[Construction of CD19-CAR retroviral vector and modification of its transduction of human T-lymphocytes].
OBJECTIVE
To improve the MigR1-CD19-CAR (chimeric antigen receptor) that contains a single chain variable region (scFv) which targeted to CD19 through a retroviral vector transduction efficiency of T-lymphocytes.


METHODS
Insert the CD19-CAR fragment into the retroviral vector (MigR1) through recombinant DNA technology, after transfecting plat-A packaging cell lines, viral supernatant was collected to transduce K562 cell line and activated human T-lymphocytes. We used flow cytometry to determine the transduction efficiency and RT-PCR to confirm the transcription of CD19-CAR gene. The ability of the transduced T cells to produce IFN-γ and TNF-α in a CD19-specific manner was measured in an enzyme-linked immunosorbent (ELISA) assay.


RESULTS
(1)Using MigR1-CD19-CAR retroviral vector to produce the high titer retrovirus. (2)MigR1-CD19-CAR transduction efficiency of K562 cell line was significantly higher than human T-lymphocytes (P<0.01). (3)120 min centrifugation could significantly improve transduction efficiency of T-lymphocytes to (54.5±14.6)%. (4)Transduction efficiency could be improved by deciding transduce time according to T-lymphocytes proliferation fold in vitro individually, and the highest transduction efficiency in the study was 69.3%. The CD19-CAR gene sequence was transcripted specificly with high efficiency. (5) IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased to (13 230±1 543) pg/ml and (4 217±211) pg/ml when coculture with CD19-K562 cells.


CONCLUSIONS
We have successfully constructed a second generation CAR which targeted to CD19 through a retroviral vector called MigR1 (MigR1-CD19-CAR). Deciding transduce time according to T-lymphocytes proliferation fold in vitro individually and 120 min centrifugation could improve the CAR transduction efficiency of T-lymphocytes. RT-PCR confirmed that the CD19-CAR gene was specificly transcripted with high efficiency. IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased when activated by target cells.
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Yang
Y
Wang
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Gusheng
G
Tang
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Lili
L
Xu
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Jie
J
Ruan
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Hui
H
Cheng
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Hong
H
Zhou
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Yifei
Y
Hua
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Xiaoxia
X
Hu
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Haihui
H
Gu
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Baohua
B
Qian
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Jianmin
J
Wang
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Jianmin
J
Yang
D018941:Antigens, CD19; D049109:Cell Proliferation; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D051603:Recoverin; D012190:Retroviridae; D013601:T-Lymphocytes; D014162:Transfection
D018941:Antigens, CD19; C496731:RCVRN protein, human; D051603:Recoverin