Expression of secreted platelet-derived growth factor-B by recombinant nonreplicating and noncytopathic vaccina virus.
OBJECTIVE
The purpose of this study was to develop a noncytopathic vector for transient delivery of biologically active platelet-derived growth factor (PDGF) to wounds.


BACKGROUND
Topical application of the protein PDGF-B has improved wound healing in experimental studies of healing-impaired wounds. However, use of PDGF-B has been limited by availability of recombinant protein, short half-life, and inability to reliably apply to the wounded area. One approach to supply local PDGF-B is through transient gene transfer and expression.


METHODS
Treatment of vaccinia virus with psoralen and long-wave ultraviolet irradiation makes it noncytopathic and nonreplicative. The authors inserted various transgenes encoding different forms of PDGF into recombinant vaccinia virus at the hemaglutinin locus by homologous recombination. Because the PDGF-B expressed from full length cDNA is not secreted because of the membrane retention sequence at the C-terminal end of the polypeptide, the authors inserted a 3'-truncated form of human PDGF-B cDNA in recombinant vaccinia virus to achieve secretion. To avoid interference in bioassays by a virally encoded epidermal growth factor homologue called vaccinia growth factor (VGF) in wild type vaccinia virus (CR-19), we used a VGF-negative strain to express PDGF-B (vSC20PDGF-B). Biologic activity of PDGF was tested by measuring proliferation of a 3T3 fibroblast cell line.


RESULTS
Supernatant from CR-19-infected cells (VGF+) and from truncated vSC20PDGF infected cells caused mild and marked proliferation of 3T3 cells, respectively, whereas supernatant from full-length vSC20PDGF virally infected cells did not. Furthermore, in vitro infection of a confluent 3T3 monolayer with noncytopathic and nonreplicative vaccinia encoding either VGF or truncated PDGF also caused similar proliferation.


CONCLUSIONS
These results provide important preliminary evidence for the ability to treat nonhealing wounds with nonreplicating and noncytopathic recombinant vaccinia viruses encoding cytokine growth factors.
Norton
A
A
Department of Surgery, Washington University School of Medicine, St. Louis, Missouri, USA.
Peplinski
G R
GR
Tsung
K
K
D000818:Animals; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use; D020574:Proto-Oncogene Proteins c-sis; D014616:Vaccinia virus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D014779:Virus Replication; D014945:Wound Healing
D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis