Synthetic messenger RNA as a tool for gene therapy.
Transfection of human cells with DNA in biomedical applications carries the risk of insertional mutagenesis. Transfection with mRNA avoids this problem; however, in vitro production of mRNA, based on preliminary DNA template cloning in special vectors, is a laborious and time-consuming procedure. We report an efficient vectorfree method of mRNA production from polymerase chain reaction-generated DNA templates. For all cell types tested mRNA was transfected more readily than DNA, and its expression was highly uniform in cell populations. Even cell types relatively resistant to transfection with DNA could express transfected mRNA well. The level of mRNA expression could be controlled over a wide range by changing the amount of input RNA. Cells could be efficiently and simultaneously loaded with several different transcripts. To test a potential clinical application of this method, we transfected human T lymphocytes with mRNA encoding a chimeric immune receptor directed against CD19, a surface antigen widely expressed in leukemia and lymphoma. The transfected mRNA conferred powerful cytotoxicity to T cells against CD19+ targets from the same donor. These results demonstrate that this method can be applied to generate autologous T lymphocytes directed toward malignant cells.
Rabinovich
Peter M
PM
Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, and Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. peter.rabinovich@yale.edu
Komarovskaya
Marina E
ME
Ye
Zhi-Jia
ZJ
Imai
Chihaya
C
Campana
Dario
D
Bahceci
Erkut
E
Weissman
Sherman M
SM
D018941:Antigens, CD19 / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000592:standards*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000592:standards / Q000627:therapeutic use*; D013601:T-Lymphocytes / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D018941:Antigens, CD19; D012333:RNA, Messenger; D004247:DNA