METHODS AND COMPOSITIONS FOR INCREASED TRANSGENE EXPRESSION
Described herein are methods of expressing nucleic acids in T cells pre-exposed to a co-stimulatory signal and then transduced with adenoviral vectors. In some embodiments, the co-stimulation is provided by anti-CD3 and anti-CD28 antibodies and the adenoviral vector is pseudotyped for T-cell entry. The invention also relates to compositions for carrying out these methods, provided as kits or pharmaceutical compositions that can be used to treat diseases including immunological conditions and hematological malignancies.
1 . A method for increasing expression of an exogenous sequence in T cells, said method comprising:
a) activating a population of T cells with at least a first and second co-stimulatory agents; and
b) contacting the activated T cell population with an adenoviral expression vector comprising said exogenous sequence;
wherein the contacting results in expression of the exogenous sequence in greater than 50% of said activated T cells.
2 . The method according to claim 1 , wherein the T cell is a CD4+ cell.
3 . The method according to claim 1 , wherein the T cell is a CD8+ cell.
4 . The method according to claim 1 , wherein at least one of the co-stimulatory agents comprises an anti-CD3 antibody.
5 . The method according to claim 4 , wherein the second co-stimulatory agent comprises an antiCD28 antibody.
6 . The method according to claim 5 , wherein the antibodies are provided on a bead.
7 . The method according to claim 4 , wherein the second co-stimulatory agent comprises IL-15.
8 . The method according to claim 4 , wherein the second co-stimulatory agent comprises a feeder cell.
9 . The method according to claim 1 , wherein the adenoviral expression vector is pseudotyped.
10 . The method according to claim 9 , wherein the pseudotyped adenovirus expression vector comprises sequences from Ad5 and Ad35 adenoviruses.
11 . The method according to claim 10 , wherein the Ad35 sequence is F35.
12 . The method according to claim 9 , wherein the pseudotyped adenovirus expression vector comprises sequences from Ad5 and Ad 11 adenoviruses.
13 . The method according to claim 1 wherein said adenoviral expression vector further comprises a sequence encoding at least one zinc finger nuclease.
14 . The method according to claim 13 , wherein the zinc finger nuclease binds to a target site in CCR5.
15 . A kit for expressing an exogenous sequence in T cells comprising at least one co-stimulatory agent and an adenoviral vector.
16 . A method for cleaving an endogenous gene, the method comprising
expressing at least one zinc finger nuclease in a T cell according to the method of claim 1 , such that the zinc finger nuclease cleaves the endogenous gene.
17 . The method of claim 16 , wherein the endogenous gene is a CCR5 gene.
18 . The method of claim 16 , wherein the endogenous gene is a GR gene.
19 . A pharmaceutical composition comprising at least one co-stimulatory agent and an adenovirus vector comprising an exogenous sequence.
20 . A pharmaceutical composition comprising:
T-cells obtained by activation and transduction of T cells with a pharmaceutical composition according to claim 19 and
a pharmaceutically acceptable carrier.