Crystal Structure of the Fab Fragment of an Anti-factor IX Antibody 10C12

10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intrinsic pathway of blood coagulation cascade, and interfere with membrane anchoring of Factor IX, thus inhibiting blood coagulation function. 10C12 has been demonstrated as an effective anti-coagulant in attenuating thrombosis in several different animal models. Here, we report the crystal structure of the Fab fragment of 10C12. The crystal contains two Fab molecules in the asymmetric unit with identical conformation, forming a lattice with large cavities. In addition, comparison of this free Fab with the antigen-bound structure of 10C12 shows no change in CDR conformations and the relative disposition of the variable subunits of H and L chains, suggesting the rigid conformation of this 10C12 Fab and a lock-and-key mechanism of antibody-antigen recognition for 10C12.

Producing and secreting human factor IX with high efficiency bymurine primary and C2C12 muscle cells carrying human factor IX gene

objective:To study systematically the abilities of skeletal muscle cells in synthesizing,processing and secreting recombinant proteins in vitro.Methods:Primary murine myoblasts from SCID mice (SCID-MB) and C2C12,a muse myoblast cell line,were trans fected with muscle specific (pdLMe4bAh ff m) or non-specif ic (LIXSN) vectors encoding human factor IX (FIX). The capacities of the transgenic cells in producing re combinant FIX were compared in the levels of intracellular and secreted FIX protein as well as FIX mRNA in primary muscle cells and C2C12 cells. Results:Both primary muscle cells and C2C12 cells can efficiently trans late and process FIX protein. Myotubes derived from the SCID-MB produced 1 800~2 000 ng FIX/106 cells/ 24 h in culture with specific activity of 95% ~ 103%. C2C12 cells can synthesize and process recombinant FIX as efficiently as primary muscle cells,but their secretion ability is less efficient in comparison with primary cells. Conclusion:This observation indicates that primary cells should be used in gene therapy related research projects and care should be taken while explaining the results derived from established cell lines.

Construction and application of gene targeting replacement vector formouse coagulation factor IX gene

Objective: To construct and apply a replacement targeting vector for mouse coagulation factor IX(mFIX) gene in embryonic stem (ES) cells. Methods: Based on the cloning and structural analysis of the genomic DNA fragment of coagulation factor IX gene from 129Sv mouse genomic DNA A phage library, PMFIXDEL plasmid was designed with positive-negative-selection (PNS) strategy, and constructed with commonmolecular cloning techniques. Structure of PMFIXDEL was identified by PCR and restriction analysis. Afterelectroporation with the linearized PMFIXDEL DNA, transfected ES cells were cultured in G418/GANC drugselection medium. The recombination efficacy of this vector was tested. Results: The main components ofPMFIXDEL were two copies of negative selection gene (HSV-tk expression cassette), a positive selectiongene (Neo expression cassette), long and short homologous fragments and plasmid backbone. The introduction of negative selection gene (HSV-tk ) into the construct resulted in 24-fold increase of selection.Conclusion: An effective replacement vector for mFIX gene targeting was constructed and applied in ES cell.

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 adeho-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.

Optimized human factor IX expression cassettes for hepaticdirected gene therapy of hemophilia B

Gene therapy provides a potential cure for hemophilia B, and significant progress has been achieved in liver-directed gene transfer mediated by adeno-associated viral vectors. Recent clinical trials involving the use of a self-complementary adeno-associated virus serotype 8-human codon-optimized factor IX （AAV8-hFIXco） vector demonstrated encouraging efficacy with hFIX expression stabilized at 1% to 6% of normal level in patients, but safety concerns related to high vector doses are still present. Thus, further improvement of AAV vectors and hFIX expression cassette may positively contribute to the ultimate success of hemophilia B gene therapy. In this study, to obtain a higher expression level of hFIX that potentiates the coagulant capacity of recipients, human FIX expression vector was optimized by upgrading the codon adaption index and adjusting the GC content, inserting a Kozak sequence （GCCACC）, and introducing a gain-of-function mutation, R338L （FIX Padua）. The efficiency of the published and the presently constructed cassettes was compared through in vivo screening. In addition, the regulatory elements that control the FIX gene expression in these cassettes were screened for liver-specific effectiveness. Among all the constructed cassettes, scAAV-Pre-hFIXco-SIH-R338L, which was the construct under the control of the prothrombin enhancer and prealbumin promoter, resulted in the highest level of coagulant activity, and the expression levels of two constructed cassettes （scAAV-Chi-hFIXco-SIH-R338L and scAAV-Pre- hFIXco-SIH-R338L） were also higher than that of the published cassette （scAAV-LPI-hFIXco-SJ）. In summary, our strategies led to a substantial increase in hFIX expression at the protein level or a remarkably elevated coagulant activity. Thus, these reconstructs of hFIX with AAV vector may potentially contribute to the creation of an efficacious gene therapy of hemophilia B.

Hybrid retroviral vector with MCK enhancers inserted in LTR for stable and specific expression of human factor IX in skeletal muscle

Background Retroviral vectors have been widely used to introduce foreign into various target cells in vitro,thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo. Methods FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK,Me2) were inserted in forward or reverse orientation at NheI site of 3’ long terminal repeat (LTR),resulting in two hybrid vectors,which were designated as LMe2IXm_2SN(F) and LMe2IXm_2SN(R),respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor IX expression with SCID mice. Results Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm_2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm_2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2. Conclusions LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo,and MCK enhancer should be positioned in the same orientation as that of LTR promoter.

ACTIVATION OF BOVINE FACTOR IX BY THE REACTION PRODUCT OF BOVINE FACTOR VII AND HUMAN TISSUE FACTOR

A study was carried out on the alternate activation of factor IX （FIX） by bovine FVII and human tissue factor （TF） rather than by activated factor XI （FXI）. The reaction product of bovine FVII and human TF functioned as a FIX activator in the assay system used. Published studies suggest that in the presence of Caions, the complex of human FVII-TF readily activates both human FIX and human FX,and at low TF concentrations, FIX appears to be the preferred substrate for the reaction product of FVII and TF. This may explain the discrepancy between the mild bleeding of hereditary FXI deficiency and the severe bleeding of hereditery FIX deficieney. The results obtained with bovine FVII and a crude human TF preparation confirm that at low TF concentrations,bovine FIX is the preferred substrate rather than FX. At higher TF concentrations, bovine FX was rapidly activated.

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

Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX 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.

Molecular mechanism of microRNA125 regulating human coagulation factor IX gene with nonsense mutation

Objective To construct human coagulation factorⅨmini-gene(Mini-h F9)and some nonsense mutants,detect the levels of the Mini-h F9 mRNA,and analyze the molecular mechanism of microRNA125 regulating F9gene with nonsense mutation.Methods Three nonsense mutants were obtained by using PCR mutagenesis to ana-

HIGH EXPRESSION OF HUMAN FACTOR IX cDNA DRIVEN BY HCMV PROMOTER IN MAMMALIAN CELLS

Ⅰ. INTRODUCTION Clotting factor Ⅸ is an essential member in the intrinsic clotting pathway, and the deficiency of factor Ⅸ may cause hemophilia B. An important subject on gene therapy for hemophilia B is to increase the protein products of factor Ⅸ in human cells. In our earlier studies, we have constructed several recombinant retroviral vectors with factor

Expression of human clotting factor IX with EBV shuttle vector

Culture of patient somatic cells, transfected with therapeutic genes into genomic DNA with retroviral vectors, followed by colony selection, amplification, and reimplantation of transformed cells into patient, has been Widely used for clinical trial of gene therapy in the past years. But the disadvantages of this protocol are obvious. (i) To a great extent the expression level of the transfected cells depends on the different integration sites,which cause the various expression rates from different colonies; besides, the life span

Polymerase Chain Reaction (PCR) in Detection of Transgenic Mice Harbouring Human Clotting Factor IX cDNA

DNA-DNA molecular hybridization is commonly used in the detection of transferred foreign gene in transgenic mice, which is an effective tool in the detection of transgenic mice due to its high sensitivity and reliability. The method, however, is very laborious and time-consuming, and requires a large number of DNA samples

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

Vector GtiIX 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 GTiIX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTiIX were obtained. At the same time, previous normal adenoviral vector pAdSPiIX containing viral genome and hFIX mini-gene was constructed, and then previous ade-novirus (pre-Ad) AdSPiIX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTiIX was less than 0.8%. 3T3 cells were transfected with AdGTiIX and AdSPiIX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 mg /106·24 h and 1.6±0.3 mg/106·24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 11010pfu AdGTiIX or AdSPiIX. 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 mL to 60 mL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTiIX was obviously weaker than that triggered by AdSPiIX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector sys-tem prolonged the expression time of hFIX and reduced immune response, thus offering a prom-ising result for further pre-clinical study.

High Expression of Human Factor IX cDNA in Rabbits by Implants of Genetically Modified Rabbit Skin Fibroblasts

Factor IX is an important component in human clotting system.Its deficiency ordysfunction leads to hemophilia B.Recently,with the advance of efficient gene transfertechniques,great progress has been made in the gene therapy of hemophilia B.It hasbeen moved from labs to clinics and positive results have been reported.In this study,a double-copy retroviral vector(N2CMVIX)and a recombinant plasmid(pCMVIX),bothcontaining human factor IX cDNA,were constructed.The vectors were transferred into cul-tured rabbit skin fibroblast(RSF).The cells capable of secreting high level of human fac-tor IX protein were embedded in collagen matrix and implanted in rabbits allograft

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.

RAPID DETECTION OF EXOGENOUS HUMAN CLOTTING FACTOR IX cDNA IN TRANSFERRED CELLS BY POLYMERASE CHAIN REACTION

Polymerase chain reaction (PCR) is a recently devoloped molecular biological technique, using specific oligonucleotide primers and DNA polymerase to amplify the specific fragment of DNA in vitro. This technique has been successfully introduced in gene diagnosis, mutation detection, polymorphism analysis and nucleotide sequencing. In the study of gene transfer and gene therapy, it is necessary to analyse DNA in the

High expression of human clotting factor IX cDNA in the bone marrow stroma cells of hemophilia B patient

Hemophilia B, a serious bleeding disorder, is an inherited X chromosome-linked diseasecaused by the deficiency or inactiveness of human clotting factor IX (FIX). The conven-tional clinical treatment of plasma infusion is expensive and associated with a high risk

Regulation of human clotting factor IX cDNA expression in transgenic mice

To study the expression of human dotting factor IX cDNA in transgenic mice,Which is an es-sential work on gene therapy for hemophilia B,3 recombinant constructions containing different lengths ofhuman dotting factor IX cDNA have been introduced into the cultured cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These results show that the expression of the human clotting factor IX cDNA in the transgenic mice canbe determined by cis regulatory element(s).As compared With the results from other related works,it is sug-gested that the cis regulatory element(s)is resided in the 5’-end non-coding region.

Construction and high expression of retroviral vector with human clotting factor IX cDNA in vitro

The construction of the high liter and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCTX, LIXSN and LCTXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporalion. Human dolling factor IX was delected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNC1X was 800000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3μg per 106 cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5μg per 106 cells in 24 h in hemophilia B patients' skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral liter and expression of human FIX were increased, and the construction of retroviral vector backbone was improved and the safety was guaranteed as compared to those vectors used previously. These vectors may produce a sufficient quantily of factor IX proteins to cause the phenotypic modification for hemophilia B patients.

人凝血IX因子基因乳腺组织特异性表达载体的构建及其在奶山羊乳腺中的分泌性表达

人凝血ＩＸ因子基因乳腺组织特异性表达载体的构建及其在奶山羊乳腺中的分泌性表达张克忠卢大儒邱信芳（复旦大学遗传学研究所上海２００４３３）黄英黄淑帧（上海市儿童医院医学遗传研究所上海２０００４０）血友病Ｂ（ＨｅｍｏｐｈｉｌｉａＢ）是由于人凝血ＩＸ因子（ｈ...