INTRACELLULAR DELIVERY OF COMPLEXES

• US 20190017072A1
• Filed: 01/11/2017
• Published: 01/17/2019
• Est. Priority Date: 01/12/2016
• Status: Abandoned Application

• Current Assignee: SQZ Biotechnologies, Co.
• Original Assignee: SQZ Biotechnologies, Co.
• Inventors: DITOMMASO, Tia, BERNSTEIN, Howard, SHAREI, Armon R., GILBERT, Jonathan B.
• Application Number:
  US16/068,631
• Publication Number:
  US 20190017072A1
• Time in Patent Office: Days
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 35/04 Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2501/71 Oxidoreductases (EC 1.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2527/00 Culture process characterised by the use of mechanical forces, e.g. strain, vibration

Abstract

The present invention provides methods for delivering a transient and/or reversible complex into a cell including passing a cell suspension through a constriction, wherein said constriction deforms the cell, thereby causing a perturbation of the cell such that the complex enters the cell.

Claims

1. A method for delivering a complex of two or more molecules into a cell, the method comprising passing a cell suspension through a constriction, wherein said constriction deforms the cell, thereby causing a perturbation of the cell such that the complex of molecules enters the cell, wherein said cell suspension is contacted with the complex of molecules.

2. The method of claim 1, wherein formation of the complex of molecules is reversible.

3. The method of claim 1 or 2, wherein at least two or more molecules of the complex associate by noncovalent interactions.

4. The method of any one of claims 1-3, wherein at least two molecules in the complex have a binding affinity in the complex ranging from about 1 μ

M to about 1 pM.

5. The method of any one of claims 1-4, wherein at least two molecules in the complex have a binding affinity in the complex ranging from about 1 μ

M to about 1 nM or from about 1 nM to about 1 pM.

6. The method of any one of claims 1-5, wherein the complex has a half-life in the cell suspension of about 1 minute to about 48 hours.

7. The method of claim 6, wherein the complex has a half-life in the cell suspension of about 1 minute to about 20 minutes, about 20 minutes to about 40 minutes, about 40 minutes to about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 12 hours, about 12 hours to about 24 hours, about 24 hours to about 36 hours, or about 36 hours to about 48 hours.

8. The method of any one of claims 1-7, wherein the complex dissociates in the presence of a detergent.

9. The method of claim 8, wherein the complex dissociates in the presence of a detergent at a concentration of about 0.1% (w/v) to about 10% (w/v).

10. The method of claim 8 or 9, wherein the complex dissociates in the presence of a detergent at a concentration of about 0.1% (w/v) to about 1% (w/v), about 1% (w/v) to about 5% (w/v), or about 5% (w/v) to about 10% (w/v).

11. The method of any one of claims 1-10, wherein the cell suspension is contacted with the complex of molecules at a temperature ranging from about 0°

C. to about 40°

C.

12. The method of claim 11, wherein the complex of molecules dissociates at a temperature greater than the temperature at which the cell suspension is contacted with the complex of molecules.

13. The method of claim 11 or 12, wherein the complex of molecules dissociates at a temperature of about 50°

C. to about 70°

C.

14. The method of any one of claims 11-13, wherein the complex of molecules dissociates at a temperature of about 50°

C. to about 60°

C., or about 60°

C. to about 70°

C.

15. The method of any one of claims 1-14, wherein the cell suspension is contacted with the complex of molecules at an ionic strength ranging from about 50 mM to about 300 mM.

16. The method of claim 15, wherein the complex of molecules dissociates at an ionic strength greater than the ionic strength at which the cell suspension is contacted with the complex of molecules.

17. The method of claim 15 or 16, wherein the complex of molecules dissociates at an ionic strength of about 350 mM to about 1000 mM.

18. The method of claim 17, wherein the complex of molecules dissociates at an ionic strength of about 350 mM to about 400 mM, about 400 mM to about 500 mM, about 500 mM to about 600 mM, about 700 mM to about 800 mM, about 800 mM to about 900 mM, or about 900 mM to about 1000 mM.

19. The method of claim 15, wherein the complex of molecules dissociates at an ionic strength less than the ionic strength at which the cell suspension is contacted with the complex of molecules.

20. The method of claim 15 or 19, wherein the complex of molecules dissociates at an ionic strength of about 0 mM to about 50 mM.

21. The method of claim 20, wherein the complex of molecules dissociates at an ionic strength of about 0 mM to about 10 mM, about 10 mM to about 20 mM, about 20 mM to about 30 mM, about 30 mM to about 40 mM, or about 40 mM to about 50 mM.

22. The method of any one of claims 1-14, wherein the cell suspension is contacted with the complex of molecules at an osmolarity ranging from about 100 mOsm/L to about 500 mOsm/L.

23. The method of claim 22, wherein the complex of molecules dissociates at an osmolarity greater than the ionic strength at which the cell suspension is contacted with the complex of molecules.

24. The method of claim 22 or 23, wherein the complex of molecules dissociates at an osmolarity of about 600 mOsm/L to about 1000 mOsm/L.

25. The method of claim 24, wherein the complex of molecules dissociates at an osmolarity of about 600 mOsm/L to about 700 mOsm/L, about 700 mOsm/L to about 800 mOsm/L, about 800 mOsm/L to about 900 mOsm/L, or about 900 mOsm/L to about 1000 mOsm/L.

26. The method of claim 22, wherein the complex of molecules dissociates at an osmolarity less than the osmolarity at which the cell suspension is contacted with the complex of molecules.

27. The method of claim 22 or 26, wherein the complex of molecules dissociates at an osmolarity of about 0 mOsm/L to about 100 mOsm/L.

28. The method of claim 27, wherein the complex of molecules dissociates at an osmolarity of about 0 mOsm/L to about 20 mOsm/L, about 20 mOsm/L to about 20 mOsm/L, about 20 mOsm/L to about 40 mOsm/L, about 40 mOsm/L to about 60 mOsm/L, about 60 mOsm/L to about 80 mOsm/L, or about 80 mOsm/L to about 100 mOsm/L.

29. The method of any one of claims 1-28, wherein the cell suspension is contacted with the complex of molecules at a pH ranging from about 5.5 to about 8.5.

30. The method of claim 29, wherein the complex of molecules dissociates at a pH greater or lower than the pH at which the cell suspension is contacted with the complex of molecules.

31. The method of claim 29 or 30, wherein the complex of molecules dissociates at a pH of about 4.0 to about 5.5 or at a pH of about 8.5 to about 10.

32. The method of claim 31, wherein the complex of molecules dissociates at a pH of about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 8.5 to about 9.0, about 9.0 to about 9.5, or about 9.5 to about 10.0.

33. The method of any one of claims 1-32, wherein the shear force as the cell passes through the constriction ranges from about 1 kPa to about 10 kPa.

34. The method of claim 33, wherein the complex dissociates at a shear force of about 10 kPa to about 100 kPa.

35. The method of claim 33 or 34, wherein the complex dissociates at a shear force of about 10 kPa to about 25 kPa, about 25 kPa to about 50 kPa, about 50 kPa to about 75 kPa, or about 75 kPa to about 100 kPa.

36. The method of any one of claims 1-35, wherein the complex of molecules comprises

a) one or more polypeptides,

b) one or more nucleic acids,

c) one or more lipids,

d) one or more carbohydrates,

e) one or more small molecules,

f) one or more metal-containing compounds,

g) one or more polypeptides and one or more nucleic acids,

h) one or more polypeptides and one or more lipids,

i) one or more polypeptides and one or more carbohydrates,

j) one or more polypeptides and one or more small molecules,

k) one or more polypeptides and one or more metal-containing compounds,

l) one or more nucleic acids and one or more lipids,

m) one or more nucleic acids and one or more carbohydrates,

n) one or more nucleic acids and one or more small molecules,

o) one or more nucleic acids and one or more metal-containing compounds,

p) one or more lipids and one or more carbohydrates,

q) one or more lipids and one or more small molecules,

r) one or more lipids and one or more metal-containing compounds,

s) one or more carbohydrates and one or more small molecules,

t) one or more carbohydrates and one or more metal-containing compounds,

u) one or more small molecules and one or more metal-containing compounds,

v) one or more polypeptides, one or more nucleic acids and one or more lipids,

w) one or more polypeptides, one or more nucleic acids and one or more carbohydrate,

x) one or more polypeptides, one or more nucleic acids and one or more small molecules,

y) one or more polypeptides, one or more nucleic acids and one or more metal-containing compounds,

z) one or more polypeptides, one or more lipids and one or more carbohydrates,

aa) one or more polypeptides, one or more lipids and one or more small molecules,

ab) one or more polypeptides, one or more lipids and one or more metal-containing compounds,

ac) one or more polypeptides, one or more carbohydrates and one or more small molecules,

ad) one or more polypeptides, one or more carbohydrates and one or more metal-containing compounds,

ae) one or more polypeptides, one or more small molecules and one or more metal-containing compounds,

af) one or more nucleic acids, one or more lipids, and one or more carbohydrates,

ag) one or more nucleic acids, one or more lipids, and one or more small molecules,

ah) one or more nucleic acids, one or more lipids, and one or more metal-containing compounds,

ai) one or more nucleic acids, one or more carbohydrates, and one or more small molecules,

aj) one or more nucleic acids, one or more carbohydrates, and one or more metal-containing compounds,

ak) one or more nucleic acids, one or more small molecules, and one or more metal-containing compounds,

al) one or more lipids, one or more carbohydrates and one or more small molecules,

am) one or more lipids, one or more carbohydrates and one or more metal-containing compounds,

an) one or more lipids, one or more small molecules and one or more metal-containing compounds,

ao) one or more carbohydrates, one or more small molecules and one or more metal-containing compounds,

ap) one or more polypeptides, one or more nucleic acids, one or more lipids, and one or more carbohydrates,

aq) one or more polypeptides, one or more nucleic acids, one or more lipids, and one or more small molecules,

ar) one or more polypeptides, one or more nucleic acids, one or more lipids, and one or more metal-containing compounds,

as) one or more polypeptides, one or more nucleic acids, one or more carbohydrates, and one or more small molecules,

at) one or more polypeptides, one or more nucleic acids, one or more carbohydrates, and one or more metal-containing compounds,

au) one or more polypeptides, one or more nucleic acids, one or more small molecules, and one or more metal-containing compounds,

av) one or more polypeptides, one or more lipids, one or more carbohydrates, and one or more small molecules,

aw) one or more polypeptides, one or more lipids, one or more carbohydrates, and one or more metal-containing compounds,

ax) one or more polypeptides, one or more lipids, one or more small molecules, and one or more metal-containing compounds,

ay) one or more polypeptides, one or more carbohydrates, one or more small molecules, and one or more metal-containing compounds,

az) one or more nucleic acids, one or more lipids, one or more carbohydrates, and one or more small molecules,

ba) one or more nucleic acids, one or more lipids, one or more carbohydrates, and one or more metal-containing compounds,

bb) one or more nucleic acids, one or more lipids, one or more small molecules, and one or more metal-containing compounds,

bc) one or more nucleic acids, one or more carbohydrates, one or more small molecules, and one or more metal-containing compounds,

bd) one or more lipids, one or more carbohydrates, one or more small molecules, and one or more metal-containing compounds,

be) one or more polypeptides, one or more nucleic acids, one or more lipids, one or more carbohydrates, and one or more small molecules,

bf) one or more polypeptides, one or more nucleic acids, one or more lipids, one or more carbohydrates, and one or more metal-containing compounds,

bg) one or more polypeptides, one or more nucleic acids, one or more lipids, one or more small molecules, and one or more metal-containing compounds,

bh) one or more polypeptides, one or more nucleic acids, one or more carbohydrates, one or more small molecules, and one or more metal-containing compounds,

bi) one or more polypeptides, one or more lipids, one or more carbohydrates, one or more small molecules, and one or more metal-containing compounds,

bj) one or more nucleic acids, one or more lipids, one or more carbohydrates, one or more small molecules, and one or more metal-containing compounds, or

bk) one or more polypeptides, one or more nucleic acids, one or more lipids, one or more carbohydrates, one or more small molecules, and one or more metal-containing compounds.

37. The method of any one of claims 1-36, wherein the complex comprises an antibody.

38. The method of any one of claims 1-36, wherein the complex comprises one or more transcription factors.

39. The method of any one of claims 1-36, wherein the complex comprises a ribosome and an mRNA.

40. The method of any one of claims 1-36, wherein the complex comprises a proteasome, a holoenzyme, an RNA polymerase, a DNA polymerase, a spliceosome, a vault cytoplasmic ribonucleoprotein, a small nuclear ribonucleic protein (snRNP), a telomerase, a nucleosome, a death signaling complex (DISC), a mammalian target of rapamycin complex 1 (mTORC1), a mammalian target of rapamycin complex 2 (mTORC2), or a class I phosphoinositide 3 kinase (Class I PI3K), RNA-induced silencing complex (RISC), histone-DNA complex, toll-like receptor (TLR)-agonist complex, transposase/transposon complex, tRNA ribosome complex, polypeptide-protease complex, or an enzyme-substrate complex.

41. The method of any one of claims 1-40, wherein the cell suspension is contacted with the complex after the cell suspension passes through the constriction.

42. The method of any one of claims 1-40, wherein the cell suspension is contacted with the complex before the cell suspension passes through the constriction.

43. The method of any one of claims 1-40, wherein the cell suspension is contacted with the complex at the same time the cell suspension passes through the constriction.

44. The method of any one of claims 1-42, wherein the complex is formed prior to contact with the cell suspension.

45. The method of claim 44, wherein the complex is formed about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, or about 6 hours prior to contact with the cell suspension.

46. The method of any one of claims 1-45, wherein the complex is purified prior to contact with the cell suspension.

47. The method of claim 40, wherein the complex is formed in the cell suspension.

48. The method of claim 47, wherein one or more of the molecules of the complex are purified prior to contact with the cell suspension.

49. The method of any one of claims 1-48, wherein the cell suspension comprises a mixed cell population.

50. The method of any one of claims 1-48, wherein the cell suspension comprises a purified cell population.

51. The method of any one of claims 1-48, wherein the cell suspension comprises prokaryotic or eukaryotic cells.

52. The method of any one of claims 1-51, wherein the cell suspension comprises bacterial cells, archael cells, yeast cells, fungal cells, algal cells, plant cells or animal cells.

53. The method of any one of claims 1-52, wherein the cell suspension comprises vertebrate cells.

54. The method of any one of claims 1-53, wherein the cell suspension comprises mammalian cells.

55. The method of any one of claims 1-54, wherein the cell suspension comprises human cells.

56. The method of any one of claims 1-55, wherein the constriction is contained within a microfluidic channel.

57. The method of any one of claims 1-55, wherein the constriction is a pore or contained within a pore.

58. The method of claim 57, wherein the pore is contained in a surface.

59. The method of claim 58, wherein the surface is a filter.

60. The method of claim 58, wherein the surface is a membrane.

61. The method of any one of claims 57-60, wherein the pore size is about 0.4 μ

m, about 1 μ

m, about 2 μ

m, about 3 μ

m, about 4 μ

m, about 5 μ

m, about 6 μ

m, about 7 μ

m, about 8 μ

m, about 9 μ

m, about 10 μ

m, about 11 μ

m, about 12 μ

m, about 13 μ

m, or about 14 μ

m.

62. The method of any one of claims 1-61, wherein the constriction size is a function of the cell diameter.

63. The method of any one of claim 1-62, wherein 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.

64. The method of any one of claims 1-63, wherein the constriction has a length of about 30 μ

m and a width of about 3 μ

m to about 8 μ

m.

65. The method of any one of claims 1-64, wherein the constriction has a length of about 10 μ

m and a width of about 3 μ

m to about 8 μ

m.

66. The method of any one of claims 1-65, wherein the method further comprises the step of contacting the cell suspension and complex with an electric field generated by at least one electrode.

73. The system of any one of claims 67-72, wherein the system is used to deliver a complex comprising two or more molecules into a cell by the method of any one of claims 1-66.

74. A cell comprising a complex of two or more molecules, wherein the complex of two or more molecules was delivered into the cell by the method of any one of claims 1-66.

67. A system for delivering a complex of two or more molecules into a cell, the system comprising a microfluidic channel comprising a constriction, a cell suspension comprising the cell, and the complex of two or more molecules;

wherein the constriction is configured such that the cell can pass through the constriction wherein the constriction deforms the cell thereby causing a perturbation of the cell such that the complex of two or more molecules enters the cell.

70. The system of any one of claims 67-69, wherein the system further comprises at least one electrode to generate an electric field.

71. The system of any one of claims 67-70, wherein formation of the complex of molecules is reversible.

72. The system of any one of claims 67-71, wherein at least two or more molecules of the complex associate by noncovalent interactions.

68. A system for delivering a complex of two or more molecules into a cell, the system comprising a surface with pores, a cell suspension comprising the cell, and the complex of two or more molecules;

wherein the surface with pores is configured such that the cell can pass through the pore wherein the pore deforms the cell thereby causing a perturbation of the cell such that the complex of two or more molecules enters the cell.

69. The system of claim 68, wherein the surface is a filter or a membrane.