Pa·s or about 8.9x 10 -4 Pa·s to about 1.0x 10 -3Pa·s. In some aspects, the viscosity is between about 0.89cP and about 4.0cP, between about 0.89cP and about 3.0cP, between about 0.89cP and about 2.0cP, or between about 0.89cP and about 1.0cP. In some aspects, a shear thinning effect is observed, wherein the viscosity of the cell suspension is reduced under shear strain conditions. The viscosity can be measured by any suitable method known in the art, including but not limited to a viscometer, such as a glass capillary viscometer or a rheometer. The viscometer measures the viscosity under a flow condition, and the rheometer is used to measure the viscosity that varies with the flow condition. In some aspects, the viscosity of a shear thinning solution (such as blood) is measured. In some aspects, 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), above 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 at a temperature between these exemplary temperatures.

[0105] III.B. Payload

[0106] As described herein and demonstrated, the delivery method provided in the present disclosure is particularly useful for delivering the saRNA encoding reprogramming factors (such as reprogramming factors that can induce cell differentiation into neurons) in cells. In some aspects, the delivery method provided herein may also include contacting cells with one or more additional payloads so that multiple payloads can enter cells by disturbance when contacting cells. In some aspects, the additional payload can be delivered to cells simultaneously with saRNA as described herein (for example, saRNA encoding reprogramming factors). For example, in some aspects, the additional payload can be present in the cell suspension with saRNA as described herein before, during and / or after the cell suspension is passed through a narrow passing step. In some aspects, the cell suspension 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 additional payloads. When multiple payloads are involved, in some aspects, multiple payloads can be delivered to cells as a single unit (for example, a single saRNA encoding multiple payloads). In some aspects, multiple payloads can be delivered to a unit as multiple units. For example, the delivery methods provided herein can be used to deliver a 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).

[0107] As further described elsewhere in the present disclosure, in some aspects, a cell suspension can pass through multiple stenosis. In such aspects, when cells are passed through one or more stenosis in multiple stenosis, a payload can be loaded into the cell. In some aspects, each time a cell is passed through one or more stenosis in multiple stenosis, a payload is loaded into the cell. When multiple payloads are involved, each payload can be the same. In some aspects, one or more payloads are different. The present disclosure provides additional disclosures relating to the combined delivery of saRNA described herein and one or more additional payloads (also referred to herein as "multiple delivery") elsewhere.

[0108] As is clear 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 saRNA 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 aspects, small molecules include impermeable small molecules. As used herein, "impermeable small molecules" refer to small molecules that do not naturally pass through the cell membrane of a cell. In some aspects, additional payloads that can be delivered to cells using the delivery methods provided herein include nucleic acids. In some aspects, nucleic acids include DNA, RNA, or both. In some aspects, DNA includes recombinant DNA, cDNA, genomic DNA, or a combination thereof. In some aspects, RNA includes siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, self-amplification mRNA (saRNA), or a combination thereof. In some aspects, RNA is mRNA. In some aspects, RNA is siRNA. In some aspects, RNA is shRNA. In some aspects, RNA is miRNA. In some aspects, RNA is saRNA. For example, in some aspects, the delivery methods described herein may 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., iPSC), wherein the first saRNA and the second saRNA are delivered to the cell using an extrusion process. As further described herein, in some aspects, the first saRNA and the second saRNA may be delivered to the cell simultaneously. In some aspects, the first saRNA and the second saRNA may be delivered to the cell in sequence. In some aspects, the delivery methods provided herein include delivering a first saRNA encoding a first payload (e.g., a reprogramming factor) and a second payload, wherein the second payload is a non-self-amplifying RNA encoding a second payload (e.g., a reprogramming factor).

[0109] In some respects, additional compound is the nucleic acid of the enzyme that encodes antibiotic resistance.Not bound by any theory, by adding these compounds into the cell suspension comprising multiple cells and reprogramming factors, it is possible to increase the purity of the mixture comprising modified cells (that is, reprogrammed cells / differentiated cells).By adding the specific antibiotics relevant to additional compounds, the purity of the mixture comprising modified cells (for example, comprising differentiated neurons) can be increased by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times or at least about 50 times.The limiting examples of this additional compound include puromycin-N-acetyltransferase and antibiotic is puromycin.

[0110] III.C. Stenosis

[0111] III.C.1. Microfluidic channels

[0112] As described herein, stenosis is used to cause physical deformation of cells so as to form disturbances in the cell membrane of cells, thereby allowing payloads (e.g., reprogramming factors) to be delivered to cells. In some aspects, stenosis is in a channel (referred to herein as "microfluidic channel" or "channel") in a microfluidic device. When multiple channels are involved, in some aspects, multiple channels can be placed in parallel and / or in series in a microfluidic device. In some aspects, 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 individual strictures. In some aspects, the cells described herein pass through more than about 1,000 individual strictures. In some aspects, multiple narrowings can be a part of a single microfluidic device (e.g., multiple rows of narrow chips). In some aspects, one or more narrowings in multiple narrowings can be a part of different microfluidic devices. For example, in some aspects, cells as described herein (e.g., stem cells or PBMC) experience the first extrusion process, wherein the cell passes through the first microfluidic device (e.g., chip) in the first narrowing. Then, after the cell experiences the first extrusion process (e.g., passing through the first narrowing), the cell experiences the second extrusion process, wherein the cell passes through the second microfluidic device (e.g., chip) in the second narrowing. In some aspects, each narrowing is the same (e.g., having the same length, width and / or depth). In some aspects, one or more narrowings are different. When using multiple narrowings, multiple narrowings may include the first narrowing associated with the first reprogramming factor, and the second narrowing associated with the second reprogramming factor, wherein the cell suspension is passed through the first narrowing, so that the first reprogramming factor is delivered to one or more cells in a plurality of cells, and then the cell suspension is passed through the second narrowing, so that the second reprogramming factor is delivered to one or more cells in a plurality of cells. In some aspects, the cell suspension is passed through the second stenosis 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 passing the cell suspension through the first stenosis.

[0113] In some aspects, when a cell suspension is passed through multiple stenosis (e.g., multiple extrusion processes), the cell remains active after passing through each stenosis. As is clear from the present disclosure, in some aspects, multiple stenosis may include being present in a single microfluidic device (e.g., multiple rows of narrow chips) so that the cell passes through two or more stenosis of multiple stenosis in sequence. In some aspects, multiple stenosis is a part of a separate microfluidic device, so that the first stenosis is associated with the first microfluidic device, and the second stenosis is associated with the second microfluidic device. For example, as demonstrated herein (e.g., Example 1), in some aspects, the cell is passed through the first stenosis (i.e., the first extrusion process) associated with the first microfluidic device (e.g., chip). After the cell has been passed through the first stenosis, the cell is passed through the second stenosis (i.e., the second extrusion process) associated with the second microfluidic device (e.g., chip). In some aspects, after passing through the first stenosis, the cell is cultured in a culture medium, and then the cell is passed through the second stenosis. In some aspects, before the cell is passed through the second stenosis, the cell is 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. As is apparent from this disclosure, in some aspects, the first and second stenosis have the same length, depth, and / or width. In some aspects, the first and second stenosis can have different lengths, depths, and / or widths.

[0114] In some aspects, after passing through the stenosis, 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 making cells pass through multiple stenosis (e.g., a portion of a single microfluidic device or a separate microfluidic device), 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 after passing through each of the multiple stenosis. Any suitable method known in the art can be used to measure the viability of cells. In some aspects, Nucleocounter NC-200, Orflo Moxi Go II cell counter or both can be used to measure the viability of cells.

[0115] Exemplary microfluidic channels containing cell-deformable constrictions for use in the methods disclosed herein are described in U.S. Publication No. 2020 / 0277566A1, U.S. Publication No. 2020 / 0332243A1, U.S. 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.

[0116] In some respects, microfluidic channel as described herein (that is, including stenosis) includes lumen and is configured so that cells suspended in buffer (e.g., cell suspension) can pass through channel. Microfluidic channel that can be used for the present disclosure can be made 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 a combination thereof. In some respects, the material is silicon. The manufacture of microfluidic channel can be carried out 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 mask or a combination thereof. In some respects, manufacture is carried out using dry etching.

[0117] In some aspects, the microfluidic channel that can be used in the present disclosure includes an inlet portion, a center point, and an outlet portion. In some aspects, the cross-section of one or more of the inlet portion, the center point, and / or the outlet portion can vary. For example, the shape of the cross-section can be annular, elliptical, elongated slit-shaped, square, hexagonal, or triangular.

[0118] The inlet portion defines a narrow angle. In some aspects, by adjusting (e.g., increasing or decreasing) the narrow angle, any narrow blockage can be reduced or prevented. In some aspects, the angle of the outlet portion can also be adjusted. For example, in some aspects, the angle of the outlet portion can be configured to reduce the possibility of turbulence that can cause non-laminar flow. In some aspects, the wall of the inlet portion and / or the outlet portion is linear. In some aspects, the wall of the inlet portion and / or the outlet portion is curved.

[0119] In some aspects, the length, depth and / or width of the stenosis can vary. In some aspects, by adjusting (e.g., increasing or decreasing) the length, depth and / or width of the stenosis, the delivery efficiency of the payload can be adjusted. As used herein, the term "delivery efficiency" refers to the amount of payload delivered to the cell. For example, when the total amount of payload delivered increases, the delivery efficiency can be increased.

[0120] In some aspects, the stenosis has a length of less than about 1 μm. In some aspects, the stenosis has a length of about 0 μm to about 100 μm. In some aspects, the length of the stenosis 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 aspects, the length of the narrow 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 aspects, the length of the narrow is about 10 μm. In some aspects, the narrow has a length of about 0 μm. For example, in some aspects, the microfluidic device (e.g., chip) that can be used for the present disclosure includes a narrowness similar to two points of a rhombus that are aggregated together so that the length of the narrow is about 0 μm.

[0121] In some aspects, the width of the narrow is between about 0 μm and about 10 μm. In some aspects, the width of the narrow 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 aspects, the narrow width 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 aspects, the narrow width is between about 3 μm and about 10 μm. In some aspects, the narrow width is about 6 μm.

[0122] In some aspects, the depth of the narrow is at least about 1 μm. In some aspects, the depth of the narrow 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 aspects, the depth of the narrow is about 5 μm to about 90 μm. In some aspects, the depth of the narrow 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 aspects, the depth of the narrow is about 70 μm.

[0123] In some aspects, the length of the stenosis is about 10 μm, the width of the stenosis is about 6 μm, and the depth of the stenosis is about 70 μm. In some aspects, the length of the stenosis is 10 μm, the width of the stenosis is 6 μm, and the depth of the stenosis is 70 μm.

[0124] In some aspects, the diameter of a stenosis (e.g., a stenosis contained in a microfluidic channel) is a function of the diameter of one or more cells passing through the stenosis. Without being bound by any one theory, in some aspects, the diameter of the stenosis is smaller than the diameter of the cell, such that a deforming force is applied to the cell as the cell passes through the stenosis, thereby causing a temporary physical deformation of the cell.

[0125] Thus, in some aspects, the diameter of the stenosis (also referred to herein as the "stenosis size") is about 20% to about 99% of the diameter of the cell. In some aspects, the stenosis size 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. As is clear from this disclosure, by adjusting (e.g., increasing or decreasing) the diameter of the stenosis, the efficiency of delivering the payload to the cell can also be adjusted.

[0126] III.C.2. Surfaces with holes

[0127] In some aspects, the stenosis described herein comprises a hole contained in the surface. Non-limiting examples of holes contained in the surface that can be used for the present disclosure are described in, for example, U.S. Publication No. 2019 / 0382796A1, which is incorporated herein by reference in its entirety.

[0128] In some aspects, surfaces useful in the present disclosure (i.e., comprising one or more pores that can cause physical deformation of cells when cells pass through the pores) can be made of any suitable material available in the art and / or in any of a variety of forms. Non-limiting examples of such materials include synthetic or natural polymers, polycarbonates, silicon, glass, metals, alloys, nitrocellulose, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose esters, porcelain, ceramics, or combinations thereof.

[0129] In some aspects, the surface comprises a filter. In some aspects, the filter is a tangential flow filter. In some aspects, the surface comprises a membrane. In some aspects, the surface comprises a sponge or sponge-like matrix. In some aspects, the surface comprises a matrix. In some aspects, the surface comprises a tortuous path surface. In some aspects, the tortuous path surface comprises cellulose acetate.

[0130] The surface disclosed herein (i.e., the surface comprising one or more holes) can have any suitable shape known in the art. When the surface has a 2-dimensional shape, the surface can be, but is not limited to, an annular, elliptical, circular, square, star, triangle, polygon, pentagon, hexagon, heptagon or octagon. In some aspects, the shape of the surface is circular. When the surface has a 3-dimensional shape, in some aspects, the surface can be, but is not limited to, cylindrical, conical or rectangular.

[0131] As is clear from the present disclosure, the surface that can be used for the present disclosure (for example, the surface that comprises one or more holes) can have various cross-sectional widths and thicknesses. In some aspects, the cross-sectional width of the surface is between about 1mm and about 1m. In some aspects, the surface has a limited thickness. In some aspects, the surface thickness is uniform. In some aspects, the surface thickness is variable. For example, in some aspects, some parts of the surface are thicker or thinner than other parts of the surface. In such aspects, the thickness of different parts of the surface can vary from about 1% to about 90%. In some aspects, the surface thickness is from about 0.01 μm to about 5mm.

[0132] The cross-sectional width of the hole may depend on the cell type targeted by the payload. In some aspects, the hole size is a function of the diameter of the cell of the cell cluster to be targeted. In some aspects, the hole size causes the cell to be disturbed (i.e., physical deformation) when passing through the hole. In some aspects, the hole size is less than the diameter of the cell. In some aspects, the hole size is about 20% to about 99% of the cell diameter. In some aspects, the hole size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the cell diameter. In some aspects, the pore size is about 0.4 μm, 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 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, or about 15 μm or larger.

[0133] The entrance and exit of a hole can have a variety of angles. In some aspects, by adjusting (for example, increasing or decreasing) the hole angle, any hole blockage can be reduced or prevented. In some aspects, the flow rate (that is, the rate at which a cell or a suspension comprising cells passes through a hole) is between about 0.001 mL / cm / s and about 100 mL / cm / s. For example, the angle of an entrance or exit portion can be between about 0 and about 90 degrees. In some aspects, the hole has the same entrance angle and exit angle. In some aspects, the hole has different entrance angles and exit angles. In some aspects, the hole edge is smooth, such as round or curved. As used herein, a "smooth" hole edge has a continuous, flat and uniform surface without protrusions, ridges or uneven parts. In some aspects, the hole edge is sharp. As used herein, a "sharp" hole edge has a sharp or acutely angled thin edge. In some aspects, a hole channel is straight. As used herein, a "straight" hole channel does not contain bending, bends, angles or other irregularities. In some aspects, a hole channel is curved. As used herein, a "curved" pore channel is curved or deviates from a straight line. In some aspects, the pore channel has multiple bends, such as about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more bends.

[0134] Hole can have any shape as known in the art, including 2-dimensional or 3-dimensional shape.Hole shape (such as cross-sectional shape) can be but not limited to annular, elliptical, circular, square, star, triangle, polygon, pentagon, hexagon, heptagon and octagon.In some aspects, the shape of the cross section of hole is circular.In some aspects, the 3-dimensional shape of hole is cylindrical or conical.In some aspects, hole has the entrance and exit shape of groove shape.In some aspects, the hole shape between the hole in a given surface is homogeneous (that is, consistent or regular).In some aspects, the hole shape between the hole in a given surface is heterogeneous (that is, mixed or variable).

[0135] Surfaces useful in the present disclosure may have a single pore. In some aspects, surfaces useful in the present disclosure include a plurality of pores. In some aspects, the pores comprise about 10% to about 80% of the total surface area of ​​the surface. In some aspects, the surface contains about 1.0 x 10 5 About 1.0x 10 30 In some aspects, the surface comprises from about 10 to about 1.0 x 10 15 Holes / mm 2 between surface areas.

[0136] The holes can be distributed in a given surface in a variety of ways. In some aspects, the holes are distributed in parallel in a given surface. In some aspects, the holes are distributed side by side in the same direction in a given surface, and are at the same distance apart. In some aspects, the distribution of the holes is ordered or homogeneous. In such aspects, the holes can be distributed in a regular, systematic pattern in a given surface, or can be at the same distance apart. In some aspects, the distribution of the holes is random or heterogeneous. For example, in some aspects, the holes are distributed in an irregular, disordered pattern in a given surface, or at different distances apart.

[0137] In some aspects, multiple surfaces are used so that cells pass through multiple holes, wherein the holes are located on different surfaces. In some aspects, multiple surfaces are distributed in series. Multiple surfaces can be homogeneous or heterogeneous in terms of surface size, shape and / or roughness. Multiple surfaces can also contain holes with homogeneous or heterogeneous pore size, shape and / or quantity, so that a series of payloads can be delivered to different types of cells at the same time.

[0138] In some aspects, for example, the holes that can be used for the surface of the present disclosure have uniform width dimensions (i.e., constant width along the length of the hole channel). In some aspects, a single hole has a variable width (i.e., increasing or decreasing the width along the length of the hole channel). In some aspects, the holes in a given surface have the same single hole depth. In some aspects, the holes in a given surface have different single hole depths. In some aspects, the holes are adjacent to each other. In some aspects, the holes are spaced a certain distance apart from each other. In some aspects, the holes are spaced a distance of about 0.001 μm to about 30 mm apart from each other.

[0139] In some aspects, the surface is coated with material. The material can be selected from any material known in the art, including but not limited to Teflon, adhesive coating, surfactant, protein, adhesion molecule, antibody, anticoagulant, factor regulating cell function, nucleic acid, lipid, carbohydrate, transmembrane protein or its combination. In some aspects, the surface is coated with polyvinyl pyrrolidone. In some aspects, the material is covalently attached to the surface. In some aspects, the material is non-covalently attached to the surface. In some aspects, the surface molecule is released when the cell passes through the hole.

[0140] In some aspects, the surface has a modified chemical property. In some aspects, the surface is hydrophilic. In some aspects, the surface is hydrophobic. In some aspects, the surface is charged. In some aspects, the surface is positively charged and / or negatively charged. In some aspects, the surface may be positively charged in certain areas and negatively charged in other areas. In some aspects, the surface has an overall positive charge or an overall negative charge. In some aspects, the surface may be any of a smooth, electropolished, roughened, or plasma treated surface. In some aspects, the surface comprises zwitterions or dipole compounds. In some aspects, the surface is plasma treated.

[0141] In some aspects, the surface is contained in a larger module. In some aspects, the surface is contained in a syringe such as a plastic or glass syringe. In some aspects, the surface is contained in a plastic filter holder. In some aspects, the surface is contained in a pipette tip.

[0142] III.D. Cell Perturbations

[0143] As described herein, when a cell passes through a stenosis, it is physically deformed so that a disturbance (e.g., a hole, a crack, a cavity, a pore, a hole, a fracture, a gap, a perforation) occurs in the cell membrane of the cell. This disturbance in the cell membrane is temporary and sufficient for delivering any payload described herein (e.g., a reprogramming factor) to the cell. The cell has a self-repair mechanism that allows the cell to repair any damage on its cell membrane. See Blazek et al., Physiology (Bethesda) 30 (6): 438-48 (November 2015), which is incorporated herein by reference in its entirety. Therefore, in some aspects, once a cell has passed through a stenosis (e.g., a microfluidic channel or hole), the disturbance in the cell membrane can be reduced or eliminated so that the payload delivered to the cell does not leave the cell.

[0144] In some aspects, after the pressure is removed (e.g., the cell has passed through the stenosis), the perturbation in the cell membrane persists for about 1.0 x 10 -9 In some aspects, the cell perturbation lasts for about 1.0 x 10 -9 In some aspects, the cell perturbation lasts for about 1.0 x 10 -9 seconds to about 1.0x 10 -1 seconds, about 1.0x10 -9 seconds to about 1.0x 10 -2 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -3 seconds, about 1.0x 10 -9 seconds to about 1.0x10 -4 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -5 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -6 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -7 seconds or about 1.0x 10 -9 seconds to about 1.0x 10 -8 In some aspects, the cell perturbation lasts about 1.0 x 10 -8 seconds to about 1.0x 10 -1 seconds, about 1.0x 10 -7 seconds to about 1.0x10 -1 seconds, about 1.0x 10 -6 seconds to about 1.0x 10 -1 seconds, about 1.0x 10 -5 seconds to about 1.0x 10-1 seconds, about 1.0x 10 -4 seconds to about 1.0x 10 -1 seconds, about 1.0x10 -3 seconds to about 1.0x 10 -1 seconds or about 1.0x 10 -2 seconds to about 1.0x 10 -1 The cellular perturbations (eg, pores or holes) formed by the methods described herein are not formed due to the assembly of polypeptide subunits to form a multimeric pore structure (such as that formed by complement or bacterial hemolysin).

[0145] In some aspects, as the cell passes through the stenosis, the pressure applied to the cell temporarily causes damage to the cell membrane, causing the material to diffuse passively through the perturbation. In some aspects, the cell is deformed or perturbed for only a short period of time, such as about 100 μs or less, to minimize the chance of activating apoptotic pathways through cell signaling mechanisms, although other durations are possible (e.g., ranging from nanoseconds to hours). In some aspects, the cell is deformed less than about 1.0 x 10 -9 In some aspects, the cell deformation is less than about 1.0 x 10 -9 In some aspects, the cell deforms by about 1.0 x 10 -9 In some aspects, the cell deforms by about 1.0 x 10 -9 In some aspects, the cell deforms by about 1.0 x 10 -9 seconds to about 1.0x 10 -1 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -2 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -3 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -4 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -5 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -6 seconds, about 1.0x 10 -9 seconds to about 1.0x 10 -7 seconds or about 1.0x 10 -9 seconds to about 1.0x 10 -8 In some aspects, the cell deforms by about 1.0 x 10 -8seconds to about 1.0x 10 -1 seconds, about 1.0x 10 -7 seconds to about 1.0x 10 -1 seconds, about 1.0x 10 -6 seconds to about 1.0x 10 -1 seconds, about 1.0x 10 -5 seconds to about 1.0x 10 -1 seconds, about 1.0x 10 -4 seconds to about 1.0x 10 -1 seconds, about 1.0x 10 -3 seconds to about 1.0x 10 -1 seconds or about 1.0x 10 -2 seconds to about 1.0x 10 -1 In some aspects, deforming the cell comprises deforming the cell for a time ranging from, but not limited to, about 1 μs to at least about 750 μs, such as at least about 1 μs, at least about 10 μs, at least about 50 μs, at least about 100 μs, at least about 500 μs, or at least about 750 μs.

[0146] In some aspects, the delivery of saRNA encoding a payload (e.g., a reprogramming factor) to a cell occurs simultaneously with the passage of the cell through the stenosis. In some aspects, the delivery of saRNA encoding a payload to a cell may occur after the cell is passed through the stenosis (i.e., when the perturbation of the cell membrane still exists and before the cell membrane of the cell is restored). In some aspects, the delivery of saRNA encoding a payload to a cell occurs about several minutes after the cell is passed through the stenosis. In some aspects, the perturbation in the cell after the cell is passed through the stenosis is corrected within about five minutes after the cell is passed through the stenosis.

[0147] In some aspects, the viability of cells (e.g., stem cells or PBMCs) after passing through the stenosis is about 5% to about 100%. In some aspects, the viability of cells after passing through the stenosis 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 aspects, the viability of cells after passing through the stenosis is about 1.0x10 -2 For example, the viability of cells in the stenosis can be measured after about 1.0 x 10 -2 In some aspects, the cell viability is measured after about 1.0 x 10 cells have been passed through the stenosis. -2 seconds to about 2 hours, about 1.0x 10 -2seconds to about 1 hour, about 1.0x10 -2 seconds to about 30 minutes, about 11.0x 10 -2 seconds to about 1 minute, about 1.0x 10 -2 seconds to about 30 seconds, about 1.0x 10 -2 seconds to about 1 second or about 1.0x 10 -2 In some aspects, the cell viability is measured from about 1.5 hours to about 2 hours, about 1 hour to about 2 hours, about 30 minutes to about 2 hours, about 15 minutes to about 2 hours, about 1 minute to about 2 hours, about 30 seconds to about 2 hours, or about 1 second to about 2 hours after the cell is passed through the stenosis. In some aspects, the cell viability is measured from about 2 hours to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, or about 24 hours to about 10 days after the cell is passed through the stenosis.

[0148] III.E. Delivery Parameters

[0149] As is clear from the present disclosure, many parameters can affect the efficiency of delivering saRNA described herein (e.g., saRNA encoding reprogramming factors) to cells using the extrusion treatment method provided herein. Therefore, by adjusting (e.g., increasing or decreasing) one or more delivery parameters, the delivery of the payload to the cell can be improved. Therefore, in some aspects, the present disclosure relates to a method for increasing the delivery of a payload (e.g., a reprogramming factor) to a cell, wherein the method includes adjusting one or more parameters that allow a cell suspension to pass through a narrow, wherein the cell suspension comprises a cell group, and wherein one or more parameters increase the delivery of the payload to one or more cells in the cell group compared to a reference parameter. As described elsewhere in the present disclosure, the payload may be contacted with the cell group before, during, or after the extrusion step.

[0150] In some aspects, by adjusting one or more delivery parameters, delivery of a payload (e.g., a reprogramming factor) to one or more cells 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 compared to delivery of the payload agent to the corresponding cells using reference parameters.

[0151] In some aspects, one or more delivery parameters that can be adjusted to increase the delivery efficiency of the parameter include cell density (ie, the concentration of cells present in, for example, a cell suspension), pressure, or both. Additional examples of adjustable delivery parameters are provided elsewhere in this disclosure.

[0152] In some aspects, the cell density is about 1 x 10 7 cells / mL, approximately 2x 10 7 cells / mL, about 3x 10 7 cells / mL, about 4x 10 7 cells / mL, about 5x 10 7 cells / mL, about 6x 10 7 cells / mL, about 7x 10 7 cells / mL, about 8x 10 7 cells / mL, about 9x 10 7 cells / mL, about 1x 10 8 cells / mL, about 1.1x 10 8 cells / mL, about 1.2 x 10 8 cells / mL, about 1.3x 10 8 cells / mL, about 1.4 x 10 8 cells / mL, about 1.5x 10 8 cells / mL, about 2.0x 10 8 cells / mL, about 3.0x10 8 cells / mL, about 4.0x 10 8 cells / mL, about 5.0x 10 8 cells / mL, about 6.0x 10 8 cells / mL, about 7.0x 10 8 cells / mL, about 8.0x 10 8 cells / mL, about 9.0x 10 8 cells / mL or approximately 1.0 x 10 9 In some aspects, the cell density is about 6 x 10 7 cells / mL to about 1.2 x 10 8 cells / mL.

[0153] In some aspects, the pressure is about 20psi, about 25psi, about 30psi, about 35psi, about 40psi, about 50psi, about 55psi, about 60psi, about 65psi, about 70psi, about 75psi, about 80psi, about 85psi, about 90psi, about 95psi, about 100psi, about 110psi, about 120psi, about 130psi, about 140psi, about 150psi, about 160psi, about 170psi, about 180psi, about 190psi or about 200psi or more. In some aspects, the pressure is between about 30psi and about 90psi.

[0154] In some aspects, a particular type of device (e.g., a microfluidic chip) may also have an impact on the delivery efficiency of the payload described herein (e.g., reprogramming factors). In the case of a microfluidic chip, different chips may have different narrow parameters, such as narrow length, depth and width; inlet angle, outlet angle, length, depth and width of the approach zone, etc. As described herein, such variables may affect the delivery of the payload of the extrusion treatment method disclosed herein to the cell. In some aspects, the length of the narrow is up to 100 μm. For example, in some aspects, 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 aspects, the length of the narrow is less than 1 μm. In some aspects, the length of the stenosis 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 aspects, the stenosis has a length of about 10 μm.

[0155] In some aspects, the narrow width is up to about 10 μm. In some aspects, the narrow width 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 aspects, the width is between about 3 μm and about 10 μm. In some aspects, 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 aspects, the narrow width is about 6 μm.

[0156] In some aspects, the depth of the narrow is at least about 1 μm. In some aspects, the depth of the narrow 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 aspects, the depth is between about 5 μm and about 90 μm. In some aspects, 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 aspects, the depth of the narrow is about 70 μm.

[0157] In some aspects, the length is about 10 μm, the width is about 6 μm, and the depth is about 70 μm.

[0158] Additional examples of parameters that can affect the delivery of payload to cells include, but are not limited to, narrow size (e.g., length, width, and / or depth), narrow inlet angle, narrow surface characteristics (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity), operating flow rate, payload concentration, the amount of time for cell recovery, or a combination thereof. Other parameters that can affect the delivery efficiency of payload (e.g., reprogramming factors) can include the speed of cells in narrow, the shear rate in narrow, the viscosity of cell suspensions, the velocity component perpendicular to the flow rate, and the time of narrow. Such parameters can be designed to control the delivery of payload.

[0159] In some aspects, the temperature used in the method of the present disclosure may also have an impact on the efficiency of the payload being delivered to the cell and the viability of the cell. In some aspects, the extrusion treatment method is carried out between about -5 ° C to about 45 ° C. For example, the method can be carried out at room temperature (e.g., about 20 ° C), physiological temperature (e.g., about 37 ° C), above physiological temperature (e.g., above about 37 ° C to 45 ° C or higher) or low temperature (e.g., about -5 ° C to about 4 ° C) or at a temperature between these exemplary temperatures.

[0160] Various methods can be used to drive cells through stenosis. For example, pressure can be applied by a pump (e.g., a gas cylinder or a compressor) on the inlet side, vacuum can be applied by a vacuum pump on the outlet side, capillary action can be applied by a tube, and / or the system can be fed by gravity. A flow system based on displacement (e.g., a syringe pump, a peristaltic pump, a manual syringe or a pipette, a piston, etc.) can also be used. In some aspects, cells are passed through stenosis by positive pressure. In some aspects, cells are passed through stenosis by constant pressure or variable pressure. In some aspects, pressure is applied using a syringe. In some aspects, pressure is applied using a pump. In some aspects, the pump is a peristaltic pump or a diaphragm pump. In some aspects, pressure is applied using a vacuum. In some aspects, cells are passed through stenosis by the action of gravity. In some aspects, cells are passed through stenosis by capillary pressure.

[0161] In some aspects, the liquid flow guides the cells through the stenosis. In some aspects, before the cells are passed through the stenosis, the fluid flow is a turbulent flow. Turbulent flow is a fluid flow in which the speed at a given point varies irregularly in amplitude and direction. In some aspects, the fluid flow through the stenosis is a laminar flow. Laminar flow involves the uninterrupted flow of fluid near a solid boundary, wherein the flow direction of each point remains constant. In some aspects, after the cells are passed through the stenosis, the liquid flow is a turbulent flow. The speed at which the cells are passed through the stenosis can vary. In some aspects, the cells are passed through the stenosis at a uniform cell speed. In some aspects, the cells are passed through the stenosis at a fluctuating cell speed.

[0162] In some aspects, a combination therapy is used to deliver a payload, for example, the method described herein, followed by exposure to an electric field downstream of the stenosis. In some aspects, after passing through the stenosis, the cell is passed through an electric field generated by at least one electrode. In some aspects, the electric field helps deliver the payload to a second location in the cell, such as a nucleus. In some aspects, one or more electrodes are close to the cell deformation stenosis to generate an electric field. In some aspects, the electric field is between about 0.1kV / m and about 100MV / m. In some aspects, an integrated circuit is used to provide an electrical signal to drive the electrode. In some aspects, the cell is exposed to an electric field with a pulse width between about 1ns and about 1s and a period between about 100ns and about 10s.

[0163] III.F. Therapeutic Uses

[0164] In some aspects, the present disclosure relates to the use of cells produced using the extrusion treatment methods described herein to treat various diseases or conditions. As is clear from the present disclosure, the methods and compositions provided herein can be used for diseases and conditions for which cell replacement therapy can be used as treatment. In some aspects, by replacing damaged cells with cells produced using the methods provided herein, one or more functions associated with damaged cells can be restored, and thereby treating diseases or conditions. For example, in some aspects, neurons produced using the extrusion treatment methods provided herein can be administered to subjects suffering from neurological disorders. The administration of such neurons can be used to improve one or more symptoms associated with neurological disorders.

[0165] As used herein, the terms "neurological disorders" and "neuroimmune system disorders" can be used interchangeably and refer to diseases and disorders of the central or peripheral nervous system. Unless otherwise indicated, the terms "neurological disorders" and "neuroimmune system disorders" include all diseases or disorders of the nervous system, including autoimmune disorders. Non-limiting examples of neurological disorders treated using the present disclosure include brain tumors, neoplastic meningitis, leptomeningeal cancer disease (LMD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease (PD), Huntington's disease (HD), Alzheimer's disease (AD), or a combination thereof. In some aspects, the neurological disorder is Parkinson's disease.

[0166] IV. Compositions of the Disclosure

[0167] 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 as described herein, a cell suspension comprising a plurality of cells and a payload; wherein the stenosis is configured such that the plurality of cells can pass through the microfluidic channel, wherein the passage of the plurality of cells causes deformation and disruption of the cell membranes of the cells, thereby allowing the payload to enter the cells.

[0168] In some aspects, the present disclosure provides a system for delivering a payload, the system comprising a surface with a hole, a cell suspension comprising a plurality of cells and a payload; wherein the surface with a hole is configured so that a plurality of cells can pass through the hole, wherein the passage of a plurality of cells causes deformation and destruction of the cell membrane of the cell, thereby allowing the payload to enter the cell. 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 to generate an electric field. In some aspects, the system is used to deliver the payload to the cell by any method described herein. The system may include any aspect described for the method disclosed above, including a microfluidic channel or a surface with a hole, to provide cell deformation stenosis, cell suspension, cell disturbance, delivery parameters. In some aspects, delivery parameters are optimized, such as operating flow rate, cell and compound concentration, cell speed in stenosis, and composition of the cell suspension (e.g., osmotic pressure, salt concentration, serum content, cell concentration, pH, etc.), for delivering a payload (e.g., a reprogramming factor) to the cell.

[0169] In some aspects, the disclosure provides a kind of cell (for example, neuron), which is produced using any method provided herein.In some aspects, a cell is provided herein, which includes the disturbance in the cell membrane, wherein the disturbance is due to one or more parameters (for example, delivery parameters as described herein) that deform the cell, so as to form the disturbance in the cell membrane of the cell, so that a payload (for example, a reprogramming factor) can enter the cell.In some aspects, a cell is provided herein, which includes a payload (for example, a reprogramming factor), wherein the payload enters the cell by the disturbance in the cell membrane, and the disturbance is caused by one or more parameters (for example, delivery parameters as described herein) that deform the cell, and so as to form the disturbance in the cell membrane, so that a payload enters the cell.In some aspects, such cells may include any cell described herein (for example, stem cell or PBMC).

[0170] In some aspects, the disclosure provides a composition comprising a plurality of cells, wherein the plurality of cells are produced by any method provided herein. A composition is also provided herein, comprising a cell group and a payload (e.g., a reprogramming factor) under one or more parameters, wherein the composition causes one or more cells in the cell group to deform, thereby forming a disturbance in the cell membrane of one or more cells, and wherein the disturbance in the cell membrane allows the payload to enter one or more cells.

[0171] Also provided is a kit or article for delivering a payload as described herein (e.g., a reprogramming factor) to a cell. In some aspects, the kit includes a composition as described herein (e.g., a microfluidic channel or a surface containing a hole, a cell suspension, and / or a payload) in a suitable package. Suitable packaging materials are known in the art, and include, for example, vials (such as sealed vials), vessels, ampoules, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. These articles can be further sterilized and / or sealed.

[0172] The present disclosure also provides a kit comprising the components of the methods described herein and may also include instructions for carrying out the method to deliver a payload (e.g., a reprogramming factor) to a cell. Kits described herein may also include other materials, including other buffers, diluents, filters, needles, syringes, and package inserts, which have instructions for carrying out any method described herein; For example, instructions for delivering a payload to a cell.

[0173] The following examples are offered by way of illustration and not limitation.

[0174] Example

[0175] Example 1: Analysis of GFP-Puro saRNA expression and duration in iPSCs

[0176] To determine the duration of GFP expression and GFP-Puro saRNA, iPSCs were treated with Accutase and dissociated into single cells. 8 The prepared iPSCs were then combined with GFP-Puro saRNA (1.5 mg / ml or 0.3 mg / ml). The cell suspension was added to the mTeSR TMPlus basal medium with the following dimensions of narrow channels: length of 10μm, width of 6μm, and depth of 70μm. Cells treated with GFP mRNA (non-self-amplification) (0.125mg / ml) were used as positive controls and cells not treated with any cargo were used as negative controls. GFP-Puro saRNA and GFP mRNA concentrations were adjusted based on their size to have the same molecular concentration. After extrusion treatment, the extrusion-loaded iPSCs were transferred to mTeSR with supplements. TM Plus basal medium and incubated at 37°C. From day 1 to day 9, the GFP-Puro saRNA-delivered iPSC subset was treated with 5ug / ml puromycin. (See Figure 1A ). Cells were collected on days 1, 3, 9, 15, and 21 for analysis of green fluorescence signals by flow cytometry.

[0177] like Figure 1B-1D As shown, GFP mRNA and GFP saRNA have similar extrusion delivery efficiency. However, on the first day, the GFP MFI of saRNA was more than 20 times higher than that of mRNA. In addition, puromycin selection is very effective (95%+GFP positive) and GFP expression is extended for more than 9 days of culture. After discontinuation of puromycin, GFP% dropped from 95% (the 9th day) to 6% (the 21st day), indicating that cells lose GFP-Puro saRNA in the absence of puromycin selection. The ratio of GFP+saRNA cells to non-delivered cells decreased from the 1st day to the 3rd day, while the MFI of GFP+mRNA cells remained the same, indicating that the iPSC delivered by saRNA has a proliferation disadvantage. The MFI of GFP-positive cells is still high in the GFP-Puro saRNA on the 21st day, and is comparable to the GFP mRNA on the 1st day.

[0178] In summary, these results demonstrate that GFP saRNA is expressed at a higher level and for a longer period of time compared to GFP mRNA.

[0179] Example 2: Analysis of Ascl1 saRNA-derived cells after extrusion treatment

[0180] To evaluate whether Ascl1 saRNA delivered to cells using the squeezing treatment method described herein can induce dopamine neuron differentiation, iPSCs were treated with Accutase and dissociated into single cells. 8iPSCs of cells / mL were added and combined with one of the following conditions in a cell suspension: (1) GFP saRNA; (2) Ascl1 saRNA; (3) Ascl1 saRNA+5TF (FoxA2, ​​Lmx1a, NR4A2, Pitx3 and EN1) mRNA (non-self-amplification). The cell suspension was added to a stenosis of the same size as described in Example 1 at room temperature under a pressure of 60psi. After the cells passed through the stenosis, the cells were collected and transferred to StemFlex basal medium with supplements and incubated at 37°C for 6 hours. At 6 hours, the cells were washed with PBS to remove unwanted debris. A mixture of Stem lex basal medium and N2B medium + B27 (100X) with 5ug / ml puromycin was added at a ratio of 1:1. The mixture was incubated for 18 hours. The culture medium was then removed and a mixture of NBM culture medium + B27 (50X) + BDNF + GDNF (1:1000) was added. Total RNA was collected from cells on days 1 and 4, and the expression of general neuronal markers (NeuroD1) and dopamine lineage markers (FoxA2, ​​Pitx3, Lmx1a, NR4A2, and TH) was analyzed using RT-qPCR.

[0181] like Figure 2 and Figure 3 As shown, all markers were upregulated compared to GFP saRNA. Most markers were more expressed on day 4 than on day 1. All tested dopamine lineage markers were higher when Ascl1 saRNA was co-delivered with 5TF (FoxA2, ​​Lmx1a, NR4A2, Pitx3, and EN1) mRNA compared to Ascl1 saRNA alone, indicating an additional effect of 5TF mRNA.

[0182] Example 3: Analysis of Ascl1 saRNA-derived dopamine neurons after crush treatment

[0183] In order to evaluate whether dopamine neurons can be generated by delivering Ascl1 saRNA to cells using the extrusion treatment method described herein, iPSCs were treated with Accutase and dissociated into single cells. Then, the density was prepared to be 1x108 cells / mL of iPSC, and merged with Ascl1 saRNA+5TF (FoxA2, ​​Lmx1a, NR4A2, Pitx3 and EN1) mRNA (non-self-amplification) in a cell suspension. The cell suspension was added to a stenosis with the same size as described in Example 1 at room temperature under a pressure of 60psi. After the cells were passed through the stenosis, the cells were collected and transferred to a StemFlex basal medium with supplements and incubated at 37°C for 6 hours. At 6 hours, the cells were washed with PBS to remove unwanted debris. A mixture of Stem lex basal medium and N2B medium+B27 (100X) with 5ug / ml puromycin at a ratio of 1:1 was added. The mixture was incubated for 18 hours. The medium was then 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 replaced with NBM medium + B27 (50X) + BDNF and GDNF (1:1000). The cells were fixed on day 14 and immunofluorescence staining was performed for dopamine neuron marker tyrosine hydroxylase TH, early neuron marker TUJ1, and mature neuron marker MAP2.

[0184] like Figure 4 As shown, all neuron-like cells express the early neuronal marker TUJ1. Some cells have high expression of dopamine neuron marker tyrosine hydroxylase (green arrow), while some cells (red arrow) have lower expression. All neuron-like cells express mature neuron marker MAP2, while some cells also have high expression of dopamine neuron marker tyrosine hydroxylase TH (green arrow), while some cells have lower TH (red arrow) expression. These results further confirm the earlier data and prove that the use of extrusion treatment to deliver saRNA can effectively induce iPSC differentiation into neurons.

[0185] Example 4: Analysis of kinetic differences between mRNA (non-self-amplification) and saRNA after extrusion treatment

[0186] To evaluate the kinetic differences between mRNA and saRNA, iPSCs were treated with Accutase and dissociated into single cells. Then, a density of 1x10 8iPSCs of 10 cells / mL were added and combined with one of the following conditions in a cell suspension: (1) GFP saRNA; (2) 6TF (Ascl1, FoxA2, ​​Lmx1a, NR4A2, Pitx3 and EN1) + PAC mRNA (non-self-amplification) (3) Ascl1 saRNA; (4) Ascl1 saRNA + 6TF (Ascl1, FoxA2, ​​Lmx1a, NR4A2, Pitx3 and EN1) mRNA (non-self-amplification). The cell suspension was added to a stenosis of the same size as described in Example 1 at room temperature under a pressure of 60psi. After the cells passed through the stenosis, the cells were collected and transferred to StemFlex basal medium with supplements and incubated at 37°C for 6 hours. At 6 hours, the cells were washed with PBS to remove unwanted debris. A mixture of Stem lex basal medium and N2B medium + B27 (100X) with 5ug / ml puromycin was added at a ratio of 1:1. The mixture was incubated for 18 hours. The medium was then removed and a mixture of NBM medium + B27 (50X) + BDNF + GDNF (1:1000) was added. Total RNA was collected from cells on days 1 and 4, and the expression of general neuronal markers (NeuroD1) and dopamine lineage markers (FoxA2, ​​Pitx3, Lmx1a, NR4A2 and TH) was analyzed using RT-qPCR.

[0187] like Figure 5 and Figure 6 As shown, most markers showed that 6TF mRNA had higher target gene expression on day 1 compared to day 4, indicating the transient nature of the mRNA. For samples with saRNA, most target genes increased from day 1 to day 4, showing the effect of long-term expression of saRNA.

[0188] In summary, the above results demonstrate that saRNA encoding reprogramming factors, especially when delivered using the extrusion treatment method, allows long-term expression of the encoded reprogramming factors and efficient differentiation of iPSCs into neurons.

[0189] Incorporated by Reference

[0190] All publications, 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 publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0191] Equivalent solutions

[0192] Although various specific aspects have been illustrated and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the present disclosure. After reading this specification, many changes will become apparent to those skilled in the art.

Claims

1. A method for inducing the expression of reprogramming factors in cells, comprising passing a cell suspension comprising the cells through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes a perturbation in the cell membrane, so that a self-amplifying RNA encoding the reprogramming factor can enter the cell through the perturbation when in contact with the cell, and thereby cause the reprogramming factor to be expressed in the cell.

2. The method of claim 1, wherein 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 perturbation.

3. The method of claim 1 or 2, wherein after the self-amplifying RNA enters the cell through the perturbation, the expression of the reprogramming factor in the cell increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times.

4. A method for enhancing the expression of reprogramming factors in cells, comprising passing a cell suspension comprising the cells through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes a perturbation within the cell membrane, such that self-amplifying RNA encoding the reprogramming factor can enter the cell through the perturbation when in contact with the cell, and thereby resulting in enhanced expression of the reprogramming factor compared to the corresponding expression in a reference cell.

5. The method of claim 4, wherein the reference cell comprises a corresponding cell that passed through the stenosis under the set of parameters, and wherein the reference cell is contacted with a non-self-amplifying RNA encoding the reprogramming factor.

6. The method of claim 4, wherein the reference cell comprises a corresponding cell that did not pass through the stenosis under the set of parameters, and wherein the reference cell is contacted with the self-amplifying RNA encoding the reprogramming factor.

7. The method of any one of claims 4 to 6, wherein the enhanced expression comprises (i) an increased level of expression, (ii) a longer duration of expression, or (iii) both (i) and (ii).

8. The method of claim 7, wherein the expression level of the reprogramming factor in the cell is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, or at least about 500 times compared to the expression level in the reference cell.

9. The method of claim 7 or 8, wherein the duration of expression of the reprogramming factor in the cell is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 450 times, or at least about 500 times compared to the duration of expression in the reference cell.

10. A method for reprogramming cells, comprising passing a cell suspension comprising the cells through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes a perturbation within the cell membrane, such that a self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbation when in contact with the cell, and thereby reprogram the cell.

11. The method of claim 10, wherein 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 perturbation.

12. The method of claim 10 or 11, wherein after the self-amplifying RNA enters the cell through the perturbation, the expression of the reprogramming factor in the cell increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times.

13. A method for enhancing cell reprogramming, comprising passing a cell suspension comprising the cells through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes a perturbation within the cell membrane, such that self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbation when in contact with the cell, and thereby resulting in enhanced reprogramming of the cell compared to a reference method.

14. The method of claim 13, wherein the reference method comprises passing the cell suspension through the stenosis under the set of parameters and contacting the cell suspension with a non-self-amplifying RNA encoding the reprogramming factor.

15. The method of claim 13, wherein the reference method does not include passing the cell suspension through the stenosis under the set of parameters, and wherein the cell suspension is contacted with the self-amplifying RNA.

16. The method of any one of claims 13 to 15, wherein the enhanced reprogramming of the cells comprises (i) a greater number of cells that have been reprogrammed, (ii) a shorter duration required to reprogram cells, or (iii) both (i) and (ii).

17. The method of claim 16, wherein the number of cells that have been reprogrammed 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 as compared to the reference method.

18. The method of claim 16 or 17, wherein the duration required to reprogram the 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.

19. The method of any one of claims 10 to 18, wherein the reprogramming of the cells comprises inducing the cells to differentiate into neurons.

20. The method of any one of claims 10 to 19, wherein the reprogramming of the cell comprises inducing expression of other reprogramming factors in the cell.

21. A method for producing neurons, comprising passing a cell suspension through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes perturbations within cell membranes present in the cell suspension, such that self-amplifying RNA encoding a reprogramming factor can enter the cell through the perturbation when in contact with the cell, and thereby cause the cell to differentiate into a neuron.

22. A method for enhancing the production of neurons, comprising passing a cell suspension through a stenosis under a set of parameters, wherein passing the cell suspension through the stenosis under the set of parameters causes perturbations within the cell membranes present in the cell suspension, such that self-amplifying RNA encoding reprogramming factors can enter the cells through the perturbations when in contact with the cells, and thereby resulting in enhanced production of neurons compared to the reference method.

23. The method of claim 22, wherein the reference method comprises passing the cell suspension through the stenosis under the set of parameters and contacting the cell suspension with a non-self-amplifying RNA encoding the reprogramming factor.

24. The method of claim 22, wherein the reference method does not include passing the cell suspension through the stenosis under the set of parameters, and wherein the cell suspension is contacted with the self-amplifying RNA.

25. The method of any one of claims 1 to 14, further comprising contacting the cell suspension with the self-amplifying RNA prior to passing the cell suspension through the stenosis.

26. The method of claim 25, wherein the cells are contacted with the self-amplifying RNA prior to passing the cell suspension through the stenosis.

27. The method of any one of claims 1 to 26, further comprising contacting the cell suspension with the self-amplifying RNA during passage of the cell suspension through the stenosis.

28. The method of claim 27, wherein the cells are contacted with the self-amplifying RNA during passage of the cell suspension through the stenosis.

29. The method of any one of claims 1 to 28, further comprising contacting the cell suspension with the self-amplifying RNA after passing the cell suspension through the stenosis.

30. The method of claim 29, wherein the cells are contacted with the self-amplifying RNA after passing the cell suspension through the stenosis.

31. The method of any one of claims 1 to 30, wherein the reprogramming factor comprises a transcription factor.

32. The method of claim 31, wherein 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 a combination thereof.

33. The method of any one of claims 1 to 32, further comprising contacting the cell suspension with a payload, such that the payload can also enter the cells upon contact with the cells through the perturbation.

34. The method of claim 33, comprising contacting the cell suspension with the payload prior to passing the cell suspension through the stenosis.

35. The method of claim 34, wherein the cells are contacted with the payload prior to passing the cell suspension through the stenosis.

36. The method of any one of claims 33 to 35, comprising contacting the cell suspension with the payload during passage of the cell suspension through the stenosis.

37. The method of claim 36, wherein the cells are contacted with the payload during passage of the cell suspension through the stenosis.

38. The method of any one of claims 33 to 37, comprising contacting the cell suspension with the payload after passing the cell suspension through the stenosis.

39. The method of claim 38, wherein the cells are contacted with the payload after passing the cell suspension through the stenosis.

40. The method of any one of claims 33 to 39, wherein the self-amplifying RNA and the payload are contacted with the cell simultaneously.

41. The method of any one of claims 33 to 39, wherein the self-amplifying RNA and the payload are contacted with the cell sequentially.

42. The method of any one of claims 33 to 41, wherein the payload comprises a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, a metal-containing compound, an antibody, a transcription factor, a nanoparticle, a liposome, a fluorescently labeled molecule, or a combination thereof.

43. The method of claim 42, wherein the nucleic acid comprises DNA, RNA, or both.

44. The method of claim 43, wherein the DNA comprises recombinant DNA, cDNA, genomic DNA, or a combination thereof.

45. The method of claim 44, wherein the RNA comprises siRNA, non-self-amplifying mRNA, micro RNA (miRNA), lncRNA, tRNA, shRNA, self-amplifying mRNA (saRNA), or a combination thereof.

46. ​​The method of any one of claims 42 to 45, wherein the small molecule comprises an impermeable small molecule.

47. The method of any one of claims 33 to 46, wherein the payload comprises additional reprogramming factors.

48. The method of claim 47, wherein 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 a combination thereof.

49. The method of any one of claims 1 to 48, wherein the cells comprise stem cells, somatic cells, or both.

50. The method of claim 49, wherein the stem cell is an induced pluripotent stem cell (iPSC), an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, or a combination thereof.

51. The method of claim 50, wherein the stem cells are iPSCs.

52. The method of any one of claims 49 to 51, wherein the somatic cells comprise blood cells.

53. The method of claim 52, wherein the blood cells are PBMCs.

54. The method of claim 53, wherein the PBMCs comprise immune cells.

55. The method of claim 54, wherein the immune cells comprise T cells, B cells, natural killer (NK) cells, dendritic cells (DC), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells, or a combination thereof.

56. A method as described in any one of claims 1 to 55, wherein the set of parameters is selected from cell density; pressure; length, width and / or depth of the stenosis; diameter of the stenosis; diameter of the cells; temperature; entrance angle of the stenosis; exit angle of the stenosis; length, width and / or width of the approach zone; surface properties of the stenosis (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity); operating flow rate; payload concentration; viscosity, osmotic pressure, salt concentration, serum content and / or pH of the cell suspension; time in the stenosis; shear rate in the stenosis; type of payload or a combination thereof.

57. The method of claim 56, wherein the cell density is at least about 6 x 10 7 cells / mL, at least about 7x10 7 cells / mL, at least about 8 x 10 7 cells / mL, at least about 9 x 10 7 cells / mL, at least about 1x 10 8 cells / mL, at least about 1.1 x 10 8 cells / mL, at least about 1.2 x 10 8 cells / mL, at least about 1.3 x 10 8 cells / mL, at least about 1.4 x 10 8 cells / mL, at least about 1.5 x 10 8 cells / mL, at least about 2.0 x 10 8 cells / mL, at least about 3.0 x 10 8 cells / mL, at least about 4.0 x 10 8 cells / mL, at least about 5.0 x 10 8 cells / mL, at least about 6.0x10 8 cells / mL, at least about 7.0 x 10 8 cells / mL, at least about 8.0 x 10 8 cells / mL, at least about 9.0 x 10 8 cells / mL or at least about 1.0 x 10 9 cells / mL or greater.

58. The method of claim 56 or 57, wherein 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.

59. The method of any one of claims 1 to 58, wherein the stenosis is contained in a microfluidic chip.

60. The method of any one of claims 1 to 59, wherein the narrow 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 one or more cells in the cell population.

61. The method of any one of claims 1 to 60, wherein the stenosis has a length of between about 0 μm and about 100 μm.

62. The method of claim 61, wherein the stenosis has a length of less than about 1 μm.

63. The method of claim 61, wherein the length of the stenosis 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.

64. The method of claim 61, wherein the length of the stenosis 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.

65. The method of claim 64, wherein the stenosis is about 10 μm in length.

66. The method of any one of claims 1 to 65, wherein the stenosis has a width of between about 0 μm and about 10 μm.

67. The method of claim 66, wherein the narrow width 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.

68. The method of claim 66, wherein the stenosis has a width of between about 3 μm and about 10 μm.

69. The method of claim 68, wherein the stenosis has a width of 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.

70. The method of claim 69, wherein the stenosis has a width of about 6 μm.

71. The method of any one of claims 1 to 70, wherein the depth of the stenosis is at least about 1 μm.

72. The method of claim 71, wherein the depth of the stenosis 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.

73. The method of claim 71, wherein the stenosis has a length of about 5 μm to about 90 μm.

74. The method of claim 73, wherein the depth of the stenosis 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.

75. The method of claim 74, wherein the stenosis has a length of about 70 μm.

76. The method of any one of claims 1 to 75, wherein the length of the stenosis is about 10 μm, the width of the stenosis is about 6 μm, and the depth of the stenosis is about 70 μm.

77. The method of any one of claims 33 to 76, comprising contacting the cell suspension with a plurality of self-amplifying RNAs, a plurality of payloads, or both: (i) before passing the cell suspension through the stenosis, (ii) during passing the cell suspension through the stenosis, (iii) after passing the cell suspension through the stenosis, or (iv) any combination of (i) to (iii).

78. The method of claim 77, wherein the cell is contacted with the plurality of self-amplifying RNAs, and wherein at least two or more of the plurality of self-amplifying RNAs can enter the cell through the perturbation.

79. The method of claim 77 or 78, wherein the cell is contacted with the plurality of payloads, and wherein at least two or more of the plurality of payloads can enter the cell via the perturbation.

80. The method of any one of claims 77 to 79, wherein the plurality of self-amplifying RNAs comprises at least 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.

81. The method of any one of claims 77 to 80, wherein each of the plurality of self-amplifying RNAs is different.

82. The method of any one of claims 77 to 80, wherein at least two of the plurality of self-amplifying RNAs are identical.

83. The method of any one of claims 77 to 82, wherein the plurality of payloads comprises 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.

84. The method of any one of claims 77 to 83, wherein each of the plurality of payloads is different.

85. The method of any one of claims 77 to 83, wherein at least two of the plurality of payloads are identical.

86. The method of any one of claims 77 to 85, wherein the plurality of self-amplifying RNAs are contacted with the cell simultaneously.

87. The method of any one of claims 77 to 85, wherein the plurality of self-amplifying RNAs are contacted with the cell sequentially.

88. The method of any one of claims 77 to 85, wherein the plurality of payloads are contacted with the cell simultaneously.

89. The method of any one of claims 77 to 85, wherein the plurality of payloads are contacted with the cell sequentially.

90. The method of any one of claims 77 to 85, wherein the plurality of self-amplifying RNAs and the plurality of payloads are contacted with the cell simultaneously.

91. The method of any one of claims 77 to 85, wherein the plurality of self-amplifying RNAs and the plurality of payloads are contacted with the cell sequentially.

92. The method of any one of claims 1 to 91, comprising passing the cell suspension through a plurality of constrictions.

93. The method of claim 92, wherein the plurality of stenoses comprises 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 individual stenoses.

94. The method of claim 92 or 93, wherein each of the plurality of stenoses is identical.

95. The method of claim 92 or 93, wherein one or more of the plurality of stenoses are different.

96. The method of claim 95, wherein the one or more stenoses vary in length, depth, width, or a combination thereof.

97. The method of any one of claims 92 to 96, wherein each of the plurality of constrictions is associated with the same self-amplifying RNA.

98. The method of any one of claims 92 to 96, wherein one or more of the plurality of constrictions are associated with different self-amplifying RNAs.

99. A method as claimed in claim 98, wherein the multiple constrictions include a first constriction associated with a first self-amplifying RNA and a second constriction associated with a second amplifying RNA, wherein the cell suspension is passed through the first constriction so that the first self-amplifying RNA can enter the cells, and then the cell suspension is passed through the second constriction so that the second self-amplifying RNA can enter the cells.

100. The method of claim 99, wherein the cell suspension is passed through the second stenosis 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 is passed through the first stenosis.

101. The method of any one of claims 1 to 100, further comprising contacting the cell suspension with an additional compound.

102. The method of claim 101, wherein the additional compound comprises a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, a metal-containing compound, an antibody, a transcription factor, a nanoparticle, a liposome, a fluorescently labeled molecule, or a combination thereof.

103. The method of claim 102, wherein the additional compound is a nucleic acid encoding an enzyme that confers antibiotic resistance.

104. The method of any one of claims 101 to 103, wherein contacting the additional compound with the cell suspension occurs simultaneously with contacting the cell suspension with the self-amplifying RNA.

105. The method of any one of claims 101 to 103, wherein contacting the additional compound with the cell suspension occurs before or after contacting the cell suspension with the self-amplifying RNA.

106. The method of any one of claims 103 to 105, further comprising collecting the cell suspension that has passed through the stenosis and treating the cell suspension with the antibiotic.

107. The method of any one of claims 103 to 106, wherein the enzyme comprises puromycin-N-acetyltransferase and the antibiotic is puromycin.

108. The method of claim 106 or 107, wherein after treatment with the antibiotic, the proportion of cells present in the cell suspension that contain the self-amplifying RNA increases 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.

109. A cell population comprising reprogramming factors, wherein the cell population is produced using the method of any one of claims 1 to 9 and 25 to 108.

110. A population of reprogrammed cells produced using the method of any one of claims 10 to 20 and 25 to 108.

111. A population of neurons produced using the method of any one of claims 21 to 108.

112. A composition comprising the cell population of claim 109.

113. A composition comprising the reprogrammed cell population of claim 110.

114. A composition comprising the neuronal population of claim 111.

115. The composition of any one of claims 112 to 114, further comprising a pharmaceutically acceptable excipient.

116. A kit comprising the cell population of claim 109 and instructions for use.

117. A kit comprising the reprogrammed cell population of claim 110 and instructions for use.

118. A kit comprising the neuronal population of claim 111 and instructions for use.

119. A composition comprising a cell population and a self-amplifying RNA encoding a reprogramming factor that causes one or more cells in the cell population to deform under a set of parameters, wherein the one or more cells comprise a perturbation in the cell membrane sufficient to allow the self-amplifying RNA to enter the one or more cells.

120. The composition of claim 119, further comprising a pharmaceutically acceptable excipient.

121. A cell comprising a perturbation in the cell membrane due to a set of parameters that deform the cell, thereby causing the perturbation in the cell membrane of the cell to allow self-amplifying RNA encoding a reprogramming factor to enter the cell.

122. A cell comprising reprogramming factors, wherein the reprogramming factors enter the cell through a perturbation in the cell membrane caused by a set of parameters that deform the cell, thereby causing the perturbation in the cell membrane of the cell and allowing self-amplifying RNA encoding the reprogramming factors to enter the cell through the perturbation.

123. A composition comprising the cells of claim 121 or 122.

124. The composition of claim 123, further comprising a pharmaceutically acceptable excipient.

125. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a composition as described in any one of claims 112 to 115, 119, 120, 123 and 124.

126. The method of claim 125, wherein the disease or disorder comprises a neurological disorder.

127. The method of claim 126, wherein the neurological disorder comprises Parkinson's disease.

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