Daru Lu

Distribution of Two HIV-1–Resistant Polymorphisms (SDF1-3′A and CCR2-64I) in East Asian and World Populations and Its Implication in AIDS Epidemiology

Chemokine receptor CCR2 and stromal-derived factor (SDF-1) are involved in HIV infection and AIDS symptom onset. Recent cohort studies showed that point mutations in these two genes, CCR2-64I and SDF1-3A, can delay AIDS onset > or = 16 years after seroconversions. The protective effect of CCR2-64I is dominant, whereas that of SDF1-3A is recessive. SDF1-3A homozygotes also showed possible protection against HIV-1 infection. In this study, we surveyed the frequency distributions of the two alleles at both loci in world populations, with emphasis on those in east Asia. The CCR2-64I frequencies do not vary significantly in the different continents, having a range of 0.1-0.2 in most populations. A decreasing cline of the CCR2-64I frequency from north to south was observed in east Asia. In contrast, the distribution of SDF1-3A in world populations varies substantially, and the highest frequency was observed in Oceanian populations. Moreover, an increasing cline of the SDF1-3A frequency from north to south was observed in east Asia. The relative hazard values were computed to evaluate the risk of AIDS onset on the basis of two-locus genotypes in the east Asian and world populations.

Increment of hFIX expression with endogenous intron 1 in vitro

This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intron 1 sequence, hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5iIX, retroviral vector GINaCiIX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKG5iIX, 151 ng/106 cells/24h; PA317/G1NaCiIX, 308 ng/106 cells/24 h; C2C12/G1 NaCiIX, 186 ng/106 cells/24 h; RSF/G1NaCiIX, 1929 ng/106 cells/24 h; HSF/G1NaCiIX, 1646 ng/106 cells/24 h. These results indicated that hFIX minigene with intron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from intron splicing during viral production, a retroviral vector G1NaCiIXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/106 cells/24 h with 79 % of bioactivity. PCR detection of HT/G1NaCiIXR cells infected with PA317/G1NaCiIXR supernatant confirmed the existence of intron 1 sequence. These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.

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.

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.

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.

Effects of CIITA antisense RNA on the expression of HLA class II molecules

To study the effect of the major histocompatibility complex class II (MHC II) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class II molecules, 5′ end cDNA sequence of CIITA gene was cloned, and antisense RNA expression vector pcDNA-II was constructed. HeLa cells transfected with pcDNA-II and pcDNA3 were induced by IFN-γ for 3 d. The expression of HLA class II molecules on HeLa/pcDNA-II cells was significantly decreased, while it has no effect on the expression of HLA class I molecules. This result suggests that the CIITA antisense RNA can inhibit the expression of HLA class II molecules in HeLa cells. It also implies a promising approach to generate immune tolerance in graft transplantation.