P. Umana et al., Efficient FLPe recombinase enables scalable production of helper-dependentadenoviral vectors with negligible helper-virus contamination, NAT BIOTECH, 19(6), 2001, pp. 582-585
Helper-dependent (HD), high-capacity adenoviruses are one of the most effic
ient and safe gene therapy vectors, capable of mediating long-term expressi
on(1-12). Currently, the most widely used system for HD vector production a
voids significant contamination with helper virus by using producer cells s
tably expressing a nuclear-targeted Cre recombinase and an engineered first
-generation helper virus with parallel loxP sites flanking its packaging si
gnal(1,3-12). The system requires a final, density-based separation of HD a
nd residual helper viruses by ultracentrifugation to reduce contaminating h
elper virus to low levels. This separation step hinders large-scale product
ion of clinical-grade HD virus(13), By using a very efficient recombinase,
in vitro-evolved FLPe (ref. 14), to excise the helper virus packaging signa
l in the producer cells, we have developed a scalable HD vector production
method. FLP has previously been shown to mediate maximum levels of excision
close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD pla
smid backbone(1,7,8,10-12), the FLPe-based system reproducibly yielded HD v
irus with the same low levels of helper virus contamination before any dens
ity-based separation by ultracentrifugation. This should allow large-scale
production of HD vectors using column chromatography-based virus purificati
on(13).