METHODS FOR PRODUCING CELL POPULATIONS WITH INCREASED NUCLEIC ACID UPTAKE
Described herein are methods of producing enriched target cell populations that are susceptible to genetic engineering.
1 . A cell population comprising cells comprising a heterologous DNA, wherein:
a) a percentage of cells comprising the heterologous DNA in the cell population is at least 20% higher than a percentage of cells comprising the heterologous DNA in a buffy coat cell population; or
b) the cell population expands to comprise at least 2×10e9 T cells comprising the heterologous DNA in at least 30% less time than the buffy coat cell population when cultured in a medium comprising 50 ng/ml of IL-7 and 50 ng/ml of IL-15 in a 6-well G-Rex plate;
wherein the cell population was isolated from a sample from a subject and transduced with a viral vector comprising the heterologous DNA, and
wherein the buffy coat cell population was isolated from a similar sample from the subject by density gradient centrifugation and similarly transduced with the viral vector comprising the heterologous DNA.
2 . The cell population of claim 1 , wherein the heterologous DNA comprises an inverted terminal repeat sequence or a long terminal repeat sequence.
3 . The cell population of claim 1 , wherein the density gradient centrifugation comprises layering the sample over an aqueous solution comprising sodium diatrizoate, disodium calcium EDTA, and a neutral, highly branched, high-mass, hydrophilic polysaccharide having a density of about 1.078 g/ml [e.g. Ficoll].
4 . The cell population of claim 1 , wherein the sample is a leukopak.
5 . The cell population of claim 1 , wherein the sample is residual leukocytes from a platelet donation.
6 . The cell population of claim 1 , wherein the sample is a blood sample.
7 . The cell population of claim 6 , wherein the hematocrit of the blood sample is >2%.
8 . The cell population of claim 6 , wherein the hematocrit of the blood sample is >4%.
9 . The cell population of claim 6 , wherein the hematocrit of the blood sample is <30%.
10 . The cell population of claim 6 , wherein the sample is a leukophoresis or apheresis sample.
11 . The cell population of claim 1 , wherein the sample is an adipose sample or a bone marrow sample.
12 . The cell population of claim 1 , wherein the subject is a human.
13 . The cell population of claim 1 , wherein the subject is a healthy individual.
14 . The cell population of claim 1 , wherein the subject has a cancer.
15 . The cell population of claim 14 , wherein the cancer is a leukemia.
16 . The cell population of claim 1 , wherein the viral vector is a lentiviral vector.
17 . The cell population of claim 1 , wherein the viral vector is an adenovirus vector.
18 . The cell population of claim 1 , wherein the viral vector is an adeno-associated virus vector.
19 . The cell population of claim 1 , wherein the heterologous DNA encodes a CRISPR guide RNA.
20 . The cell population of claim 1 , wherein the heterologous DNA encodes an siRNA or a miRNA.
21 . The cell population of claim 1 , wherein the heterologous DNA encodes a polypeptide.
22 . The cell population of claim 1 , wherein the polypeptide is a chimeric antigen receptor.
23 . The cell population of claim 22 , wherein the chimeric antigen receptor is selected from the list consisting of tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, and combinations thereof.
24 . The cell population of claim 21 , wherein the polypeptide is an immunoglobulin, a T cell receptor, a cytokine, or a chemokine.
25 . The cell population of claim 1 , wherein at least 90% of the cells of the cell population are viable.