TRANSDUCTION EFFICIENCY ASSAY
Disclosed herein are lentiviral vector transduction efficiency assays for gene therapy treatments. Also disclosed herein are methods for measuring transduction efficiency of a lentiviral vector.
1 . A transduction efficiency assay comprising:
a) transducing a population of cells from a sample with a lentiviral vector comprising a polynucleotide encoding a therapeutic gene;
b) culturing the transduced cells for a period of at least three days;
c) assaying the cultured transduced cells using single cell PCR;
d) measuring presence of genomic and viral DNA sequences in the cells in the sample;
e) quantifying number of transduced cells, wherein cells are considered transduced when they include both genomic and viral DNA sequences;
f) quantifying number of untransduced cells, wherein cells are considered untransduced when they include only genomic DNA sequences; and
g) calculating efficiency of lentiviral vector transduction (percentage of transduced cells), wherein the efficiency of the transduction is measured as:
Transduction
efficiency
(
%
)
=
∑
transduced
cells
∑
transduced
and
untransduced
cells
×
100.
2 . The assay of claim 1 , wherein the cells are peripheral blood mononuclear cells.
3 . The assay of claim 1 or claim 2 , wherein the cells are PBMCs isolated from a subject that has cancer.
4 . The assay of claim 3 , wherein the cancer is multiple myeloma.
5 . The assay of claim 3 or claim 4 , wherein the lentiviral vector comprises an engineered antigen receptor.
6 . The assay of claim 5 , wherein the engineered antigen receptor is selected from the group consisting of: an engineered αβ-TCR, an engineered δγ-TCR, a chimeric antigen receptor (CAR), and a dimerizing agent regulated immunoreceptor complex (DARIC).
7 . The assay of claim 5 or claim 6 , wherein the engineered antigen receptor is an anti-BCMA CAR.
8 . The assay of claim 1 , wherein the cells are hematopoietic stem or progenitor cells.
9 . The assay of claim 8 , further comprising obtaining the hematopoietic stem or progenitor cells from a subject that has sickle cell disease or β-thalassemia.
10 . The assay of claim 8 or claim 9 , wherein the hematopoietic stem or progenitor cells comprise CD34+ cells.
11 . The assay of any one of claims 8 to 10 , wherein the hematopoietic stem or progenitor cells comprise CD133 + cells.
12 . The assay of any one of claims 8 to 11 , wherein the hematopoietic stem or progenitor cells comprise CD34 + CD38 Lo CD90 + CD45RA − cells.
13 . The assay of any one of claims 8 to 12 , wherein the hematopoietic stem or progenitor cells comprise a pair of β-globin alleles selected from the group consisting of: β E /β 0 , β C /β 0 , β 0 /β 0 , β C /β C , β E /β E , β E /β + , β C /β E , β C /β + , β 0 /β + , and β + /β + .
14 . The assay of any one of claims 8 to 13 , wherein the polynucleotide encodes a globin selected from the group consisting of a human β-globin, a human δ-globin, an anti-sickling globin, a human γ-globin, a human β A-T87Q -globin, a human β A-G16D/E22A/T87Q -globin, and a human β A-T87Q/K95E/K120E -globin protein.
15 . The assay of any one of claims 8 to 14 , wherein the lentiviral vector is an AnkT9W vector, a T9Ank2W vector, a TNS9 vector, a TNS9.3 vector, a TNS9.3.55 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, a G9 vector, a BCL11A shmir vector, or a derivative thereof.
16 . The assay of any one of claims 1 to 15 , wherein the culturing of the transduced cells occurs for a period of 3 to 10 days.
17 . The assay of any one of claims 1 to 16 , wherein a cell is considered as transduced when it is measured as having a Threshold Cycle (CO value of ≤32 for both genomic and viral DNA sequences.
18 . The assay of any one of claims 1 to 17 , wherein the viral DNA sequences is a lentiviral vector psi-gag DNA sequence.
19 . The assay of any one of claims 1 to 18 , wherein the genomic DNA sequence is a RNAseP DNA sequence.
20 . A transduction efficiency assay comprising:
a) obtaining a peripheral blood or bone marrow sample from a subject;
b) isolating nucleated cells from the peripheral blood by density gradient centrifugation;
c) assaying the isolated cells using single cell PCR;
d) measuring presence of genomic and lentiviral vector DNA sequences in the cells in the sample;
e) quantifying number of transduced cells, wherein cells are considered transduced when they include both genomic and lentiviral vector DNA sequences;
f) quantifying number of untransduced cells, wherein cells are considered untransduced when they include only genomic DNA sequences; and
g) calculating efficiency of lentiviral vector transduction, wherein the efficiency of the transduction is measured as:
Transduction
efficiency
(
%
)
=
∑
transduced
cells
∑
transduced
and
untransduced
cells
×
100.
21 . The assay of claim 20 , wherein the nucleated cells are peripheral blood mononuclear cells.
22 . The assay of claim 20 , wherein the nucleated cells are hematopoietic stem or progenitor cells.
23 . The assay of claim 22 , wherein the hematopoietic stem or progenitor cells comprise CD34+ cells.
24 . The assay of claim 22 or claim 23 , wherein the hematopoietic stem or progenitor cells comprise CD133 + cells.
25 . The assay of any one of claims 22 to 24 , wherein the hematopoietic stem or progenitor cells comprise CD34 + CD38 Lo CD90 + CD45RA − cells.
26 . The assay of any one of claims 22 to 25 , wherein the hematopoietic stem or progenitor cells comprise a pair of β-globin alleles selected from the group consisting of: β E /β 0 , β C /β 0 , β 0 /β 0 , β C /β C , β E /β E , β E /β + , β C /β E , β C /β + , β 0 /β + , and β + /β + .
27 . The assay of any one of claims 20 , and 22 to 26 , wherein the peripheral blood is obtained from a subject treated with a drug product comprising a lentiviral vector comprising a polynucleotide encoding a globin.
28 . The assay of claim 27 , wherein the globin is a human β-globin, a human δ-globin, an anti-sickling globin, a human γ-globin, a human β A-T87Q -globin, a human β A-G16D/E22A/T87Q -globin, or a human β A-T87Q/K95E/K120E -globin protein.
29 . The assay of claim 27 or claim 28 , wherein the lentiviral vector is an AnkT9W vector, a T9Ank2W vector, a TNS9 vector, a TNS9.3 vector, a TNS9.3.55 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, a G9 vector, a BCL11A shmir vector, or a derivative thereof.
30 . The assay of claim 20 or claim 21 , wherein the nucleated cells are PBMCs isolated from a subject that has cancer.
31 . The assay of claim 30 , wherein the cancer is multiple myeloma.
32 . The assay of claim 30 or claim 31 , wherein the lentiviral vector comprises an engineered antigen receptor.
33 . The assay of claim 32 , wherein the engineered antigen receptor is selected from the group consisting of: an engineered αβ-TCR, and engineered δγ-TCR, a chimeric antigen receptor (CAR), or a dimerizing agent regulated immunoreceptor complex (DARIC).
34 . The assay of claim 32 or claim 33 , wherein the engineered antigen receptor is an anti-BCMA CAR.
35 . The assay of any one of claims 20 to 34 , wherein a cell is considered as transduced when it is measured as having a Threshold Cycle (C t ) value of ≤32 for both genomic and lentiviral vector DNA sequences.
36 . The assay of any one of claims 20 to 35 , wherein the lentiviral vector DNA sequences is a lentiviral vector psi-gag DNA sequence.
37 . The assay of any one of claims 20 to 36 , wherein the genomic DNA sequence is a RNAseP DNA sequence.
38 . The assay of any one of claims 20 to 37 , wherein the subject has sickle cell disease or β-thalassemia.
39 . A method for measuring transduction efficiency comprising:
a. assaying a population of cells using single cell PCR, wherein the population of cells are transduced with a lentiviral vector comprising a polynucleotide encoding a therapeutic gene;
b. measuring presence of genomic and viral DNA sequences in the cells;
c. quantifying number of transduced cells, wherein cells are considered transduced when they include both genomic and viral DNA sequences;
d. quantifying number of untransduced cells, wherein cells are considered untransduced when they include only genomic DNA sequences; and
e. calculating efficiency of the lentiviral vector transduction, wherein the efficiency of the transduction is measured as:
Transduction
efficiency
(
%
)
=
∑
transduced
cells
∑
transduced
and
untransduced
cells
×
100.
40 . The method of claim 39 , wherein the cells are peripheral blood mononuclear cells.
41 . The method of claim 39 , wherein the cells are hematopoietic stem or progenitor cells.
42 . The method of claim 41 , further comprising obtaining the hematopoietic stem or progenitor cells from a subject that has sickle cell disease or β-thalassemia.
43 . The method of claim 41 or 42 , wherein the hematopoietic stem or progenitor cells comprise CD34+ cells.
44 . The method of any one of claims 41 to 43 , wherein the hematopoietic stem or progenitor cells comprise CD133 + cells.
45 . The method of any one of claims 41 to 44 , wherein the hematopoietic stem or progenitor cells comprise CD34 + CD38 Lo CD90 + CD45RA − cells.
46 . The method of any one of claims 41 to 45 , wherein the hematopoietic stem or progenitor cells comprise a pair of β-globin alleles selected from the group consisting of: β E /β 0 , β C /β 0 , β 0 /β 0 , β C /β C , β E /β E , β E /β + , β C /β E , β C /β + , β 0 /β + , and β + /β + .
47 . The assay of any one of claims 39 , and 41 to 45 , wherein the cells are isolated from peripheral blood obtained from a subject treated with a drug product comprising a lentiviral vector comprising a polynucleotide encoding a globin.
48 . The method of any one of claim 47 , wherein the globin is a human β-globin, a human δ-globin, an anti-sickling globin, a human γ-globin, a human β A-T87Q -globin, a human β A-G16D/E22A/T87Q -globin, or a human β A-T87Q/K95E/K120E -globin protein.
49 . The method of any one of claim 47 or 48 , wherein the lentiviral vector is an AnkT9W vector, a T9Ank2W vector, a TNS9 vector, a TNS9.3 vector, a TNS9.3.55 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, a G9 vector, a BCL11A shmir vector, or a derivative thereof.
50 . The method of claim 39 or claim 40 , wherein the cells are PBMCs isolated from a subject that has cancer.
51 . The method of claim 50 , wherein the cancer is multiple myeloma.
52 . The method of claim 50 or claim 51 , wherein the lentiviral vector comprises an engineered antigen receptor.
53 . The method of claim 52 , wherein the engineered antigen receptor is selected from the group consisting of: an engineered αβ-TCR, and engineered δγ-TCR, a chimeric antigen receptor (CAR), and a dimerizing agent regulated immunoreceptor complex (DARIC).
54 . The method of claim 52 or claim 53 , wherein the engineered antigen receptor is an anti-BCMA CAR.
55 . The method of any one of claims 39 to 54 , wherein a cell is considered as transduced when it is measured as having a Threshold Cycle (C t ) value of ≤32 for both genomic and viral DNA sequences.
56 . The method of any one of claims 39 to 55 , wherein the viral DNA sequences is a lentiviral vector psi-gag DNA sequence.
57 . The method of any one of claims 39 to 56 , wherein the genomic DNA sequence is a RNAse P DNA sequence.