MicroRNA 142-3p attenuates spread of replicating retroviral vector in hematopoietic lineage-derived cells while maintaining an antiviral immune response.
We are developing a retroviral replicating vector (RRV) encoding cytosine deaminase as an anticancer agent for gliomas. Despite its demonstrated natural selectivity for tumors, and other safety features, such a virus could potentially cause off-target effects by productively infecting healthy tissues. Here, we investigated whether incorporation of a hematopoietic lineage-specific microRNA target sequence in RRV further restricts replication in hematopoietic lineage-derived human cells in vitro and in murine lymphoid tissues in vivo. One or four copies of a sequence perfectly complementary to the guide strand of microRNA 142-3p were inserted into the 3' untranslated region of the RRV genome expressing the transgene encoding green fluorescent protein (GFP). Viral spread and GFP expression of these vectors in hematopoietic lineage cells in vitro and in vivo were measured by qPCR, qRT-PCR, and flow cytometry. In hematopoietic lineage-derived human cell lines and primary human stimulated peripheral blood mononuclear cells, vectors carrying the 142-3pT sequence showed a remarkable decrease in GFP expression relative to the parental vector, and viral spread was not observed over time. In a syngeneic subcutaneous mouse tumor model, RRVs with and without the 142-3pT sequences spread equally well in tumor cells; were strongly repressed in blood, bone marrow, and spleen; and generated antiviral immune responses. In an immune-deficient mouse model, RRVs with 142-3pT sequences were strongly repressed in blood, bone marrow, and spleen compared with unmodified RRV. Tissue-specific microRNA-based selective attenuation of RRV replication can maintain antiviral immunity, and if needed, provide an additional safeguard to this delivery platform for gene therapy applications.
1 Tocagen , San Diego, CA 92109.
Amy H
AH
Lin
Nina
N
Timberlake
Christopher R
CR
Logg
Yanzheng
Y
Liu
Shuichi
S
Kamijima
Oscar
O
Diago
Kenneth
K
Wong
Dawn K
DK
Gammon
Derek
D
Ostertag
Katrin
K
Hacke
Emily C
EC
Yang
Harry
H
Gruber
Noriyuki
N
Kasahara
Douglas J
DJ
Jolly
D000818:Animals; D001854:Bone Marrow Cells / Q000502:physiology / Q000821:virology*; D045744:Cell Line, Tumor; D015316:Genetic Therapy; D005822:Genetic Vectors; D005910:Glioma / Q000628:therapy*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007963:Leukocytes, Mononuclear; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000008:administration & dosage / Q000235:genetics*; D009368:Neoplasm Transplantation; D009928:Organ Specificity; D012190:Retroviridae / Q000502:physiology*; D014161:Transduction, Genetic; D014779:Virus Replication
C555159:MIRN142 microRNA, human; D035683:MicroRNAs; D049452:Green Fluorescent Proteins