Use of nonviral vectors for cystic fibrosis gene therapy

Over the last decade, three groups within the United Kingdom (Edinburgh, Oxford, and Imperial College, London) have undertaken key studies in the development of clinical gene therapy for cystic fibrosis. In 2001, catalyzed by the Cystic Fibrosis Trust, these groups came together to form the United Kingdom Cystic Fibrosis Gene Therapy Consortium. The Consortium has removed duplication and competition, developed core facilities playing to the respective strengths of the centers, and introduced the joint strategy described in this article. This is driven by a clinical trial program, with a product pipeline and the necessary development of novel preclinical and human assays. The program is milestone-related, has a structure that lies between the pharmaceutical industry and academia, and has as its endpoint negotiations with industry to undertake a phase III clinical trial of the identified product.     

Development of viral vectors for use in cardiovascular gene therapy

Cardiovascular disease represents the most common cause of mortality in the developed world but, despite two decades of promising pre-clinical research and numerous clinical trials, cardiovascular gene transfer has so far failed to demonstrate convincing benefits in the clinical setting. In this review we discuss the various targets which may be suitable for cardiovascular gene therapy and the viral vectors which have to date shown the most potential for clinical use. We conclude with a summary of the current state of clinical cardiovascular gene therapy and the key trials which are ongoing.     

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.     

Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy

Genetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) alpha/beta production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.     

Empirical advantages of adeno associated viral vectors in vivo gene therapy for arthritis

OBJECTIVE: To evaluate the utility of the adeno associated viral (AAV) vector for gene delivery to joint cells in vivo and in vitro, and to assess its potential as a vector for arthritis gene therapy. METHODS: A recombinant AAV (rAAV) vector expressing the bacterial beta-galactosidase (beta-gal) gene (rAAV-CMV-LacZ) was directly introduced into healthy-normal mouse knees, or arthritic knees in mice overexpressing tumor necrosis factor-alpha (hTNFalpha-Tg). Beta-gal expression levels were determined by immunohistochemistry and chemiluminescence. The transduction efficiency of this vector on primary fibroblast-like synoviocytes (FLS) in vitro was determined by FACS. The effects of UV and gamma-irradiation as well as TNF-alpha on transduction efficiency were determined using the same methods. RESULTS: We found little evidence of rAAV transduction in the joint cells of healthy mice. Target gene expression was detected in all animals at Day 3, and peaked at Day 7 before returning to baseline levels 21 days after injection. In contrast, synoviocytes, articular chondrocytes, and meniscal cells of diseased mice were transduced by rAAV-CMV-LacZ in hTNFalpha-Tg animals. Transduction efficiencies correlated with joint damage, and target gene expression was up to 10-fold greater than that seen in the normal mice. In vitro, we found that rAAV transduction of FLS can be enhanced by pretreatment with UV or gamma-irradiation and TNF-alpha stimulation. CONCLUSION: We find that rAAV vectors have several empirical advantages for in vivo gene therapy for arthritis: (1) rAAV preferentially transduces arthritic joint cells in vivo. (2) rAAV can transduce both FLS and chondrocytes in vivo. (3) rAAV transduction of FLS can be augmented by pretreatment with agents that induce DNA repair enzymes.     

Development of viral vectors for gene therapy of beta-chain hemoglobinopathies: optimization of a gamma-globin gene expression cassette

Progress toward gene therapy of beta-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of the Agamma- and beta-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that the Agamma-globin promoter could be deleted to -159 without affecting expression, while deleting the beta-globin promoter to -127 actually increased expression compared with longer fragments. Expression from the optimal beta-globin gene promoter was consistently higher than that from the optimal Agamma-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the beta-globin locus control region (termed a muLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the alpha-globin locus. All three enhancers increased expression from the beta-globin gene to roughly the same extent, while the HS-40 element was notably less effective with the Agamma-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Agamma-globin gene linked to the beta-globin promoter. Inclusion of extended 3' sequences from either the beta-globin or the Agamma-globin genes had no significant effect on expression. A 714-bp internal deletion of Agamma-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a -127 beta-globin promoter, an Agamma-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, gamma-globin expression averaged 166% of murine alpha-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine alpha-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for gamma-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.     

肝脏疾病基因治疗中非病毒性载体的研究进展

目前大部分肝脏疾病(病毒性肝炎、自身免疫性肝病、先天性代谢性肝病、原发性肝癌等)在临床上尚无有效的根治方法,作为医学研究中热点课题的基因治疗能否应用于肝脏疾病,引起了许多学者的关注.     

Development of HIV vectors for anti-HIV gene therapy.

Current gene therapy protocols for HIV infection use transfection or murine retrovirus mediated transfer of antiviral genes into CD4+ T cells or CD34+ progenitor cells ex vivo, followed by infusion of the gene altered cells into autologous or syngeneic/allogeneic recipients. While these studies are essential for safety and feasibility testing, several limitations remain: long-term reconstitution of the immune system is not effected for lack of access to the macrophage reservoir or the pluripotent stem cell population, which is usually quiescent, and ex vivo manipulation of the target cells will be too expensive and impractical for global application. In these regards, the lentivirus-specific biologic properties of the HIVs, which underlie their pathogenetic mechanisms, are also advantageous as vectors for gene therapy. The ability of HIV to specifically target CD4+ cells, as well as non-cycling cells, makes it a promising candidate for in vivo gene transfer vector on one hand, and for transduction of non-cycling stem cells on the other. Here we report the use of replication-defective vectors and stable vector packaging cell lines derived from both HIV-1 and HIV-2. Both HIV envelopes and vesicular stomatitis virus glycoprotein G were effective in mediating high-titer gene transfer, and an HIV-2 vector could be cross-packaged by HIV-1. Both HIV-1 and HIV-2 vectors were able to transduce primary human macrophages, a property not shared by murine retroviruses. Vesicular stomatitis virus glycoprotein G-pseudotyped HIV vectors have the potential to mediate gene transfer into non-cycling hematopoietic stem cells. If so, HIV or other lentivirus-based vectors will have applications beyond HIV infection.     

Viral vectors for vascular gene therapy

Vascular gene therapy is the focus of multiple experimental and clinical research efforts. While several genes with therapeutic potential have been identified, the best method of gene delivery is unknown. Viral vectors have the capacity to transfer genes at high efficiency rates. Several viral-based vectors have been used in experimental vascular gene therapy for in vivo and ex vivo gene transfer. Adenoviral-based vectors are being used for the induction of angiogenesis in phase 1 and 2 clinical trials. In the present review, the characteristics of the ‘ideal’ viral vector are discussed and the major types of viral vectors used in vascular gene transfer are reviewed. Basic knowledge of the use of viral vectors for direct in vivo gene transfer (adenoviral-based vectors, etc) and for ex vivo gene transfer (retroviral-based vectors) is provided. New developments in the field of viral vectorology, such as pseudotyping of retroviral vectors and targeting of other viral vectors to a specific cell type, will enhance the more rapid transition of vascular gene therapy from the experimental arena to the clinical setting.     

Viral vectors in gene therapy. Advantages of the adenoassociated vectors

Gene therapy has evolved due to the development of a number of biotechnology weapons, i.e., vectors that achieve for a longer expression and safer administration. Among viral vectors developed adeno-associated virus have shown promising advantages. These DNA viruses transduce a wide cell range, can integrate in host's genome and achieve for a long-period expression, besides avoiding a cellular immune response. The new technologies applied to the production and purification of these vectors had resulted in notable increases in quantity and quality of the infectious particles obtained. Actually, due to biotechnological advances, gene therapy is a potential therapeutic option.     

Tumor vascular targeting therapy with viral vectors

Tumor angiogenesis is crucial for the progression and metastasis of cancer. The vasculature of tumor tissue is different from normal vasculature. Therefore, tumor vascular targeting therapy could represent an effective therapeutic strategy with which to suppress both primary tumor growth and tumor metastasis. The use of viral vectors for tumor vascular targeting therapy is a promising strategy based on the unique properties of viral vectors. In order to circumvent the potential problems of antiviral neutralizing antibodies, poor access to extravascular tumor tissue, and toxicities to normal tissue, viral vectors need to be modified to target the tumor endothelial cells. Viral vectors that could be used for tumor vascular targeting therapy include adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, measles virus, and herpes simplex viral vectors. In this review, we will summarize the strategies available for targeting viral vectors for tumor vascular targeting therapy.     

三种非病毒载体对神经母瘤细胞进行转染的效果比较

目的以增强型绿色荧光蛋白(EGFP)为报告基因,研究Lipofectamine2000、改良纳米材料CS(N/P=10)和纳米粒子PAMAM(N/P=20)介导的基因导入系统对神经母瘤细胞SH-SY5Y的毒性及基因转染效率,为神经系统疾病的基因治疗奠定基础。方法制备Lipofectamine2000、CS和PAMAM与质粒DNA(pDNA)的复合物,分别转染在体外培养的人胚肾细胞Hek293和SH-SY5Y,利用噻唑蓝(MTT)比色实验比较三种载体的细胞毒性作用、用激光共聚焦显微镜观察转染后的细胞形态、倒置荧光显微镜检测转染细胞EGFP表达强度、流式细胞仪检测转染效率。结果①Lipofectamine2000、CS和PAMAM转染细胞存活率在Hek293细胞分别为73.09%、73.69%、69.24%,三者比较无显著差异;在SH-SY5Y细胞分别为67.69%、61.71%、42.92%,前两者均显著高于后者(P均<0.05)。②Hek293细胞经Lipofectamine2000和CS与pDNA的复合物分别转染后,细胞形态均未发生明显改变,保持梭状生长;而PAMAM/pDNA复合物转染后,细胞发生皱缩,呈圆形分布生长。经三种载体转染后的SH-SY5Y细胞均发生形态改变,其中PAMAM/pDNA复合物转染者形态改变程度相对较大。Hek293细胞和SH-SY5Y细胞中,均以Lipofectamine2000/pDNA复合物转染者EGFP表达强度最大,其次为CS/pDNA复合物、PAMAM/pDNA复合物转染者。④Lipofectamine2000、CS和PAMAM的转染效率在Hek293细胞分别为60.9%、45.2%、42.7%,前者显著高于后两者(P均<0.05);在SH-SY5Y细胞分别为25.3%、19.2%、8.9%,前两者均显著高于后者(P均<0.05)。结论 Lipofectamine2000为目前最适于神经系统疾病基因治疗研究的非病毒载体;改良后的新纳米材料CS毒性比PAMAM小、转染效率比PAMAM高,有望在神经系统疾病研究中得到进一步运用。     

Lentiviral vectors for gene therapy of heart disease

Technological advances in genetic engineering developed over the past few years have been applied to the research and treatment of cardiovascular diseases. In many animal models, gene therapy has been shown to be an effective treatment schema. Some of these gene therapy treatments are now being applied in clinical trials. Also, as the science of gene therapy has progressed, alternative vector systems such as lentiviruses have been developed and implemented. Here we focus on the emerging role of lentiviral vectors in the treatment of cardiovascular disease.     

Preclinical gene therapy studies for hemophilia using adenoviral vectors

Hemophilia A and B are hereditary coagulation defects resulting from a deficiency of factor VIII (FVIII) and factor IX (FIX), respectively. Introducing a functional FVIII or FIX gene could potentially provide a cure for these bleeding disorders. Adenoviral vectors have been used as tools to introduce potentially therapeutic genes into mammalian cells and are by far the most efficient vectors for hepatic gene delivery. Long-term expression of both FVIII and FIX has been achieved in preclinical (hemophilic) mouse models using adenoviral vectors. Therapeutic levels of FVIII and FIX also have been achieved in hemophilic dogs using adenoviral vectors and in some cases expression was long-term. The performance of earlier generation adenoviral vectors, which retained residual viral genes, was compromised by potent acute and chronic inflammatory responses that contributed to significant toxicity and morbidity and short-term expression of FVIII and FIX. The development of improved adenoviral vectors devoid of viral genes (gutless or high-capacity adenoviral vectors) was therefore warranted, which led to a significant reduction in acute and chronic toxicity and more prolonged expression of FVIII and FIX. Strategies aimed at making these vectors safer and less immunogenic and their implications for hemophilia gene therapy are discussed in this review.