Efficient transfer and expression of human clotting factor IX cDNA in neonatal hemophilia B mice mediated by VSV-G pseudotyped retrovi-rus

The feasibility of in vivo gene therapy for hemophilia B by VSV-G pseudotyped retroviral vector was introduced. The novel packaging cell line 293GPG was used to produce VSV-G/G1NaBAIX pseudotyped virus with the highest liters up to 8.5×108cfu .mL-1. In contrast to the conventional retrovirus, VSV-G pseudotyped virus was more resistant to inactivation by serum complements (P【0.001). Our results also demonstrated that VSV-G pseudotyped virus was more stable in neonatal mice serum than in adult mice serum (P【0.01). After intraperitoneal injection of different doses of virus, hFIX antigen was detected and lasted for more than 120 d, the highest level reached (72.5+6.1) ng- mL11. Moreover, the functional activity was improved to some extent in all hFIX-treated mice, the most remarkable improvement was observed in the mice treated with higher dose of virus whose clotting activity increased to (3.4±1.5)% and APTT (activated partial thromboplastin time) reduced to (43.2±7.2) s. The anti-hFIX antibody was

Expression of biologically active human clotting factor Ⅸ(hFⅨ) in the mammary gland of transgenic mice

The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F 0 pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genomes, giving an integration frequency of 3% (6/216). Two F\-0 female transgenic mice could express hFⅨ protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hFⅨ in the milk of two F\-0 mice were 44 67% and 79 43%, respectively.

Preparation of a recombinant adeno-associated viral vector with a mutation of human factor-Ⅸin large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25?012 particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitroand in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(106 cells)-1·(24h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt(2565.76?4.36) ng·(106 cells) -1·(24 h)-1 in C2C12 and453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapyresearch for hemophilia B, meanwhile, a novel packagingsystem, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation ofindustrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated withrAAV-hFⅨ.

Construction and expression of inverted configuration of retroviral vector containing intron 1 of hFIX

Intron was found to play an important role in improving gene expression. To improve the human factor IX(hFIX) expression level in hemophilia B gene therapy study, the retroviral vector containing intron 1 of hFIX gene was constructed in forwarded configuration, but the intron 1 was found spliced in virus particles by RT_PCR detection. So the inverted configuration vector G1NaPAi′IX was suggested and constructed on the basis of SNMBAIXm and transfected into PA317. Then C2C12 cells were transfected using the above virus supernatant and the G418_resistant clones were selected. PCR and RT_PCR detection found that intron 1 structure existed in C2C12 clones and retroviral particles. And the expression level of inverted vector was 3 times higher than that of forwarded vector. These results showed that the inverted configuration vector was in deed able to avoid splicing of intron 1 during the process of retroviral packaging and improved the expression level of hFIX protein.