Production of human clotting Factor IX without toxicity in mice after vascular delivery of a lentiviral vector.

Replication-deficient lentiviral vectors (LV) have been shown to enable the stable genetic modification of multiple cell types in vivo. We demonstrate here that vascular and hepatic delivery of a third-generation HIV-derived lentiviral vector encoding human Factor IX (LV-hFIX) produced potentially therapeutic serum levels of hFIX protein with no vector-mediated local or systemic toxicity of adult mice. Portal vein administration produced the highest serum levels of hFIX and demonstrated proportionally higher levels of gene transfer to the liver with up to 4% of hepatocytes expressing hFIX. Vascular delivery of a lentiviral vector encoding GFP resulted in genetic modification of up to 12% of liver cells. Cell proliferation was not required for hepatocyte transduction with either vector. Serum hFIX levels reached 4% of normal levels following vascular LV-mediated hFIX gene transfer and remained stable for months following vector administration.     

Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme

The Drosophila γ-glutamyl carboxylase (dγC) has substrate recognition properties similar to that of the vertebrate γ-carboxylase (γC), and its carboxylated product yield, in vitro, was shown to be more than that obtained with the human enzyme. However, whether the Drosophila enzyme is able to γ-carboxylate the human vitamin K-dependent (VKD) proteins, such as the human coagulation factor IX (hFIX), as synthesized in cultured Drosophila cells was not known. To examine this possibility, the Drosophila Schnider (S2) cell line was transfected with a metallothionein promoter-regulated hFIX-expressing plasmid. After induction with copper ion, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assey (ELISA) and coagulation test on the culture supernatant of the transfected S2 cells during 72 h of postinduction. In comparison with Chinese hamster ovary cell line, S2 cells showed higher (≈ 12-fold) expression level of the hFIX. The γ-carboxylation of the Drosophila-derived hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate. The biological activity of the S2 cell-derived hFIX indicated the capability of S2 cells to fulfill the required γ-carboxylation of the expressed hFIX. Coexpression of the human γ-glutamyl carboxylases (hγC) was also shown to improve both expression and γ-carboxylation of the hFIX. This is the first in vivo data to describe the ability of the dγC to recognize the human-based propeptide as substrate, which is an essential step for production of biologically active γ-carboxylated VKD proteins.     

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     

人羊水祖细胞的分离和基因修饰

探讨从孕中期羊水中分离出人羊水祖细胞的有效方法和FIX基因修饰后的效果,为血友病B的产前治疗提供可行的基础。从镜下分离出呈致密克隆生长的梭形细胞集落,经培养传代后,通过第3代慢病毒载体将hFIX导入该细胞,经酶联免疫反应(ELISA)等方法检测hFIX的表达并检测凝血活性。用这种方法得到的羊水祖细胞呈成纤维细胞样,倍增时间为39.05 h,该细胞在不仅在蛋白水平表达干细胞表面分子SSEA4,TRA1-60,在基因水平还可检测到NANOG,OCT4,SOX2mRNA的表达。羊水祖细胞导入hFIX cDNA后,能合成并分泌hFIX蛋白,传代后48 h在上清液中的浓度为20.37%±2.77%,凝血活性16.42%±1.78%。上清液中的浓度在第4天达到平台期,为50.35%±5.42%,凝血活性可达45.34%±4.67%。ELISA检测显示转染后的羊水细胞表达的hFIX蛋白的水平呈现基本稳定趋势,波动幅度较小;同时检测FIX凝血活性也与蛋白浓度呈正相关。从羊水中可以分离得到具有多能性祖细胞,转染了hFIX的羊水祖细胞在体外能持续合成有凝血功能的hFIX蛋白,为血友病B产前治疗的新方法提供了实验依据。     

Kinetics of recombinant adeno-associated virus-mediated gene transfer

Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IX (hFIX) gene transfer into human primary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduction, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold higher expression than that with a muscle-specific creatine kinase enhancer linked to beta-actin promoter. Factor IX expression from transduced myoblasts as well as myotubes reached levels as high as approximately 2 microgram of hFIX/10(6) cells/day. Southern blot analyses of high-molecular-weight (HMW) cellular genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells showed that the conversion of single-stranded vector genomes to double-stranded DNA forms, but not the level of the integrated forms in HMW DNA, correlated with increasing expression of the transgene. Together, these results indicate that rAAV can transduce both proliferating and terminally differentiated muscle cells at about the same efficiency, that expression of transgenes increases linearly over their lifetime with no initial lag phase, and that increasing expression correlates with the appearance of double-stranded episomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained.     

A novel mechanism of cooperation between c-Kit and erythropoietin receptor. Stem cell factor induces the expression of Stat5 and erythropoietin receptor, resulting in efficient proliferation and survival by erythropoietin

Optimal production of red cells in vivo requires collaboration between c-Kit, erythropoietin receptor (Epo-R), and GATA-1. However, the mechanism(s) of collaboration remain unclear. Utilizing an embryonic stem cell-derived erythroid progenitor cell line from mice deficient in GATA-1, we have examined the role of c-Kit and Epo-R in erythroid cell proliferation, survival, and differentiation. In the absence of GATA-1, we demonstrate an essential role for c-Kit in survival and proliferation of erythroid progenitors via the regulation of Bcl-2 expression. In addition, we demonstrate that Epo-R and Stat5 are regulated by a second, novel mechanism. We demonstrate that c-Kit stimulation by stem cell factor is essential for the maintenance of Epo-R and Stat5 protein expression, which results in significantly enhanced Bcl-x(L) induction and survival of erythroid progenitors in response to Epo stimulation. Restoration of GATA-1 function results in terminal erythroid maturation and up-regulation of Epo-R and Bcl-x(L) expression, leading also to significantly enhanced survival of terminally differentiating erythroid progenitors in the presence of only Epo. These results demonstrate that c-Kit and Epo-R have unique role(s) during distinct phases of erythroid maturation, and both stem cell factor and Epo contribute to the regulation of the Epo-R-Stat5-Bcl-x(L) pathway to ensure optimal survival, proliferation, and differentiation of erythroid progenitors.     

Epstein-Barr virus vectors provide prolonged robust factor IX expression in mice

We demonstrate that vectors incorporating components from Epstein-Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high-pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5', 3', and intronic sequences that provided relatively good gene expression in vivo. We demonstrated that addition of the EBV EBNA1 gene and its family of repeats binding sites provided a 10- to 100-fold increase in prolonged hFIX expression in mouse liver. A single 25-microg dose of vector DNA generated normal (>5 microg/mL) levels of hFIX throughout the 8 month duration of the experiment. Vector DNA with or without the EBV sequences was retained in liver cells, and vector replication was not a factor in these nondividing liver cells. Instead, it appears that enhancement of stable hFIX expression by the EBV components was responsible for the increased level and duration of therapeutic gene expression. The EBV sequences also significantly enhanced stable expression of a vector carrying the full genomic hFIX gene delivered to mouse liver. These results underline the crucial importance of appropriate gene expression signals on gene therapy vectors and the utility of EBV sequences in particular for increasing stable gene expression.     

Encapsulated human primary myoblasts deliver functional hFIX in hemophilic mice

BACKGROUND: Hemophilia B is a bleeding disorder caused by defective factor IX (FIX), currently treated by regular infusions of plasma-derived or recombinant FIX. We propose a gene therapy strategy based on the implantation of cells secreting FIX enclosed in alginate microcapsules as a highly desirable alternative treatment. We have reported sustained delivery of human factor IX (hFIX) in immunocompetent mice implanted with encapsulated primary mouse myoblasts engineered to secrete hFIX. As a step towards the treatment of human patients, in this study we report the implantation of encapsulated human primary myoblasts secreting hFIX in hemophilia B mice. METHODS: Human primary myoblasts were transfected with plasmids pKL4M-hFIX, pLNM-betaIXL, pMFG-hFIX, and transduced with retrovirus MFG-hFIX. Two human primary myoblast clones secreting approximately 1 microg hFIX/10(6) cells/day were enclosed in biocompatible alginate microcapsules and implanted intraperitoneally into SCID and hemophilic mice. RESULTS: Circulating hFIX (peak of approximately 120 ng/ml) was detected in hemophilia B mice on day 1 after implantation. Human FIX delivery was transient, however, becoming undetectable on day 14. Concurrently, anti-hFIX antibodies were detected. At the same time, activated partial thromboplastin time (APTT) was reduced from 94 s before treatment to 78-80 s. Tail bleeding time decreased from 15 min to 1.5-7 min after treatment, some mice being normalised. These findings indicate that the delivered hFIX is biologically active. Similarly treated NOD/SCID mice had circulating hFIX levels of 170 ng/ml on day 1 that remained detectable for 1 month, albeit at low levels. Cell viability of microcapsules retrieved on day 60 was below 5%. CONCLUSIONS: Our findings indicate that encapsulated human primary myoblasts secrete functional hFIX. Furthermore, implantation of encapsulated human primary myoblasts can partially correct the phenotype of hemophilia B mice, supporting the feasibility of this gene therapy approach for hemophilia B. However, the long-term viability of the encapsulated human myoblasts must first be improved.     

Sustained and therapeutic levels of human factor IX in hemophilia B mice implanted with microcapsules: key role of encapsulated cells

BACKGROUND: A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein was explored in this study. In order to prevent immune rejection of the delivered cells, they were enclosed in non-antigenic biocompatible alginate microcapsules prior to being implanted intraperitoneally into mice. We have shown that encapsulated C2C12 myoblasts can temporarily deliver therapeutic levels of factor IX (FIX) in mice, but the C2C12 myoblasts elicited an immune response to FIX. In this study we report the use of mouse fetal G8 myoblasts secreting hFIX in hemophilia mice. METHODS: Mouse G8 myoblasts were transduced with MFG-FIX vector. A pool of recombinant G8 myoblasts secreting approximately 1500 ng hFIX/10(6) cells/24 h in vitro were enclosed in biocompatible alginate microcapsules and implanted intraperitoneally into immunocompetent C57BL/6 and hemophilic mice. RESULTS: Circulating levels of hFIX in treated mice reached approximately 400 ng/ml for at least 120 days (end of experiment). Interestingly, mice treated with encapsulated G8 myoblasts did not develop anti-hFIX antibodies. Activated partial thromboplastin time (APTT) of plasmas obtained from treated hemophilic mice was reduced from 107 to 82 sec on day 60 post-treatment, and whole blood clotting time (WBCT) was also corrected from 7-9 min before treatment to 3-5 min following microcapsule implantation. Further, mice were protected against bleeding following major trauma. Thus, the FIX delivery in vivo was biologically active. CONCLUSIONS: Our findings suggest that the type of cells encapsulated play a key role in the generation of immune responses against the transgene. Further, a judicious selection of encapsulated cells is critical for achieving sustained gene expression. Our findings support the feasibility of encapsulated G8 myoblasts as a gene therapy approach for hemophilia B.     

Lipocalin 2-mediated growth suppression is evident in human erythroid and monocyte/macrophage lineage cells

Lipocalin 2 (LCN2), a secreted protein of the lipocalin family, induces apoptosis in some types of cells and inhibits bacterial growth by sequestration of the iron-laden bacterial siderophore. We have recently reported that LCN2 inhibits the production of red blood cells in the mouse. Here we analyzed the role of LCN2 in human hematopoiesis. Expression of LCN2 was observed not only in mature cells such as those of the granulocyte/macrophage and erythroid lineages but also in hematopoietic stem/progenitor cells. We also examined expression of two candidate receptors for LCN2, brain type organic cation transporter (BOCT) and megalin, in various cell types. BOCT showed relatively high levels of expression in erythroid and hematopoietic stem/progenitor cells but lower levels in granulocyte/macrophage and T lymphoid cells. Megalin was expressed at high levels in T lymphoid and erythroid cells but at lower levels in granulocyte/macrophage lineage cells. LCN2 suppressed the growth of erythroid and monocyte/macrophage lineages in vitro, but did not have this effect on cells of other lineages. In addition, immature hematopoietic stem/progenitor cells were not sensitive to LCN2. These results demonstrate a lineage-specific role for LCN2 in human hematopoiesis that is reminiscent of its effects upon mouse hematopoiesis and strongly suggest an important in vivo function of LCN2 in the regulation of human hematopoiesis.     

Diminishing adenovirus gene expression and viral replication by promoter replacement.

The adenovirus E4 promoter was replaced by a synthetic promoter composed of a minimal TATA box and five consensus 17-mer yeast GAL4-binding-site elements. The viral vectors, which also contained human factor IX (hFIX) cDNA driven by Rous sarcoma virus long terminal repeat in the E1 region, were then constructed and expanded in 293 cells permanently expressing GAL4/VP16 fusion protein. Viral replication and expression of adenovirus E4 genes and late genes (hexon and fiber) were evaluated in vitro in the human lung carcinoma cell line H1299. Viral replication and viral gene expression were dramatically reduced in the cells transduced by vectors with a replaced E4 promoter compared to the levels in the cells transduced by vectors with the wild-type E4 promoter. The levels of transgene (hFIX) expression remained similar between vectors with or without E4 promoter replacement. These results indicate that diminution of viral gene expression and viral replication is achievable by promoter replacement.     

Expression and regulation of hFIX minigene and cDNA driven by β-casein gene in mouse mammary gland

Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β-casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and Lac2 gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ-carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β-casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β-casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector.     

Proteomic analysis reveals higher demand for antioxidant protection in embryonic stem cell-derived smooth muscle cells

Embryonic stem (ES) cells can differentiate into vascular smooth muscle cells (SMCs), but differences in protein composition, function and behaviour between stem cell-derived and mature SMCs remain to be characterized. Using differential in gel electrophoresis (DIGE) and MS, we identified 146 proteins that differed between ES cell-derived SMCs (esSMCs) and aortic SMCs, including proteins involved in DNA maintenance (higher in esSMCs), cytoskeletal proteins and calcium-binding proteins (higher in aortic SMCs). Notably, esSMCs showed decreased expression of mitochondrial, but a compensatory increase of cytosolic antioxidants. Subsequent experiments revealed that mitochondrial-derived reactive oxygen species (ROS) were markedly increased in esSMCs. Despite a three-fold rise in glutathione (GSH) reductase activity, esSMCs had lower levels of reduced GSH, and depletion of GSH by diethyl maleate or inhibition of GSH reductase by carmustine (BCNU) resulted in more pronounced cell death compared to aortic SMCs, while addition of antioxidants improved the viability of esSMCs. We present the first proteomic analysis of esSMCs demonstrating that stem cell-derived SMCs are more sensitive to oxidative stress due to increased generation of mitochondrial-derived ROS and require additional antioxidant protection for survival.     

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 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.