Xinfang Qiu

Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer

The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor IX (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is evaluated. The muscle creatine kinase enhancer (MCK) and β-actin promoter (βA) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high titer (2.5 × 1011 vector genomes/mL) of rAAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-mediated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.

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

The DNA of human factor IX (hF IX) gene vector pMC IX m, which had been proven to be able to express inin vitro and living cells, was introduced into 586 zygotes of Kunming Whlte 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, 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 genornes, giving an integration frequency of 3% (6/216). Two F0 female transgenic mice could express hF IX protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hF IV in the milk of two F0 mike were 44.67 % and 79.43 %, respectively.

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%±5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.

INJECTING SKIN FIBROBLASTS INTO MUSCLES TO EXPRESS HUMAN CLOTTING FACTOR IX CDNA

Primary rabbit skin fibroblasts (RSF) containing lacZ reporter gene or human clotting factor IX (hFIX) cDNA could long-termly exist and be expressed after being implanted in rabbit muscles. Among them, the beta-glactosidase expression lasted at least 3 months, and hFIX expression lasted 1 month. These results will be helpful to organically combine the advantages of both skin fibroblasts and muscle, and provide a new way for somatic gene therapy of hemophilia B and other genetic diseases.

High expression of human clotting factor IX cDNA in myoblasts C2C12 cells and C3H mice

Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and G1NaCIX, G1NaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector G1Na. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are G1NaMCIX> G1NaCIX> LNCIX> XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with G1NaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly suggested that a myoblast-mediated gene delivery system had the potential to be optimized as a safe and effective therapeutic modality for hemophilia B.

Muscle injection of rAAV/mFIX to secrete clotting factor IX corrects the hemorrhagic tendencies in hemophilia B mice

Recombinant AAV particles of high titer (>1013 virus genome/mL) were prepared according to the rHSV/AAV helper virus method. After intramuscular injection of viral vectors in the hind limb, a sustained elevated level (>370 ng/mL) of murine FIX expression in the plasma of hemophilia B mouse was detected and persisted for more than 350 days. The biological activity reached 30% of normal levels, and bleeding symptoms in the treated mice were significantly alleviated. No anti-FIX antibody (inhibitor) was detected, though anti-AAV antibodies were found at a very low level after single injection. Repeated injection with rAAV/mFIX led to a variation in anti-AAV antibody levels between the two groups which had received different doses. Results from tissue analysis confirmed the skeletal muscle as the origin for circulating functional mFIX. Our results suggest that AAV-mediated gene transfer offers a promising method of gene therapy for hemophilia B.

A study of gene transfer and expression of human clotting factor IX in hemophilia B mice mediated by mini-adenoviral vector

Vector Gti’IX containing human clotting factor IX cDNAwith intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with GTi’IX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTi’IX were obtained. At the same time, previous normal adenoviral vector pAdSPi’IX containing viral genome and hFIX mini-gene was constructed, and then previous adenovirus (pre-Ad) AdSPi’IX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTi’IX was less than 0.8%. 3T3 cells were transfected with AdGTi’IX and AdSPi’IX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4 ±0.2 μg /106 · 24 h and 1.6 ±0.3 μg/106 · 24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 1x1010 pfu AdGTi’IX or AdSPi’IX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 μL to 60 μL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTi’IX was obviously weaker than that triggered by AdSPi’IX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector system prolonged the expression time of hFIX and reduced immune response, thus offering a promising result for further pre-clinical study.

Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

Constitutive expression of hFIX protein in nonhepatocytes was studied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenoushFIX promoter was replaced with anhCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression ofhFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested thathFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.

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

The feasibility ofin 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/GlNaBAIX pseudotyped virus with the highest titers up to 8.5×108 cfu · 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 · mL−1. 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 not detected by the method of Bethesda, no germ line transmission and any side effects associated with gene transfer were found. Our results indicated that neonatal gene therapy for hemophilia B mice by VSV-G pseudotyped retrovirus is promising.

Preparation of a recombinant adeno-associated viral vector with a mutation of human factor IX in large scale and its expressionin vitro andin 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-hF IX R338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFKR338A was more than 1.25×1012 particle/mL, and then, a mammalian cell line, C2C12 and the factor IX knock-out mice were transfected with the rAAV-hFIXR338Ain vitro andin 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 · (24 h)−1 in C2C12 cellin vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFIX R338A, which was as high as the expression of rAAV-hFIX-wt (2565.76±64.36) ng · (106 cells)−1 · (24 h)−1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFIX-wt)in vitro andin 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 therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFIX R338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFK.