Granted Patent
B2
US 12,723,265 · App. 15/761,783 · Granted Sep 1, 2026
Method for high level and stable gene transfer in lymphocytes
Assignee:
Julius-Maximilians-Universität Würzburg
C12N15/90A61K40/11A61K40/31A61K40/4204A61K40/4211A61K48/005C07K14/70503C07K14/7051C07K14/70578C07K14/71C07K16/2803C12N5/0636C12N7/00C12N9/1241C12Y207/07A61K2239/31A61K2239/38A61K2239/48C07K2317/622C07K2319/02C07K2319/30C12N2740/15043C12N2800/50C12N2800/90
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Abstract
The method disclosed herein describes a novel technology offering unparalleled efficiency, flexibility, utility and speed for the stable integration of transgenes into lymphocytes and other mammalian cells. The novel method is based on the use of an mRNA-encoded transposase (e.g. sleeping beauty transposase) in combination with a minicircle DNA-encoded transposable element. The novel method enables higher gene-transfer rates and is at the same time less toxic than the conventional approach, which is the use of plasmid DNA-encoded transposase in combination with a plasmid DNA-encoded transposable element. Applications of the invention include but are not limited to the stable integration of a transgene encoding an immune receptor (e.g. a T-cell receptor or synthetic chimeric antigen receptor) into human T lymphocytes, with the immune receptor conferring specificity for a molecule expressed by a tumor cell. The transposase mRNA and transposon minicircle DNA may be introduced into lymphocytes by methods including but not limited to electrotransfer such as electroporation and nucleofection.
Claims (21)
1 . A non-viral method for administering a recombinant lymphocyte that expresses an engineered T-cell receptor or chimeric antigen receptor to a subject, the method comprising:
(a) introducing (i) a DNA minicircle comprising a transposable element and (ii) an in vitro transcribed RNA encoding a transposase into a human primary lymphocyte,
wherein the DNA minicircle does not comprise a selectable marker;
wherein the transposable element comprises an expression cassette comprising a promoter and a coding sequence for the engineered T-cell receptor or the chimeric antigen receptor,
wherein introduction of the DNA minicircle and in vitro transcribed RNA into the human primary lymphocyte results in transposition of the transposable element into the genome of the cell and expression of the engineered T-cell receptor or chimeric antigen receptor on the human primary lymphocyte; and
wherein the in vitro transcribed RNA and the DNA minicircle are introduced into the human primary lymphocyte in a weight ratio of 4:1 to 8:1
(b) administering a therapeutically effective dose of the cells produced in step (a) as an adoptive immunotherapy to a human subject that has cancer,
wherein steps (a) and (b) and any steps that are performed between steps (a) and steps (b) are done without selecting for recombinant lymphocytes that express the engineered T-cell receptor or chimeric antigen receptor.
2 . The method according to claim 1 , wherein the engineered T-cell receptor or chimeric antigen receptor is specific for CD19, CD20, CD22, CD33, CD44v6, CD123, CD135, EpCAM, EGFR, an EGFR variant, GD2, ROR1, ROR2, CD269, CD319, CD38, or CD138.
3 . The method according to claim 1 , wherein:
I) the coding sequence encodes an a/b or g/d T-cell receptor;
II the introducing is done by electroporation;
III) the transposase is Sleeping Beauty, PiggyBac, Frog Prince, Himarl, Passport, Minos, hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, and Hsmar1; and/or
IV) the DNA minicircle encoding the transposable element and the in vitro transcribed RNA are introduced into the human primary lymphocyte together.
4 . The method of claim 1 , wherein the transposase encoded by the in vitro transcribed RNA is the 100X hyperactive Sleeping Beauty transposase (SB100X).
5 . The method of claim 1 , wherein the lymphocyte is a T lymphocyte or natural killer (NK) cell.
6 . The method of claim 1 , wherein the method comprises:
transfecting a population of human primary lymphocytes with the DNA minicircle and in vitro transcribed RNA;
incubating the transfected population of human primary lymphocytes so as to provide for expression of the engineered T-cell receptor or chimeric antigen receptor on some of the human primary lymphocytes; and
after the incubating step, administering the human primary lymphocytes to a subject without selecting for cells that express the engineered T-cell receptor or chimeric antigen receptor.
7 . The method of claim 1 , wherein the introducing is done by electrotransfer or chemotransfer.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 1, 2018
From: HUDECEK, MICHAEL; IVICS, ZOLTAN
To: JULIUS-MAXIMILIANS-UNIVERSITÄT WÜRZBURG
Reel/Frame 045681/0488 →
Priority Claims (2)
EP 15002732
· Sep 22, 2015
· regional
EP 16153490
· Jan 29, 2016
· regional
Continuity (1)
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Communication pursuant to Rule 114(2) EPC—Third Part Observations, issued for European Application No. 16770015.2, Sep. 17, 2025.
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