EXPRESSION OF THE HUMAN MULTIDRUG-RESISTANCE AND GLUCOCEREBROSIDASE CDNAS FROM ADENOASSOCIATED VECTORS - EFFICIENT PROMOTER ACTIVITY OF AAVSEQUENCES AND IN-VIVO DELIVERY VIA LIPOSOMES
M. Baudard et al., EXPRESSION OF THE HUMAN MULTIDRUG-RESISTANCE AND GLUCOCEREBROSIDASE CDNAS FROM ADENOASSOCIATED VECTORS - EFFICIENT PROMOTER ACTIVITY OF AAVSEQUENCES AND IN-VIVO DELIVERY VIA LIPOSOMES, Human gene therapy, 7(11), 1996, pp. 1309-1322
Recombinant adeno-associated viruses (rAAV) are attractive tools for g
ene therapy, We designed plasmids in which the human multidrug resista
nce gene (hMDR1) cDNA was placed downstream from portions of the 5' en
d of AAV including either a 234-bp cassette or the entire AAV p5 promo
ter, The drug-resistant phenotype conferred by the P-glycoprotein (Pgp
) efflux pump encoded by the hMDR1 cDNA was used to select NIH-3T3 cel
ls transfected with these plasmids, The 234-bp region alone showed pro
moter activity similar in strength to that of the entire p5 promoter o
r the retroviral Harvey murine sarcoma virus long terminal repeat (LTR
); this result demonstrates that the 234-bp cassette might be used as
a small and efficient promoter in rAAV designed to express large genes
approaching the packaging limit of AAV particles, After transfection
of AAV-MDR1 vectors, the integration of MDR1 sequences into the host c
ell genome was demonstrated by fluorescent in situ hybridization (FISH
), In addition, Southern analysis of low-molecular-weight DNA extracte
d from drug-resistant cells grown under continuous selection pressure
indicated the persistence of nonintegrated AAV-MDR1 plasmids, Coordina
te expression of Pgp and human glucocerebrosidase (hGC) was observed i
n drug-selected NIH-3T3 cells transfected with a bicistronic vector in
which MDR1 cDNA was linked to hGC cDNA via the encephalomyocarditis i
nternal ribosome entry site sequence, Moreover, following a single int
ravenous injection of the bicistronic vector complexed to cationic lip
osomes into recipient mice, delivery of MDR1 and GC cDNAs was achieved
in all the organs we tested. Our results demonstrate that the efficie
ncy of liposomes as vehicles for in vitro and in vivo gene delivery, t
he advantages of AAV-vectors, and the use of MDR1 as a selectable mark
er might be successfully combined in gene therapy protocols.