Secretory expression of the α-subunit of human coagulation factor XIII in the yeast Pichia pastoris

Pro-FXIIIa (the -subunit of FXIII with activation peptide, which must be removed to produce the active form of FXIIIa), cloned from human placenta cDNA library, was overexpressed in the methylotrophic yeast Pichia pastoris GS115 (his4) and secreted into the culture medium to yield the recombinant pro-FXIIIa subunit with a predicted molecular mass of approximately 83 kDa. The gene was located immediately downstream of the strong yeast alcohol oxidase promoter (AOX1). In shake flask culture, recombinant pro-FXIIIa (rFXIIIa) was secreted into the culture medium at above 50 mg l^–1. The fibrin-stabilizing activity of the recombinant pro-FXIIIa, after thrombin activation, was confirmed using fibrin cross-linking patterns, and analyzed by SDS-PAGE.     

In vitro differentiation of mouse ES cells into hepatocytes with coagulation factorsⅧandⅨexpression profiles

Coagulation factorsⅡ,Ⅴ,Ⅶ,Ⅷ,ⅨandⅩare produced by hepatocytes. So factorsⅧandⅨdeficiencies, which result in hemophilia A and B, have the potential to respond to cellular replacement therapy. Embryonic stem (ES) cells provide a unique source for therapeutic applications. Here, E14 mouse ES cells have been induced into hepatocytes in vitro. Morphology revealed that ES-derived hepatic-like cells were round or polyhedral shaped with distinct boundary of individual cells, and some arranged in trabeculae. These cells expressed endodermal- or liver-specific mRNA——transthyretin (TTR),α1-anti-trypsin (AAT),α-fetoprotein (AFP), albumin (ALB), glucose-6- phoshpatase (G6P) and tyrosine aminotransferase (TAT). Approximately (85.1±0.5)% of the ES-derived cells was stained positive green with ICG uptake. These cells were also stained magenta as a result of PAS reaction. In this paper, expression of coagulation factorsⅧandⅨmRNA in the ES-derived cells is documented. Therefore, ES cells might be developed as substitute donor cells for the therapy of coagulation factor deficiencies.     

Protein trans-splicing based dual-vector delivery of the coagulation factor Ⅷ gene

A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.     

Knockdown of mouse adult β-globin gene expression in MEL cells by retrovirus vector-mediated RNA interference

RNA interference (RNAi) efficiently induces sequence-specific gene silencing in mammalian cells through short interfering RNA (siRNA) of 21–23 nucleotides synthesized in vitro or expressed by DNA-based vector. However, introduction of siRNA into mammalian cells by transfection limits the application of RNAi, especially when it is necessary to generate long-term gene silencing in vivo. Virus vector-mediated RNAi provides an alternative to transfection. In the present study, we investigated such transduction system and showed that retrovirus vector-mediated RNAi can substantially down-regulate expression of mouse adult β-globin gene in MEL cells. The results suggest that retrovirus vector-delivered RNAi may find its use in functional genomics and in gene therapy.     

Effect of human interferon β gene transfer upon human glioma, transplanted into nude mouse brain, involves induced natural killer cells

We investigated the immunological responses induced by human interferon β (IFNβ) gene transfer in human gliomas produced in the brains of nude mice. A suspension of human glioma U251-SP cells was injected into the brains of nude mice. The IFNβ gene was transferred by intratumoral injection with cationic liposomes or cationic liposomes associated with anti-glioma monoclonal antibody (immunoliposomes). When intratumoral injection of liposomes or immunoliposomes containing the human IFNβ gene was performed every second day for a total of six injections, starting 7 days after tumor transplantation, complete disappearance of the tumor was observed in six of seven mice that had received liposomes and in all seven mice receiving immunoliposomes. In addition, experimental gliomas injected with immunoliposomes were much smaller than those injected with ordinary liposomes following delayed injections beginning 14 days after transplantation. An immunohistochemical study of the treated nude mouse brains revealed a remarkable induction of natural killer (NK) cells expressing asialoGM1 antigen. To investigate the significance of NK cells in the antitumor effect, we injected liposomes or immunoliposomes containing the human IFNβ gene into tumors in nude mice depleted of NK cells by irradiation and anti-asialoGM1 antibody administration. The antitumor effect of the liposomes or immunoliposomes was abolished. Subsequent subcutaneous glioma challenge of the nude mice after intracerebral tumor implantation and gene transfer resulted in no subcutaneous tumor growth. These results suggest that the induction of NK cells is important in the cytocidal effect of liposomes or immunoliposomes containing the human IFNβ gene upon experimental gliomas. Received: 10 February 1998 / Accepted: 1 September 1998     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of thehα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the hα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

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.     

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

Hemophilia B is an X-linked disorder caused by the deficiency of clotting factor IX (FIX) activity. Compared with conventional treatment, gene therapy offers an attractive approach by which the goal of prophylactic hemostasis may be achieved without extreme costs, infectious and thrombotic risks. It is the key for?successful somatic gene therapy to effectively and safely ex-press target gene in cell, animal or human as well mediated by appropriate vectors. Adenoviral vectors are currently a…     

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 cDNA with 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 1×1010pfu 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.     

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-associ-ated 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 deve     

Utility of intraperitoneal administration as a route of AAV serotype 5 vector-mediated neonatal gene transfer

BACKGROUND: Gene transfer into a fetus or neonate can be a fundamental approach for treating genetic diseases, particularly disorders that have irreversible manifestations in adulthood. Although the potential utility of this technique has been suggested, the advantages of neonatal gene transfer have not been widely investigated. Here, we tested the usefulness of neonatal gene transfer using adeno-associated virus (AAV) vectors by comparing the administration routes and vector doses. METHODS: To determine the optimal administration route, neonates were subjected to intravenous (i.v.) or intraperitoneal (i.p.) injections of AAV5-based vectors encoding the human coagulation factor IX (hfIX) gene, and the dose response was examined. To determine the distribution of transgene expression, vectors encoding lacZ or luciferase (luc) genes were used and assessed by X-gal staining and in vivo imaging, respectively. After the observation period, the vector distribution across tissues was quantified. RESULTS: The factor IX concentration was higher in i.p.-injected mice than in i.v.-injected mice. All transgenes administered by i.p. injection were more efficiently expressed in neonates than in adults. The expression was confined to the peritoneal tissue. Interestingly, a sex-related difference was observed in transgene expression in adults, whereas this difference was not apparent in neonates. CONCLUSIONS: AAV vector administration to neonates using the i.p. route was clearly advantageous in obtaining robust transgene expression. Vector genomes and transgene expression were observed mainly in the peritoneal tissue. These findings indicate the advantages of neonatal gene therapy and would help in designing strategies for gene therapy using AAV vectors.     

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 GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. 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 GlNaMCIX>GlNaCIX> 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 GlNaMCIX 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 sug     

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-associ- ated 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.     

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.