A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia

Many approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Recombinant adenoviral vectors offer high-efficiency transfer of an episomal vector but have been plagued by the cytotoxicity/immunogenicity of early-generation vectors that contain viral genes. In our current study, we have used a nonintegrating helper-dependent (HD) adenoviral vector for liver-directed gene transfer to achieve hemostatic correction in a dog with hemophilia B. We measured plasma canine factor IX (cFIX) concentrations at a therapeutic range for up to 2.5 months and normalization of the whole blood clotting time (WBCT) for about a month. This was followed by a decrease and stabilized partial correction for 4.5 months. Hepatic gene transfer of a slightly lower dose of the HD vector resulted in WBCTs that were close to normal for 2 weeks, suggesting a dose threshold effect in dogs. In sharp contrast to other studies using first- or second-generation adenoviral vectors, we observed no vector-related elevation of liver enzymes, no fall in platelet counts, and normal liver histology. Taken together, this study demonstrates that injection of an adenoviral HD vector results in complete but transient phenotypic correction of FIX deficiency in canine models with no detectable toxicity.     

Total correction of hemophilia A mice with canine FVIII using an AAV 8 serotype

Despite the popularity of adeno-associated virus 2 (AAV2) as a vehicle for gene transfer, its efficacy for liver-directed gene therapy in hemophilia A or B has been suboptimal. Here we evaluated AAV serotypes 2, 5, 7, and 8 in gene therapy of factor VIII (FVIII) deficiency in a hemophilia A mouse model and found that AAV8 was superior to the other 3 serotypes. We expressed canine B domain-deleted FVIII cDNA either in a single vector or in 2 separate AAV vectors containing the heavy- and light-chain cDNAs. We also evaluated AAV8 against AAV2 in intraportal and tail vein injections. AAV8 gave 100% correction of plasma FVIII activity irrespective of the vector type or route of administration.     

Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs

Canine hemophilia A closely mimics the human disease and has beenused previously in the development of factor VIII (FVIII) proteinreplacement products. FVIII-deficient dogs were studied to evaluate anin vivo gene therapy approach using an E1/E2a/E3-deficient adenoviralvector encoding canine FVIII. Results demonstrated a high level ofexpression of the canine protein and complete phenotypic correction ofthe coagulation defect in all 4 treated animals. However, FVIIIexpression was short-term, lasting 5 to 10 days following vectorinfusion. All 4 dogs displayed a biphasic liver toxicity, a transientdrop in platelets, and development of anticanine FVIII antibody. CanineFVIII inhibitor development was transient in 2 of the 4 treatedanimals. These data demonstrate that systemic delivery of attenuatedadenoviral vectors resulted in liver toxicity and hematologic changes.Therefore, the development of further attenuated adenoviral vectorsencoding canine FVIII will be required to improve vector safety andreduce the risk of immunologic sequelae, and may allow achievement ofsustained phenotypic correction of canine hemophilia A.     

Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)-mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 x 10(12) vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.     

Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1

Adeno-associated viral (AAV) vectors (serotype 2) efficiently transduce skeletal muscle, and have been used as gene delivery vehicles for hemophilia B and for muscular dystrophies in experimental animals and humans. Recent reports suggest that AAV vectors based on serotypes 1, 5, and 7 transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2-fold to 1000-fold. We sought to determine whether this increased efficacy could be observed in species other than mice. In immunodeficient mice we saw 10- to 20-fold higher levels of human factor IX (hF.IX) expression at a range of doses, and in hemophilic dogs we observed approximately 50-fold higher levels of expression. The increase in transgene expression was due partly to higher gene copy number and a larger number of cells transduced at each injection site. In all immunocompetent animals injected with AAV-1, inhibitory antibodies to F.IX developed, but in immunocompetent mice treated with high doses of vector, inhibitory antibodies eventually disappeared. These studies emphasize that the increased efficacy of AAV-1 vectors carries a risk of inhibitor formation, and that further studies will be required to define doses and treatment regimens that result in tolerance rather than immunity to F.IX.     

A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice

A longstanding goal for the treatment of hemophilia B is the development of a gene transfer strategy that can maintain sustained production of clotting factor IX (F.IX) in the absence of an immune response. To this end, we have sought to use lentiviral vectors (LVs) as a means for systemic gene transfer. Unfortunately, initial evaluation of LVs expressing F.IX from hepatocyte-specific promoters failed to achieve sustained F.IX expression in hemophilia B mice due to the induction of an anti-F.IX cellular immune response. Further analysis suggested that this may be a result of off-target transgene expression in hematopoietic-lineage cells of the spleen. In order to overcome this problem, we modified our vector to contain a target sequence for the hematopoietic-specific microRNA, miR-142-3p. This eliminated off-target expression in hematopoietic cells, and enabled sustained gene transfer in hemophilia B mice for more than 280 days after injection. Treated mice had more than 10% normal F.IX activity, no detectable anti-F.IX antibodies, and were unresponsive to F.IX immunization. Importantly, the mice survived tail-clip challenge, thus demonstrating phenotypic correction of their bleeding diathesis. This work, which is among the first applications to exploit the microRNA regulatory pathway, provides the basis for a promising new therapy for the treatment of hemophilia B.     

Complete short-term correction of canine hemophilia A by in vivo gene therapy

Hemophilia A is a severe bleeding disorder caused by a deficiency in clotting factor VIII (FVIII). A canine model that closely mimics the human disease was used to determine if an adenoviral vector expressing a human FVIII cDNA could be used to correct the hemophilia A phenotype. Within 48 hours after peripheral vein administration of the vector to FVIII-deficient dogs, the hemophilic phenotype was corrected, based on determination of the activated clotting time, the activated partial thromboplastin time, and the cuticle bleeding time. Direct measurement of human FVIII in the dog plasma showed FVIII expression at amounts well above the human therapeutic level. FVIII expression in treated dogs was short-term, lasting 1 to 2 weeks, due to the development of a human FVIII-specific inhibitor antibody response. These data provide the first demonstration of in vivo gene therapy of hemophilia A.     

Sustained correction of bleeding disorder in hemophilia B mice by gene therapy

Mice generated by disrupting the clotting factor IX gene exhibit severe bleeding disorder and closely resemble the phenotype seen in hemophilia B patients. Here we demonstrate that a single intraportal injection of a recombinant adeno-associated virus (AAV) vector encoding canine factor IX cDNA under the control of a liver-specific enhancer/promoter leads to a long-term and complete correction of the bleeding disorder. High level expression of up to 15-20 microgram/ml of canine factor IX was detected in the plasma of mice injected with 5.6 x 10(11) particles of an AAV vector for >5 months. The activated partial thromboplastin time of the treated mice was fully corrected to higher than normal levels. Liver-specific expression of canine factor IX was confirmed by immunofluorescence staining, and secreted factor IX protein was identified in the mouse plasma by Western blotting. All treated mice survived the tail clip test without difficulty. Thus, a single intraportal injection of a recombinant adeno-associated virus vector expressing factor IX successfully cured the bleeding disorder of hemophilia B mice, proving the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.     

Self-complementary adeno-associated viral vectors for gene therapy of hemophilia B: progress and challenges

Therapies currently used for hemophilia involve injection of protein concentrates that are expensive, invasive and associated with side effects such as development of neutralizing antibodies (inhibitors) that diminish therapeutic efficacy. Gene transfer is an attractive alternative to circumvent these issues. However, until now, clinical trials using gene therapy to treat hemophilia have failed to demonstrate sustained efficacy, although a vector based on a self-complementary adeno-associated virus has recently shown promise. This article will briefly outline a novel gene-transfer approach using self-complementary adeno-associated viral vectors using hemophilia B as a target disorder. This approach is currently being evaluated in the clinic. We will provide an overview of the development of self-complementary adeno-associated virus vectors as well as preclinical and clinical data with this vector system.     

Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy

Hemophilia B, also known as Christmas disease, arises from mutations in the factor IX (F9) gene. Its treatment in humans, by recombinant protein substitution, is expensive, thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of his disease without development of immune reactions by introduction of an HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent hemophiliac and normal outbred mice. Plasma factor IX antigen remained at around 9%, 13%, and 16% of normal in the 3 hemophilia B mice, respectively, until the last measurement at 14 months. Substantial improvement in blood coagulability as measured by coagulation assay was seen in all 3 mice and they rapidly stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes, or vector spread to the germline or maternal circulation were detected.     

A single injection of double-stranded adeno-associated viral vector expressing GH normalizes growth in GH-deficient mice

GH is secreted by the somatotropic cells of the pituitary gland, and its deficiency (GHD) impairs longitudinal growth. Due to its short half-life, GH therapy needs administration of GH injections daily. Adeno-associated viral vectors (AAV) can deliver gene therapy to animals with possible future applications in humans. The new generation of double-stranded AAV vectors (dsAAV) provides widespread, strong, and stable transgene expression without toxicity and immune response. To determine whether such a new system could be used to deliver GH to a mouse model of isolated GHD due to ablation of the GHRH knock-out gene (GHRHKO), we have created AAV viral particles containing mouse GH cDNA driven by a cytomegalovirus promoter (dsAAV8-CMV-GH), and tested them in male GHRHKO mice. GHRHKO animals received either a single (low dose) or two (high dose) i.p. injections of dsAAV8-CMV-GH (1x10(11) particles) at the 10th and 11th days of age, or a placebo injection, and were followed up to the 6th or 24th week of life. A single dsAAV8-GH injection caused body length and weight normalization. At week 6, serum GH was higher in mice receiving both virus doses compared with controls, while it was normal at week 24. Serum IGF-1 increased in both virus-treated groups, and it was normal at 24 weeks. GH mRNA expression was detected in liver, skeletal, and heart muscle of virus-injected animals. These data show that normalization of longitudinal growth can be reached in GHD mice using a single injection of a double-stranded adeno-associated virus expressing GH.     

Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII

Neonatal gene therapy is a promising strategy for treating a number of congenital diseases diagnosed shortly after birth as expression of therapeutic proteins during postnatal life may limit the pathologic consequences and result in a potential "cure." Hemophilia A is often complicated by the development of antibodies to recombinant protein resulting in treatment failure. Neonatal administration of vectors may avoid inhibitory antibody formation to factor VIII (FVIII) by taking advantage of immune immaturity. A helper-dependent adenoviral vector expressing human factor VIII was administered i.v. to neonatal hemophilia A knockout mice. Three days later, mice produced high levels of FVIII. Levels declined rapidly with animal growth to 5 wk of age with stable factor VIII expression thereafter to >1 y of age. Decline in factor VIII expression was not related to cell-mediated or humoral responses with lack of development of antibodies to capsid or human factor VIII proteins. Subsequent readministration and augmentation of expression was possible as operational tolerance was established to factor VIII without development of inhibitors; however, protective immunity to adenovirus remained.     

The phenotypic heterogeneity of severe hemophilia

It has been long recognized that 10 to 15% of patients with "phenotypically characterized" severe hemophilia (< 1% clotting factor activity) have relatively mild disease clinically. Not all these patients have frequent spontaneous bleeding, and even among those who bleed, the extent of joint damage tends to vary considerably. The basis for this difference has not been completely understood. This article reviews the literature on possible determinants of phenotypic variation in patients with severe hemophilia. Apart from the well-recognized associations of the level of residual clotting factor activity, pharmacokinetics of administered clotting factor concentrates, and presence of prothrombotic markers, there is evidence to suggest that variations in other coagulation proteins as assessed in tests of global hemostasis as well as the fibrinolytic system can affect the clinical severity of bleeding. We also hypothesize that mediators of the inflammatory response in the synovium are likely to impact the severity of joint damage in these patients. One of the major issues in the management of hemophilia today is to decide on ways in which therapy, particularly the initiation and intensity of prophylaxis, can be individualized. A detailed understanding of all factors that may contribute to joint damage in severe hemophilia could help us in tailoring therapy for these individuals.     

Preclinical gene therapy studies for hemophilia using adeno-associated virus (AAV) vectors

Gene therapy offers a potential cure for hemophilia and several gene transfer vectors have been evaluated for their ability to treat this disease. This article reviews the studies that have been performed to evaluate the ability of recombinant adeno-associated virus (AAV) vectors to achieve safely the sustained expression of clotting factors following intramuscular, intravenous, and intrahepatic delivery to several animal models. These routes of administration are all effective in providing sustained and therapeutic levels of factor IX (FIX), although the levels vary. Intrahepatic delivery is more efficacious than intravenous administration, which is superior to intramuscular delivery. The recent development of efficient factor VIII (FVIII) expression cassettes has made AAV-based gene therapy for hemophilia A also within reach. Although no acute toxicity has been observed with any route of administration, an increased risk of antibody formation against FIX has been noted following intramuscular delivery. Biodistribution studies concluded that the vector disseminates to most tissues in a dose-dependent and time-dependent manner, but the majority of the vector resides in the targeted tissue. In addition, the risk of germline transmission has been shown to be low or absent. The relatively recent isolation of new AAV serotypes has resulted in the identification of vectors that have enhanced tropism for certain tissues. This combined with the potential of these new vectors to evade the immune response to AAV2, makes them attractive candidates for gene therapy. Although much progress has been made using AAV to treat hemophilia, there are several outstanding issues that need to be addressed. Delivery of AAV to large animals has not been reproducible, which could be due to nonoptimized delivery and/or immune responses to the vector or transgene product. In addition, a complete understanding of the biology of these vectors is required to assess their long-term safety. Solving these issues will lead to the development of a successful gene therapy product.     

腺相关病毒为载体的基因治疗在肝病应用中的进展

基因治疗是目前医学研究的热点课题。将目的基因转入细胞的方法有很多，包括脂质体包裹转染、电打孔、微注射等，最近以重组病毒为载体的基因治疗因其独特的优势受到关注。比较常用的三大病毒载体是逆转录病毒、腺病毒和腺相关病毒（adeno—associated virus，AAV）。各种病毒载体都有各自的优点和不足，在细胞和动物实验研究中均有大量的应用。但是在临床应用中，逆转录病毒为载体的基因治疗由于在法国的临床试验出现2例白血病病历，     

腺相关病毒介导心肌肌浆网三磷酸腺苷酶基因治疗心力衰竭犬的安全性分析

目的研究心肌注射重组腺相关病毒(rAAV)_(2/1)介导心肌肌浆网Ca~(2+)-ATP酶(SERCA2a)基因治疗慢性心力衰竭犬的安全性。方法选取成年比格犬8只,随机分为对照组和SERCA2a基因转导组(转导组),每组4只。分别采用PCR、RT PCR和Western blot法从DNA、RNA和蛋白水平检测rAAV_(2/1)SERCA2a转导后在犬体内的分布;采用双夹心ELISA法检测转导组犬的血清。结果除注射rAAV_(2/1)的心肌区域外,未注射rAAV_(2/1)的心肌区域、主要脏器、骨骼肌和性腺中均未检测到rAAV_(2/1)-SERCA2a。对照组与转导组犬心肌、腰大肌、咀嚼肌和睾丸表现出相同的SERCA2a蛋白表达谱,未在其他组织中检测到。转导组犬血清内未检测到抗rAAV_(2/1)抗体。结论 rAAV_(2/1)介导的基因转导是相对安全的,未整合到心脏以外的其他组织,也未在犬体内引发体液免疫。     

重组腺相关病毒2型载体-血管内皮生长因子165改善小型猪慢性心肌缺血的研究

目的应用重组腺相关病毒2型载体(recombinant adenovirus-associated virus2,rAAV2)介导血管内皮生长因子165(vascular endothelial growth factor,VEGF165)基因转染,观察其促进小型猪慢性缺血心肌血管生成并改善心肌血流灌注和心功能的有效性。方法小型猪左冠状动脉回旋支(LCX)放置血管缩窄环,建立慢性心肌缺血模型。5周后行心电图、冠状动脉造影和心脏核磁共振成像检查确认LCX闭塞或相应心肌的缺血。动物随机分为实验组和对照组,每组8只,分别在心肌内直接注射rAAV2-VEGF165(1×1012virus genome)或磷酸盐缓冲液。治疗后3个月和6个月,观察心肌VEGF mRNA和蛋白的表达;6个月后,观察心肌毛细血管和小动脉密度,行冠状动脉造影进行LCX血流分级,应用心脏核磁共振成像观察心肌灌注及左心室功能。结果放置血管缩窄环后5周,所有动物均出现LCX完全/次全闭塞或LCX支配区域的心肌缺血。基因治疗后3个月,实验组心肌VEGF mRNA和蛋白表达显著高于对照组;6个月时,实验组VEGF表达水平较3个月时下降。基因治疗后6个月,VEGF组心肌毛细血管密度和小动脉密度[分别为(1404.06±250.48)/mm2和(167.81±36.29)/mm2]均高于对照组[分别为(976.88±344.79)/mm2和(116.56±34.48)/mm2](P<0.05);潘生丁负荷后心肌灌注成像显示VEGF组心肌灌注明显优于对照组(P<0.05),且较治疗前有改善(P<0.05);两组左心室功能在治疗前后均无明显变化。结论在小型猪慢性心肌缺血模型中,心肌内注射rAAV2-VEGF165后外源VEGF基因的表达至少可持续3个月;rAAV2-VEGF165能够促进缺血心肌毛细血管和小动脉生成并改善心肌灌注。     

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microRNA cluster

RNA interference (RNAi) is being evaluated as an alternative therapeutic strategy for hepatitis C virus (HCV) infection. The use of viral vectors encoding short hairpin RNAs (shRNAs) has been the most common strategy employed to provide sustained expression of RNAi effectors. However, overexpression and incomplete processing of shRNAs has led to saturation of the endogenous miRNA pathway, resulting in toxicity. The use of endogenous microRNAs (miRNAs) as scaffolds for short interfering (siRNAs) may avoid these problems, and miRNA clusters can be engineered to express multiple RNAi effectors, a feature that may prevent RNAi-resistant HCV mutant generation. We exploited the endogenous miRNA-17-92 cluster to generate a polycistronic primary miRNA that is processed into five mature miRNAs that target different regions of the HCV genome. All five anti-HCV miRNAs were active, achieving up to 97% inhibition of Renilla luciferase (RLuc) HCV reporter plasmids. Self-complementary recombinant adeno-associated virus (scAAV) vectors were chosen for therapeutic delivery of the miRNA cluster. Expression of the miRNAs from scAAV inhibited the replication of cell culture-propagated HCV (HCVcc) by 98%, and resulted in up to 93% gene silencing of RLuc-HCV reporter plasmids in mouse liver. No hepatocellular toxicity was observed at scAAV doses as high as 5 × 10(11) vector genomes per mouse, a dose that is approximately five-fold higher than doses of scAAV-shRNA vectors that others have shown previously to be toxic in mouse liver. Conclusion: We have demonstrated that exogenous anti-HCV miRNAs induce gene silencing, and when expressed from scAAV vectors inhibit the replication of HCVcc without inducing toxicity. The combination of an AAV vector delivery system and exploitation of the endogenous RNAi pathway is a potentially viable alternative to current HCV treatment regimens.