Development of lentiviral vectors for gene therapy for human diseases

Retroviral vectors derived from murine retroviruses are being used in several clinical gene therapy trials. Recently, progress has been made in the development of vectors based on the lentivirus genus of retroviruses, which ironically includes a major human pathogen, human immunodeficiency virus (HIV). As these vector systems for clinical gene transfer are developed, it is important to understand the rationale behind their design and development. This article reviews the fundamental features of retrovirus replication and of the elements necessary for development of a retroviral vector system, and it discusses why vector systems based on HIV or other lentiviruses have the potential to become important tools in clinical gene therapy. (Blood. 2000;95:2499-2504)     

逆转录病毒载体介导双靶区乙型肝炎病毒反义RNA模型的构建

构建在真核细胞内转录表达乙型肝炎病毒(HBV)双靶区反义核酸重组载体用以抗HBV基因治疗的研究。方法为合成互补于HBV(ayw亚型)X区核苷酸X片段以及互补于HBV P区的P片段,利用基因重组技术将X、P片段分别正向、反向插入逆转录病毒载体pLXSN的相应酶切位点构建成单靶区重组载体质粒。在构建单靶区载体质粒的基础上,类似方法再构建双靶区重组载体质粒。经酶切电泳、PCR扩增和DNA测序鉴定成功构建了HBV双靶区反义核酸重组载体。为探索在真核细胞内转录表达HBV反义RNA的方法及研究多基因区抗病毒治疗和抗变异病毒打下基础。     

Gene transfer with ultrasound and microbubbles (optison) as a potential treatment for cardiovascular diseases

Cardiovascular diseases are the leading causes of mortality and morbidity in developed countries. Most conventional therapy is inefficient and tends to treat the symptoms rather than the underlying causes of the disorder. Gene therapy based on ultrasound with microbubbles offers a novel approach for the prevention and treatment of cardiovascular diseases. The major development of gene transfer has importantly contributed to intense investigation of the potential of gene therapy in cardiovascular medicine. The amazing advances in molecular biology have provided a dramatic improvement of the technology that is necessary to transfer target genes into somatic cells. Gene transfer methods have been surprisingly improved. In fact, some of them (retroviral vectors, adenoviral vectors or liposome based vectors, etc) have been used in clinical trials already. However, some severe side effects were reported in clinical gene therapy using such viral vector, so people wish for safe and efficient clinical gene therapy. Transfection with ultrasound and microbubbles has been reported as a new powerful tool in a new class for gene therapy.     

Gene therapy： Regulations, ethics and its practicalities in liver disease

Gene therapy is a new and promising approach which opens a new door to the treatment of human diseases. By direct transfer of genetic materials to the target cells, it could exert functions on the level of genes and molecules. It is hoped to be widely used in the treatment of liver disease, especially hepatic tumors by using different vectors encoding the aim gene for anti-tumor activity by activating primary and adaptive immunity, inhibiting oncogene and angiogenesis. Despite the huge curative potential shown in animal models and some pilot clinical trials, gene therapy has been under fierce discussion since its birth in academia and the public domain because of its unexpected side effects and ethical problems. There are other challenges arising from the technique itself like vector design, administration route test and standard protocol exploration. How well we respond will decide the fate of gene therapy clinical medical practice.     

逆转录病毒载体感染原代大鼠肝细胞的研究

目的研究逆转录病毒载体高效转移并表达于大鼠原代肝细胞．方法采用表皮生长因子（ＥＧＦ）刺激大鼠原代肝细胞增殖,以表达β半乳糖苷酶的双顺反子逆转录病毒载体（ｐＧＣＥＮ／βｇａｌ）感染肝细胞．采用Ｘｇａｌ原位染色方法检测肝细胞内ＬａｃＺ的表达．结果肝细胞表达β半乳糖苷酶基因,转导效率为３％～２２％．结论双顺反子逆转录病毒载体（ｐＧＣＥＮ／βｇａｌ）可有效介导βｇａｌ基因表达于原代大鼠肝细胞,提示该载体可用于标记大鼠肝细胞,有利于研究肝细胞移植后在体内的生物学特性和肝病的体外基因治疗．     

A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.

Retroviral vectors have been central components in many studies leading to human gene therapy. However, the generally low titers and inefficient infectivity of retroviral vectors in human cells have limited their use. We previously reported that the G protein of vesicular stomatitis virus can serve as the exclusive envelope protein component for one specific retroviral vector, LGRNL, that expresses vesicular stomatitis virus G. We now report a more useful general transient transfection scheme for producing very high-titer vesicular stomatitis virus G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector without having to rely on the expression of the cytotoxic G protein from the retroviral vector itself. We also demonstrate very high efficiency of infection with a pseudotyped lacZ vector in primary mouse hepatocytes. We suggest that pseudotyped retroviral vectors carrying reporter genes will permit genetic studies in many previously inaccessible vertebrate and invertebrate systems. Furthermore, because these vectors represent retroviral vectors of sufficiently high titer to allow efficient direct retroviral-mediated in vivo gene transfer, we also suggest that pseudotyped vectors carrying potentially therapeutic genes will become useful to test the potential for in vivo gene therapy.     

Gene therapy for severe combined immunodeficiency caused by adenosine deaminase deficiency: improved retroviral vectors for clinical trials

Severe combined immunodeficiency (SCID) caused by adenosine deaminase deficiency (ADA-) is the first genetic disorder to be treated with gene therapy. Since 1990 when the first trial started for 2 patients with ADA- SCID, five clinical trials enrolling 11 patients have been conducted with different clinical approaches and the results obtained from these trials have recently been reported. According to these reports, T cell-directed gene transfer was useful in the treatment of ADA- SCID whereas the retroviral-mediated gene transfer to hematopoietic stem cells was insufficient for achievement of clinical benefits. This chapter reviews several crucial problems inherent in the current retroviral technology based on the clinical data observed in these pioneering ADA gene therapy trials and presents our new retroviral vector system for the next stem cell gene therapy.     

Gene therapy targeting hematopoietic cells: better not leave it to chance

Gene therapy targeting hematopoietic cells has arrived at a new stage of potency. While the potential for curing inherited disorders of the immune system has been demonstrated in clinical trials, we were also confronted with the first serious adverse events related to random insertion of foreign DNA into cellular chromosomes. As it is likely that the manifestation of severe side effects results from a multifactorial process, it will be of crucial importance to define the significance of the individual risk factors involved. The future of the field will depend on our ability to define risk classifications of clinical approaches, to continuously improve transgene technologies, and to introduce new concepts for targeted selection of transgenic cells. Interestingly, correction of genetic disorders by homologous gene repair in defined stem cell clones is on the horizon, but far from being available for clinical use.     

The impact of retroviral suicide gene transduction procedures on T cells

Retroviral vectors encoding the herpes simplex thymidine kinase gene have been used to render T cells sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used in clinical trials for their graft-versus-leukaemia effects following allogeneic haematopoietic stem cell transplantation. In the event of graft-versus-host disease (GVHD) the cells were susceptible to elimination through exposure to ganciclovir. We have investigated the impact of T-cell activation, required for successful retrovirus-mediated gene transfer, on T-cell receptor repertoire profile, subset distribution and antiviral potential. Using a combination of antibodies against CD3 and CD28, T cells were transduced at high efficiency when exposed to retrovirus between 48 and 72 h later. Lymphocytes had undergone up to seven cycles of cell division by the end of the procedure. Although the T-cell receptor Vbeta repertoire was not altered after retroviral transduction, there were notable shifts in subset profiles with an increased proportion of CD45RO cells in transduced populations. T cells continued to proliferate for several days after transduction and were difficult to sustain under the extended culture conditions required to generate virus-specific T cells. These observations may explain the lower than expected levels of GVHD and poor antiviral immunity reported in recent trials.     

The use of nonhuman primate models to improve gene transfer into haematopoietic stem cells

Primitive haematopoietic progenitor and stem cells (HSC) have been pursued as highly desirable targets for genetic therapy as technology allowing safe and controllable transfer of exogenous genes into eukaryotic cells was developed a decade ago. Retroviral vectors have been used for the majority of preclinical and clinical studies directed at these cells, because these vectors have a number of the necessary properties, including chromosomal integration, helper-free production systems, and lack of toxicity. Until recently, however, results with these vectors in clinical trials and large animal models indicated efficiency of gene transfer as a major hurdle to be overcome. We have focused on using the rhesus macaque autologous transplantation model to optimize gene transfer to primitive haematopoietic cells, and investigate questions regarding in vivo stem cell behaviour, in a system with proven predictive value for human haematopoiesis. By optimization of transduction conditions using standard vectors, gene transfer efficiency to primitive repopulating cells has reached the clinically relevant range of 5-20% long-term. Alternative vector systems, have also yielded promising results. We have also found that relatively simple manipulation of cell cycle status prior to reinfusion of marked cells results in significantly improved engraftment of transduced cells: this finding may have an impact particularly in the nonablative setting. The high level marking has permitted insertion site analysis and clonal tracking in vivo. Inverse PCR and/or a ligation-mediated PCR procedure have demonstrated that a large number of transduced clones (over 50) contribute to multiple lineages in vivo for up to at least 2 years post-transplantation. Thus far we have little evidence for rapid clonal succession or lineage-restricted engraftment of transduced cells. These and other advances should result in successful gene therapy for a variety of acquired and congenital disorders affecting HSCs and their progeny lineages.     

Retroviral-mediated transfer and amplification of a functional human factor VIII gene

Hemophilia A results from a deficiency in factor VII (FVIII), a cofactor in the intrinsic pathway of blood coagulation. As an approach toward genetic therapy of this disease, we constructed a retroviral vector encoding human FVIII and a selectable and amplifiable genetic marker, human adenosine deaminase (Ada). A retrovirus packaging line was transfected with this vector and stable transformants were selected for Ada expression. Isolated transformants produced both FVIII activity in the conditioned medium and retrovirus capable of transferring the Ada selectable marker and FVIII expression to the mouse 3T3 fibroblasts. Selection of virus-producer cell lines for increasing levels of Ada expression yielded a 20-fold increase in both FVIII expression and viral titer. Similarly, selection of infected 3T3 fibroblasts for Ada gene amplification yielded a 20-fold increase in FVIII expression. The results demonstrate the feasibility of retrovirus- mediated transfer of human FVIII, and also the utility of selection for gene amplification to increase retrovirus titers in producer cell lines as well as expression levels in infected cells.     

Emerging biotechnological strategies for non-viral antiangiogenic gene therapy

Angiogenesis has emerged as a promising target of cancer treatment. With the development of biotechnology, major progress has been made in the exploring effective therapies on targeting tumor angiogenesis over the last 20?years. Gene therapy has attracted considerable interest by virtue of the capabilities of expressing sustained levels of therapeutic agents within cells of the patients. However, the major challenge of gene therapy is the efficient delivery of therapeutic gene to the target site. Compared with viral strategies, non-viral strategies were more acceptable by their widely recognized security and lower side effects. This paper reviews the basic biology of angiogenesis, the potential advantages of antiangiogenic gene therapy, the therapeutic genetic drugs developed through biotechnology, as well as the biotechnological strategies that enhancing non-viral gene therapy targeting to tumor angiogenesis in a more controlled manner, with great respect to RNA interference, ligand-directed vascular targeting strategies, vascular endothelial growth factor pathway and tumor associated macrophages targeting. In conclusion, antiangiogenic gene therapy holds great promise in advancing cancer therapy. Developing better non-viral biotechnological platforms will benefit antiangiogenic targeted cancer gene therapeutic methods, support their evaluation in human clinical trials and realize the actual utilization in the near future.     

Gene therapy for lung disease: hype or hope?

Gene therapy, the treatment of any disorder or pathophysiologic state on the basis of the transfer of genetic information, was a high-priority goal in the 1990s. The lung is a major target of gene therapy for genetic disorders, such as cystic fibrosis and alpha1-antitrypsin deficiency, and for other diseases, including lung cancer, malignant mesothelioma, pulmonary inflammation, surfactant deficiency, and pulmonary hypertension. This paper examines general concepts in gene therapy, summarizes the results of published clinical trials, and highlights areas of research aimed at overcoming challenges in the field. Although progress has been slower than anticipated, gene transfer has been safely achieved in patients with lung diseases. Recent advancements in understanding of the molecular basis of lung disease and the development of improved vector systems make it likely that gene therapy will be an important tool for the 21st-century clinician.     

Gene therapy for the treatment of hepatoma by retroviral-mediated gene transfer of the herpes simplex virus thymidine kinase

We previously demonstrated that a foreign gene transferred by means of a retroviral vector can be expressed selectively in hepatoma cells when a liver-specific promoter was used to direct its expression. We now describe an approach for the treatment of hepatoma by the introduction of herpes simplex virus thymidine kinase (HSV-TK) gene. Expression of HSV TK gene in hepatoma cells was evaluated as an elimination system for a potential use in therapies. A murine retroviral vector was constructed in which the HSV-TK gene was expressed under control of the murine albumin enhancer and promoter elements. Replication-defective vector viral particles were obtained by transfer of the vector DNA into the ecotropic packaging cell line psi2 and were used to infect murine hepatoma cells. The introduction of the HSV-TK gene into hepatoma cells by infection of the recombinant retrovirus did not affect their proliferation at all. The sensitivity of those infected cells to the toxic effects of the nucleoside analog ganciclovir was found to be significantly increased by transfer of the HSV-TK gene. The difference in sensitivity between infected and uninfected cells to ganciclovir concentrations should give the utility for a clinical application indicating the feasibility of gene therapy toward hepatoma by the retroviral-mediated HSV-TK gene transfer.     

Side effects of retroviral gene transfer into hematopoietic stem cells

Recent conceptual and technical improvements have resulted in clinically meaningful levels of gene transfer into repopulating hematopoietic stem cells. At the same time, evidence is accumulating that gene therapy may induce several kinds of unexpected side effects, based on preclinical and clinical data. To assess the therapeutic potential of genetic interventions in hematopoietic cells, it will be important to derive a classification of side effects, to obtain insights into their underlying mechanisms, and to use rigorous statistical approaches in comparing data. We here review side effects related to target cell manipulation; vector production; transgene insertion and expression; selection procedures for transgenic cells; and immune surveillance. We also address some inherent differences between hematopoiesis in the most commonly used animal model, the laboratory mouse, and in humans. It is our intention to emphasize the need for a critical and hypothesis-driven analysis of "transgene toxicology," in order to improve safety, efficiency, and prognosis for the yet small but expanding group of patients that could benefit from gene therapy.     

Insertional oncogenesis by non-acute retroviruses: implications for gene therapy

Retroviruses cause cancers in a variety of animals and humans. Research on retroviruses has provided important insights into mechanisms of oncogenesis in humans, including the discovery of viral oncogenes and cellular proto-oncogenes. The subject of this review is the mechanisms by which retroviruses that do not carry oncogenes (non-acute retroviruses) cause cancers. The common theme is that these tumors result from insertional activation of cellular proto-oncogenes by integration of viral DNA. Early research on insertional activation of proto-oncogenes in virus-induced tumors is reviewed. Research on non-acute retroviruses has led to the discovery of new proto-oncogenes through searches for common insertion sites (CISs) in virus-induced tumors. Cooperation between different proto-oncogenes in development of tumors has been elucidated through the study of retrovirus-induced tumors, and retroviral infection of genetically susceptible mice (retroviral tagging) has been used to identify cellular proto-oncogenes active in specific oncogenic pathways. The pace of proto-oncogene discovery has been accelerated by technical advances including PCR cloning of viral integration sites, the availability of the mouse genome sequence, and high throughput DNA sequencing. Insertional activation has proven to be a significant risk in gene therapy trials to correct genetic defects with retroviral vectors. Studies on non-acute retroviral oncogenesis provide insight into the potential risks, and the mechanisms of oncogenesis.     

Liver as an ideal target for gene therapy: expression of CTLA4Ig by retroviral gene transfer

BACKGROUND AND AIMS: Liver has been a target organ for gene therapy as it plays a central role in metabolism and production of serum proteins. Many metabolic disorders result from a deficiency of liver-derived protein products. In transplantation settings, modulation of the immune responses caused by CTLA4Ig protein has been shown to be an attractive direction. In this study, we investigated the efficacy of hepatocyte transduction via introduction of the exogenous CTLA4Ig gene to the rat liver graft by retrovirus vector, and examined the presence of target serum protein after gene transfers. METHODS: We constructed a replication defective retroviral vector that contained the CTLA4Ig gene. The liver graft regeneration index was first examined by 5-bromo-2-deoxyuridine, Ki-67 and proliferating cell nuclear antigen antibodies to determine the optimal time of gene transduction. The liver graft was then perfused with the retroviral vector, and animals were killed at constant time points to examine for the presence of CTLA4 protein in the graft and peripheral blood. RESULTS: CTLA4 protein was detected on postoperative days 5, 9 and 14, with liver graft tissue transduction indexes of 7.2, 10.9 and 1.8, respectively. Blood protein levels were at 151.6, 26.5 and 21.4 rhog/mL, respectively. A transduction index reaching 22.1 was observed in the graft with the most rapid liver regeneration. CONCLUSIONS: We had established the gene delivery model in rat with auxiliary partial liver transplantation. Expression of the exogenous gene delivered by retrovirus was demonstrated in the liver with secretion of diffusible protein in the bloodstream. The present study provides important information for gene transfer using the liver to produce the target protein in situ and as serum protein. This will also be applicable to the treatment of other metabolic diseases.     

Gene therapy for inherited disorders of haematopoietic cells

Over the last decade, the majority of inborn errors of haematopoiesis has been elucidated on a molecular level. For some of these diseases, gene transfer into transplantable cells offers new therapeutic perspectives. Improved retroviral or lentiviral techniques allow stable gene transfer in >10% of repopulating cells cultured in vitro. However, severe impediments are still encountered with respect to achieving sufficient and long-lasting transgene expression levels and appropriate numbers of transgenic cells in vivo. Improving the techniques for manipulation of stem cells in vitro, and the development of regimens promoting engraftment and selection of gene-modified cells are important areas of current research. Further activities address the level and persistence of transgene expression within individual cell clones, and the functional characteristics of progeny cells in vivo. Promises with respect to the potential impact of gene therapy for patients suffering from inherited disorders are often triggered by 'proof of concept' in preclinical disease models. However, recent observations of side effects related to random vector insertion or transgene expression indicate that even more careful quantitative and qualitative investigations of efficiency and toxicity may be needed for individual transgenes before approaching clinical trials.     

Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.

Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.