Modified hematopoietic stem cells and uses thereof

Abstract

The present disclosure provides methods of generating modified HSCs, the methods comprising passing a cell suspension comprising cells and a payload through a constriction, which deforms the cells, thereby causing perturbation of the cells and allowing the payload to enter the cells. In some embodiments, the payload enhances one or more properties of the HSCs, thereby enabling the HSCs to be successfully engrafted into the bone marrow of a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 296,544, filed January 5, 2022, and No. 63 / 374,622, filed September 6, 2022, each of which is incorporated by reference in its entirety herein.

[0002] Reference to Electronically Submitted Sequence Listing The contents of the electronically submitted sequence listing in .XML file format (Name: 4821_007PC02_Seqlisting_ST26, Size: 20,966 bytes, and Creation Date: January 3, 2023) submitted with the application are hereby incorporated by reference in their entirety.

[0003] The present disclosure relates generally to methods for producing hematopoietic stem cells (HSCs) with enhanced properties (eg, increased resistance to mobilizing and / or apoptotic factors). [Background technology]

[0004] Hemoglobinopathies are a group of inherited monogenic blood cell disorders (e.g., sickle cell disease and thalassemia syndromes) characterized by abnormal production or structure of the hemoglobin molecule. Modell et al., Bull World Health Organ 86:480-487 (2008); and Kohne et al., Dtsch Arztebl Int 108:532 (2011). Such disorders can be associated with severe pain and multiorgan ischemic damage, increased risk of infection, and even premature death. Current treatment options include small molecule-based therapies such as hydroxyurea, L-glutamine, and voxerotol, all of which have shown limited efficacy and are not curative. Hematopoietic stem cell transplantation (HSCT) has also shown some success, but such treatment regimens require the use of toxic myeloablative therapies that deplete the endogenous stem cell compartment to make room for transplantation of therapeutically modified HSCs into the bone marrow niche. Busulfan, a commonly used myeloablative agent, is associated with severe short-term and long-term clinical adverse events (e.g., cerebellar hemorrhage, myelodysplastic syndrome, veno-occlusive disease). Morales-Ramirez et al., Arch Med Res 37:316-321 (2006); Iwamoto et al., Cancer Sci 95:454-458 (2004); and Finazii et al., Br J Haematol 110:577-583 (2000). Thus, there remains a need for more efficient and safe approaches to HSC-based treatment regimens for the treatment of hemoglobinopathies and other similar disorders. Summary of the Invention

[0005] Disclosed herein are methods of producing modified hematopoietic stem cells (HSCs), the methods comprising passing a cell suspension comprising a population of HSCs through a constriction under one or more parameters, where passing the cell suspension through the constriction under the one or more parameters allows a payload to enter the HSCs, and the payload modifies the HSCs such that the HSCs exhibit (i) increased resistance to a mobilizing factor, (ii) increased resistance to an apoptotic factor, (iii) increased resistance to a depleting factor, (iv) increased expression of a homing factor, or (v) a combination thereof.

[0006] In some embodiments, after modification, resistance of the HSCs to mobilization factors is increased by 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 corresponding HSCs that have not been passed through a constriction under one or more parameters. In some embodiments, after modification, resistance of the HSCs to apoptotic factors is increased by 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 corresponding HSCs that have not been passed through a constriction under one or more parameters. In some embodiments, after modification, resistance of the HSCs to depletion factors is increased by 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 corresponding HSCs that have not been passed through a constriction under one or more parameters. In some embodiments, expression of homing factors on the HSCs is increased by 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 corresponding HSCs that have not been passed through a constriction under one or more parameters.

[0007] Also provided herein is a method of increasing homing of hematopoietic stem cells (HSCs) to the bone marrow in a subject in need of HSCs, the method comprising passing a cell suspension comprising HSCs through a constriction under one or more parameters, where passing the cell suspension through the constriction under the one or more parameters allows a payload to enter the HSCs, and the payload modifies the HSCs such that, when the HSCs are administered to the subject, the HSCs exhibit increased homing to the bone marrow.

[0008] In some embodiments, after modification, the HSCs exhibit increased expression of a homing receptor. In some embodiments, expression of a homing receptor on the HSCs is increased by 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, as compared to corresponding HSCs that have not been passed through a constriction under one or more parameters.

[0009] The disclosure further provides a method of increasing survival of HSCs in a subject in need of hematopoietic stem cells, the method comprising passing a cell suspension comprising HSCs through a constriction under one or more parameters, where passing the cell suspension through the constriction under the one or more parameters allows a payload to enter the HSCs, where the payload modifies the HSCs such that, when the HSCs are administered to the subject, the HSCs exhibit increased survival.

[0010] In some embodiments, after modification, the HSCs exhibit increased resistance to apoptotic factors, hi some embodiments, the resistance of the HSCs to apoptotic factors is increased by 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, as compared to corresponding HSCs that have not been passed through the constriction under one or more parameters.

[0011] Provided herein are methods for promoting engraftment of hematopoietic stem cells (HSCs) in the bone marrow of a subject in need thereof, the methods comprising passing a cell suspension comprising HSCs through a constriction under one or more parameters, where passing the cell suspension through the constriction under the one or more parameters allows a payload to enter the HSCs, where the payload modifies the HSCs such that, upon administration of the HSCs to the subject, the HSCs are capable of engrafting into the bone marrow of the subject.

[0012] In some embodiments, after modification, the HSCs exhibit (i) increased resistance to mobilization factors, (ii) increased resistance to apoptotic factors, (iii) increased resistance to depletion factors, (iv) increased expression of homing factors, or (v) a combination thereof.

[0013] In some embodiments, the resistance of the HSCs to a mobilization factor is increased by 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 a corresponding HSC that has not been passed through a stenosis under one or more parameters. In some embodiments, the resistance of the HSCs to an apoptotic factor is increased by 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 a corresponding HSC that has not been passed through a stenosis under one or more parameters. In some embodiments, resistance of the HSCs to depletion factors is increased by 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 corresponding HSCs that have not been passed through a constriction under one or more parameters. In some embodiments, expression of homing factors on the HSCs is increased by 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 corresponding HSCs that have not been passed through a constriction under one or more parameters.

[0014] Also provided herein is a method of producing modified hematopoietic stem cells (HSCs). The method includes intracellular delivery of a payload to HSCs, where the payload modifies the HSCs such that the HSCs exhibit (i) increased resistance to a mobilizing factor, (ii) increased resistance to an apoptotic factor, (iii) increased resistance to a depleting factor, (iv) increased expression of a homing factor, or (v) a combination thereof. Also provided herein is a method of increasing homing of hematopoietic stem cells (HSCs) to bone marrow in a subject in need of HSCs. The method includes intracellular delivery of a payload to HSCs, where the payload modifies the HSCs such that the HSCs exhibit increased homing to bone marrow when administered to the subject. Also provided herein is a method of increasing survival of hematopoietic stem cells (HSCs) in a subject in need of HSCs. The method includes intracellular delivery of a payload to HSCs, where the payload modifies the HSCs such that the HSCs exhibit increased survival when administered to the subject. Also provided herein are methods of promoting engraftment of hematopoietic stem cells (HSCs) in the bone marrow of a subject in need thereof. The methods include intracellular delivery of a payload to the HSCs, where the payload modifies the HSCs such that, upon administration of the HSCs to the subject, the HSCs are capable of engrafting in the bone marrow of the subject. In some embodiments, the payload is transiently expressed in the modified HSCs.

[0015] In some embodiments, the payload comprises a homing receptor, a cytokine, a growth factor, a cell adhesion molecule, a proliferation agent, a survival factor, a combination thereof, or a regulator thereof.

[0016] In some embodiments, the homing receptor comprises CXCR4, CXCR2, or both. In some embodiments, CXCR4 comprises an amino acid sequence different from the corresponding wild-type amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of CXCR4 comprises one of the following mutations: R334X, A175F, H113A, D171N, D262N, I284A, H281A, Q200W, Q200A, or a combination thereof.

[0017] In some embodiments, the cytokine comprises stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L), or both. In some embodiments, the growth factor comprises thrombopoietin (TPO). In some embodiments, the cell adhesion molecule comprises an integrin, a selectin, or both. In some embodiments, the cell adhesion molecule comprises VLA-4, VLA-5, LFA-1, or a combination thereof. In some embodiments, the proliferation agent comprises an activator of a signaling pathway involved in cell proliferation. In some embodiments, the signaling pathway comprises PI3K-ATK, Ras-ERK, or both. In some embodiments, the survival factor comprises Bcl-2, Bcl-xL, MCL-1, Ced-9, bfl-1, or a combination thereof.

[0018] In some embodiments, Bcl-2 comprises an amino acid sequence that differs from the corresponding wild-type amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the amino acid sequence of Bcl-2 comprises a G101V mutation, a D103Y mutation, or both.

[0019] In some embodiments, the regulator can increase the expression and / or activity of a homing receptor, a cytokine, a growth factor, a cell adhesion molecule, a proliferation agent, a survival factor, or a combination thereof. In some embodiments, the regulator comprises a prostaglandin (e.g., prostaglandin E2 (PGE2)). In some embodiments, the regulator can reduce or prevent (e.g., knock down) the activity of an inhibitor of a homing receptor, a cytokine, a growth factor, a cell adhesion molecule, a proliferation agent, a survival factor, or a combination thereof.

[0020] In some embodiments, the inhibitor comprises GPRASP1 / 2, CD26, or both.

[0021] In some embodiments, the recruitment factor comprises Plerixaflor, AMD3465, GROβ, G-CSF, anti-CD117 antibody, or a combination thereof. In some embodiments, the apoptotic factor comprises a Bcl-2 inhibitor (e.g., venetoclax), an MCL-1 inhibitor (e.g., S63845 or S64315), a BCL-XL inhibitor, or a combination thereof.

[0022] In some embodiments, the payload comprises a nucleic acid. In some embodiments, the nucleic acid comprises DNA, RNA, or both. In some embodiments, the RNA comprises mRNA, siRNA, miRNA, lncRNA, tRNA, shRNA, self-amplifying mRNA (saRNA), PNA, locked nucleic acid (LNA), or a combination thereof.

[0023] In some embodiments, the method includes contacting the HSCs with the payload (I) before the cell suspension passes through the constriction, (ii) while the cell suspension passes through the constriction, (iii) after the cell suspension passes through the constriction, or (iv) a combination thereof. In some embodiments, the HSCs are contacted with the payload before the cell suspension passes through the constriction. In some embodiments, the HSCs are contacted with the payload while the cell suspension passes through the constriction. In some embodiments, the HSCs are contacted with the payload after the cell suspension passes through the constriction.

[0024] In some aspects, HSCs are CD34+, CD90+, CD45RA-, and lineage marker negative (e.g., CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b, and CD235).

[0025] In some embodiments, the constriction is in a microfluidic chip.

[0026] In some embodiments, the one or more parameters are selected from cell density; pressure; length, width and / or depth of the constriction; diameter of the constriction; diameter of the cells; temperature; entrance angle of the constriction; exit angle of the constriction; length, width and / or width of the approach region; surface characteristics of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity); operating flow rate; payload concentration; viscosity, osmolality, salt concentration, serum content and / or pH of the cell suspension; time at the constriction; shear rate at the constriction; type of payload; or combinations thereof.

[0027] In some embodiments, the cell density is at least about 1×10 5 Cells / mL, at least approximately 2 x 10 5 Cells / mL, at least approximately 3 x 10 5 Cells / mL, at least approximately 4 x 10 5 Cells / mL, at least approximately 5 x 10 5 Cells / mL, at least approximately 6 x 10 5 Cells / mL, at least approximately 7 x 10 5Cells / mL, at least approximately 8 x 10 5 Cells / mL, at least approximately 9 x 10 5 Cells / mL, at least approximately 1 x 10 6 Cells / mL, at least approximately 2 x 10 6 Cells / mL, at least approximately 3 x 10 6 Cells / mL, at least approximately 4 x 10 6 Cells / mL, at least approximately 5 x 10 6 Cells / mL, at least approximately 6 x 10 6 Cells / mL, at least approximately 7 x 10 6 Cells / mL, at least approximately 8 x 10 6 Cells / mL, at least approximately 9 x 10 6 Cells / mL, at least approximately 1 x 10 7 Cells / mL, at least approximately 2 x 10 7 Cells / mL, at least approximately 3 x 10 7 Cells / mL, at least approximately 4 x 10 7 Cells / mL, at least approximately 5 x 10 7 Cells / mL, at least approximately 6 x 10 7 Cells / mL, at least approximately 7 x 10 7 Cells / mL, at least approximately 8 x 10 7 Cells / mL, at least approximately 9 x 10 7 Cells / mL, at least approximately 1 x 10 8 Cells / mL, at least approximately 1.1 x 10 8 Cells / mL, at least approximately 1.2 x 10 8 Cells / mL, at least approximately 1.3 x 10 8 Cells / mL, at least approximately 1.4 x 10 8 Cells / mL, at least approximately 1.5 x 10 8 Cells / mL, at least approximately 2.0 x 10 8 Cells / mL, at least approximately 3.0 x 10 8 Cells / mL, at least approximately 4.0 x 10 8 Cells / mL, at least approximately 5.0 x 10 8 Cells / mL, at least approximately 6.0 x 10 8 Cells / mL, at least approximately 7.0 x 10 8 Cells / mL, at least approximately 8.0 x 10 8 Cells / mL, at least approximately 9.0 x 108 cells / mL, or at least about 1.0 x 10 9 cells / mL or more.

[0028] In some embodiments, the pressure is at least about 20 psi, at least about 25 psi, 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.

[0029] In some embodiments, the diameter of the constriction is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the diameter of the HSC.

[0030] In some embodiments, the length of the constriction is up to about 100 μm. In some embodiments, the length of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2.5 μm, less than about 5 μm, less than about 7.5 μm, less than about 10 μm, less than about 12.5 μm, less than about 15 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, or less than about 100 μm. In some embodiments, the length of the constriction is about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 2.5 μm, about 5 μm, about 7.5 μm, about 10 μm, about 12.5 μm, about 15 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm.

[0031] In some embodiments, the width of the constriction is up to about 10 μm. In some embodiments, the width of the constriction is less than about 1 μm, less than about 2 μm, less than about 3 μm, less than about 4 μm, less than about 5 μm, less than about 6 μm, less than about 7 μm, less than about 8 μm, less than about 9 μm, or less than about 10 μm. In some embodiments, the width of the constriction is about 1 μm to about 10 μm. In some embodiments, the width of the constriction is about 1 μm, about 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.7 μm, about 1.8 μm, about 1.9 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.

[0032] In some embodiments, the depth of the constriction is at least about 1 μm. In some embodiments, the depth of the constriction is at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 110 μm, or at least about 120 μm. In some embodiments, the depth of the constriction is about 5 μm to about 90 μm. In some embodiments, the depth 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.

[0033] In some embodiments, the constriction comprises a width and a depth, wherein the width of the constriction is about 1 μm, about 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.7 μm, about 1.8 μm, about 1.9 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, and the depth of the constriction is about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or about 90 μm.

[0034] In some embodiments, contacting the HSCs with multiple payloads (i) before the cell suspension passes through the constriction, (ii) while the cell suspension passes through the constriction, (iii) after the cell suspension passes through the constriction, or (iv) a combination thereof, allows at least two or more of the multiple payloads to enter the HSCs by passing the cell suspension through the constriction under one or more parameters.

[0035] In some embodiments, 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 embodiments, at least two or more of the plurality of payloads enter the cell simultaneously. In some embodiments, at least two or more of the plurality of payloads enter the cell sequentially.

[0036] In some embodiments, the methods provided herein include passing a cell suspension through a plurality of constrictions. In some embodiments, the plurality of constrictions includes at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1,000 or more distinct constrictions.

[0037] In some embodiments, each constriction of the plurality of constrictions is the same. In some embodiments, one or more constrictions of the plurality of constrictions are different.

[0038] In some embodiments, one or more of the constrictions differ in length, depth, width, or a combination thereof. In some embodiments, each constriction of the plurality of constrictions is associated with the same payload. In some embodiments, one or more of the plurality of constrictions are associated with different payloads.

[0039] In some embodiments, the multiple constrictions are contained within a single microfluidic chip. In some embodiments, the multiple constrictions are contained within a plurality of microfluidic chips, each of the plurality of microfluidic chips comprising a constriction. In some embodiments, each of the plurality of microfluidic chips are the same. In some embodiments, one or more of the plurality of microfluidic chips are different.

[0040] In some embodiments, the time interval between passage of the cell suspension through a first constriction and a second constriction of the plurality of constrictions is less than about 1 μs, less than about 1 second, less than about 1 minute, less than about 30 minutes, less than about 1 hour, less than about 6 hours, less than about 12 hours, less than about 1 day, less than about 2 days, less than about 3 days, less than about 4 days, or less than about 5 days.

[0041] In some embodiments, the plurality of constrictions includes a first constriction associated with a first payload and a second constriction associated with the payload, where the cell suspension passes through the first constriction to allow the first payload to enter the HSCs, and then the cell suspension passes through the second constriction to allow the second payload to enter the HSCs.

[0042] Provided herein is a population of hematopoietic stem cells (HSCs) produced using any of the methods disclosed herein.Also provided herein is a composition comprising the population of HSCs produced herein and a pharma- ceutically acceptable carrier.Further provided herein is a kit comprising the population of HSCs produced herein and instructions for use.

[0043] Provided herein is a composition comprising a population of modified hematopoietic stem cells (HSCs).The modified HSCs comprise a payload, which can (i) increase the resistance of the modified HSCs to a mobilizing factor, (ii) increase the resistance of the modified HSCs to an inhibitor of an anti-apoptotic factor, (iii) increase the resistance of the modified HSCs to a depleting factor, (iv) increase the expression of a homing factor on the modified HSCs, or (v) a combination thereof.

[0044] In some embodiments, the resistance of the modified HSCs to a mobilization factor is increased by 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 a corresponding HSC that has not been modified to include a payload. In some embodiments, the resistance of the modified HSCs to an inhibitor of an anti-apoptotic factor is increased by 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 a corresponding HSC that has not been modified to include a payload. In some embodiments, the resistance of the modified HSCs to depletion factors is increased by 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 corresponding HSCs that have not been modified to include a payload. In some embodiments, the expression of a homing receptor on the modified HSCs is increased by 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 corresponding HSCs that have not been modified to include a payload.

[0045] In some embodiments, the payload is transiently expressed in the modified HSC. In some embodiments, the payload comprises a homing receptor, a cytokine, a growth factor, a cell adhesion molecule, a proliferation agent, a survival factor, a combination thereof, or a regulator thereof. In some embodiments, the homing receptor comprises CXCR4, CXCR2, or both. In some embodiments, the CXCR4 comprises an amino acid sequence that differs from the corresponding wild-type amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid sequence of CXCR4 comprises one of the following mutations: R334X, A175F, H113A, D171N, D262N, I284A, H281A, Q200W, Q200A, or a combination thereof. In some embodiments, the cytokine comprises stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L), or both. In some embodiments, the growth factor comprises thrombopoietin (TPO). In some embodiments, the cell adhesion molecule comprises an integrin, a selectin, or both. In some embodiments, the cell adhesion molecule comprises VLA-4, VLA-5, LFA-1, or a combination thereof. In some embodiments, the proliferation agent comprises an activator of a signaling pathway involved in cell proliferation. In some embodiments, the signaling pathway comprises PI3K-ATK, Ras-ERK, or both. In some embodiments, the survival factor comprises Bcl-2, Bcl-xL, MCL-1, Ced-9, bfl-1, or a combination thereof. In some embodiments, Bcl-2 comprises an amino acid sequence that differs from the corresponding wild-type amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the amino acid sequence of Bcl-2 comprises a G101V mutation, a D103Y mutation, or both. In some embodiments, the regulator can increase the expression and / or activity of a homing receptor, a cytokine, a growth factor, a cell adhesion molecule, a proliferation agent, a survival factor, or a combination thereof. In some embodiments, the regulator comprises a prostaglandin (e.g., prostaglandin E2 (PGE2)).In some embodiments, the regulator can reduce or prevent (e.g., knock down) the activity of an inhibitor of a homing receptor, a cytokine, a growth factor, a cell adhesion molecule, a proliferation agent, a survival factor, or a combination thereof. In some embodiments, the inhibitor comprises GPRASP1 / 2, CD26, or both. In some embodiments, the recruitment factor comprises Plerixaflor, AMD3465, GROβ, G-CSF, an anti-CD117 antibody, or a combination thereof. In some embodiments, the apoptotic factor comprises a Bcl-2 inhibitor (e.g., venetoclax), an MCL-1 inhibitor (e.g., S63845 or S64315), a BCL-XL inhibitor, or a combination thereof. In some embodiments, the payload comprises a nucleic acid. In some embodiments, the nucleic acid comprises DNA, RNA, or both. In some embodiments, the RNA comprises an mRNA, an siRNA, a miRNA, an lncRNA, a tRNA, a shRNA, a self-amplifying mRNA (saRNA), a PNA, a locked nucleic acid (LNA), or a combination thereof.

[0046] Disclosed herein are methods of treatment in a subject in need of treatment for a disease or disorder, the methods comprising administering to the subject any of the populations of HSCs generated herein or any of the compositions described herein.

[0047] In some embodiments, the disease or disorder comprises a hematological disorder, hi some embodiments, the hematological disorder comprises sickle cell disease (SCD), thalassemia syndrome, severe aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, congenital amegakaryocytic thrombocytopenia, or a combination thereof.

[0048] In some embodiments, the disease or disorder comprises an immune disorder, hi some embodiments, the immune disorder comprises severe combined immunodeficiency, Wiskott-Aldrich syndrome, or both.

[0049] In some embodiments, the disease or disorder comprises a metabolic disorder, hi some embodiments, the metabolic disorder comprises Krabbe disease (GLD), Hurler syndrome, adrenoleukodystrophy, metachromatic leukodystrophy, Fabry disease, Gaucher disease, cystinosis, Hunter syndrome, Pompe disease, or a combination thereof.

[0050] In some embodiments, the subject does not undergo a myeloablative conditioning regimen prior to administration of the population or composition of HSCs. In some embodiments, the myeloablative conditioning regimen comprises radiation, chemotherapy, a small molecule, an antibody, or a combination thereof. [Brief description of the drawings]

[0051] [Figure 1] Engraftment of squeezed human CD34+ HSCs in bone marrow of NSG (top) and NBSGW (bottom) mice (no pretreatment) 6 weeks after adoptive transfer is shown. Engraftment of HSCs is expressed as a percentage of total CD45+ bone marrow cells measured using flow cytometry. "hCD45" = human CD45 (i.e., adoptively transferred human HSCs) and "mCD45" = mouse CD45 (i.e., endogenous mouse HSCs). [Diagram 2] Figure 2 shows the frequency of adoptively transferred human HSCs present in the peripheral blood (Figure 2A), spleen (Figure 2B) and bone marrow (Figure 2C) of NSG and NBSGW mice 6 weeks after transfer. Cell frequencies are expressed as percentages of total CD45+ cells. "NBSGW NC" = HSCs squeezed without any payload and administered to NBSGW mice; "NBSGW SQZ" = HSCs squeezed with GFP mRNA and administered to NBSGW mice; "NSG NC" = HSCs squeezed without any payload and administered to NSG mice; and "NSG SQZ" = HSCs squeezed with GFP mRNA and administered to NSG mice, i.e., groups A, B, C and D in Table 5, respectively (see Example 1). [Figure 3A]Figure 2 shows the expression kinetics of different CXCR4 variants in human CD34+HSCs squeezed with CXCR4 variant mRNA. CXCR4 expression measured using flow cytometry at 4, 24, 48 and 72 hours after squeeze is displayed. In A, CXCR4 expression is shown as geometric mean fluorescence intensity. In B, expression of CXCR4 variants is shown as fold increase over the corresponding expression in control HSCs (i.e. no payload and no squeeze treatment). The different groups shown are as follows: (1) no payload and no squeezing ("NC"), (2) squeezed without any payload ("Empty"), (3) squeezed with wild-type CXCR4 mRNA ("CXCR4 wt"), (4) squeezed with CXCR4-A175F variant mRNA ("CXCR4 A175F"), (5) squeezed with CXCR4-A175F+R334X variant mRNA ("CXCR4 A175F R334X"), and (6) squeezed with CXCR4-R334X variant mRNA ("CXCR4 R334X"), i.e., groups A to F in Table 6, respectively (see Example 2). [Figure 3B]Figure 2 shows the expression kinetics of different CXCR4 variants in human CD34+HSCs squeezed with CXCR4 variant mRNA. CXCR4 expression measured using flow cytometry at 4, 24, 48 and 72 hours after squeeze is displayed. In A, CXCR4 expression is shown as geometric mean fluorescence intensity. In B, expression of CXCR4 variants is shown as fold increase over the corresponding expression in control HSCs (i.e. no payload and no squeeze treatment). The different groups shown are as follows: (1) no payload and no squeezing ("NC"), (2) squeezed without any payload ("Empty"), (3) squeezed with wild-type CXCR4 mRNA ("CXCR4 wt"), (4) squeezed with CXCR4-A175F variant mRNA ("CXCR4 A175F"), (5) squeezed with CXCR4-A175F+R334X variant mRNA ("CXCR4 A175F R334X"), and (6) squeezed with CXCR4-R334X variant mRNA ("CXCR4 R334X"), i.e., groups A to F in Table 6, respectively (see Example 2). [Figure 3C] Comparison of CXCL12 signaling inhibition caused by Plerixafor in human CD34+ HSCs squeezed with wild-type CXCR4 mRNA (middle bar) or CXCR4 variant (containing A175F modification) mRNA (right bar). Non-squeezed HSCs (i.e., treated with Plerixafor alone) were used as control (left bar). [Figure 3D](D) CXCR4 expression in squeezed human CD34+ HSCs after adoptive transfer into NSG mice. (D) Schematic diagram of the experimental design. Prior to transfer, human CD34+ HSCs were squeezed with no payload (i.e., empty squeeze, "ES") or with mRNA encoding CXCR4 (i.e., a bone marrow homing receptor) as indicated. (E) Comparison of CXCR4 expression (geometric mean fluorescence intensity) by adoptively transferred cells in bone marrow 24 hours after adoptive transfer. [Figure 3E] (D) CXCR4 expression in squeezed human CD34+ HSCs after adoptive transfer into NSG mice. (D) Schematic diagram of the experimental design. Prior to transfer, human CD34+ HSCs were squeezed with no payload (i.e., empty squeeze, "ES") or with mRNA encoding CXCR4 (i.e., a bone marrow homing receptor) as indicated. (E) Comparison of CXCR4 expression (geometric mean fluorescence intensity) by adoptively transferred cells in bone marrow 24 hours after adoptive transfer. [Figure 4A] Figure 1 shows the expression kinetics of Bcl-2 variants in human CD34+ HSCs squeezed with Bcl-2 variant mRNA. Bcl-2 expression is shown at 4, 24, 48 and 72 hours after squeeze, measured using flow cytometry. In A, Bcl-2 expression is shown as the geometric mean fluorescence intensity. In B, Bcl-2 expression is shown as the fold increase over the corresponding expression in control HSCs (i.e., no payload and no squeeze). The different groups shown are: (1) no payload and no squeeze ("NC"); (2) squeezed without any payload ("Empty"); (3) squeezed with wild-type Bcl-2 mRNA ("Bcl-2 wt"); and (4) squeezed with Bcl-2-G101V variant mRNA ("Bcl-2 G101V"). That is, Groups A, B, G and H in Table 6, respectively (see Example 2). [Figure 4B]Figure 1 shows the expression kinetics of Bcl-2 variants in human CD34+ HSCs squeezed with Bcl-2 variant mRNA. Bcl-2 expression is shown at 4, 24, 48 and 72 hours after squeeze, measured using flow cytometry. In A, Bcl-2 expression is shown as the geometric mean fluorescence intensity. In B, Bcl-2 expression is shown as the fold increase over the corresponding expression in control HSCs (i.e., no payload and no squeeze). The different groups shown are: (1) no payload and no squeeze ("NC"); (2) squeezed without any payload ("Empty"); (3) squeezed with wild-type Bcl-2 mRNA ("Bcl-2 wt"); and (4) squeezed with Bcl-2-G101V variant mRNA ("Bcl-2 G101V"). That is, Groups A, B, G and H in Table 6, respectively (see Example 2). [Diagram 5] Figure 1 shows Bcl-2 expression in squeezed human CD34+ HSCs after adoptive transfer into NSG mice. A shows a schematic of the experimental design. Prior to transfer, human CD34+ HSCs were squeezed with control mRNA or Bcl-2 variant mRNA (i.e., containing the G101V amino acid modification) as indicated. B shows a comparison of Bcl-2 expression (geometric mean fluorescence intensity) by adoptively transferred cells in bone marrow 24 hours after adoptive transfer. [Figure 6]Competitive engraftment following adoptive transfer of human CD34+ HSCs squeezed with GFP mRNA ("GFP HSCs") or Bcl-2 variant mRNA (i.e., containing a G101V amino acid modification) ("Bcl-2 variant HSCs") into NSG mice. A shows a schematic of the experimental design. As indicated, GFP HSCs and Bcl-2 variant HSCs were administered to mice at a 1:1 ratio, and some mice were additionally administered Bcl-2 and MCL-1 inhibitors (i.e., venetoclax and S63845, respectively). B shows a comparison of the frequency of adoptively transferred HSCs (as a percentage of human cells) in the bone marrow of animals 24 hours after adoptive transfer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The present disclosure is generally directed to a method for producing hematopoietic stem cells (HSCs) that exhibit one or more enhanced properties. Specifically, the methods provided herein include passing a cell suspension containing a population of HSCs through a constriction under one or more parameters. In some embodiments, this results in a transient perturbation of the cell membrane of the HSCs, through which a payload can enter, thereby enhancing one or more properties of the HSCs (e.g., increased resistance to mobilizing factors, increased resistance to apoptotic factors, increased resistance to depleting factors, increased expression of homing receptors, or a combination thereof). Non-limiting examples of various embodiments are provided in the present disclosure.

[0053] I. Overall Technology Some of the techniques and procedures described or referenced herein are generally well understood and commonly employed by those of skill in the art using conventional techniques, examples of which include widely used procedures described, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., 2004). thed.、Cold Spring Harbor Laboratory Press、Cold Spring Harbor、N.Y.、2012);Current Protocols in Molecular Biology (F.M. Ausubel、et al. eds.、2003);the series Methods in Enzymology (Academic Press、Inc.);PCR 2: A Practical Approach(M.J. MacPherson、B.D. Hames and G.R. Taylor eds.、1995);Antibodies、A Laboratory Manual (Harlow and Lane、eds.、1988);Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications(R.I. Freshney、6 thed.、J. Wiley and Sons、2010);Oligonucleotide Synthesis(M.J. Gait、ed.、1984);Methods in Molecular Biology、Humana Press;Cell Biology:A Laboratory Notebook(J.E. Cellis、ed.、Academic Press、1998);Introduction to Cell and Tissue Culture(J.P. Mather and P.E. Roberts、Plenum Press、1998);Cell and Tissue Culture:Laboratory Procedures(A. Doyle、J.B. Griffiths、and D.G. Newell、eds.、J. Wiley and Sons、1993-8);Handbook of Experimental Immunology(D.M. Weir and C.C. Blackwell、eds.、1996);Gene Transfer Vectors for Mammalian Cells(J.M. Miller and M.P. Calos、eds.、1987);PCR:The Polymerase Chain Reaction、(Mullis et al.、eds.、1994);Current Protocols in Immunology(J.E. Coligan et al.、eds.、1991);Short Protocols in Molecular Biology(Ausubel et al.、eds.、J. Wiley and Sons、2002);Immunobiology(C.A. Janeway et al.、2004);Antibodies(P. Finch、1997);Antibodies:A Practical Approach(D. Catty.、ed.、IRL Press、1988-1989);Monoclonal Antibodies:A Practical Approach(P. Shepherd and C. Dean、eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 2011). .

[0054] II. Definition For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth herein shall control. Additional definitions are set forth throughout the detailed description.

[0055] As used herein, the singular terms "a," "an," and "the" entity refer to one or more entities, unless otherwise indicated. Thus, the terms "a" (or "an" or "the"), "one or more," and "at least one" can be used interchangeably herein.

[0056] It is understood that the embodiments of the disclosure described herein include the embodiments "comprising," "consisting of," and "consisting essentially of." It is also understood that when an embodiment is described herein with the word "comprising," other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0057] Furthermore, as used herein, "and / or" is to be construed as meaning that each of the two specifically mentioned features or components is specifically disclosed with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: 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 (single); B (single); and C (single).

[0058] In all compositions described herein, and in all methods of using compositions described herein, the compositions can either include the recited components or steps, or can "consist essentially of" the recited components or steps. When a composition is described as "consisting essentially of" the recited components, the composition includes the recited components and can further include other components that do not substantially affect the disclosed method, but does not include any other components other than the explicitly recited components that substantially affect the disclosed method, or when the composition includes extra components other than those recited that substantially affect the disclosed method, the composition does not include the extra components in a concentration or amount sufficient to substantially affect the disclosed method. When a method is described as "consisting essentially of" the recited steps, the method includes the recited steps and can further include other steps that do not substantially affect the disclosed method, but does not include any other steps other than the explicitly recited steps that substantially affect the disclosed method. As a non-limiting specific example, when a composition is described as "consisting essentially of" a certain component, the composition can additionally include any amount of pharma- ceutically acceptable carriers, excipients or diluents, and other such components that do not substantially affect the disclosed methods.

[0059] Units, prefixes, and symbols are denoted in the form accepted by the International System of Units (SI). Numerical ranges are intended to be inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of the disclosure, which can be had by reference to the entire specification. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0060] The term "about" as used herein means approximately, roughly, around, or within a region thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value by, for example, a 10 percent increase or decrease (higher or lower) change above or below the set forth value.

[0061] As used herein, the term "apoptosis" refers to the death of cells due to the activation of programmed cell death pathways. Generally, cells undergoing apoptosis can be characterized by morphological and biochemical changes, examples of which include DNA fragmentation, chromatin condensation, chromosome shifting, nuclear margination, formation of apoptotic bodies, mitochondrial swelling, enlargement of mitochondrial cristae, opening of the mitochondrial permeability transition pore, loss of mitochondrial proton gradient, caspase activity, or combinations thereof. The term "apoptotic factor" refers to any agent capable of inducing apoptosis in a cell (e.g., HSC). In some embodiments, an apoptotic factor can directly act on a cell to induce apoptosis. In some embodiments, an apoptotic factor acts indirectly by inhibiting the activity of an anti-apoptotic factor. As used herein, the term "anti-apoptotic factor" refers to any molecule capable of reducing or preventing apoptosis of a cell. Non-limiting examples of anti-apoptotic factors include Bcl-2, MCL-1, BCL-XL, and combinations thereof. Non-limiting examples of apoptotic factors include inhibitors of Bcl-2 ("Bcl-2 inhibitors") (e.g., venetoclax), inhibitors of MCL-1 ("MCL-1 inhibitors") (e.g., S63845 and S64315), inhibitors of BCL-XL ("BCL-XL inhibitors"), or combinations thereof. Such inhibitors are also referred to herein as "inhibitors of anti-apoptotic factors."

[0062] The term "constriction" as used herein refers to a narrowed passage. In some embodiments, the constriction is a microfluidic channel, such as one contained in a microfluidic device. In some embodiments, the constriction is a pore or is contained within a pore. When the constriction is a pore, in some embodiments, the pore is contained within a surface. Unless otherwise indicated, the term constriction refers to both microfluidic channels and pores, and other suitable constrictions available in the art. Thus, where applicable, disclosure related to microfluidic channels can also be applied to pores and / or other suitable constrictions available in the art. Similarly, where applicable, disclosure related to pores can equally be applied to microfluidic channels and / or other suitable constrictions available in the art.

[0063] As used herein, the terms "deform" and "transformation" (including derivatives thereof) refer to a physical change within a cell. As described herein, when a cell passes through a constriction (such as that disclosed herein), the cell experiences various forces due to the compressive physical environment, including, but not limited to, mechanical deformation and / or shear forces that cause perturbation in the cell membrane. As used herein, "perturbation" in a cell membrane refers to any opening in the cell membrane that is not present under normal steady state conditions (e.g., when no deformation forces are applied to the cell). Perturbation can include holes, tears, cavities, openings, pores, tears, gaps, perforations, or combinations thereof.

[0064] As used herein, the term "depletion factor" refers to any agent capable of inducing depletion (i.e., removal or deletion) of HSCs from a stem cell niche (e.g., bone marrow) of a subject. As will be apparent from the present disclosure, in some embodiments, HSCs can be depleted from the stem cell niche by mobilizing HSCs (e.g., from bone marrow to peripheral blood). Non-limiting examples of depletion factors include CD117 antibody drug conjugates. In some embodiments, HSCs can be depleted by inducing cell death (e.g., by inducing apoptosis of HSCs). Thus, in some embodiments, the term "depletion factor" can be used interchangeably with the terms "mobilization factor" and / or "apoptotic factor". Non-limiting examples of additional mobilization and apoptotic factors are described elsewhere in this disclosure.

[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 that contains pores.

[0066] As used herein, the term "hematopoietic stem cells" or "HSC" refers to a subset of multipotent stem cells that give rise to all blood or immune cell types, including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells, mast cells), and lymphoid (e.g., innate lymphoid cells, T cells, B cells, NKT cells, NK cells) lineages, and has multilineage hematopoietic differentiation potential and sustained self-renewal activity. Methods for identifying HSCs that can be used in the present disclosure are known in the art. For example, in some embodiments, HSCs useful in the present disclosure can be identified based on their phenotypic expression (e.g., using flow cytometry) because they are CD34+, CD90+, CD45RA- and lack expression of any lineage markers (CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b, and CD235).

[0067] The term "heterogeneous" as used herein refers to something that is mixed or not uniform in structure or composition. For example, when used to describe the pores present in the constrictions provided herein, such a term can refer to pores having a variety of sizes, shapes, or distributions within a given surface.

[0068] As used herein, the term "homing factor" refers to any agent capable of inducing homing (i.e., migration) of cells to a particular compartment within a subject. For example, in some embodiments, the homing factors provided herein can induce HSCs to migrate to the bone marrow when administered to a subject. Non-limiting examples of homing factors include homing receptors. A "homing receptor" refers to a molecule that is expressed on the surface of a cell (e.g., HSC) and exhibits affinity for a ligand such that cells expressing the homing receptor preferentially migrate toward the ligand. As further described elsewhere in this disclosure, in some embodiments, the homing receptor is a chemokine receptor, such as CXCR4 and CXCR2.

[0069] The term "homogeneous" as used herein refers to something being uniform or uniform in structure or composition throughout. For example, when used to describe the pores present in the constrictions provided herein, such a term can refer to pores having a uniform size, shape, or distribution within a given surface.

[0070] The term "membrane" as used herein refers to a selective barrier or sheet that contains pores. The term includes, but is not limited to, flexible sheet-like structures that function as a boundary or lining. In some aspects, the term refers to a surface or filter that contains pores. This term differs from the term "cell membrane," which refers to a semi-permeable membrane that surrounds the cytoplasm of a cell.

[0071] As used herein, the term "mobilization" (or grammatical derivatives thereof) refers to the recruitment of HSCs from a first location (e.g., a stem cell niche, e.g., bone marrow) to a second location (e.g., a tissue, peripheral blood, or an organ). In some embodiments, the first location is bone marrow and the second location is peripheral blood. As used herein, the term "mobilization factor" includes any agent capable of inducing mobilization of HSCs (e.g., from bone marrow to peripheral blood). Non-limiting examples of mobilizing factors include Plerixafor (e.g., MOZOBIL®), AMD3465 (CXCR4 antagonist), Groβ (CXCR2 agonist, induces MMP-9 secretion), granulocyte colony-stimulating factor (G-CSF), anti-CD117 (c-kit) antibodies, chemotherapy (e.g., cyclophosphamide), etoposide (e.g., TOPOSAR® and ETOPOPHOS®), POL6326 (CXCR4 antagonist), TG-0054 (CXCR4 antagonist), BKT140 (anti-SDF-1), bortezomib (proteasome inhibitor, downregulates VLA4 / VCAM-1 axis) (e.g., VELCADE®), PTH (PTH receptor ... HSC proliferation), CDX-301 (FLT3 agonist), LY2510924 (CXCR4 antagonist), natalizumab (VLA-4 antagonist) (e.g., TYSABRI®), meloxicam (nonsteroidal anti-inflammatory drug) (e.g., VIVLODEX®, MOBIC®, COMFORT®), eltrombopag (TPO receptor agonist) (e.g., PROMACTA®), ALX-0651 (anti-CXCR4 nanobody), and combinations thereof. See, e.g., Domingues et al., Int J Hematol 105:141-152 (2017); Bakanay et al., Bone Marrow Transplantation 47:1154-1163 (2012), both of which are incorporated herein by reference in their entirety. or combinations thereof.

[0072] The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Thus, the 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 containing purine and pyrimidine bases, or other naturally occurring, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide can include sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide can include polymers of synthetic subunits such as phosphoramidates, and thus can be oligodeoxynucleotide phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. Additionally, double-stranded polynucleotides can be obtained from the single-stranded polynucleotide products of chemical synthesis by either synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using DNA polymerase with an appropriate primer.

[0073] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may include, but are not limited to, natural or non-natural amino acid residues, and may include peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition encompasses both full-length proteins and fragments thereof. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host producing the protein or errors during PCR amplification.

[0074] The term "pore" as used herein refers to an opening, including, but not limited to, a hole, a crevice, a cavity, an opening, a breach, a gap, or a perforation in a material. In some aspects (where indicated), the term refers to a pore in a surface of a microfluidic device, such as those described in this disclosure. In some aspects (where indicated), the pore can refer to a pore in a cell wall and / or cell membrane.

[0075] As used herein, the term "reference" HSC refers to HSC that is not modified using the squeeze treatment method provided herein.For example, in some embodiments, reference HSC refers to the corresponding HSC that is exposed to payload but uses alternative delivery methods known in the art (e.g., electroporation or lipofection).In some embodiments, reference HSC refers to the corresponding HSC that has undergone squeeze treatment but does not use any payload described herein.In some embodiments, reference HSC refers to the corresponding HSC that is not modified (i.e., not squeezed and not delivered any payload).

[0076] As used herein, the term "survival factor" refers to any agent that can promote the survival of a cell (e.g., HSC). Such agents are also referred to herein as "anti-apoptotic factors." For example, in some embodiments, a survival factor can reduce or prevent a cell from undergoing apoptosis. Non-limiting examples of survival factors include Bcl-2, Bcl-xL, MCL-1, Ced-9, bfl-1, or combinations thereof. As is evident from the present disclosure, modifying HSCs to increase expression of a survival factor by the squeeze treatment method provided herein can result in increased survival of HSCs compared to reference HSCs.

[0077] III. Method of Disclosure In some aspects, the present disclosure relates to a method for generating HSCs with enhanced properties by delivering a payload to the HSCs, where the payload can enhance one or more properties of the HSCs. As further described herein, such a method includes passing a cell suspension including a population of HSCs through a constriction under one or more parameters. Upon passing through the constriction, the cells are temporarily deformed, thereby perturbing the cell membrane of the HSCs. Perturbation in the cell membrane can allow the payload to enter or be loaded into the cell (e.g., through diffusion). The particular process in which the cells are temporarily deformed upon passing through the constriction is referred to herein as a "squeezing process" or "squeezing".

[0078] As further described herein, the squeezing method of the present disclosure has certain unique properties that are not shared by other delivery methods known in the art. For example, in addition to improving the ability to deliver various types of payloads into cells, the squeezing method described herein has minimal lasting effects on cells. Compared to traditional delivery methods such as electroporation, the squeezing method of the present disclosure maintains both the structural and functional integrity of squeezed cells. In contrast to the delivery methods provided herein, electroporation can induce extensive and persistent alterations in gene expression, which can lead to non-specific activation of cells (e.g., human T cells) and delayed proliferation upon antigen stimulation. In this method, any alterations of cells (e.g., perturbation of cell membrane) are temporary and are quickly repaired once the cells are removed from the constriction.

[0079] As demonstrated herein, such improved delivery methods can be used to modify various aspects of HSCs, such that the HSCs described herein are structurally and functionally distinct from their naturally occurring counterparts in nature. For example, in some embodiments, by using payloads (e.g., CXCR4 and / or Bcl-2) that play an important role in the engraftment of HSCs in the bone marrow of a subject (e.g., promoting homing to the bone marrow and / or increasing survival), the squeezing methods provided herein can be used to express (e.g., overexpress) such payloads in HSCs, thereby allowing for better engraftment of the HSCs when administered to a subject.

[0080] Furthermore, in some embodiments, by using a payload that can increase resistance to mobilization factors, the squeeze treatment method provided herein can enhance the ability of HSCs to resist the effects of mobilization factors. Similarly, in some embodiments, by using a payload that can increase resistance of HSCs to apoptotic factors, the squeeze treatment method provided herein can enhance the ability of HSCs to resist the effects of apoptotic factors. Also, by using a payload that can increase resistance to HSC depletion factors, the squeeze treatment method provided herein can be used to generate HSCs with increased resistance to such depletion factors. As further described elsewhere in this disclosure, such enhanced properties may be particularly useful since they can be transplanted into the bone marrow of a subject without the need for harsh and toxic myeloablative conditioning.

[0081] While the present disclosure generally discloses the use of payloads that can increase the particular properties described above (i.e., resistance of HSCs to mobilizing factors, resistance of HSCs to apoptotic factors, resistance of HSCs to depleting factors, and / or increased expression of homing factors), it will be apparent to one of skill in the art that the disclosure relating to such payloads applies equally to other types of payloads, e.g., those that enhance one or more other properties of HSCs.

[0082] In some embodiments, provided herein are methods of generating modified HSCs, comprising passing a cell suspension through a constriction under one or more parameters, the cell suspension comprising a population of HSCs, the passing of the cell suspension through the constriction under one or more parameters allowing a payload to enter the HSCs (e.g., by transient perturbation of the cell membrane of the HSCs as the cell suspension passes through the constriction), and the payload modifies the HSCs such that the HSCs exhibit (i) increased resistance to mobilization factors, (ii) increased resistance to apoptotic factors, (iii) increased resistance to depletion factors, (iv) increased expression of homing factors, or (v) any combination thereof. Non-limiting examples of such payloads are described elsewhere in this disclosure.

[0083] In some embodiments, compared to a reference HSC, the modified HSCs generated using the methods provided herein are 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 more resistant to a mobilization factor. In some embodiments, compared to a reference HSC, the HSCs generated using the methods provided herein are 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 more resistant to an apoptotic factor. In some embodiments, compared to a reference HSC, the HSCs generated using the methods provided herein are 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 more resistant to depletion factors. In some embodiments, compared to a reference HSC, the HSCs generated using the methods provided herein have increased expression of homing factors. In some embodiments, the expression of homing factors is increased by 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.

[0084] Thus, in some embodiments, the methods provided herein can be used to increase homing of HSCs when administered to a subject in need thereof (e.g., as a result of increased resistance to mobilizing factors, increased resistance to depleting factors, and / or increased expression of homing factors). In some embodiments, such methods include passing a cell suspension comprising HSCs through a constriction under one or more parameters such that a payload enters the HSCs (e.g., by transient perturbation of the cell membrane of the HSCs as the cell suspension passes through the constriction), where the payload modifies the HSCs such that, when administered to a subject, the HSCs exhibit increased homing to bone marrow.

[0085] Additionally, in some embodiments, the methods provided herein can be used to increase survival of HSCs when administered to a subject in need thereof (e.g., as a result of increased resistance to apoptotic factors). In some embodiments, such methods include passing a cell suspension comprising HSCs through a constriction under one or more parameters such that a payload enters the HSCs (e.g., by transient perturbation of the cell membrane of the HSCs as the cell suspension passes through the constriction), where the payload modifies the HSCs such that the HSCs exhibit increased survival when administered to a subject.

[0086] As will be apparent to one of skill in the art and as further described elsewhere in this disclosure (see, e.g., the section entitled "Therapeutic Applications"), the above methods have a variety of important clinical implications. For example, in some embodiments, enhancing one or more properties of HSCs described herein can improve engraftment of HSCs when administered to a subject. Thus, in some embodiments, the present disclosure is directed to a method of promoting engraftment of HSCs in the bone marrow of a subject in need thereof, comprising passing a cell suspension comprising HSCs through a constriction under one or more parameters such that a payload enters the HSCs (e.g., by transient perturbation of the cell membrane of the HSCs as the cell suspension passes through the constriction), where the payload modifies the HSCs such that the HSCs are capable of engrafting in the bone marrow of the subject when administered to the subject.

[0087] The methods provided herein can result in increased engraftment of the administered HSCs as compared to the reference HSCs, hi some embodiments, engraftment of the administered HSCs is increased by 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 as compared to engraftment observed following administration of the reference HSCs.

[0088] In some embodiments, the above method further comprises contacting the HSCs with a payload (e.g., as described herein) before passing the cell suspension containing the HSCs through the constriction. As is evident from the present disclosure, in some embodiments, contacting the HSCs with the payload before squeezing can improve delivery efficiency since the payload can enter the cells as soon as the squeezing process causes perturbation of the cell membrane. In some embodiments, before passing the cell suspension through the constriction, the method provided herein comprises contacting the HSCs with the payload to generate a cell suspension. In some embodiments, the method provided herein comprises contacting the HSCs with the payload as the cell suspension passes through the constriction. In some embodiments, the HSCs first contact the payload as the cell suspension passes through the constriction. In some embodiments, the HSCs are in contact with the payload both before and during the passing step (i.e., the cell suspension passes through the constriction). In some embodiments, the method provided herein comprises contacting the HSCs with the payload after the cell suspension passes through the constriction. In some embodiments, the cells first contact the payload after the cell suspension has passed through the constriction. In some embodiments, the HSCs contact the payload before, during, and / or after the passage step. As further described elsewhere in this disclosure, when the HSCs contact the payload after the passage step, the contact occurs immediately after the HSCs have passed through the constriction, so that perturbation still occurs in the cell membrane.

[0089] As used herein, "contact" that can occur between HSCs and a payload (e.g., as described herein) includes that the cell can contact the payload once perturbation has occurred in the cell membrane, so long as the payload is able to enter the cell. For ease of explanation, in some embodiments, the cell and the payload can be said to be in contact when both are present in the same cell suspension.

[0090] III.A. Cell Suspension In some embodiments, the cell suspensions described herein include any suitable HSCs known in the art and that can be modified (e.g., by introducing a payload) using the squeezing method described herein. While the present application generally describes the use of HSCs, it will be apparent to one of skill in the art that the disclosure provided herein is also applicable to other types of stem cells that may be useful in treating diseases or disorders (e.g., blood disorders) described herein. As used herein, the term "stem cells" refers to cells that have the ability to not only self-renew, but also to differentiate into other types of cells. In some embodiments, stem cells useful in the present disclosure include induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), tissue-specific stem cells (e.g., liver stem cells, cardiac stem cells, or neural stem cells), mesenchymal stem cells, hematopoietic stem cells (HSCs), or combinations thereof. In some embodiments, the stem cells are HSCs.

[0091] As demonstrated herein, the delivery of a payload to cells can be regulated through one or more parameters of the process of passing a cell suspension through a constriction. In some embodiments, certain properties of the cell suspension can affect the delivery of a payload to cells. Such properties include, but are not limited to, osmolality, salinity, serum content, cell concentration, pH, temperature, or combinations thereof. Additional parameters relevant to this disclosure are provided elsewhere in this disclosure.

[0092] In some embodiments, the cell suspension comprises a homogenous population of cells (e.g., a purified population of HSCs). In some embodiments, the cell suspension comprises a heterogenous population of cells (e.g., whole blood or a mixture of cells including HSCs in, e.g., saline or a physiological medium other than blood). When the cell suspension comprises a heterogenous population of cells, in some embodiments, the HSCs can comprise about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, or about 90% of the total population of cells. In some embodiments, the heterogenous population of cells undergoes a concentration step, such that the HSCs represent a greater percentage of the total cell population.

[0093] In some embodiments, the cell suspension comprises an aqueous solution. In some embodiments, the aqueous solution comprises cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, bulking agents, surfactants, lubricants, vitamins, polypeptides, agents that affect actin polymerization, or combinations thereof. In some embodiments, the cell culture medium comprises DMEM, OptiMEM, EVIDM, RPMI, XVivo10, or combinations thereof. Additionally, the solution buffer can include one or more lubricants (pluronic or other surfactants) that can be designed to reduce or eliminate clogging of surfaces and improve cell viability. Exemplary surfactants include, but are not limited to, poloxamers, polysorbates, sugars such as mannitol, serum from animals, and albumin proteins.

[0094] When the suspension contains a particular type of cell, in some embodiments, the cells can be treated with a solution that aids in the delivery of a payload to the interior of the cell. In some embodiments, the solution contains an agent that affects actin polymerization. In some embodiments, the agent that affects actin polymerization includes latrunculin A, cytochalasin, colchicine, or a combination thereof. For example, in some embodiments, the cells can be incubated in a depolymerizing solution such as latrunculin A for about 1 hour before passing the cells through a constriction to depolymerize the actin cytoskeleton. In some embodiments, the cells can be incubated in colchicine (Sigma) for about 2 hours before passing the cells through a constriction to depolymerize the microtubule network.

[0095] In some embodiments, a property of a cell suspension that may affect the delivery of a payload to a cell is the viscosity of the cell suspension. As used herein, the term "viscosity" refers to the internal resistance to flow that a fluid exhibits. In some embodiments, the viscosity of the cell suspension is about 8.9×10 -4 Pa·s~approx. 4.0×10 -3 Pa·s, approx. 8.9×10 -4 Pa·s~approx.3.0×10 -3 Pa·s, approx. 8.9×10 -4 Pa·s~approx. 2.0×10 -3 Pa s, or approximately 8.9 × 10 -4 Pa·s~approx. 1.0×10 -3Pa·s. In some embodiments, the viscosity is about 0.89 cP to about 4.0 cP, about 0.89 cP to about 3.0 cP, about 0.89 cP to about 2.0 cP, or about 0.89 cP to about 1.0 cP. In some embodiments, a shear-thinning effect is observed in which the viscosity of the cell suspension decreases under conditions of shear strain. 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. A viscometer measures the viscosity under one flow condition, whereas a rheometer is used to measure the viscosity as it changes with flow conditions. In some embodiments, the viscosity is measured for a shear-thinning solution such as blood. In some embodiments, the viscosity is measured between about 0°C and about 45°C. For example, the viscosity of a cell suspension can be measured at room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), higher than physiological temperature (e.g., greater than about 37°C to about 45°C or higher), low temperature (e.g., about 0°C to about 4°C), or a temperature between these exemplary temperatures.

[0096] III.B. Payload As described herein, in some embodiments, the cell suspension additionally comprises one or more payloads, examples of which can improve one or more properties of the HSCs such that they are better able to engraft when administered to a subject (e.g., increase resistance of the HSCs to mobilizing factors, increase resistance of the HSCs to apoptotic factors, increase resistance of the HSCs to depleting factors, increase expression of homing factors in the HSCs, or combinations thereof). Non-limiting examples of payloads useful in the present disclosure include homing factors (e.g., homing receptors), cytokines, growth factors, cell adhesion molecules, proliferation agents, survival factors, or combinations thereof. In some embodiments, payloads that can be used in the present disclosure include regulators of such payloads. The term "regulator" when used to describe payloads useful in the present disclosure refers to any agent that can modulate (e.g., increase and / or decrease) the expression and / or activity of other payloads described herein. For example, in some embodiments, payloads useful in the present disclosure include regulators of homing factors. In some embodiments, payloads that can be used in the present disclosure include regulators of cytokines. In some embodiments, the payload comprises a regulator of a growth factor. In some embodiments, the payload comprises a regulator of a cell adhesion molecule. In some embodiments, the payload comprises a regulator of a proliferation agent. In some embodiments, the payload comprises a regulator of a survival factor. Additional embodiments of such payloads are further described below.

[0097] As is evident from the present disclosure, in some embodiments, the payloads useful in the present disclosure are expressed in the modified HSCs transiently, such that any modification to one or more properties of the HSC mediated by the payload is not permanent (i.e., expressed transiently). For example, in some embodiments, the payload is expressed in the HSCs for less than about 1 day, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days, less than about 7 days, less than about 8 days, less than about 9 days, or less than about 10 days. In some embodiments, the payload is expressed in the HSCs for less than about 1 day, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days, less than about 7 days, less than about 8 days, less than about 9 days, or less than about 10 days.

[0098] As described and demonstrated herein, in some embodiments, the payload that can be used to generate the modified HSCs described herein comprises a homing factor. For example, in some embodiments, the payload comprises a homing receptor that is involved in the homing and repopulation of HSCs in the bone marrow. Non-limiting examples of homing receptors useful in the present disclosure include CXCR4, CXCR2, or both. In some embodiments, the homing receptor is CXCR4. CXCR4 interacts with CXCL12, which is secreted from mesenchymal stromal cells that reside primarily in the bone marrow niche. This interaction has been described as important in both homing and retention of HSCs in the bone marrow niche. See, e.g., Karpova et al., Stem Cell 33:2391-2399 (2015); and Sugiyama et al., Immunity 25:977-988 (2006).

[0099] Thus, in some embodiments, the squeeze treatment methods provided herein can be used to increase the expression of CXCR4 on HSCs compared to reference HSCs. In some embodiments, the expression of CXCR4 on HSCs is increased by 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 the corresponding expression on reference HSCs. In some embodiments, increasing CXCR4 expression on HSCs can aid in increasing homing and / or retention of HSCs within the bone marrow niche.

[0100] In some embodiments, CXCR4 can be modified to help further improve one or more properties of HSCs generated using the methods provided herein. For example, as will be apparent to one of skill in the art, in order to facilitate engraftment of any adoptively transferred HSCs (e.g., as part of an HSC transplantation therapy), it is necessary to deplete / reduce the endogenous stem cell compartment to make room for the adoptively transferred HSCs. Plerixafor is an FDA-approved small molecule (i.e., a mobilization factor) that inhibits the interaction between CXCR4 and CXCL12, thereby promoting the mobilization of HSCs from bone marrow to peripheral blood. Thus, in some embodiments, payloads useful in the present disclosure include CXCR4 variants that include one or more mutations that can confer resistance to Plerixafor or any other similar mobilization factor. A non-limiting example of such a CXCR4 variant includes the mutation A175F compared to the corresponding wild-type amino acid sequence set forth in SEQ ID NO:1 (see Tables 1 and 2). [Table 1] JPEG2025503608000002.jpg45159 [Table 2] JPEG2025503608000004.jpg239159 JPEG2025503608000005.jpg231159 JPEG2025503608000006.jpg95159

[0101] It will be apparent to one of skill in the art that by modifying HSCs to express such CXCR4 variants, in some embodiments, Plerixafor can be used to selectively mobilize unmodified HSCs (e.g., endogenous HSCs) from the bone marrow, thereby allowing improved engraftment of adoptively transferred modified HSCs.

[0102] In some embodiments, the CXCR4 variants can include one or more mutations that enhance the function of CXCR4 (e.g., enhance the interaction between CXCR4 and CXCL12). A non-limiting example of such a CXCR4 variant includes the mutation R334X compared to the corresponding wild-type amino acid sequence set forth in SEQ ID NO: 1 (see Table 1). In some embodiments, the CXCR4 variants include both the A175F mutation and the R334X mutation compared to the corresponding wild-type amino acid sequence set forth in SEQ ID NO: 1. Non-limiting examples of additional mutations of the CXCR4 variants described herein include H113A, D171N, D262N, I284A, H281A, Q200W, Q200A, or combinations thereof.

[0103] In some embodiments, the homing receptor is CXCR2 or its variant.In some embodiments, the squeeze treatment method provided herein can be used to increase the expression of CXCR2 on HSC.In some embodiments, compared with reference HSC, the expression of CXCR2 can be increased 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.

[0104] In some embodiments, payloads useful in the present disclosure include survival factors, examples of which, when expressed (or overexpressed) in HSCs, can increase survival of the HSCs. Non-limiting examples of survival factors include Bcl-2, Bcl-xL, MCL-1, Ced-9, bfl-1, or combinations thereof.

[0105] In some embodiments, the payload comprises Bcl-2. Qing et al., Blood 123:1002 (2014). Thus, in some embodiments, the squeeze treatment method provided herein can be used to increase the expression of Bcl-2 on HSCs compared to reference HSCs. In some embodiments, the expression of Bcl-2 on HSCs is increased 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 the corresponding expression on reference HSCs.

[0106] In some embodiments, Bcl-2 can be modified to help further improve one or more properties of HSCs generated using the methods provided herein. Venetoclax is an FDA-approved BH3 mimetic that specifically targets and inhibits Bcl-2. Thus, in some embodiments, payloads useful in the present disclosure include Bcl-2 variants that contain one or more mutations that can confer resistance to venetoclax or any other similar apoptotic agent. Non-limiting examples of such Bcl-2 variants include the mutations G101V, D103Y, or both, compared to the corresponding wild-type amino acid sequence set forth in SEQ ID NO: 9 (see Tables 3 and 4). It will be apparent to one of skill in the art that by modifying HSCs to express such Bcl-2 variants, in some embodiments, venetoclax can be used to selectively deplete unmodified HSCs (e.g., endogenous HSCs) from bone marrow, thereby allowing for improved engraftment of adoptively transferred modified HSCs. [Table 3] [Table 4] JPEG2025503608000009.jpg41159

[0107] In some embodiments, the payload comprises MCL1 apoptosis regulator (MCL-1). MCL-1 has been shown to be important for the survival of HSCs and other hematopoietic progenitor cells. Bohler et al., Haematologica 106:3136-3148 (2021); and Chin et al., Front Cell Dev 9:704547 (2021). Thus, in some embodiments, the squeeze treatment methods provided herein can be used to increase expression of MCL-1 on HSCs compared to reference HSCs. In some embodiments, expression of MCL-1 on HSCs is increased by 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 the corresponding expression on reference HSCs.

[0108] In some embodiments, the payload comprises Bcl-xL. In some embodiments, the squeeze treatment method provided herein can be used to increase the expression of Bcl-xL on HSCs compared to reference HSCs. In some embodiments, the expression of Bcl-xL on HSCs is increased 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 the corresponding expression on reference HSCs.

[0109] In some embodiments, the payload comprises Ced-9. In some embodiments, the squeeze method provided herein can be used to increase the expression of Ced-9 on HSCs compared to reference HSCs. In some embodiments, the expression of Ced-9 on HSCs is increased 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 the corresponding expression on reference HSCs.

[0110] In some embodiments, the payload comprises bfl-1. In some embodiments, the squeeze method provided herein can be used to increase the expression of bfl-1 on HSCs compared to reference HSCs. In some embodiments, the expression of bfl-1 on HSCs is increased 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 the corresponding expression on reference HSCs.

[0111] In some embodiments, payloads useful in the present disclosure include cytokines or growth factors, examples of which play a role in the engraftment of HSCs within the bone marrow of a subject. In some embodiments, such payloads include stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L), thrombopoietin (TPO), or combinations thereof.

[0112] In some embodiments, using the squeeze treatment methods provided herein, HSCs can be modified to exhibit higher levels of cytokines or growth factors described herein, with higher expression allowing the HSCs to be better engrafted into the bone marrow of a subject. In some embodiments, compared to reference HSCs, the expression of SCF in HSCs can be increased by 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. In some embodiments, expression of Flt3L can be increased by 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, as compared to a reference HSC. In some embodiments, expression of TPO in HSC can be increased by 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, as compared to a reference HSC.

[0113] In some embodiments, payloads useful in the present disclosure include cell adhesion molecules, examples of which are involved in the transport of HSCs to the bone marrow of a subject. For example, in some embodiments, the cell adhesion molecules include integrins, selectins, or both. Non-limiting examples of suitable integrins and selectins are known in the art, and include, for example, VLA-4, VLA-5, LFA-1, and combinations thereof. In some embodiments, the squeeze treatment method provided herein can be used to increase the expression of cell adhesion molecules on HSCs compared to reference HSCs. In some embodiments, the expression of cell adhesion molecules can be increased by 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 reference HSCs.

[0114] In some embodiments, the payload that can be used in the present disclosure includes a proliferation agent. As used herein, the term "proliferation agent" refers to any molecule that can induce proliferation of cells (e.g., HSCs). In some embodiments, the proliferation agent induces activation of signaling pathways involved in regulating cell proliferation, such as PI3K-ATK, Ras-ERK, or both. Thus, in some embodiments, the squeeze treatment methods provided herein can be used to increase expression and / or activity of the proliferation agent in HSCs compared to reference HSCs. In some embodiments, the increase in expression and / or activity of the proliferation agent is associated with an increase in proliferation of HSCs. In some embodiments, the proliferation of HSCs generated using the present disclosure is increased by 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 reference HSCs.

[0115] As described herein, in some embodiments, payloads useful in the present disclosure include agents (i.e., regulators) that can modulate the expression and / or activity of other payloads described herein. For example, G protein-coupled receptor associated sorting proteins 1 and 2 (GPRASP1 / 2) signaling has been described to inhibit CXCR4 expression. Knockdown of GPRASP1 / 2 has been shown to increase CXCR4 expression in HSCs. Morales-Hernandez et al., Blood 135:1111-1123 (2020); and Hernandez et al., Exp Hematol 64:S88 (2018). Thus, in some embodiments, payloads useful in the present disclosure can increase the expression of CXCR4 in HSCs by reducing and / or inhibiting the expression and / or activity of GPRASP1 / 2. For example, in some embodiments, the payload can include siRNA (or any other gene editing tool) that selectively targets GPRASP1 / 2. Similarly, CD26 has also been shown to disrupt CXCR4 expression and / or activity, thereby inducing HSC mobilization. Fang et al., Stem Cell Res Ther 12(1):17(2021). Thus, in some embodiments, payloads that can be used in the present disclosure can increase CXCR4 expression by reducing and / or inhibiting CD26 expression and / or activity (e.g., siRNA targeting CD26).

[0116] In addition to the above, payloads that can reduce or prevent the expression and / or activity of endogenous genes involved in HSC engraftment are also useful in the present disclosure. For example, such payloads can be used to mobilize endogenous HSCs by reducing the expression of homing receptors (e.g., CXCR4) naturally expressed on endogenous HSCs, thus allowing for greater engraftment of therapeutic HSCs that have been modified to express higher levels of the homing receptor.

[0117] Any of the payloads described herein can be present in the cell suspension before, during, and / or after the passing step in which the cell suspension passes through the constrictions. In some embodiments, the cell suspension 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. As further described elsewhere in this disclosure, in some embodiments, the cell suspension can be passed through multiple constrictions. In such embodiments, the payload can be loaded into the cells as the cells pass through one or more of the multiple constrictions. In some embodiments, the payload is loaded into the cells each time the cells pass through one or more of the multiple constrictions. When multiple payloads are involved, each payload can be the same. In some embodiments, one or more payloads are different.

[0118] Thus, as is evident from the present disclosure, in some embodiments, multiple payloads can be delivered to cells using the squeezing method described herein. In some embodiments, multiple payloads can be delivered to cells using a single squeezing process (e.g., a cell suspension includes multiple payloads that are delivered to cells in combination, i.e., "co-delivery"). In some embodiments, multiple payloads can be delivered to cells sequentially. As used herein, the term "sequential delivery" refers to the delivery of multiple payloads to cells, where a first payload is delivered to a cell, and then a second (or subsequent) payload is delivered to the cell. In some embodiments, the first payload, the second payload, or both the first and second payloads can be delivered to a cell using a squeezing process. For example, in some embodiments, the first payload can be delivered to a cell using a squeezing process, and the second payload can be delivered to a cell using a non-squeezing process (e.g., transfection). In some embodiments, a first payload can be delivered to a cell using a non-squeezing process (e.g., transfection), and a second payload can be delivered to a cell using a squeezing process. In some embodiments, a first payload can be delivered to a cell using a first squeeze, and then a second payload can be delivered to a cell using a second squeeze (also referred to herein as "sequential squeeze" or "sequential squeeze process"). Thus, sequential delivery useful in the present disclosure can include multiple squeeze processes. In some embodiments, each of the multiple squeeze processes delivers a separate payload to a cell. In some embodiments, one or more of the multiple squeeze processes does not involve the delivery of a payload.For example, in some embodiments, the sequential delivery methods described herein include a first squeeze, a second squeeze, and a third squeeze, where the first squeeze includes passing the cells without any payload through a first constriction, the second squeeze includes passing the cells from the first squeeze through a second constriction to deliver a first payload to the cells, and the third squeeze includes passing the cells from the second squeeze through a third constriction to deliver a second payload to the cells. Without being bound to any one theory, in some embodiments, passing the cells without any payload through the first constriction (i.e., the first squeeze) can serve to prepare the cells for subsequent payload delivery, e.g., improve the efficiency of delivery of the first and / or second payload.

[0119] In some embodiments, the squeezing methods described herein can be used to repeatedly deliver multiple payloads to a cell (e.g., at least two times, at least three times, at least four times, at least five times, or more). As further described elsewhere in this disclosure (see, e.g., the section entitled "Constriction"), in some embodiments, each of the multiple squeezing methods can be the same (e.g., the same parameters). In some embodiments, one or more of the multiple squeezing methods can be different (e.g., one or more delivery parameters described herein are different).

[0120] As is evident from the present disclosure, the payloads useful in the present disclosure are not particularly limited, so long as the payloads modify the HSCs such that the HSCs can exhibit one or more improved properties after modification. Non-limiting examples of suitable payloads include nucleic acids, polypeptides, lipids, carbohydrates, small molecules, metal-containing compounds, antibodies, transcription factors, nanoparticles, liposomes, fluorescently tagged molecules, or combinations thereof. In some embodiments, the nucleic acid comprises DNA, RNA, or both. In some embodiments, the DNA comprises recombinant DNA, cDNA, genomic DNA, or combinations thereof. In some embodiments, the RNA comprises siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, self-amplifying mRNA (saRNA), PNA, LNA, or combinations thereof. In some embodiments, the RNA is mRNA.

[0121] As further described and demonstrated herein, the squeezing method provided herein can be used to deliver payloads to HSCs alone or in combination (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least about eight, at least about nine, or all of the exemplary transcription factors listed). In some embodiments, when a combination of payloads is involved, they can be delivered to HSCs using a single squeezing process (e.g., simultaneous delivery). In some embodiments, a combination of payloads can be repeatedly delivered to HSCs. For example, in some embodiments, a combination of payloads is delivered to HSCs in a first squeezing process, and then a combination of payloads is delivered to the cells again in a second squeezing process. In some embodiments, the first squeezing process includes a microfluidic device (e.g., chip) having multiple rows of constrictions, allowing the squeezing process to be performed on a single microfluidic device (e.g., chip). As further described herein, in some embodiments, the second squeeze process can be performed shortly after the cells have undergone a first squeeze process (e.g., shortly after the cells have passed through the constriction of the first squeeze process). In some embodiments, the second squeeze process can be performed at a time after the first squeeze 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 cells have passed through the constriction of the first squeeze process).

[0122] Although the present disclosure primarily refers to the use of constrictions to deliver payloads to HSCs as described herein, it will be apparent to one of skill in the art that in some embodiments, payloads can be delivered to HSCs using any suitable delivery method known in the art. Non-limiting examples of such delivery methods include electroporation, vortex ejection forces (e.g., INDEE), lipid transfection, nanoparticles, sonoporation, and combinations thereof.

[0123] III.C. Stenosis III.C.1. Microfluidic Channels As described herein, a constriction is used to induce a physical deformation in a cell, resulting in perturbation in the cell's plasma membrane, allowing delivery of a payload into the cell. In some embodiments, the constriction is present in a channel (referred to herein as a "microfluidic channel" or "channel") contained within a microfluidic device. When multiple channels are involved, in some embodiments, the multiple channels can be arranged in parallel and / or series within the microfluidic device. In some embodiments, the cells described herein can be passed through at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1,000 or more distinct constrictions. In some embodiments, the cells described herein pass through more than about 1,000 distinct constrictions.

[0124] In some embodiments, the multiple constrictions can be part of a single microfluidic device (e.g., a multi-row constriction chip). In some embodiments, one or more of the multiple constrictions can be part of different microfluidic devices. For example, in some embodiments, cells (e.g., HSCs) described herein undergo a first squeezing process, where the cells pass through a first constriction in a first microfluidic device (e.g., chip). Then, after the cells have undergone the first squeezing process (e.g., passing through the first constriction), the cells undergo a second squeezing process, where the cells pass through a second constriction in a second microfluidic device (e.g., chip). In some embodiments, each constriction is the same (e.g., has the same length, width, and / or depth). In some embodiments, one or more of the constrictions are different. When multiple constrictions are used, the multiple constrictions can include a first constriction associated with a first payload (e.g., an mRNA encoding CXCR4 or a variant thereof) and a second constriction associated with a second payload (e.g., an mRNA encoding Bcl-2 or a variant thereof), where the cell suspension passes through the first constriction to deliver the first payload to one or more cells of the plurality of cells, and then the cell suspension passes through the second constriction to deliver the second payload to one or more cells of the plurality of cells. In some embodiments, the cell suspension passes through the second constriction at least about 1 minute, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, or at least about 1 day after the cell suspension passes through the first constriction.

[0125] In some embodiments, when the cell suspension passes through multiple constrictions (e.g., multiple squeeze processes), the cells remain viable after passing through each constriction. As is evident from the present disclosure, in some embodiments, the multiple constrictions include two or more constrictions present in a single microfluidic device (e.g., a multi-row constriction chip), allowing the cells to pass through the multiple constrictions sequentially. In some embodiments, the multiple constrictions are part of separate microfluidic devices, such that a first constriction is associated with the first microfluidic device and a second constriction is associated with the second microfluidic device. For example, as demonstrated herein, in some embodiments, the cells pass through a first constriction (i.e., a first squeeze process) associated with a first microfluidic device (e.g., a chip). After the cells pass through the first constriction, the cells pass through a second constriction (i.e., a second squeeze process), which is associated with a second microfluidic device (e.g., a chip). In some embodiments, after passing through the first constriction, the cells are cultured in a medium before passing through the second constriction. In some embodiments, before passing through the second constriction, the cells are cultured for at least about 1 minute, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, or at least about 1 day. As will be apparent from the present disclosure, in some embodiments, the first constriction and the second constriction have the same length, depth, and / or width. In some embodiments, the first constriction and the second constriction can have different lengths, depths, and / or widths.

[0126] In some embodiments, after passing through a constriction, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cells remain viable. If the cells pass through multiple constrictions (e.g., part of a single microfluidic device or separate microfluidic devices), 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 constrictions. Any suitable method known in the art can be used to measure cell viability. In some embodiments, cell viability can be measured using a Nucleocounter NC-200, an Orflo Moxi Go II Cell Counter, or both.

[0127] Exemplary microfluidic channels containing cell-deforming 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 by reference in its entirety herein.

[0128] In some embodiments, the microfluidic channels described herein (i.e., including the constriction) include a lumen and are configured to allow cells suspended in a buffer solution (e.g., a cell suspension) to pass through the channel. Microfluidic channels useful in the present disclosure can be fabricated using any suitable material available in the art, including, but not limited to, silicon, metal (e.g., stainless steel), plastic (e.g., polystyrene), ceramic, glass, crystalline substrate, amorphous substrate, polymer (e.g., polymethylmethacrylate (PMMA), PDMS, cyclic olefin copolymer (COC)), or combinations thereof. In some embodiments, the material is silicon. Fabrication of the microfluidic channels can be performed by any method known in the art, including, but not limited to, dry etching (e.g., deep reactive ion etching), wet etching, photolithography, injection molding, laser ablation, SU-8 mask, or combinations thereof. In some embodiments, fabrication is performed using dry etching.

[0129] In some embodiments, a microfluidic channel useful in the present disclosure includes an inlet portion, a center point, and an outlet portion. In some embodiments, the cross-section of one or more of the inlet portion, center point, and / or outlet portion can vary. For example, the cross-section can be circular, elliptical, elongated slit, square, hexagonal, or triangular in shape.

[0130] The inlet portion defines a constriction angle. In some embodiments, the constriction angle can be adjusted (e.g., increased or decreased) to reduce or prevent any clogging of the constriction. In some embodiments, the angle of the outlet portion is also adjustable. For example, in some embodiments, the angle of the outlet portion can be configured to reduce the possibility of turbulence that can result in non-laminar flow. In some embodiments, the walls of the inlet portion and / or outlet portion are straight. In some embodiments, the walls of the inlet portion and / or outlet portion are curved.

[0131] In some embodiments, the length, depth, and / or width of the constriction can vary. In some embodiments, the delivery efficiency of the payload can be adjusted by adjusting (e.g., increasing or decreasing) the length, depth, and / or width of the constriction. As used herein, the term "delivery efficiency" refers to the amount of payload delivered into a cell. For example, an increase in delivery efficiency can occur when the total amount of payload delivered is increased.

[0132] In some embodiments, the length of the constriction is less than about 1 μm. In some embodiments, the length of the constriction is about 0.1 μm to about 100 μm. In some embodiments, the length of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2.5 μm, less than about 5 μm, less than about 7.5 μm, less than about 10 μm, less than about 12.5 μm, less than about 15 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, or less than about 100 μm. In some embodiments, the length of the constriction is about 0.5 μm. In some embodiments, the length of the constriction is about 10 μm. In some embodiments, the length of the constriction is about 70 μm.

[0133] In some embodiments, the depth of the constriction is about 5 μm to about 90 μm. In some embodiments, the depth of the constriction is about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, or about 120 μm or more. In some embodiments, the depth of the constriction is about 10 μm. In some embodiments, the depth of the constriction is about 20 μm. In some embodiments, the depth of the constriction is about 70 μm. In some embodiments, the depth of the constriction is about 10 μm.

[0134] In some embodiments, the width of the constriction is about 1 μm to about 10 μm. In some embodiments, the width of the constriction is about 1 μm, about 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.7 μm, about 1.8 μm, about 1.9 μm, about 2 μm, about 3 μm, about 4 μm, about 4.5 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In some embodiments, the width of the constriction is about 6 μm. In some embodiments, the length of the constriction is 10 μm, the width is 6 μm, and the depth is 70 μm. In some embodiments, the width of the constriction is about 3.5 μm. For example, in some embodiments, a constriction that can be used in the present disclosure is 10 μm deep, 3.5 μm wide, and 70 μm long.

[0135] In some embodiments, the diameter of a constriction (e.g., contained within a microfluidic channel) is a function of the diameter of one or more cells passing through the constriction. Without being bound to any one theory, in some embodiments, the diameter of the constriction is smaller than the diameter of the cell, such that a deformation force is applied to the cell as it passes through the constriction, resulting in a temporary physical deformation of the cell.

[0136] Thus, in some embodiments, the diameter of the constriction (also referred to herein as "constriction size") is about 20% to about 99% of the diameter of the cell. In some embodiments, the constriction 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. As is evident from the present disclosure, by adjusting (e.g., increasing or decreasing) the diameter of the constriction, the efficiency of delivery of a payload into a cell can also be tuned.

[0137] III.C.2. Porous Surfaces In some embodiments, the constrictions described herein comprise pores contained in the surface. Non-limiting examples of pores contained in the surface that can be used in the present disclosure are described, for example, in U.S. Publication No. 2019 / 0382796 A1, which is incorporated herein by reference in its entirety.

[0138] In some embodiments, surfaces useful in the present disclosure (i.e., surfaces that contain one or more pores that induce physical deformation in cells as they pass through the pores) can be made using any suitable material available in the art and / or can take any one of several forms. Non-limiting examples of such materials include synthetic or natural polymers, polycarbonate, silicon, glass, metals, alloys, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose esters, porcelain, ceramic, or combinations thereof.

[0139] In some embodiments, the surface comprises a filter. In some embodiments, the filter is a tangential flow filter. In some embodiments, the surface comprises a membrane. In some embodiments, the surface comprises a sponge or a sponge-like matrix. In some embodiments, the surface comprises a matrix. In some embodiments, the surface comprises a meandering path surface. In some embodiments, the meandering path surface comprises cellulose acetate.

[0140] The surface disclosed herein (i.e., the surface that comprises one or more pores) can have any suitable shape known in the art.When the surface has a two-dimensional shape, the surface can be, but is not limited to, circular, elliptical, round, square, star, triangular, polygonal, pentagonal, hexagonal, heptagonal or octagonal.In some embodiments, the surface is round.When the surface has a three-dimensional shape, in some embodiments, the surface can be, but is not limited to, cylindrical, conical or rectangular.

[0141] As will be apparent from the present disclosure, surfaces useful in the present disclosure (e.g., surfaces containing one or more pores) can have a variety of cross-sectional widths and thicknesses. In some embodiments, the cross-sectional width of the surface is between about 1 mm and about 1 m. In some embodiments, the surface has a defined thickness. In some embodiments, the thickness of the surface is uniform. In some embodiments, the thickness of the surface is variable. For example, in some embodiments, certain portions of the surface are thicker or thinner than other portions of the surface. In such embodiments, the thickness of different portions of the surface can vary from about 1% to about 90%. In some embodiments, the thickness of the surface is between about 0.01 μm and about 5 mm.

[0142] The cross-sectional width of the pores may depend on the type of cell targeted by the payload. In some embodiments, the pore size is a function of the diameter of the cell or cell cluster to be targeted. In some embodiments, the pore size is such that the cell is perturbed (i.e., physically deformed) as it passes through the pore. In some embodiments, the pore size is smaller than the diameter of the cell. In some embodiments, the pore size is about 20% to about 99% of the diameter of the cell. In some embodiments, the pore 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. In some embodiments, the pore size is about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, or about 15 μm or more.

[0143] The inlet and outlet of the pores may have various angles. In some embodiments, the pore angle can be adjusted (e.g., increased or decreased) to reduce or prevent any clogging of the pores. In some embodiments, the flow rate (i.e., the rate at which the cells, or the suspension containing the cells, pass through the pores) is about 0.001 mL / cm / sec to about 100 L / cm / sec. For example, the angle of the inlet or outlet portion can be between about 0 degrees and about 90 degrees. In some embodiments, the pores have the same inlet and outlet angles. In some embodiments, the pores have different inlet and outlet angles. In some embodiments, the edges of the pores are smooth, e.g., rounded or curved. As used herein, a "smooth" pore edge has a continuous, flat, uniform, no-step, no-ridge, no-roughness surface. In some embodiments, the edges of the pores are sharp. As used herein, a "sharp" pore edge has a narrow edge that is pointed or at an acute angle. In some embodiments, the pore passage is straight. As used herein, a "straight" pore passage does not include curves, bends, corners, or other irregularities. In some embodiments, the pore passage is curved. As used herein, a "curved" pore passage is bent or deviates from a straight line. In some embodiments, the pore passage has multiple curves, such as, for example, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more curves.

[0144] The pores can have any shape known in the art, including two-dimensional or three-dimensional shapes. The pore shapes (e.g., cross-sectional shapes) can be, but are not limited to, circular, elliptical, round, square, star, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. In some embodiments, the cross-sectional shape of the pores is round. In some embodiments, the three-dimensional shape of the pores is cylindrical or conical. In some embodiments, the pores have grooved inlet and outlet shapes. In some embodiments, the pore shapes are homogeneous (i.e., uniform or regular) among the pores within a given surface. In some embodiments, the pore shapes are heterogeneous (i.e., mixed or varied) among the pores within a given surface.

[0145] Surfaces useful in the present disclosure may have a single pore. In some embodiments, surfaces useful in the present disclosure include a plurality of pores. In some embodiments, the pores comprise between about 10% and about 80% of the total surface area of ​​the surface. In some embodiments, the surface comprises between about 1.0×10 5 ~Approx. 1.0×10 30 In some embodiments, the surface comprises a surface area of ​​1 mm 2 Approximately 10 to 1.0 x 10 per 15 The micropores are

[0146] Within a given surface, the pores can be distributed in a number of ways. In some embodiments, the pores are distributed in parallel within a given surface. In some embodiments, the pores are distributed side by side in the same direction and are spaced apart by the same distance within a given surface. In some embodiments, the distribution of the pores is ordered or homogeneous. In such embodiments, the pores can be distributed in a regular and systematic pattern or spaced apart by the same distance within a given surface. In some embodiments, the distribution of the pores is random or non-homogeneous. For example, in some embodiments, the pores are distributed in an irregular and chaotic pattern or spaced apart by different distances within a given surface.

[0147] In some embodiments, the use of multiple surfaces allows cells to pass through multiple pores present on different surfaces. In some embodiments, the multiple surfaces are distributed in an array. The multiple surfaces can be homogenous or heterogeneous in surface size, shape, and / or roughness. The multiple surfaces can further include pores with homogenous or heterogeneous pore size, shape, and / or number, thereby enabling simultaneous delivery of a series of payloads to different cell types.

[0148] In some embodiments, for example, the individual pores of a surface that can be used in the present disclosure have a uniform width dimension (i.e., a constant width along the length of the pore passage). In some embodiments, the individual pores have a variable width (i.e., a width that increases or decreases along the length of the pore passage). In some embodiments, the pores within a given surface have the same individual pore depth. In some embodiments, the pores within a given surface have different individual pore depths. In some embodiments, the pores are directly adjacent to each other. In some embodiments, the pores are separated from each other by a distance of about 0.001 μm to about 30 mm.

[0149] In some embodiments, the surface is coated with a material. The material can be selected from any material known in the art, including, but not limited to, Teflon, adhesive coating, surfactants, proteins, adhesion molecules, antibodies, anticoagulants, factors that regulate cell function, nucleic acids, lipids, carbohydrates, transmembrane proteins, or combinations thereof. In some embodiments, the surface is coated with polyvinylpyrrolidone. In some embodiments, the material is covalently attached to the surface. In some embodiments, the material is non-covalently attached to the surface. In some embodiments, the surface molecules are released when the cells pass through the pores.

[0150] In some embodiments, the surface has modified chemical properties. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged. In some embodiments, the surface is positively and / or negatively charged. In some embodiments, the surface can be positively charged in some areas and negatively charged in other areas. In some embodiments, the surface has an overall positive charge or an overall negative charge. In some embodiments, the surface can be any one of smooth, electropolished, rough, or plasma treated. In some embodiments, the surface comprises a zwitterionic or dipolar compound. In some embodiments, the surface is plasma treated.

[0151] In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe, such as a plastic or glass syringe. In some embodiments, the surface is contained within a plastic filter holder. In some embodiments, the surface is contained within a pipette tip.

[0152] III.D. Cell Perturbation As described herein, when a cell passes through a constriction, the cell is physically deformed, resulting in perturbation (e.g., holes, tears, cavities, openings, pores, tears, gaps, perforations) in the cell's plasma membrane. Such perturbation in the plasma membrane is temporary and sufficient to allow any of the payloads described herein to be delivered into the cell. Cells have self-repair mechanisms that allow the cell to repair any disruption in its plasma membrane. See Blazek et al., Physiology (Bethesda) 30(6):438-48 (Nov. 2015), which is incorporated by reference in its entirety. Thus, in some embodiments, once the cell passes through a constriction (e.g., a microfluidic channel or pore), perturbation of the plasma membrane can be reduced or eliminated, thereby preventing the payload delivered into the cell from exiting the cell.

[0153] In some embodiments, perturbation of the cell membrane occurs after pressure is removed (e.g., after the cells pass through the constriction) at a rate of about 1.0×10 -9 In some embodiments, cell perturbation lasts from about 1.0×10 -9 In some embodiments, cell perturbation lasts for about 1.0×10 seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. -9 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -2 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -3 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -4 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -5 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -6 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -7 seconds, or approximately 1.0 x 10 -9 seconds ~ approx. 1.0×10 -8 In some embodiments, cell perturbation lasts for about 1.0×10 seconds. -8 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -7 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -6 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -5 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -4 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -3 seconds ~ approx. 1.0×10 -1 seconds, or approximately 1.0 x 10 -2 seconds ~ approx. 1.0×10 -1The cell perturbations (e.g., pores or holes) produced by the methods described herein are not formed as a result of assembly of polypeptide subunits that form multimeric pore structures such as those produced by complement hemolysins or bacterial hemolysins.

[0154] In some embodiments, as the cells pass through the constriction, pressure applied to the cells causes temporary damage to the cell membrane, which causes passive diffusion of material through perturbation. In some embodiments, the cells are deformed or perturbed for only a short period of time, e.g., on the order of 100 μs or less, to minimize the possibility of activating apoptotic pathways through cell signaling mechanisms, although other durations (e.g., ranging from nanoseconds to hours) are possible ... -9 In some embodiments, the cells are transformed for less than about 1.0×10 -9 In some embodiments, the cells are deformed for less than about 1.0×10 seconds to less than about 1 second, less than about 1 second to less than about 1 minute, or less than about 1 minute to less than about 1 hour. -9 In some embodiments, the cells are transformed for between about 1.0×10 seconds and about 2 hours. -9 In some embodiments, the cells are deformed for about 1.0×10 seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. -9 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -2 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -3 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -4 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -5 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -6 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -7 seconds, or approximately 1.0 x 10 -9 seconds ~ approx. 1.0×10 -8In some embodiments, the cells are deformed for about 1.0×10 -8 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -7 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -6 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -5 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -4 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -3 seconds ~ approx. 1.0×10 -1 seconds, or approximately 1.0 x 10 -2 seconds ~ approx. 1.0×10 -1 In some embodiments, transforming the cells includes transforming the cells for a time range of, but not limited to, about 1 μs to at least about 750 μs, examples of which include 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.

[0155] In some embodiments, the delivery of the payload into the cell occurs simultaneously with the cell passing through the constriction. In some embodiments, the delivery of the payload into the cell can occur after the cell passes through the constriction (i.e., when the cell membrane perturbation is still present and before the cell membrane is restored). In some embodiments, the delivery of the payload into the cell occurs on the order of minutes after the cell passes through the constriction. In some embodiments, the perturbation in the cell after the cell passes through the constriction is corrected within about 5 minutes after the cell passes through the constriction.

[0156] In some embodiments, the viability of cells (e.g., stem cells or PBMCs) after passing through the constriction is about 5% to about 100%. In some embodiments, the viability of cells after passing through the constriction is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, the viability of cells is about 1.0×10 after passing through the constriction. -2 For example, the cell viability is measured for about 1.0 × 10 cells per second to at least about 10 days after passing through the constriction. -2 The cell viability can be measured from about 1.0×10 2 to about 1 sec, from about 1 sec to about 1 min, from about 1 min to about 30 min, or from about 30 min to about 2 hr. In some embodiments, the cell viability can be measured from about 1.0×10 2 to about 1 sec after the cells have passed through the constriction. -2 seconds to about 2 hours, about 1.0 x 10 -2 seconds to about 1 hour, about 1.0 x 10 -2 seconds to approximately 30 minutes, approximately 11.0×10 -2 seconds to approximately 1 minute, approximately 1.0×10 -2 seconds ~ approx. 30 seconds, approx. 1.0 x 10 -2 seconds to about 1 second, or about 1.0 x 10 -2 In some embodiments, the cell viability is measured 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 cells have passed through the constriction. In some embodiments, the cell viability is measured 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 cells have passed through the constriction.

[0157] III.E. Delivery Parameters As is evident from the present disclosure, multiple parameters can affect the efficiency of delivery of a payload to a cell using the squeezing method provided herein. Thus, by adjusting (e.g., increasing or decreasing) one or more delivery parameters, the delivery of a payload to a cell can be improved. Thus, in some embodiments, the present disclosure relates to a method of increasing the delivery of a payload to a cell, the method comprising adjusting one or more parameters of a cell suspension passing through a constriction, the cell suspension comprising a population of cells, the one or more parameters increasing the delivery of the payload to one or more cells of the population of cells compared to a reference parameter. As described elsewhere in the present disclosure, the payload can be contacted with the population of cells before, during, or after the squeezing step.

[0158] In some embodiments, by adjusting one or more of the delivery parameters, delivery of the payload 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 a corresponding cell using the reference parameters.

[0159] In some embodiments, one or more delivery parameters that can be adjusted to increase the efficiency of delivery of the parameter include cell density (i.e., the concentration of cells present, e.g., in a cell suspension), pressure, or both. Additional examples of adjustable delivery parameters are provided elsewhere in this disclosure.

[0160] In some embodiments, the cell density is about 1×10 5 cells / mL, approximately 2×10 5 cells / mL, approximately 3×10 5 cells / mL, approximately 4×10 5cells / mL, approximately 5×10 5 cells / mL, approximately 6×10 5 cells / mL, approximately 7×10 5 cells / mL, approximately 8×10 5 cells / mL, approximately 9×10 5 cells / mL, approximately 1×10 6 cells / mL, approximately 2×10 6 cells / mL, approximately 3×10 6 cells / mL, approximately 4×10 6 cells / mL, approximately 5×10 6 cells / mL, approximately 6×10 6 cells / mL, approximately 7×10 6 cells / mL, approximately 8×10 6 cells / mL, approximately 9×10 6 cells / mL, approximately 1×10 7 cells / mL, approximately 2×10 7 cells / mL, approximately 3×10 7 cells / mL, approximately 4×10 7 cells / mL, approximately 5×10 7 cells / mL, approximately 6×10 7 cells / mL, approximately 7×10 7 cells / mL, approximately 8×10 7 cells / mL, approximately 9×10 7 cells / mL, approximately 1×10 8 cells / mL, approximately 1.1×10 8 cells / mL, approximately 1.2×10 8 cells / mL, approximately 1.3×10 8 cells / mL, approximately 1.4×10 8 cells / mL, approximately 1.5×10 8 cells / mL, approximately 2.0×10 8 cells / mL, approximately 3.0×10 8 cells / mL, approximately 4.0×10 8 cells / mL, approximately 5.0×10 8 cells / mL, approximately 6.0×10 8 cells / mL, approximately 7.0×10 8 cells / mL, approximately 8.0×10 8 cells / mL, approximately 9.0×10 8 cells / mL, or approximately 1.0 x 10 9 In some embodiments, the cell density is about 6×10 7 cells / mL ~ approx. 1.2×108 In cells / mL.

[0161] In some embodiments, the pressure is about 20 psi, about 25 psi, about 30 psi, about 35 psi, about 40 psi, about 50 psi, about 55 psi, about 60 psi, about 65 psi, about 70 psi, about 75 psi, about 80 psi, about 85 psi, about 90 psi, about 95 psi, about 100 psi, about 110 psi, about 120 psi, about 130 psi, about 140 psi, about 150 psi, about 160 psi, about 170 psi, about 180 psi, about 190 psi, or about 200 psi or more. In some embodiments, the pressure is about 30 psi to about 90 psi. In some embodiments, the pressure is about 20 psi.

[0162] In some embodiments, the particular type of device (e.g., microfluidic chip) may also affect the efficiency of delivery of the payload described herein. In the case of microfluidic chips, different chips may have different constriction parameters, examples of which include the length, depth and width of the constriction; the entrance angle, exit angle, length, depth and width of the approach region, etc. As described herein, such variables may affect the delivery of payloads to cells using the squeezing method of the present disclosure.

[0163] In some embodiments, the length of the constriction is up to 100 μm. For example, in some embodiments, the length is about 0 μm, 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, 10 μm, about 12.5 μm, about 15 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. In some embodiments, the length of the constriction is less than 1 μm. In some embodiments, the length of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2.5 μm, less than about 5 μm, less than about 7.5 μm, less than about 10 μm, less than about 12.5 μm, less than about 15 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, or less than about 100 μm. In some embodiments, the length of the constriction is about 10 μm. In some embodiments, the length of the constriction is about 0.5 μm. In some embodiments, the length of the constriction is about 70 μm. In some embodiments, the length of the constriction is about 0 μm. For example, in some embodiments, a microfluidic device (e.g., a chip) useful in the present disclosure includes a constriction in which the two ends of a diamond come together, resulting in a length of the constriction of about 0 μm.

[0164] In some embodiments, the width of the constriction is up to about 10 μm. In some embodiments, the width of the constriction is less than about 1 μm, less than about 2 μm, less than about 3 μm, less than about 4 μm, less than about 5 μm, less than about 6 μm, less than about 7 μm, less than about 8 μm, less than about 9 μm, or less than about 10 μm. In some embodiments, the width is about 1 μm to about 10 μm. In some embodiments, the width is about 1 μm, about 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.7 μm, about 1.8 μm, about 1.9 μm, about 2.0 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In some embodiments, the width of the constriction is about 6 μm. In some embodiments, the width of the constriction is about 3.5 μm.

[0165] In some embodiments, the depth of the constriction is at least about 1 μm. In some embodiments, the depth of the constriction is at least about 1 μm, at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 110 μm, or at least about 120 μm. In some embodiments, the depth is about 5 μm to about 90 μm. In some embodiments, the depth is about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or about 90 μm. In some embodiments, the depth of the constriction is about 20 μm. In some embodiments, the depth of the constriction is about 70 μm. In some embodiments, the depth of the constriction is about 10 μm.

[0166] In some embodiments, the depth is about 10 μm, the width is about 3.5 μm, the length is about 70 μm, and the pressure is about 20 psi.

[0167] In some embodiments, a constriction useful in the present disclosure comprises a width and a depth, the width of the constriction being any distance described herein, and the depth of the constriction being any distance described herein. In some embodiments, a constriction comprises a width and a depth, the width of the constriction being about 1 μm, about 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.7 μm, about 1.8 μm, about 1.9 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, and the depth of the constriction being about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, or about 90 μm. In some embodiments, such a constriction can include a length of about 0 μm (e.g., a constriction in which the two ends of a diamond come together, making the length of the constriction about 0 μm).

[0168] Additional examples of parameters that can affect the delivery of payload to cells include, but are not limited to, the dimensions of the constriction (e.g., length, width and / or depth), the entrance angle of the constriction, the surface characteristics of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity), the operating flow rate, the payload concentration, the time for cells to recover, or combinations thereof. Further parameters that can affect the efficiency of payload delivery can include the velocity of the cells within the constriction, the shear rate within the constriction, the viscosity of the cell suspension, the velocity component perpendicular to the flow rate, and the time within the constriction. Such parameters can be designed to control the delivery of the payload.

[0169] In some embodiments, the temperature used in the methods of the present disclosure can also affect the efficiency of delivery of the payload to the cells and the viability of the cells. In some embodiments, the squeezing method is carried out at about -5°C to about 45°C. For example, these methods can be carried out at room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), higher than physiological temperature (e.g., greater than about 37°C to 45°C or higher), or low temperature (e.g., about -5°C to about 4°C), or a temperature between these exemplary temperatures.

[0170] A variety of methods can be used to drive the cells through the constriction. For example, pressure can be applied with a pump (e.g., a gas cylinder or compressor) on the inlet side, a vacuum can be applied with a vacuum pump on the outlet side, capillary action can be applied through tubing, and / or the system can be gravity fed. Positive displacement flow systems can also be used (e.g., syringe pumps, peristaltic pumps, manual syringes or pipettes, pistons, etc.). In some embodiments, the cells are passed through the constriction by positive pressure. In some embodiments, the cells are passed through the constriction by constant or variable pressure. In some embodiments, pressure is applied using a syringe. In some embodiments, pressure is applied using a pump. In some embodiments, the pump is a peristaltic pump or a diaphragm pump. In some embodiments, pressure is applied using a vacuum. In some embodiments, the cells are passed through the constriction by gravity. In some embodiments, the cells are passed through the constriction by capillary pressure.

[0171] In some embodiments, the fluid flow guides the cells through the constriction. In some embodiments, the fluid flow before the cells pass through the constriction is turbulent. Turbulent flow is a fluid flow in which the magnitude and direction of velocity at a given point changes randomly. In some embodiments, the fluid flow through the constriction is laminar. Laminar flow involves an uninterrupted flow in a fluid near a solid boundary, where the direction of flow remains constant at all points. In some embodiments, after the cells pass through the constriction, the fluid flow becomes turbulent. The speed at which the cells pass through the constriction can be varied. In some embodiments, the cells pass through the constriction with a uniform cell velocity. In some embodiments, the cells pass through the constriction with a fluctuating cell velocity.

[0172] In some embodiments, a combination treatment is used to deliver a payload, for example, exposure to an electric field downstream of the constriction following the methods described herein. In some embodiments, the cells pass through the constriction and then through an electric field generated by at least one electrode. In some embodiments, the electric field assists in delivery of the payload to a second location within the cell, such as the cell nucleus. In some embodiments, one or more electrodes generate an electric field in proximity to the cell deformation constriction. In some embodiments, the electric field is about 0.1 kV / m to about 100 MV / m. In some embodiments, an integrated circuit is used to provide electrical signaling to drive the electrodes. In some embodiments, the cells are exposed to the electric field with a pulse width of about 1 ns to about 1 s and a period of about 100 ns to about 10 s.

[0173] III.F. Therapeutic applications In some embodiments, the present disclosure relates to the use of modified HSCs generated using the squeeze processing methods described herein to treat various diseases or disorders. As is evident from the present disclosure, the methods and compositions provided herein may be useful for diseases and disorders in which cell replacement therapy can be used as a treatment. By replacing damaged cells with cells generated using the methods provided herein, in some embodiments, one or more functions associated with the damaged cells may be restored, thereby treating the disease or disorder. For example, in some embodiments, modified HSCs generated using the squeeze processing methods provided herein may be administered to a subject suffering from a blood disorder (e.g., a hemoglobinopathy). Administration of such HSCs may be useful in improving one or more symptoms associated with the blood disorder. Non-limiting examples of blood disorders that can be treated using the methods provided herein include sickle cell disease (SCD), thalassemia syndrome, severe aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, congenital amegakaryocytic thrombocytopenia, or combinations thereof. In some embodiments, the disease or disorder that can be treated by the present disclosure includes an immune disorder. Non-limiting examples of immune disorders include severe combined immunodeficiency, Wiskott-Aldrich syndrome, or both. In some embodiments, the disease or disorder that can be treated by the present disclosure includes a metabolic disorder. Non-limiting examples of metabolic disorders include Krabbe disease (GLD), Hurler syndrome, adrenoleukodystrophy, metachromatic leukodystrophy, Fabry disease, Gaucher disease, cystinosis, Hunter syndrome, Pompe disease, or a combination thereof.

[0174] As is evident from the present disclosure, compared to other HSC-based treatments available in the art, the therapeutic methods provided herein can be used without the need to pretreat the subject to be treated (e.g., with toxic myeloablative therapy such as busulfan administration). Non-limiting examples of such myeloablative conditioning regimens include radiation, chemotherapy, small molecules, antibodies, or combinations thereof. As described herein, through the use of mobilizing agents (e.g., plerixaflor) and / or apoptotic agents (e.g., venetoclax) without any harsh side effects, the therapeutic methods provided herein allow a much safer and more efficient approach to promoting the engraftment of therapeutically modified HSCs.

[0175] IV. Disclosed Compositions In some aspects, the disclosure provides a system for delivering a payload to a cell (e.g., HSC), the system comprising a microfluidic channel as described herein and a cell suspension comprising a plurality of cells and a payload, wherein the constriction is configured to allow passage of the plurality of cells through the microfluidic channel, where passage of the plurality of cells causes deformation and disruption of a cell membrane of the cells, allowing the payload to enter the cells.

[0176] In some aspects, the disclosure provides a system for delivering a payload, the system comprising a surface having pores and a cell suspension comprising a plurality of cells and a payload, the surface having pores configured to allow the plurality of cells to pass through the pores, which causes deformation and disruption of the cell membrane of the cells, allowing the payload to enter the cells. In some aspects, the surface is a filter or membrane. In some of the above aspects, the system further comprises at least one electrode for generating an electric field. In some aspects, the system is used to deliver a payload into a cell by any of the methods described herein. The system can include any of the aspects described in the above disclosed methods, including a cell deformation constriction, a cell suspension, cell perturbation, a microfluidic channel for providing delivery parameters, or a surface having pores. In some aspects, delivery parameters such as operating flow rate, cell and compound concentration, cell velocity within the constriction, and composition of the cell suspension (e.g., osmolality, salt concentration, serum content, cell concentration, pH, etc.) are optimized for delivery of the payload into the cell.

[0177] In some aspects, the disclosure provides cells (e.g., HSCs) generated using any of the methods provided herein. In some aspects, provided herein are cells comprising perturbation of the cell membrane. The perturbation is due to one or more parameters (e.g., delivery parameters described herein) that transform the cell, thereby causing perturbation of the cell membrane of the cell, thereby allowing the payload to enter the cell. In some aspects, provided herein are cells comprising a payload. The payload enters the cell by perturbation of the cell membrane, the perturbation is due to one or more parameters (e.g., delivery parameters described herein) that transform the cell, thereby causing perturbation of the cell membrane of the cell, thereby allowing the payload to enter the cell. In some aspects, such cells can comprise modified HSCs (e.g., exhibiting increased resistance to mobilizing and / or apoptotic factors) as described herein.

[0178] In some aspects, the disclosure provides a composition comprising a plurality of cells, the plurality of cells being generated by any of the methods provided herein. Also provided herein is a composition comprising a population of cells and a payload under one or more parameters such that one or more cells of the population of cells are deformed, thereby causing perturbation of a cell membrane of the one or more cells, and the perturbation of the cell membrane allows the payload to enter the one or more cells.

[0179] In some embodiments, the present disclosure further provides a composition comprising a population of modified hematopoietic stem cells (HSCs), the modified HSCs comprising a payload that can (i) increase the resistance of the modified HSCs to a mobilizing factor, (ii) increase the resistance of the modified HSCs to an inhibitor of an anti-apoptotic factor, (iii) increase the resistance of the modified HSCs to a depleting factor, (iv) increase the expression of a homing factor on the modified HSCs, or (v) a combination thereof. As further described elsewhere in this disclosure, in some embodiments, the payload is delivered to the HSCs using a method other than the squeeze delivery method provided herein. Non-limiting examples of such methods are provided elsewhere in this disclosure.

[0180] Also provided are kits or articles of manufacture for use in delivering the payloads described herein into cells. In some embodiments, the kits include the compositions described herein (e.g., microfluidic channels or surfaces containing pores, cell suspensions and / or payloads) in suitable packaging. Suitable packaging materials are known in the art and include, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar bags or sealed plastic bags), and the like. These articles of manufacture can be further sterilized and / or sealed.

[0181] The present disclosure also provides kits that include components of the methods described herein and can further include instruction(s) for carrying out the methods for delivering a payload into a cell. The kits described herein can further include other materials, including other buffers, diluents, filters, needles, syringes, and inserts with instructions for carrying out any of the methods described herein (e.g., instructions for delivering a payload to a cell).

[0182] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0183] Example 1: Analysis of the effect of squeeze treatment on HSC transplantation efficiency To assess any potential impact on engraftment efficiency, CD34+ HSCs were loaded with mRNA encoding GFP using the squeezing method provided herein and adoptively transferred into NSG and NBSGW recipient mice. The specific method used is described below.

[0184] Squeeze Processing Two 5M (i.e., 5×10 6 ) A vial of cells was thawed and diluted in FACS buffer. The cells were centrifuged at 400rcf at room temperature to 2M / ml. The cells were transferred to a 12-well non-treated plate and cultured overnight at 37°C in complete CD34+ medium. The supernatant was aspirated and the cells were resuspended in 3ml of CD34+ medium. The cells were diluted to 2M / ml, plated and cultured overnight at 37°C. The cells were prepared for squeezing. Table 5 shows a description of the various test groups and the specific cell suspension solutions used for squeezing. [Table 5]

[0185] As shown in Table 5, groups A and C were squeezed without mRNA at room temperature using a microfluidic constriction (10 μm deep, 3.5 μm wide, 70 μm long) at 20 psi. The cells were then transferred to a 15 ml conical containing 5 mL of CD34+ medium. Groups B and D were squeezed with GFM mRNA at room temperature using a microfluidic constriction (10 μm deep, 3.5 μm wide, 70 μm long) at 20 psi. The cells were transferred to a 15 ml conical containing 5 mL of CD34+ medium. The cells of the different groups were centrifuged separately at 500 rcf for 5 min at room temperature. The supernatant was aspirated and the cells were resuspended in 1 mL of sterile PBS.

[0186] Adoption All mice received tail vein injections. Each mouse received 100 ul of 250K HSC cells according to the groups specified above.

[0187] Bone marrow collection and analysis Six weeks after injection, for all mice, bone marrow was harvested from the femur and tibia of each mouse. The tibia and femur were cut near the knee joint and placed in a microcentrifuge tube. 25 μL of FACs buffer was added to an Eppendorf tube and the microcentrifuge tube containing the sample was placed in it. The sample was centrifuged at 10,000 rcf for 15 seconds. The cells were then treated with 2 ml of ACK lysis buffer for 1-2 minutes to remove red blood cells. After lysis, the cells were washed and resuspended using FACs buffer.

[0188] Spleen collection and analysis Six weeks after injection, spleens were collected from all mice and each was placed into an Eppendorf tube containing 1 ml of FACs buffer. Each spleen sample was crushed and passed through a 70 μm filter into a 50 ml conical. Samples were washed with 10 ml of FACs buffer. Samples were spun at 400 rcf for 5 minutes and then resuspended in 2 ml of ACK buffer. Samples were incubated for 1-2 minutes and then quenched with 10 ml of FACs buffer. Samples were then filtered through a 70 μm filter, washed, and then resuspended in FACs buffer.

[0189] Peripheral blood sampling Six weeks after injection, blood was collected from all mice. Each blood sample was diluted (1:1) with PBS and then layered on top of 500ul of Ficoll Paque. The cells were spun at 800rcf for 25 minutes at room temperature to collect the white blood cells at the interface between the Ficoll layer and the aqueous layer. The cells were washed and then resuspended in 0.5ml of FACs buffer.

[0190] FACS analysis Two to five million cells were centrifuged at 400rcf for 4 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 200μL of L / D NIR (1:200 dilution) and FC block (1:50 dilution). The cells were then stained with anti-human CD45 and anti-mouse CD45 antibodies. The percentage of human CD45 cells versus mouse CD45 cells was then analyzed using an Attune flow cytometer.

[0191] result As shown in Figures 1 and 2C, squeezed HSCs did not have a detrimental effect on engraftment potential. A significant percentage of squeezed adoptively transferred human HSCs was observed in the bone marrow of both NSG and NBSGW mice. Similar results were observed in the peripheral blood (Figure 2A) and spleen (Figure 2B).

[0192] These results indicate that the squeeze treatment method provided herein does not have any negative effect on the engraftment potential of HSCs.

[0193] Example 2: Analysis of CXCR4 and Bcl-2 overexpression after squeeze treatment To assess whether expression of specific payloads described herein can help promote engraftment of HSCs, the squeeze treatment method provided herein was used to load mRNA encoding CXCR4 variants or Bcl-2 into CD34+ HSCs. The specific method used is described below.

[0194] Squeeze Processing One vial of 5M cells was thawed and diluted with FACs buffer. Cells were centrifuged at 400rcf at room temperature, then washed and resuspended at 2M / ml in complete CD34+ medium (StemSpan™ SFEM II with 100ng / ml SCF, TPO, Flt3-L). Cells were transferred to 12-well non-treated plates and cultured at 37°C. To expand HSCs, cells were cultured in complete CD34+ medium for 4 days.

[0195] The cells were prepared for squeezing. The cells were counted, washed, and the concentration was adjusted to 10 M / mL with OptiMem. The following buffer solutions were prepared for each sample, as shown in Table 6: [Table 6]

[0196] As shown in Table 6 above, Group A was used as a control (i.e., not contacted with any payload and not squeezed). Group B was squeezed without any payload at room temperature at 30 psi using a microfluidic constriction (depth 10 μm, width 3.5 μm, length 70 μm). Groups C-H were squeezed at room temperature with one of the payloads shown above, i.e., Group C: wild-type CXCR4 mRNA, Group D: CXCR4-A175F variant mRNA, Group E: CXCR4-R334X+A175F variant mRNA, Group F: CXCR4-R334X variant mRNA, Group G: wild-type Bcl-2 mRNA, and Group H: Bcl-2-G101V variant mRNA. Groups C–H were squeezed using the same conditions as group B (i.e., microfluidic constriction: depth 10 μm, width 3.5 μm, length 70 μm, pressure 30 psi).

[0197] Cells from each group A-H were then transferred separately into their respective 5 ml conicals containing 2 mL of CD34+ medium. Cells from all groups were separately centrifuged at 500 rcf for 5 min at room temperature. The supernatant was aspirated and the cell populations were separately resuspended in 1 mL of CD34+ medium and cultured at 37°C for 72 h.

[0198] FACS analysis After 4, 24, 48, and 72 hours, 200ul of cells were aspirated and centrifuged at 400rcf for 4 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 50μL of L / D aqua (diluted 1:200 in FACs buffer) and FC block (diluted 1:50 in FACs buffer). The cells were incubated in the dark for 20 minutes at room temperature and the cells were spun down at 400rcf for 4 minutes at room temperature. The cells were then incubated with fluorescent anti-human CXCR4 antibody for 30 minutes at 4°C. After incubation, 200μL of FACs separation buffer was added to each sample and the samples were spun at 400rcf for 4 minutes. The cells were then fixed with paraformaldehyde for 20 minutes and subsequently stained with fluorescent anti-human BCL2 antibody for 30 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 200μL of FACs separation buffer. The cells were then analyzed using an Attune flow cytometer.

[0199] result As shown in Figures 3A, 3B, 4A and 4B, the squeeze treatment induced high expression of the delivered mRNA. All CXCR4 variants showed expression above background for at least 48 hours as measured in vitro (Figures 3A and 3B). Furthermore, as shown in Figure 3C, CD34+ HSCs squeezed with CXCR4-A175F variants showed increased resistance to Plerixafor (i.e., mobilization factor) compared to corresponding CD34+ HSCs squeezed with wild-type CXCR4 mRNA (as evidenced by CXCL12 signaling). Without being bound to any one theory, such results suggest that modified HSCs are more resistant to the effects of mobilization factors (which promote mobilization of cells from bone marrow to peripheral blood), thereby allowing improved engraftment within the bone marrow. Finally, all BCL2 variants also showed expression above background for at least 72 hours (Figures 4A and 4B).

[0200] Collectively, the results provided herein demonstrate that the squeeze treatment methods provided herein can be used to generate HSCs with enhanced engraftment potential.

[0201] Example 3: In vivo analysis of CXCR4 expression in squeezed CD34+ HSCs To further evaluate whether modifying HSCs to express specific homing receptors could improve engraftment in the bone marrow, human CD34+ HSCs were cultured overnight and prepared for squeezing as generally described in Example 1. As shown in FIG. 3D, CD34+ HSCs were squeezed either without payload (squeezing alone) or with mRNA encoding wild-type CXCR4 and then adoptively transferred into NSG mice. After approximately 24 hours, mice were sacrificed and CXCR4 expression was assessed in the bone marrow.

[0202] As shown in FIG. 3E, significantly higher expression of CXCR4 was observed in the bone marrow of animals receiving HSCs squeezed with CXCR4 mRNA compared to animals receiving HSCs squeezed without mRNA. These results confirm the in vivo expression of CXCR4 in HSCs squeezed with CXCR4 mRNA after adoptive transfer. Such results further demonstrate that using the squeeze treatment methods provided herein, HSCs can be modified to express a specific homing receptor (e.g., CXCR4) to improve homing of the modified HSCs to the bone marrow, thereby enabling improved engraftment.

[0203] Example 4: In vivo analysis of Bcl-2 expression in squeezed CD34+ HSCs To further evaluate the effect of the survival factors described herein on engraftment of HSCs, human CD34+ HSCs were cultured overnight and prepared for squeezing as generally described in Example 1. As shown in FIG. 5A, CD34+ HSCs were squeezed with either control mRNA or mRNA encoding a Bcl-2 variant (i.e., G101V) and then adoptively transferred into NSG mice. After approximately 24 hours, mice were sacrificed and engraftment of the transferred HSCs in the bone marrow was assessed by measuring Bcl-2 expression using flow cytometry.

[0204] As shown in Figure 5B, mice that received HSCs squeezed with control mRNA had minimal expression of Bcl-2 in the bone marrow. In contrast, significant Bcl-2 expression was observed in the bone marrow of mice that received CD34+ HSCs modified to contain Bcl-2 variant mRNA, confirming that these cells can express Bcl-2 in vivo.

[0205] These results further demonstrate that survival factors described herein (e.g., Bcl-2 G101V) can be useful in enhancing engraftment of HSCs, for example, when the survival factors are delivered to the HSCs using the squeeze treatment method described herein.

[0206] Example 5: Analysis of resistance of CD34+ HSCs containing Bcl-2 variant mRNA to apoptotic factors As described herein, modified HSCs of the present disclosure (e.g., squeezed to include survival factors, e.g., Bcl-2 variants) exhibit improved resistance to various apoptotic factors. To further demonstrate such improved properties, human CD34+ HSCs were cultured overnight and prepared for squeezing as generally described in Example 1. As shown in FIG. 6A, CD34+ HSCs were split and labeled with different fluorescent markers (either CELLTRACE® Violet or CELLTRACE® Far Red). Cells labeled with the Violet fluorescent marker were then squeezed with GFP mRNA, and cells labeled with the Far Red fluorescent marker were squeezed with mRNA encoding a Bcl-2 variant (i.e., G101V). Cells from each group were mixed 1:1 and then adoptively transferred into NSG mice. Some mice were also administered a Bcl-2 inhibitor and an MCL-1 inhibitor (i.e., venetoclax and S63845, respectively) (i.e., inhibitors of anti-apoptotic factors). Approximately 24 hours after transfer, the mice were sacrificed to assess engraftment of the transferred cells in the bone marrow.

[0207] As shown in Fig. 6B , the bone marrow of mice that received apoptotic factors contained a significantly higher frequency of transferred HSCs modified to contain Bcl-2 variant mRNA compared with the frequency of transferred HSCs modified to contain GFP mRNA.

[0208] These results confirm the improved ability of the disclosed modified HSCs to resist the effects of anti-apoptotic inhibitors. Furthermore, without being bound to any one theory, the results further suggest that the combination of anti-apoptotic inhibitors with the modified HSCs described herein may be particularly useful in enhancing engraftment by selectively depleting unmodified endogenous HSCs from the bone marrow and allowing improved engraftment of the transferred modified HSCs.

[0209] Incorporation by Reference All publications, patents, patent applications, and other documents cited in this application are herein 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.

[0210] Equivalent While various specific embodiments have been illustrated and described, the above specification is not intended to be limiting. It will be understood that various changes can be made without departing from the spirit and scope of the disclosure. Many variations will become apparent to those skilled in the art upon review of this specification.

Claims

1. 1. A method for producing modified hematopoietic stem cells (HSCs), comprising passing a cell suspension comprising a population of HSCs through a constriction; passing the cell suspension through the constriction allows a payload to enter the HSCs, thereby producing modified HSCs; The modified HSCs exhibit one or more of: (i) increased resistance to mobilizing factors; (ii) increased resistance to apoptotic factors; (iii) increased resistance to depleting factors; or (iv) increased expression of homing factors, compared to corresponding HSCs that have not been forced through the constriction. method.

2. The method described in claim 1, wherein (i) the resistance of the modified HSC to the mobilizing factor, the apoptotic factor or the depleting factor; and / or (ii) the expression of the homing factor on the modified HSC is increased by at least two-fold compared to corresponding HSC that has not been forced through the stenosis under one or more parameters. (i) the payload is transiently expressed in the HSCs; and / or (ii) the payload comprises a homing receptor, cytokine, growth factor, cell adhesion molecule, proliferation agent, survival factor, or regulator thereof; The method of claim 1. (i) the homing receptor comprises CXCR4, CXCR2, or both; (ii) the cytokine comprises stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L), or both; (iii) the growth factor comprises thrombopoietin (TPO); (iv) the cell adhesion molecule comprises an integrin, a selectin, or both; (v) the proliferation agent comprises an activator of a signal transduction pathway involved in cell proliferation; (vi) the survival factor comprises Bcl-2, Bcl-xL, MCL-1, Ced-9, or bfl-1; (vii) the regulator is capable of increasing the expression and / or activity of one or more of the homing receptor, the cytokine, the growth factor, the cell adhesion molecule, the proliferation agent, or the survival factor; or (viii) the regulator is capable of reducing or preventing (e.g., knocking down) the activity of one or more inhibitors of the homing receptor, the cytokine, the growth factor, the cell adhesion molecule, the proliferation agent, or the survival factor; The method of claim 1. (i) the CXCR4 comprises an amino acid sequence that differs from the corresponding wild-type amino acid sequence set forth in SEQ ID NO:1, or the amino acid sequence of the CXCR4 comprises one of the following mutations: R334X, A175F, H113A, D171N, D262N, I284A, H281A, Q200W, Q200A, or a combination thereof; or (ii) the Bcl-2 comprises an amino acid sequence that differs from the corresponding wild-type amino acid sequence set forth in SEQ ID NO:9, or the amino acid sequence of the Bcl-2 comprises a G101V mutation, a D103Y mutation, or both; The method of claim 4.

6. the recruitment factor comprises plerixaflor, AMD3465, GROβ, G-CSF, or an anti-CD117 antibody; and / or the apoptotic factor comprises a Bcl-2 inhibitor (e.g., venetoclax), an MCL-1 inhibitor (e.g., S63845 or S64315), or a BCL-XL inhibitor; The method of claim 1.

7. 2. The method of claim 1, wherein the payload comprises a nucleic acid, wherein the nucleic acid comprises DNA, RNA, or both, and wherein the RNA comprises mRNA, siRNA, miRNA, lncRNA, tRNA, shRNA, self-amplifying mRNA (saRNA), PNA, or locked nucleic acid (LNA), or a combination thereof.

8. 2. The method of claim 1, comprising contacting the HSCs with the payload (i) before passing the cell suspension through the constriction, (ii) while passing the cell suspension through the constriction, or (iii) after passing the cell suspension through the constriction.

9. The method of claim 1, further comprising passing the cell suspension through the constriction under one or more parameters, the one or more parameters are selected from cell density; pressure; length, width, depth of the constriction; and / or diameter of the constriction; (i) the cell density is 1 x 10 6 cells / mL or more, 2 x 10 6 cells / mL or more, 3 x 10 6 cells / mL or more, 4 x 10 6 cells / mL or more, 5 x 10 6 cells / mL or more, 6 x 10 6 cells / mL or more, 7 x 10 6 cells / mL or more, 8 x 10 6 cells / mL or more, 9 x 10 6 cells / mL or more, or 1 x 10 7 cells / mL or more; (ii) the pressure is between 20 psi and 90 psi; (iii) the diameter of the constriction is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the diameter of the HSC; (iv) the length of the constriction is 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm; (v) the width of the constriction is between 1 μm and 10 μm; and / or (vi) the depth of the constriction is 5 μm to 90 μm; method.

10. 9. The method of claim 8, wherein the HSCs are contacted with multiple payloads (i) before passing the cell suspension through the constriction, (ii) while passing the cell suspension through the constriction, or (iii) after passing the cell suspension through the constriction, such that passing the cell suspension through the constriction allows at least two or more of the multiple payloads to enter the HSCs.

11. 10. The method of claim 1, comprising passing the cell suspension through a plurality of constrictions, (i) each constriction of the plurality of constrictions is the same, or one or more constrictions of the plurality of constrictions are different; or (ii) each constriction of the plurality of constrictions is associated with the same payload, or one or more of the plurality of constrictions is associated with a different payload; method.

12. 1. A composition comprising a population of modified hematopoietic stem cells (HSCs), comprising: the modified HSC comprises a payload; The payload is capable of (i) increasing the resistance of the modified HSC to a mobilizing factor, (ii) increasing the resistance of the modified HSC to an inhibitor of an anti-apoptotic factor, (iii) increasing the resistance of the modified HSC to a depleting factor, or (iv) increasing the expression of a homing factor on the modified HSC, compared to a corresponding HSC that has not been modified to include the payload. composition.

13. The composition described in claim 12, wherein (i) the resistance of the modified HSC to a mobilizing factor, an inhibitor of an anti-apoptotic factor, or a depleting factor and / or (ii) the expression of a homing factor on the modified HSC is increased by at least two-fold compared to a corresponding HSC that has not been modified to contain the payload.

14. (i) the payload is transiently expressed in the modified HSC; and / or (ii) the payload comprises a homing receptor, cytokine, growth factor, cell adhesion molecule, proliferation agent, survival factor, or regulator thereof; The composition of claim 12.

15. (i) the homing receptor comprises CXCR4, CXCR2, or both; (ii) the cytokine comprises stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L), or both; (iii) the growth factor comprises thrombopoietin (TPO); (iv) the cell adhesion molecule comprises an integrin, a selectin, or both; (v) the proliferation agent comprises an activator of a signal transduction pathway involved in cell proliferation; (vi) the survival factor comprises Bcl-2, Bcl-xL, MCL-1, Ced-9, bfl-1, or a combination thereof; (vii) the regulator is capable of increasing the expression and / or activity of one or more of the homing receptor, the cytokine, the growth factor, the cell adhesion molecule, the proliferation agent, or the survival factor; or (viii) the regulator is capable of reducing or preventing (e.g., knocking down) the activity of one or more inhibitors of the homing receptor, the cytokine, the growth factor, the cell adhesion molecule, the proliferation agent, or the survival factor; 15. The composition of claim 14. (i) the CXCR4 comprises an amino acid sequence that differs from the corresponding wild-type amino acid sequence set forth in SEQ ID NO:1, or the amino acid sequence of the CXCR4 comprises one of the following mutations: R334X, A175F, H113A, D171N, D262N, I284A, H281A, Q200W, Q200A, or a combination thereof; or (ii) the Bcl-2 comprises an amino acid sequence that differs from the corresponding wild-type amino acid sequence set forth in SEQ ID NO:9, or the amino acid sequence of the Bcl-2 comprises a G101V mutation, a D103Y mutation, or both; 16. The composition of claim 15.

17. The composition of claim 12 for use in a method of treating a disease or disorder in a subject in need thereof, comprising: The method includes administering to the subject the composition; The disease or disorder comprises a blood disorder, an immune disorder, or a metabolic disorder. composition.

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