In vitro assembly of SV40 virions and pseudovirions: vector development for gene therapy.
SV40 is an attractive potential vector with high-efficiency gene transfer into a wide variety of human tissues, including the bone marrow, a critical target organ for the cure of many diseases. In the present study, the three SV40 capsid proteins, VP1, VP2, and VP3, were produced in Spodoptera frugiperda (Sf9) insect cells. Their co-production led to spontaneous assembly of SV40-like particles. Nuclear extracts containing the three proteins were allowed to interact with purified SV40 DNA, or with plasmid DNA produced and purified from Escherichia coli. The experiments demonstrated a physical association between the DNA and capsid proteins, protection from DNase I digestion, and the formation of infectious particles. The results indicate that intact, supercoiled DNA is being packaged and transmitted into the target cells. The transmitted DNA is biologically functional in gene expression and replication. The process, which utilizes naked DNA, is not dependent on the SV40 packaging signal ses. The procedure allows packaging of plasmids significantly larger than SV40 and permits the inclusion of potent regulatory signals, such as beta-globin locus control region (LCR) elements. These studies are the first step in the development of purified, in vitro-constructed pseudovirions for experimental and medical use.
Sandalon
Z
Z
Department of Hematology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Dalyot-Herman
N
N
Oppenheim
A B
AB
Oppenheim
A
A
D000818:Animals; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D002457:Cell Extracts; D002460:Cell Line; D002467:Cell Nucleus; D002522:Chlorocebus aethiops; D004279:DNA, Viral; D005814:Genes, Viral; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology*; D018411:Spodoptera / Q000166:cytology; D014771:Virion; D019065:Virus Assembly
D036022:Capsid Proteins; D002457:Cell Extracts; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; C442543:VP2 protein, Polyomavirus; C085902:VP3 protein, Simian virus 40