In vitro and in vivo characterization of microRNA-targeted alphavirus replicon and helper RNAs.
Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans.
Kamrud
Kurt I
KI
AlphaVax, Inc., Research Triangle Park, North Carolina 27709, USA. Kamrud@alphavax.com
Coffield
V McNeil
VM
Owens
Gary
G
Goodman
Christin
C
Alterson
Kim
K
Custer
Max
M
Murphy
Michael A
MA
Lewis
Whitney
W
Timberlake
Sarah
S
Wansley
Elizabeth K
EK
Berglund
Peter
P
Smith
Jonathan
J
D000524:Alphavirus / Q000235:genetics* / Q000254:growth & development*; D000818:Animals; D002522:Chlorocebus aethiops; D005260:Female; D018390:Gene Targeting; D005822:Genetic Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014709:Vero Cells
D035683:MicroRNAs; D012367:RNA, Viral