METHODS

1 . A method for assessing hemoglobin A (HbA) formation in cells comprising:

a) modifying a population of cells to express β-globin;

b) lysing the cells under non-denaturing conditions, thus forming cell lysates;

c) analyzing the cell lysates with ion exchange (IEX) chromatography comprising:

i) passing the cell lysates through an IEX chromatographic column;

ii) detecting heme groups associated with HbF and/or HbA hemoglobin multimers at 418 nm;

d) calculating HbA expression.

2 . The method of claim 1 , wherein the modifying comprises introducing a vector encoding a β-globin gene into the population of cells.

3 . The method of claim 2 , wherein the vector is a viral vector or a non-viral vector.

4 . The method of any one of the preceding claims , wherein the vector is introduced by transfection, transduction, or electroporation.

5 . The method of claim 1 , wherein the modifying comprises introducing into the population of cells:

a) an endonuclease or polynucleotide encoding an endonuclease;

b) and a donor repair template encoding a β-globin.

6 . The method of claim 5 wherein the endonuclease is selected from the group consisting of:

a) a homing endonuclease, or functional variant thereof;

b) a megaTAL, or functional variant thereof;

c) a CRISPR-associated nuclease, or functional variant thereof;

d) a zinc-finger nuclease, or functional variant thereof; and

e) transcription activator-like effector nuclease (TALEN), or functional variant thereof.

7 . The method of claim 5 or claim 6 , wherein the endonuclease or polynucleotide encoding an endonuclease is introduced by transfection, transduction, or electroporation.

8 . The method of any one of claims 5-7 , wherein the donor repair template is introduced by transfection, transduction, or electroporation.

9 . The method of any one of the preceding claims , wherein the method further comprises culturing the cells for about 24 to about 96 hours post-modifying.

10 . A method for assessing potency of a viral vector encoding a β-globin gene comprising:

a) transducing a population of cells that do not express hemoglobin A (HbA) with a vector encoding a β-globin gene;

b) lysing the cells under non-denaturing conditions, thus forming cell lysates;

c) analyzing the cell lysates with ion exchange (IEX) chromatography comprising:

i) passing the cell lysates through an IEX chromatographic column;

ii) detecting heme groups associated with HbF and/or HbA hemoglobin multimers at 418 nm;

d) calculating HbA expression relative to HbA expression in a cell introduced with a reference standard vector.

11 . The method of claim 4 , wherein the potency is a relative potency.

12 . The method of claim 10 or 11 , wherein the method further comprises culturing the population of cells for 24 to 96 hours post-transduction.

13 . The method of any one of the preceding claims , wherein the population of cells do not endogenously express HbA.

14 . The method of any one of the preceding claims , wherein the population of cells have been genetically edited to not express HbA.

15 . The method of any one of the preceding claims , wherein the population of cells express fetal hemoglobin (HbF).

16 . The method of any one of the preceding claims , wherein the population of cells are a myelogenous leukemia cell line.

17 . The method of any one of the preceding claims , wherein the population of cells are K562 cells.

18 . The method of any one of the preceding claims , wherein the cells are plated at a cell density of about 0.5×10 6 cells/ml, about 1.0×10 6 cells/ml, about 1.5×10 6 cells/ml, about 2.0×10 6 cells/ml, about 2.5×10 6 cells/ml, or about 3.0×10 6 cells/ml prior to modification or transduction.

19 . The method of any one of claims 1-18 , wherein the cells are plated at a cell density of about 0.5×10 6 cells/ml to about 3.0×10 6 cells/ml prior to modification or transduction.

20 . The method of any one of claims 1-18 , wherein the cells are plated at a cell density of about 0.5×10 6 cells/ml to about 2.5×10 6 cells/ml prior to modification or transduction.

21 . The method of any one of claims 1-18 , wherein the cells are plated at a cell density of about 0.5×10 6 cells/ml to about 2.0×10 6 cells/ml prior to modification or transduction.

22 . The method of any one of claims 1-18 , wherein the cells are plated at a cell density of about 0.5×10 6 cells/ml to about 1.5×10 6 cells/ml prior to modification or transduction.

23 . The method of any one of claims 1-18 , wherein the cells are plated at a cell density of about 0.6×10 6 cells/ml to about 1.4×10 6 cells/ml prior to modification or transduction.

24 . The method of any one of claims 1-18 , wherein the cells are plated at a cell density of about 0.7×10 6 cells/ml to about 1.3×10 6 cells/ml prior to modification or transduction.

25 . The method of any one of claims 1-18 , wherein the cells are plated at a cell density of about 0.8×10 6 cells/ml to about 1.2×10 6 cells/ml prior to modification or transduction.

26 . The method of any one of claims 1-18 , wherein the cells are plated at a cell density of about 0.9×10 6 cells/ml to about 1.1×10 6 cells/ml prior to modification or transduction.

27 . The method of any one of claims 1-18 , wherein the cells are plated at a cell density of about 1.0×10 6 cells/ml prior to modification or transduction.

28 . The method of any one of claims 1-27 , wherein the cells are plated in tissue culture flasks.

29 . The method of any one of claims 1-27 , wherein the cells are plated in a 12-well plate.

30 . The method of any one of claims 1-27 , wherein the cells are plated in a 24-well plate.

31 . The method of claim 29 or claim 30 , wherein the cells are plated in a total volume of about 1 ml.

32 . The method of claim 29 or claim 30 , wherein the cells are plated in a total volume of about 2 ml.

33 . The method of any one of claims 1-32 , wherein the cells are transduced in the presence of polybrene.

34 . The method of claim 33 , wherein the cells are transduced in the presence of about 2 μg/ml to about 8 μg/ml polybrene.

35 . The method of claim 33 , wherein the cells are transduced in the presence of about 8 μg/ml polybrene.

36 . The method of any one of claims 1-35 , wherein the cells are cultured for about 48 to about 96 hours post-modification or -transduction.

37 . The method of any one of claims 1-35 , wherein the cells are cultured for about 60 to about 84 hours post-modification or -transduction.

38 . The method of any one of claims 1-35 , wherein the cells are cultured for about 48, about 60, about 72, about 84, or about 96 hours post-modification or -transduction.

39 . The method of any one of claims 1-35 , wherein the cells are cultured for about 72±2 hours post-modification or -transduction.

40 . The method of any one of claims 1-35 , wherein the cells are cultured for about 72 hours post-modification or -transduction.

41 . The method of any one of claims 1-40 , wherein the cells are frozen after lysis and prior to analyzing the cell lysates with ion exchange (IEX) chromatography.

42 . The method of any one of claims 1-41 , wherein the HbA comprises α and β globin chain dimers or tetramers.

43 . The method of any one of claims 7-42 , wherein the HbF comprises α and γ globin chain dimers or tetramers.

44 . The method of any one of claims 1-43 , wherein the β-globin is a human β-globin.

45 . The method of any one of claims 1-44 , wherein the β-globin is β A-T87Q globin, a β A-G16D/E22A/T87Q -globin, or a β A-T87Q/K95E/K120E -globin.

46 . The method of any one of claims 1-45 , wherein the vector is a lentiviral vector.

47 . The method of any one of claims 1-46 , wherein the vector is an AnkT9W vector, a T9Ank2W vector, a TNS9 vector, a lentiglobin HPV569 vector, a lentiglobin BB305 vector, a BG-1 vector, a BGM-1 vector, a d432βAγ vector, a mLARβΔγV5 vector, a GLOBE vector, a G-GLOBE vector, a βAS3-FB vector, a V5 vector, a V5m3 vector, a V5m3-400 vector, and a G9 vector, or a derivative thereof.

48 . The method of claim 47 , wherein the vector is bb305.

49 . The method of any one of claims 1-48 , wherein the transducing comprises transduction of vector at a multiplicity of infection (MOI) of about 5 to about 40, about 5 to about 30, about 10 to about 40, or about 10 to about 30.

50 . The method of any one of claims 1-48 , wherein the transducing comprises transduction of vector at a multiplicity of infection (MOI) of 5-40, 5-30, 10-40, or 10-30.

51 . The method of any one of claims 1-48 , wherein the transducing comprises transduction of vector at a multiplicity of infection (MOI) of about 5, about 10, about 15, about 20, about 25, about 30, about 35, and/or about 40.

52 . The method of any one of claims 1-48 , wherein the transducing comprises transduction with vector at a multiplicity of infection (MOI) of about 20.

53 . The method of any one of claims 1-52 , wherein the transducing comprises transduction with vector at one or more MOIs in different wells or plates.

54 . The method of any one of claims 1-53 , wherein the transducing comprises transduction with vector at one or more MOIs in different wells or plates in duplicate.

55 . The method of any one of claims 1-54 , wherein the transducing comprises transduction with vector at one or more MOIs in different wells or plates in triplicate.

56 . The method of any one of claims 1-55 , wherein the transducing comprises transduction with vector at MOIs of 10, 15, 20, 25, and 30.

57 . The method of any one of claims 1-56 , wherein the IEX chromatography is IEX HPLC.

58 . The method of any one of claims 1-56 , wherein the IEX chromatography is IEX UPLC.

59 . The method of any one of claims 1-56 , wherein the IEX chromatography is IEX UHLPC.

60 . The method of any one of claims 1-59 , wherein the IEX chromatography comprises liquid-based first and second mobile phases.

61 . The method of any one of claims 1-60 , wherein the column comprises a solid phase comprising aspartic acid chains covalently linked to a substrate.

62 . The method of any one of claims 1-61 , wherein the column comprises a solid phase comprising sulfonic acid ligands covalently lined to a substrate.

63 . The method of any one of claims 1-62 , wherein the substrate is a silica substrate.

64 . The method of any one of claims 1-62 , wherein the substrate is a polymer.

65 . The method of any one of claims 1-64 , wherein the chromatography comprises a tunable ultraviolet (TUV) detector.

66 . The method of any one of claims 1-64 , wherein the chromatography comprises a photodiode array ultraviolet (PDA UV) detector.

67 . The method of any one of claim 1-66 , wherein the chromatography separates HbF multimers from HbA multimers.

68 . The method of any one of claims 1-67 , wherein chromatographic identification of HbA and HbF is made based on the matched retention time of the analyte peaks relative to a hemoglobin standard.

69 . The method of claim 68 , wherein the standard is AFSC.

70 . The method of any one of claims 1-69 , wherein the calculating comprises determining an HbA peak and measuring the area under the curve (AUC).

71 . The method of any one of claims 1-70 , wherein the calculating comprises determining an HbF peak and measuring the area under the curve (AUC).

72 . The method of any one of claims 1-71 , wherein the calculating comprises determining HbA expression as a percentage of HbA relative to the sum of HbA and HbF.

73 . The method of any one of claims 1-72 , wherein the calculating further comprises fitting a log-dose response curve to the calculated HbA expression.

74 . The method of any one of claims 1-73 , wherein the calculating further comprises fitting a linear log-dose response curve to a reference standard and the vector.

75 . The method of claim 73 or claim 74 , wherein the log-dose is a log 10 dose.

76 . The method of any one of claim 73-75 , wherein the fitting comprises a parallel line approach to determine a relative potency.

77 . The method of claim 76 , wherein the relative potency is determined by the formula:

Relative

⁢

potency

=

antilog

⁡

(

Test

⁢

intercept

-

Reference

⁢

Intercept

Common

⁢

Slope

)

78 . The method of any one of claims 73-75 , wherein the fitting comprises an interpolation approach.

79 . The method of claim 78 , wherein the interpolation approach comprises a linear fit applied to the reference standard log-dose response and the % HbA responses of the vector are used to interpolate MOI from the reference curve fit.

80 . The method of any one of the preceding claims , wherein the method is an in vitro method.

81 . The method of any one of the preceding claims , wherein the method is an ex vivo method.