Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats

AIM: To test the hypothesis that enhancement of the activity of heme oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heme oxygense-1 in a CCl4-induced micronodular cirrhosis model. METHODS: Recombinant adeno-associated viruses carrying rat HO-1 or GFP gene were generated. 1×1012 vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fibrosis. RESULTS: Conditioning the rat liver with over-expression of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The development of micronodular cirrhosis was significantly inhibited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to controls, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with improved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells (HSCs) in rAAV/ HO-1-transduced livers. CONCLUSIONS: Enhancement of HO activity in the livers suppresses the development of cirrhosis.     

One year transgene expression with adeno-associated virus cardiac gene transfer

BACKGROUND: Adeno-associated virus (AAV) has shown promise as a vector for cardiac gene transfer given its ability to stably integrate into the host genome and its lack of immune reactivity. This study examined the feasibility of AAV-mediated myocardial gene transfer in mice, the animal which, because of transgenic technology, has become the disease model of choice for cardiovascular research. METHODS: AAV encoding the cytomegalovirus promoter driven LacZ reporter gene (10(7) LacZ-forming units per animal) or vehicle control was injected into the hearts of young adult C57Bl/6 mice by a transdiaphragmatic approach. At one, two, three, six, and twelve months post-injection, cardiac function was assessed by transthoracic echocardiography and hearts were assayed by X-gal histochemical staining. RESULTS: Echocardiography revealed normal left ventricular function in both AAV and control groups at all time points. X-gal staining of cryostat sections of hearts revealed uniform LacZ expression at all time points. There were minimal signs of immunologic infiltration by hematoxylin and eosin staining. CONCLUSIONS: AAV-mediated myocardial gene transfer by transdiaphragmatic injection can be conducted safely and results in long-term expression of the LacZ gene for at least one year without causing significant inflammatory response or adversely affecting LV systolic function.     

Clinical gene transfer studies for hemophilia B

Hemophilia B, a deficiency of functional factor IX (FIX), has been extensively explored as a model for gene transfer. Two U.S. Food and Drug Administration-approved clinical studies for hemophilia B have been undertaken, both using adeno-associated viral vectors (AAV). AAV vectors have tropism for liver, muscle, central nervous system, and the respiratory tract; both skeletal muscle and liver have been used as target tissues in the hemophilia B studies. In both studies, proof of principle was first established in the hemophilia B dog model, with long-term expression of canine FIX at therapeutic levels achieved before clinical studies were initiated. In the AAV-FIX muscle trial, vector was introduced into skeletal muscle of the upper and lower extremities of eight human patients by direct intramuscular injection. Muscle biopsies taken 2 to 10 months postinjection demonstrated gene transfer and expression (by Southern blot and immunofluorescence, respectively) in all patients, but circulating FIX levels were generally not >1%, and escalation of dose to levels that proved therapeutic in animals was thwarted by feasibility issues regarding the number of injections required. Nevertheless, the study demonstrated that parenteral injection of AAV-FIX was safe at the doses tested, and could result in long-term expression of the transgene. Moreover, the general characteristics of transduction of human muscle were similar to those observed in other animal models. The safety and efficacy data established in the first trial formed the basis for a second trial in which AAV-FIX is administered systemically to target the liver. The liver study is currently ongoing, with six patients enrolled to date.     

Non-catheter associated venous thrombosis in hemophilia A and B. A critical review of all reported cases

All reported cases of non-catheter induced venous thrombosis in patients with hemophilia A or B have been carefully evaluated. A total of 27 cases were reported,12 patients with hemophilia A and 15 patients with hemophilia B. The age of patients varied between 9 and 67 years. There were 10 cases of deep vein thrombosis, 8 patients with pulmonary embolism accompanied or not by deep vein thrombosis, 5 cases of superficial vein thrombosis. In addition, there were 3 cases of thrombosis in unusual sites (1 retinal central vein thrombosis and 2 portal vein thrombosis). Finally, in one case, venous thrombosis was multiple. There was a fatality in a hemophilia B patient with pulmonary embolism. The most frequent risk or triggering factor in hemophilia A was the administration of Feiba or rFVIIa concentrates in patients with inhibitors. Surgery together with Prothrombin Complex concentrates was the most frequent cause in hemophilia B patients. Congenital associated prothrombotic risk factors were present in two patients. No or very few therapeutic procedures were initiated in these patients but for a suspension or reduction of concentrates infusion. In a few instances low molecular weight heparin was given for a few days. The frequent association of venous thrombosis with infusion of concentrates indicates the need for a careful evaluation of patients about to receive such therapy.     

腺相关病毒载体在肺疾病基因治疗中的应用

腺相关病毒（adeno-associated virus,AAV）载体具有安全性好、免疫原性低、能感染分裂细胞和非分裂细胞、能介导基因的长期稳定表达等优点。因此,作为一种基因导入系统,重组AAV载体在基因治疗的研究和开发中受到越来越多的关注和重视。本文着重就重组AAV载体在肺部疾病基因治疗中的应用以及改进重组AAV载体的策略作简要综述。     

Adeno-associated virus-mediated osteoprotegerin gene transfer protects against particulate polyethylene-induced osteolysis in a murine model

OBJECTIVE: Osteoprotegerin (OPG), a natural negative regulator of osteoclastogenesis and bone resorption, may be a potential therapeutic agent for treatment of osteolysis-associated prosthetic joint loosening. Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study was designed to evaluate the protective effects of OPG transgene against orthopedic wear debris-induced bone loss in a murine model of osteolysis. METHODS: Bone tissue was implanted into established pouches on BALB/c mice, followed by the introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles to provoke inflammation and osteolysis. The viruses encoding human OPG gene (rAAV-hOPG) or beta-galactosidase marker gene (rAAV-LacZ) were injected into the air pouches, and the tissue was harvested 7 days after viral infection for histologic and molecular analyses. RESULTS: Successful transgene expression was confirmed by the detection of OPG by enzyme-linked immunosorbent assay and positive X-Gal staining of pouch tissue (LacZ). Real-time polymerase chain reaction indicated significant diminishment of messenger RNA expression of osteoclast markers in OPG-transduced pouches compared with rAAV-LacZ-transduced pouches. The transduction and expression of OPG also markedly decreased the gene copies of the biologic receptor activator of nuclear factor kappaB. The expression of OPG in the bone-implanted pouch reduced bone calcium release by a mean of 39% compared with the calcium release in the other 2 groups. Computerized image analysis revealed that expression of OPG significantly protected against bone collagen loss. CONCLUSION: OPG gene transfer mediated by rAAV effectively protects against particulate polyethylene-induced bone resorption in this experimental model. Data suggest that gene transfer using rAAV-OPG may be a feasible and effective therapeutic candidate to treat or prevent wear debris-associated osteolysis and aseptic loosening.     

Hemophilia B with associated factor VII deficiency: A distinct variant of hemophilia B with low factor VII activity and normal factor VII antigen

Summary Factor VII activity and cross-reacting material was assayed in fresh and deep frozen non-contacted plasma in 43 patients with Hemophilia B belonging to different kindreds.Factor VII activity was found to be slightly decreased (about of 50% normal) in 12 patients, regardless of the thromboplastin used. In an additional patient (hemophilia B_m) factor VII was slightly decreased in 1 10 diluted plasma but was normal in further diluted plasma. In the remaining 30 patients factor VII activity was normal. No significant variation was found between fresh and deep frozen plasmas. Factor VII antigen or cross-reacting material was normal.These patients with associated factor VII defect represent a distinct variant of hemophilia B. The defect seems to be due to a faulty activation of factor VII but the underlying mechanism remains unclear.This study was supported in part by a grant from the M.P.I., Rome (grant 1592-77) and by a grant from the Venetian Region, Venice.     

Myocardial infarction during factor IX infusion in hemophilia B: case report and review of the literature

There are very few reports in the literature of acute myocardial infarction (MI) occurring during infusion of factor concentrates, particularly cryosupernatant in patients with hemophilia B. We describe a case of a 61-year-old man with hemophilia B who suffered an acute MI while receiving cryosupernatant infusion as factor replacement therapy. Cryosupernatant is rich in coagulation factor IX and contains low levels of fibrinogen and von Willebrands factor. Factor IX and other factors present in cryosupernatant can possibly become activated during the manufacturing process causing thrombus formation in patients who are prone to it.     

Successful esophagectomy in a patient with combined esophageal cancer and hemophilia B

Patients with esophageal cancer often require esophagectomy with esophagogastrostomy. However, the incidence of complications, such as hemorrhage, during operations for esophageal cancer is high, even with minimally invasive surgery. Without the appropriate interventions, the risk of major intraoperative and postoperative hemorrhage is very high in patients with esophageal cancer and hemophilia. We report the case of a 45-year-old man with esophageal cancer and hemophilia B who underwent a successful hybrid, minimally invasive Ivor-Lewis esophagectomy with appropriate perioperative management.     

Prolonged preservation of nerve function in diabetic neuropathy in mice by herpes simplex virus-mediated gene transfer

Aims/hypothesis The aim of this study was to determine whether prolonged expression of neurotrophin-3 (NT-3) in mice, achieved by herpes simplex virus (HSV)-mediated gene transfer with gene expression under the control of an HSV latency promoter, can provide protection against the progression of diabetic neuropathy over a 6 month period. Materials and methods Mice with diabetes induced by streptozotocin were inoculated s.c. into both hind feet with a non-replicating HSV vector containing the coding sequence for NT-3 under the control of the HSV latency-associated promoter 2 (LAP2) elements or with a control vector. Nerve function was evaluated by electrophysiological and behavioural measures over the course of 6 months after the onset of diabetes. Results Animals inoculated with the NT-3-expressing vector, but not animals inoculated with control vector, showed preservation of sensory and motor nerve amplitude and conduction velocity measured electrophysiologically, small fibre sensory function assessed by withdrawal from heat, autonomic function measured by pilocarpine-induced sweating, skin innervation assessed by protein gene product 9.5 staining of axons, and density of calcitonin gene-related peptide terminals in the spinal cord measured by immunohistochemistry 5.5 months after vector inoculation. Conclusions/interpretation These results indicate that the continuous production of NT-3 by LAP2-driven expression of the transgene from an HSV vector over a 6 month period protects against progression of diabetic neuropathy in mice, and provide a proof-of-principle demonstration for the development of a novel therapy for preventing the progression of diabetic neuropathy.     

腺相关病毒介导心肌肌浆网Ca2+-ATPase 2a基因转导治疗大鼠慢性心力衰竭

目的 研究腺相关病毒为载体的心肌肌浆网Ca^2＋ -ATPase 2a（sarcoplasmic reticulum Ca^2＋ -ATPase,SERCA2a）基因转导对慢性心力衰竭（HF）大鼠的治疗作用,并探讨其多种可能的机理.方法 采用腹主动脉缩窄术建立HF大鼠模型,应用经腹心包腔内注射术分别将生理盐水、携带eGFP基因和携带SERCA2a基因的重组腺相关病毒导入HF、HF＋EGFP和HF＋SERCA2a组大鼠心脏.于导入30天,检测各组大鼠的心脏功能、SERCA2a蛋白表达和活性;比较HF组和HF＋SERCA2a组大鼠心肌蛋白质组表达的差异;检测各组大鼠心肌肌球蛋白重链（MHC）亚型的表达.结果 HF大鼠心脏内转导入SERCA2a基因30天,心脏收缩和舒张功能达到对照组大鼠水平,并且HF＋SERCA2a组左室重/体重比值显著降低;SERCA2a蛋白表达和活性明显升高至对照组大鼠水平;多种能量代谢酶表达明显增加;α-MHC、β-MHC的表达以及α-MHC/β-MHC恢复至对照组大鼠水平.结论 以重组腺相关病毒2作为载体,SERCA2a基因转导可以增强衰竭心脏的SERCA2a功能,增加心脏能量代谢,纠正MHC亚型的异常表达;在临床方面表现为显著改善心脏收缩和舒张功能,可能能够减轻心脏肥厚等病理性结构改变.     

Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene inhibits protein synthesis of rat ventricular cardiomyocytes

We investigated whether nitric oxide (NO) synthase gene transfer could attenuate growth of cultured cardiac myocytes. First, we investigated the effects of exogenous NO and cGMP analog on protein synthesis of cultured neonatal rat cardiac myocytes. The NO donor 3-morpholino-sydnonimine-hydrochloride (SIN-1) and 8-bromo-cGMP caused concentration-dependent decreases in phenylephrine-stimulated incorporation of ³H-leucine into cardiac myocytes. We then transferred endothelial constitutive NO synthase (ecNOS) gene into cultured neonatal rat cardiac myocytes using adeno-associated virus (AAV) vectors. ecNOS gene transfer into cardiac myocytes induced 140 kD ecNOS protein expression and significantly increased cGMP contents of myocytes compared with control cells. ecNOS gene transfer inhibited ³H-leucine incorporation into cardiac myocytes in response to phenylephrine, which was significantly recovered in the presence of the NOS inhibitor N^G-monomethyl-L-arginine acetate. These results indicate that endogenously generated NO by ecNOS gene transfer using AAV vectors inhibits the -adrenergic agonist-induced cardiac protein synthesis at least partially via cGMP production.     

脂质体－白细胞介素－2基因复合物瘤体内注射治疗肝细胞癌的实验研究

以阳离子脂质体ｌｉｐｏｆｅｃｔｉｎ介导含人白细胞介素一２（ＩＬ－２）基因的真核表达质粒ｐＭＥ１８Ｓ－ＩＬ－２转染体外培养和体内成瘤的小鼠肝癌ＨＡＣ细胞，观察ＩＬ－２基因转染的肝癌细胞表达ＩＬ－２情况及对肝癌的治疗作用。结果：体外转染ＩＬ－２基因４８小时后在培养上清中可测得人ＩＬ－２活性，９６小时达最高峰（７６Ｕ／ｍｌ）；瘤体内转染后，发现治疗组小鼠生存时间明显延长（３２．１±９．４ｄ比２０．９±７．５ｄ，Ｐ＜０．０１），肿瘤体积明显小于对照组，瘤组织内可检出ＩＬ－２的ｍＲＮＡ。实验表明，脂质体－ＩＬ－２基因复合物直接注射人瘤体后可获局部ＩＬ－２基因表达，从而诱生机体抗肿瘤效应，这为肝癌基因治疗提供了一种简便实用的模式。     

A common G10430A mutation (Gly 60 Ser) in the factor IX gene describes the presence of moderate and mild hemophilia B in the majority of the Gujarati population

Hemophilia B is an X-linked recessively inherited bleeding disorder afflicting humans across all socio-economic as well as racial groups. A wide range of mutations showing high heterogeneity has been reported in different populations. Thus, it has been difficult to adopt a cost-effective strategy for the genetic diagnosis of hemophilia B families. We report the presence of a common G10430A mutation in exon d of the factor IX gene, wherein the highly conserved Gly 60 residue of the first epidermal growth like domain was changed to Ser in 22 out of 22 moderately severe to mild hemophilia B patients originating from Gujarat. None of the eight Gujarati severe hemophilia B patients, 30 normal Gujarati men, and 20 moderately severe to mild hemophilia B patients belonging to other communities showed the presence of this mutation. This mutation occurred in the same haplotype background thereby suggesting a ‘founder effect.’ The direct detection of this G10430A mutation can be used for accurate carrier detection and prenatal diagnosis in mild to moderate factor-IX-deficient patients belonging to the Gujarat state of western India.     

乙型肝炎基因治疗的研究进展

基因治疗是指通过基因水平的操纵达到治疗或预防疾病的疗法,分体内疗法和体外疗法.前者是将外源基因直接导入人体发挥作用;     

Carrier detection and prenatal diagnosis of hemophilia B with more advanced techniques

Summary We used the PCR to amplify three polymorphic regions of Factor IX gene on 35 Italian families: DdeI intron 1, MnlI exon f, and the polymorphism HhaI located 8 kb at the 3 end of FIX gene. We analyzed the MnlI and HhaI markers on DGGE and DdeI polymorphism on agarose gel. We reached an informativity of 78% and we found one mutation at codon 145 (exon f) during the screening for MnlI polymorphism. Furthermore, we performed 16 prenatal diagnoses on chorionic villus samples; five were female and 11 male. Four were uninformative three healthy and one affected male fetus were recognized by PCR techniques, two healthy and one affected fetus by Southern analysis. In three pregnant women examined for the first time during pregnancy, the PCR technique allowed us to perform a rapid diagnosis of noncarrier status, avoiding the fetal sampling procedures.     

Cellular immune response to cryptic epitopes during therapeutic gene transfer

The immune response has been implicated as a critical factor in determining the success or failure of clinical gene therapy trials. Generally, such a response is elicited by the desired transgene product or, in some cases, the delivery system. In the current study, we report the previously uncharacterized finding that a therapeutic cassette currently being used for human investigation displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T lymphocyte (CTL) response. In particular, we tested the hypothesis that antigenic epitopes derived from an ARF in coagulation factor IX (F9) cDNA can induce CTL reactivity, subsequently killing F9-expressing hepatocytes. One peptide (p18) of 3 candidates from an ARF of the F9 transgene induced CD8⁺ T cell reactivity in mice expressing the human MHC class I molecule B0702. Subsequently, upon systemic administration of adeno-associated virus (AAV) serotype 2 vectors packaged with the F9 transgene (AAV2/F9), a robust CD8⁺ CTL response was elicited against peptide p18. Of particular importance is that the ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but not AAV2/F9 codon-optimized (AAV2/F9-opt)-transduced liver cells in which p18 epitope was deleted. These results demonstrate a previously undiscovered mechanism by which CTL responses can be elicited by cryptic epitopes generated from a therapeutic transgene and have significant implications for all gene therapy modalities. Such unforeseen epitope generation warrants careful analysis of transgene sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical gene therapy protocols.     

Parvovirus-mediated gene transfer for the haemophilias

Summary.  Gene therapy may revolutionize the treatment of haemophilia. Effective gene therapy requires sustained therapeutic levels of factors IX (FIX) and VIII. Adeno-associated virus (AAV) is a member of the parvovirus family, is a nonpathogenic virus with a broad host cell range, and does not provoke a significant immune response upon infection. These favourable characteristics make AAV a suitable gene transfer vector for factor deficient patients. A new understanding of AAV biology coupled with novel AAV vector designs suggest that the goal of effective gene transfer is within reach. We review here recent advances in AAV vectors used for gene transfer of the haemophilias.     

双位点核酶抑制细胞内乙型肝炎病毒表达的研究

目的 观察针对ＨＢＶＣ区双位点核酶在细胞内阻断Ｃ区基因表达的作用。方法 采用垩 克隆技术,从ｐＧＥＭ－核酶１２３（含针对ＨＢＶＣ区２０２９－２０３１及２０６３－２０６５双位点核酶）上切下双位点核酶的片段,定向克隆于真核表达载体ｐＢＢＳ２１２中,利用脂质体介导,将重组质粒ｐＢＢＳ２１２－Ｒｚ与Ｐ１,２Ⅱ（含乙型肝炎病毒全序列）花园要入人肝癌细胞（ＨＨＣＣ）中,采用原位杂交观察核酶的表达,ＥＬＩＡＳＡ     

Inhibitory effect of adeno-associated virus-mediated gene transfer of human endostatin on hepatocellular carcinoma

AIM: To investigate the effect of adeno-associated virus-mediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC). METHODS: HCC cell line Hep3B was infected with recombinant adeno-associated virus containing human endostatin gene (rAAV2-hEndo). The results of transfection were detected by RT-PCR and SDS-PAGE assay. MTT assay was used to observe the effects of supernatant of transfected cells on ECV304 cell proliferation. An animal model of HCC was established by injecting Hep3B cells subcutaneously into the back of nude mice. Intratumoral injection of rAAV2-hEndo, empty virus and phosphate-buffered saline were given sequentially. Serum endostatin was determined by ELISA, the inhibitory effect of endostatin on the growth of xenograft was assessed in 3 wk. RESULTS: The results of RT-PCR and SDS-PAGE assay confirmed that rAAV2-hEndo successfully transfected Hep3B cells, and endostatin was secreted from Hep3B cells to medium. The supernatant of transfected cells markedly inhibited the proliferation of ECV304 cells (P<0.01). Intratumoral injection of rAAV2-hEndo (2×1010 v.g.) led to a sustained serum endostatin level of approximately (86.71±5.19) ng/mL. The tumor volume and microvessel density were less in rAAV2-hEndo group than in control groups(P<0.01). CONCLUSION: Human endostatin can be stably expressed by adeno-associated virus-mediated gene transfer and effectively inhibit the growth of HCC.