Vesicular stomatitis virus glycoprotein: a transducing coat for SFV-based RNA vectors.
BACKGROUND
Semliki Forest virus (SFV) vectors have a great potential for the induction of protective immunity in a large number of clinical conditions including cancer. Such a potential accounts for the huge efforts made to improve the in vivo expression from SFV vectors. It is noteworthy that efficient in vivo expression strongly relies on the ability to deliver high-titre vectors. To achieve this, the generation of recombinant SFV particles, using independent expression systems for structural SFV genes, has been proposed. However, despite several modifications in the production process, a risk of contamination with replication-competent, or partially recombined, virus has remained.


METHODS
Here, we exploit the ability of the vesicular stomatitis virus glycoprotein (VSV-G), expressed in trans, to hijack full-length genomic SFV RNA into secreted virus-like particles (VLPs). To allow SFV vector mobilisation, we designed a CMV driven SFV vector in which the internal 26S promoter has been extensively mutated. With this vector, mobilisation events were monitored using the Green Fluorescent Protein (GFP). The production procedure involves a sequential transfection protocol, of plasmids expressing the VSV-G and the SFV vector respectively.


RESULTS
We show that the VLPs are effective for cellular delivery of SFV vectors in a broad range of human and non-human cellular targets. Furthermore, production of VLPs is easy and allows, through concentration, the harvest of high-titre vector.


CONCLUSIONS
The present paper describes a convenient process aimed at mobilising full length SFV vectors. A major issue to consider, while developing clinically relevant gene transfer vectors, is the risk of undesirable generation of replication competent by-products. Importantly, as the VSV-G gene shares no homology with the SFV genome, our VLPs offer a strong guarantee of biosafety.
Dorange
Fabien
F
D3M, The Vector Group, Faculté de médecine de Tours, 2 Bd Tonnelle, 37000 Tours, France.
Piver
Eric
E
Bru
Thierry
T
Collin
Christine
C
Roingeard
Philippe
P
Pagès
Jean-Christophe
JC
D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D002645:Chickens; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics* / Q000627:therapeutic use; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000378:metabolism; D011995:Recombination, Genetic; D012672:Semliki forest virus / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism; D019065:Virus Assembly; D014779:Virus Replication
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins