Recently,
gene therapy has been become a promising approach to
cure hemophilia A, a most common recessive
bleeding disease. The aim of this study was to determine the perspective of lentiviral vector in
hemophilia A gene therapy in vitro and in NOD/
SCID mice.
Lentivirus transfer vector pXZ9/BDDFVIII containing
human B-domain-deleted
Factor VIII-IRES-eGFP
coding sequence and mock control pXZ9 were constructed.
Lentivirus was prepared by co-transfecting 3
plasmids into 293FT
cells. 293FT, HLF,
human bone marrow mesenchymal stem cells and Chang-
liver cells were transfected with the prepared
virus.
Coagulant activity of
human FVIII,
human FVIII
antigen,
human FVIII
mRNA transcription and genomic integration were assayed by
ELISA, one-step
method, RT-PCR and
PCR after
infection. Lentiviral particles were concentrated by
ultracentrifugation and NOD/
SCID mice were transfected via
portal vein injection.
Human FVIII
antigen in
mouse blood plasma was analyzed by
ELISA. eGFP expression was observed by fluorescent
microscopy and
human FVIII transcription in
mouse liver was analyzed by RT-PCR at one month after transduction. The results showed that the high titer of recombinant
virus was prepared and used to efficiently transduce the target
cells in vitro. At 72 h after
transfection, high levels of FVIII activity and FVIII
antigen were detected.
Human FVIII
gene transcription could be detected in the
liver of NOD/
SCID mice received lentiviral particles
carrying FVIII
gene.
Mouse hepatocytes were transfected with recombinant
lentivirus efficiently in vivo.
Human FVIII level in
mouse blood plasma reached to (49 ± 6) mU, (54 ± 8) mU and (23 ± 4) mU at 72 h, one week and one month after
transfection respectively. It is concluded that the lentiviral particles
carrying BDDhFVIII
gene can high efficiently transfect the target
cells both
in vitro and in vivo, and the transfected target
cells can secrete hFVIII efficiently. The sustained expression of
human FVIII in NOD/
SCID mice is observed after
lentivirus transfection via
portal vein injection.