[Human coagulation factor IX gene therapy in murine hemophilia B by hydrodynamic delivery and site-specific genomic integration].
OBJECTIVE
To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase.


METHODS
The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR.


RESULTS
The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes.


CONCLUSIONS
Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.
State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, CAMS & PUMC, Tianjin 300020, China.
Lei
L
Zhang
Dong-Sheng
DS
Gu
Wei-Ting
WT
DU
Peng-Xia
PX
Liu
Shi-Hong
SH
Lu
Ren-Chi
RC
Yang
D000818:Animals; D005164:Factor IX / Q000235:genetics; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D023281:Genomics; D002836:Hemophilia B / Q000628:therapy; D006801:Humans; D057446:Hydrodynamics; D051379:Mice
D005164:Factor IX