基因转移治疗血友病B的动物试验和安全性探讨

本文报道了皮肤成纤维细胞基因治疗血友病Ｂ的新西兰大白兔试验及安全性检测。转有人凝血因子ＩＸｃＤＮＡ的兔皮肤成纤维细胞包埋于胶原基质中,然后进行不同方式的自体或同种异体移植,移植兔血浆中人ＩＸ因子蛋白有高水平的表达且持续稳定,腹腔移植有ＲＳＦ－ｐＣＭＶＩＸ转化细胞的兔血浆中人ＩＸ因子表达水平达１００ｎｇ／ｍｌ．持续５个月以上,腹腔移植有ＲＳＦ－ｐＣＭＶＩＸｌ转化细胞的兔血中人ＩＸ因子表达水平达２９５     

Growth of recombinant fibroblasts in alginate microcapsules

To develop a novel strategy of nonautologous somatic gene therapy, we now demonstrate the feasibility of culturing genetically modified fibroblasts within an immunoprotective environment and the optimal conditions required for their continued survival in vitro. When mouse Ltk(-) fibroblasts transfected with the human growth hormone gene were enclosed within permselective microcapsules fabricated from alginate-polylysine-alginate, they continued to secrete human growth hormone at the same rates as the nonencapsulated cells. They also continued to proliferate in vitro for at least 1 month even though their viability gradually declined to about 50%. The viability can be improved by controlling for (a) temperature during encapsulation, (b) duration of treatment with polylysine, (c) duration of liquefying the core alginate with sodium citrate, and (d) cell density at the time of encapsulation. The best conditions leading to improved survival and maximum proliferation of cells within the microcapsules were obtained by encapsulating the cells at 4 to 10 degrees C instead of room temperature, coating the microspheres with polylysine for 6 to 10 min instead of 20 min, liquefying the core alginate by treating with citrate for 20 min instead of 6 to 10 min, and using a concentration of 2 x 10(6) cells/mL of alginate for encapsulation. Under such conditions, normally adherent and genetically engineered mouse fibroblasts survived and proliferated optimally within the microcapsule environment. The encapsulated fibroblasts maintained their level of transgene expression while recombinant gene products such as human growth hormone could diffuse through the microcapsule membrane without impediment. The demonstration that genetically modified fibroblasts can survive and continue to deliver recombinant gene products from within these microcapsules and the optimization for their maximal viability and growth within microcapsules should increase the potential for success in using such microencapsulated recombinant cells for somatic gene therapy. (c) 1994 John Wiley & Sons, Inc.     

Encapsulated transgene cells attenuate hypertension, cardiac hypertrophy and enhance renal function in Goldblatt hypertensive rats

BACKGROUND: The success of any gene-therapy approach depends on the survival of the genetically engineered cells that are implanted in the patient to deliver the therapeutic product. Immunoisolation of nonautologous cells within a microcapsule is a unique approach for gene therapy. METHODS: We employed an immunoisolation device that protects nonautologous cells from destruction, to implant human atrial natriuretic peptide (hANP)-producing Chinese hamster ovary (CHO) cells in two-kidney, one-clip (2K1C) hypertensive rats. CHO cells transfected with the plasmid CMV-cANP were encapsulated in biocompatible polycaprolactone (PCL) capsules, and then the PCL capsules were implanted into 2K1C hypertensive rats intraperitoneally. RESULTS: The implantation of encapsulated hANP-producing cells caused a significant delay of blood pressure (BP) increase 2 weeks post-implantation and the effect lasted for more than 5 months. The implantation of encapsulated hANP-producing cells also caused significant increases in renal blood flow (RBF), glomerular filtration rate (GFR), sodium output, urine excretion, and urinary cGMP levels. These beneficial effects were reflected morphologically by an attenuation of the glomerular sclerotic lesions, reduction in cardiomyocyte size, tubular injury and renal arterial thickening. Immunoreactive hANP can be detected in the blood of 2K1C rats after implantation of the PCL capsules containing hANP-producing cells. CONCLUSIONS: This study demonstrates the usefulness of encapsulated ANP gene transfected cells as a new tool for ANP gene delivery in studying renovascular hypertension and cardiovascular diseases. Thus, our results may have important implications for clinical use of transgene cells as therapeutic agents in the treatment of cardiovascular diseases.     

Iron does not cause arrhythmias in the guinea pig model of transfusional iron overload

Cardiac events, including heart failure and arrhythmias, are the leading cause of death in patients with beta thalassemia. Although cardiac arrhythmias in humans are believed to result from iron overload, excluding confounding factors in the human population is difficult. The goal of the current study was to determine whether cardiac arrhythmias occurred in the guinea pig model of secondary iron overload. Electrocardiograms were recorded by using surgically implanted telemetry devices in guinea pigs loaded intraperitoneally with iron dextran (test animals) or dextran alone (controls). Loading occurred over approximately 6 wk. Electrocardiograms were recorded for 1 wk prior to loading, throughout loading, and for approximately 4 wk after loading was complete. Cardiac and liver iron concentrations were significantly increased in the iron-loaded animals compared with controls and were in the range of those reported for humans with thalassemia. Arrhythmias were rare in both iron-loaded and control guinea pigs. No life-threatening arrhythmias were detected in either group. These data suggest that iron alone may be insufficient to cause cardiac arrhythmias in the iron-loaded guinea pig model and that arrhythmias detected in human patients with iron overload may be the result of a complex interplay of factors.     

Stimulation and inhibition of the growth of mice carrying the human growth hormone gene

The human growth hormone gene, containing mouse metallothionein gene promotor, was injected into the male pronuclei of fertilized mouse ova. The progeny of transgenic mice included animals with both accelerated and inhibited growth. Radioimmunochemical analysis has revealed human growth hormone synthesis in both groups of transgenic mice. The molecular weight of the hormone synthesized in liver cells was 25,000 daltons. A possible mechanisms of foreign hormone effect on the growth of transgenic mice is discussed.     

Regulated expression of human growth hormone genes in mouse cells

We have asked whether there are sequences around the human growth hormone gene that render this gene responsive to induction by glucocorticoid hormones. Recombinant clones encoding human growth hormone were introduced into the chromosome of murine fibroblasts by cotransformation. Exposure of cotransformants to glucocorticoids results in a three to five fold induction of human growth hormone mRNA and a similar induction in secreted human growth hormone protein. The DNA sequences required for induction reside within 500 nucleotides of 5′-flanking DNA. Fusion of this segment of 5′-flanking DNA to the structural gene sequences of a hormone-insensitive gene, such as thymidine kinase, now renders this gene responsive to glucocorticoid induction.     

Recombinant gene expression in pulmonary vascular endothelial cells: polarized secretion in vivo

A unique, spontaneously immortalized rat pulmonary endothelial cell line was transfected with a human growth hormone (hGH) fusion gene generating a line of stably transfected cells that expresses high levels of hGH (Ec/XGH-1). These cells produced significant serum levels of hGH when implanted intraperitoneally, subcutaneously, or intravenously into nude mice. Implantation of Ec/XGH-1 cells under the renal capsule resulted in the formation of large cysts that contained concentrations of hGH that were several thousand times greater than those in serum assayed simultaneously from the same animals. This study presents a new technique for in vivo gene expression using a convenient line of pulmonary vascular endothelial cells as gene carriers. In addition, this system demonstrates the special physiological features of transfected endothelial cells in forming large cysts.     

Effects of thymus grafts in nude mice transplated with human malignant tumors

The effect of neonatal thymus grafts implanted in nude mice previously transplanted with three different human malignant tumors - an adenocarcinoma of the colon, a malignant melanoma and a Burkitt's lymphoma - were studied. In all immunologically reconstituted animals tumord were rejected. Tumor rejection stated 2-3 weeks after thymus implantation, and was completed after 30-6 weeks. Histological examination of lymphoid tissues showed a correlation between immunological reconstitution and tumor rejection. The rejection process showed a characteristic histologic picture with 3 phases - an early, an intermediate and a late phase - these were similar in the three tumor types examined. The possible mechanisms of reconstitution and tumor rejection are discussed.     

Recruitment of human umbilical vein endothelial cells and human primary fibroblasts into experimental tumors growing in SCID mice

Generation of a vascular network is a hallmark of solid tumor growth, and attempts to switch off the tumor angiogenic phenotype are promising. However, this angiogenic potential might also be exploited to obtain incorporation into tumor vessels of genetically modified third-party cells, which could behave as targets of immunologic or pharmacologic attack. With this in mind, we addressed the efficiency and selectivity of third-party cell recruitment into experimental tumors generated in severe combined immunodeficiency mice. The animals were inoculated intraperitoneally with human ovarian carcinoma cell lines and with beta-galactosidase (beta-gal)-transduced human umbilical vein endothelial cell (HUVEC) or human fibroblasts. Transgenic HUVEC were scattered in tumors, but not in normal mouse tissues; immunohistochemical analysis revealed their selective homing to tumor vascular structures, over 50% of which contained beta-gal(+) cells. Injection of beta-gal-transduced human fibroblasts was also associated with transgenic cell incorporation into tumor masses; however, beta-gal(+) fibroblasts did not home to tumor blood vessels and were only localized within the tumor stroma. These findings show that the recruitment of primary third-party cells into the different compartments of experimentally induced tumors is an efficient and selective phenomenon and indicate possible alternative ways of confronting the tumor angiogenic potential in cancer therapy.     

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.     

Transgene expression in mammary glands of newborn rats

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals. © 1996 Wiley-Liss, Inc.     

Fate and functions of human adult lymphoid cells in immunodeficient mice

Laboratory models enabling to study in vivo human leukocyte functions have been developed. Most of the models consist of human immunocytes transferred to mice homozygous for the scid mutation. Mice with additional immunodeficient-prone genetic background or with immunodeficiency-induced conditioning have also been used. Human grafts mainly consisted of human immune cells in suspension injected intraperitoneally, or in pieces of human organs containing immunocytes implanted subcutaneously. Cells in suspension could be easily manipulated in vitro before transfer to the animal, but disseminated within the mouse body. In opposition, human cells mostly remained within implantation areas of animals given human organ pieces. This favorizes cell interactions and helps for cell recovery after their in vivo passage. Moreover, the diversity of antibodies in animals transplanted with human lymphoid organ pieces appeared broader than that of mice transferred with lymphocytes in suspension. Spontaneous recall antibody and autoantibody productions have been generally observed in animals transferred with cells from donors with such antibodies. In vivo boosting of recall antibody by antigen has been most successful, but such a manipulation inconstantly boosted autoantibodies. Primary human T and B cell responses were difficult to obtain in xenochimeric animals, and success has been generally obtained by optimizing human immune response parameters, such as antigen presentation.     

Retroviral marking of human bone marrow fibroblasts: in vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo

Amplification of multipotential stem cells, with or without ex vivo gene transfer, offers the potential for their use for beneficial repopulation of a host in which there is specific cellular deficiency or functional impairment. The aims of the current study were to immunoselect, genetically mark, and determine the fate of fibroblastic progenitor cells in vivo. A monoclonal antibody, HOP-26, which has high reactivity with a cell surface antigen present on human osteoprogenitors in bone marrow fibroblast populations, was used to select these cells by immunopanning. Following culture in 10% FCS in alphaMEM containing ascorbate-2-phosphate and dexamethasone the amplified cells expressed the osteoblast phenotype as determined by expression of osteocalcin protein determined immunohistochemically, and Type I collagen and osteocalcin mRNA expressions determined by RT-PCR analysis. The selected cells were genetically labeled using a murine leukemia virus (MuLV) encoding a reporter gene (lacZ) with a selective marker gene (neo(r)) using a triple transient transfection protocol. Transfected cells were implanted in CB17 scid/scid mice by local subcutaneous injection over the calvariae. Localization of the genetically marked cells within the calvarial tissues was detected by beta-galactosidase histochemistry and immunocytochemistry. Genetically marked cells were observed within the periosteal layer in close association with the osteoblast layer, covering mineralized bone surfaces and within bone osteoid at 5 and 7 days after injection. This study demonstrates the successful selection, expansion, and retroviral-marking of human osteoprogenitors and their migration and localization within calvariae of SCID mice following in vivo implantation. These basic studies indicate the migration of these cells to skeletal sites and support possibilities for future uses of human osteoprogenitors in therapy of bone deficiency diseases and the potential for development of gene therapy procedures in these conditions.     

Integration and germ line transmission of foreign genes microinjected into fertilized trout eggs

Persistence, integration into host genome, germ line transmission and expression of foreign genes microinjected into cytoplasm of fertilized rainbow trout eggs has been examined. Foreign DNA persisted as large random concatenates in approximately 50% of 6 to 12 month-old trout and exhibited a mosaic pattern between tissues. In some cases, free concatenates were observed indicating that extrachromosomal replication occurred in trout. Approximately 50% of the males had the foreign sequences in sperm DNA and all the examined animals transmitted these sequences to their progeny. The percentage of transgenic offsprings ranged from 10 to 30% and putative junction fragments were identified in Southern blot analysis in some of them. These results strongly support the hypothesis that the injected genes became integrated into the genome host, most likely after the first round of chromosomal replication. We also examined the expression of the microinjected plasmids which contained viral or mammalian promoters linked to human or rat growth hormone gene. In no case could exogenous growth hormone be detected.     

High-level expressing YAC vector for transgenic animal bioreactors

The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.     

Disruption of the Myostatin Gene in Porcine Primary Fibroblasts and Embryos Using Zinc-Finger Nucleases

Myostatin represses muscle growth by negatively regulating the number and size of muscle fibers. Myostatin loss-of-function can result in the double-muscling phenotype and increased muscle mass. Thus, knockout of myostatin gene could improve the quality of meat from mammals. In the present study, zinc finger nucleases, a useful tool for generating gene knockout animals, were designed to target exon 1 of the myostatin gene. The designed ZFNs were introduced into porcine primary fibroblasts and early implantation embryos via electroporation and microinjection, respectively. Mutations around the ZFNs target site were detected in both primary fibroblasts and blastocysts. The proportion of mutant fibroblast cells and blastocyst was 4.81% and 5.31%, respectively. Thus, ZFNs can be used to knockout myostatin in porcine primary fibroblasts and early implantation embryos.     

Slow release cortisol implants result in impaired innate immune responses and higher infection prevalence following experimental challenge with infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar) parr

Stress can affect the immune system and increase susceptibility to various diseases but knowledge of the underlying mechanisms is scarce. There is a complex interaction between the immune system and the endocrine system of vertebrates. In fish, cortisol is a key hormone regulating stress response and recent studies have also suggested that this hormone can affect the immune system, where cortisol is mainly regarded as an immunosuppressive factor. The aim of the present study was to examine the impact of chronically elevated levels of cortisol on the immune response and susceptibility to experimental infection with infectious pancreatic necrosis virus (IPNV). Further, the effect of IPNV challenge on circulating levels of cortisol was investigated. Atlantic salmon parr were implanted intraperitoneally with sustained-release implants of bovine of cortisol (50 μg cortisol g(-1) body weight in an implant based on vegetable lipids). Vehicle implants were used as control (sham-injected). At 45 days after implantation (DAI), fish were challenged with a low virulent isolate of IPNV (by immersion). Samples of plasma, liver and head kidney was taken from fish before and 24 h, 48 h, 7 days week and 21 days post infection (DPI). Cortisol level in plasma was measured using radioimmunoassay and gene expression in liver and head kidney was analyzed with real-time PCR (RT-PCR). Infection prevalence in infected fish was assessed by virus culture and RT-PCR of head kidney samples. Cortisol implantation compared with sham-implanted fish had increased levels of plasma cortisol at 45 DAI. The relative expression of Interferon alpha-1 (IFNα-1), Myxo virus-1 Mx, Heat-shock protein 70 (HSP70), Serum amyloid A (SAA), Glucocorticoid receptor (GR) and Heat-shock protein 90 (HSP90) tends to be down-regulated by cortisol implantation. There was a higher prevalence of fish with detectable levels of IPNV, as measured by cell culture and RT-PCR, in the cortisol-implanted group challenged with IPNV (0 = 0.0305) relative to the group that received a sham implantation. Further, cortisol seems to delay the induction of the antiviral IFNα-1 pathway and Mx mRNA expression. This study shows that elevated plasma cortisol level leads to an impaired innate immune response, and higher virus (IPNV) prevalence in Atlantic salmon parr.