The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery

Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo.     

反转录病毒基因治疗载体及其安全性评价

反转录病毒载体是目前基因治疗中应用最广泛的基因运载工具之一,具有能稳定整合入宿主细胞、持久表达目的基因、感染细胞范围广、基因容量较大等优点,但其生物安全性还存在争议。在简要介绍反转录基因治疗病毒载体的基础上,对反转录病毒载体中外源目的基因的表达调控及反转录病毒载体的安全性评价进行了综述。     

Characterization of a semi-replicative gene delivery system allowing propagation of complementary defective retroviral vectors

BACKGROUND: Recently, several cancer gene therapy studies have shown that replication-competent retroviral vectors represent a major improvement over replication-defective ones in terms of transgene propagation efficiency. However, this positive effect is somewhat spoiled by the increased risk of dissemination and oncogenesis that replication-competent retroviral vectors entail. To enhance both their integral safety and their transgene capacity, we developed a semi-replication-competent retroviral vector system. METHODS: The semi-replication-competent retroviral vector system is based on two transcomplementing replication-defective retroviral vectors termed gag-pol vector (GPv) and env vector (Ev). Vector propagation was monitored in vitro and in solid tumors in vivo, using different reporter transgenes for GPv and Ev. Systemic vector dissemination and leukemogenesis was assessed by direct intravenous vector injection and subsequent bone marrow transplantation, in MLV-sensitive mice. RESULTS: In vitro and in vivo the semi-replication-competent retroviral vectors propagate transgenes almost as efficiently as replication-competent ones. The semi-replication-competent retroviral vector system does not lead to detectable dissemination or leukemogenesis as does the replication-competent vector or the parental virus. Additionally, the vector duo allows co-propagation of different transgenes as well as mobilization of a third replication-defective vector. CONCLUSIONS: This study is an initial proof of principle for the use of complementary retroviral vectors to deliver and propagate transgenes in vitro and in solid tumors in vivo, but with reduced pathogenicity compared to its parental virus. In-between replication-defective and replication-competent retroviral vectors, this semi-replicative system offers good grounds for its application in in vitro studies and allows envisioning its further development for cancer gene therapy.     

Purification of retroviral vectors for clinical application: biological implications and technological challenges

For centuries mankind led a difficult battle against viruses, the smallest infectious agents at the surface of the earth. Nowadays it is possible to use viruses for our benefit, both at a prophylactic level in the production of vaccines and at a therapeutic level in the promising field of gene therapy. Retroviruses were discovered at the end of the 19th century and constitute one of the most effective entities for gene transfer and insertion into the genome of mammalian cells. This attractive feature has intensified research in retroviral vectors development and production over the past years, mainly due to the expectations raised by the concept of gene therapy. The demand for high quality retroviral vectors that meet standard requisites from the regulatory agencies (FDA and EMEA) is therefore increasing, as the technology has moved into clinical trials. The development of safer producer cell lines that can be used in large-scale production will result in the production of large quantities of retroviral stocks. Cost-efficient and scalable purification processes are essential for production of injectable-grade preparations to achieve final implementation of these vectors as therapeutics. Several preparative purification steps already established for proteins can certainly be applied to retroviral vectors, in particular membrane filtration and chromatographic methods. Nevertheless, the special properties of these complex products require technological improvement of the existing purification steps and/or development of particular purification steps to increase productivity and throughput, while maintaining biological activity of the final product. This review focuses on downstream process development in relation to the retroviral vectors characteristics and quality assessment of retroviral stocks for intended use in gene therapy.     

Targeted vectors for gene therapy of cancer and retroviral infections

Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections.     

Gene therapy using retroviral vectors

Gene therapy is a novel approach for treating various congenital and acquired genetic disorders, including cancer, heart disease, and acquired immune deficiency syndrome. Amongst possible gene delivery systems, retroviral vector mediated gene transfer has been the most extensively studied and has been approved for use in over 40 current Phase I/II clinical trials for the treatment of various disorders, primarily cancers. Recent technological improvements include the optimization of vector production by concentration and lyophilization, resulting in high titers of vectors, as well as the large-scale production of vector-produced cells for the treatment of brain cancer. Present clinical protocols require specialized care centers with expertise in molecular biology and cell transplantation. Considerable effort is under way to develop retroviral vectors that can be both injected directly into the body and targeted to specific cell types within the body. Such vectors could be administered to patients by physicians in their offices. Successful development of this new technology would greatly expand the clinical potential of gene therapy.     

Retroviral infection of hES cells produces random-like integration patterns

Retroviral integration provides us with a powerful tool to realize prolonged gene expressions that are often critical to gene therapy. However, the perturbation of gene regulations in host cells by viral genome integration can lead to detrimental effects, yielding cancer. The oncogenic potential of retroviruses is linked to the preference of retroviruses to integrate into genomic regions that are enriched in gene regulatory elements. To better navigate the double-edged sword of retroviral integration we need to understand how retroviruses select their favored genomic loci during infections. In this study I showed that in addition to host proteins that tether retroviral pre-integration complexes to specific genomic regions, the epigenetic architecture of host genome might strongly affect retroviral integration patterns. Specifically, retroviruses showed their characteristic integration preference in differentiated somatic cells. In contrast, retroviral infections of hES cells, which are known to display decondensed chromatin, produced random-like integration patterns lacking of strong preference for regulatory-element-rich genomic regions. Better identification of the cellular and viral factors that determine retroviral integration patterns will facilitate the design of retroviral vectors for safer use in gene therapy.     

Targeted retroviral gene delivery using ultrasound

BACKGROUND: Achieving specificity of delivery represents a major problem limiting the clinical application of retroviral vectors for gene therapy, whilst lack of efficiency and longevity of gene expression limit non-viral techniques. Ultrasound and microbubble contrast agents can be used to effect plasmid DNA delivery. We therefore sought to evaluate the potential for ultrasound/microbubble-mediated retroviral gene delivery. METHODS: An envelope-deficient retroviral vector, inherently incapable of target cell entry, was combined with cationic microbubbles and added to target cells. The cells were exposed to pulsed 1 MHz ultrasound for 5 s and subsequently analysed for marker gene expression. The acoustic pressure profile of the ultrasound field, to which transduction efficiency was related, was determined using a needle hydrophone. RESULTS: Ultrasound-targeted gene delivery to a restricted area of cells was achieved using virus-loaded microbubbles. Gene delivery efficiency was up to 2% near the beam focus. Significant transduction was restricted to areas exposed to > or = 0.4 MPa peak-negative acoustic pressure, despite uniform application of the vector. An acoustic pressure-dependence was demonstrated that can be exploited for targeted retroviral transduction. The mechanism of entry likely involves membrane perturbation in the vicinity of oscillating microbubbles, facilitating fusion of the viral and cell membranes. CONCLUSIONS: We have established the basis of a novel retroviral vector technology incorporating favourable aspects of existing viral and non-viral gene delivery vectors. In particular, transduction can be controlled by means of ultrasound exposure. The technology is ideally suited to targeted delivery following systemic vector administration.     

Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility

Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire gag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.     

Mutations in p53 cDNA sequence introduced by retroviral vector

The high mutation rates of retroviruses are a potential problem with retroviral vectors. We studied the mutation rates and spectra of p53 sequences transduced with a retroviral vector in a cancer gene therapy model. When p53-deficient H358 non-small cell lung cancer cells were treated with a retroviral vector carrying normal p53 cDNA, most of transduced cells were killed by apoptosis. However, a small number of clones escaped p53-mediated apoptosis. We examined the p53 cDNA structure in these resistant clones. PCR-based analysis showed that 88/102 clones had detectable mutations in p53, including gross rearrangements, deletions/insertions, and base substitutions. To study the mutation rate of the p53 sequence in all transduced clones, the retroviral vector containing the non-functional p53 gene and the Neo-resistant marker gene was introduced into H358 cells. Only one of 95 isolated clones showed a base substitution. These results indicate that the mutation rate of p53 is not particularly high, but there is a significant risk that cancer cells will resist p53 gene therapy as a result of retroviral replication errors.     

逆转录病毒载体在血友病A基因治疗中的研究进展

基因治疗是彻底治愈血友病A的最理想方法,逆转录病毒是最为常用的载体之一,本文对逆转录病毒在血友病A基因治疗中的研究进展作一综述。     

肿瘤基因治疗的靶向策略

对肿瘤组织的靶向性可以提高基因治疗的效果 ,避免对正常组织的损伤 ,并且能降低作为载体的微生物对机体的危害。对于瘤内注射的给药方法 ,靶向性似乎显得不是特别重要 ,但是如果要系统给药 ,靶向性是很关键的一个问题。靶向基因治疗肿瘤可以通过靶向基因导入和靶向基因表达来实现。近年来 ,在靶向基因导入方面的研究有很多进展 ,例如 ,用双亲性的桥连分子协助腺病毒和逆转录病毒靶向转导 ;在各种病毒载体的衣壳蛋白中插入靶向性的小肽或较大的多肽靶向结构域 ;增殖病毒作为一种很有前途的抗肿瘤制剂可有效地靶向杀伤肿瘤细胞。受体介导的DNA或DNA 脂质体复合物的靶向系统和其他一些靶向性的有疗效的载体 ,如细菌 ,也处于研究中。其中的一些载体已经进入临床实验。为了实现基因的靶向可调控表达 ,组织或肿瘤特异性的启动子和人工合成的可调控表达系统被用来调控治疗基因的表达。反义核酸、核酶以及脱氧核酶 (DNAzyme)被用来靶向抑制与肿瘤发生密切相关基因的表达。     

Antibody-directed targeting of retroviral vectors via cell surface antigens

Targeted stable transduction of specific cells is a highly desirable goal for gene therapy applications. We report an efficient and broadly applicable approach for targeting retroviral vectors to specific cells. We find that the envelope of the alphavirus Sindbis virus can pseudotype human immunodeficiency virus type 1- and murine leukemia virus-based retroviral vectors. When modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody. Unlike previous targeting strategies for retroviral transduction, the virus titers are relatively high and stable and can be further increased by ultracentrifugation. This study provides proof of principle for a targeting strategy that would be generally useful for many gene therapy applications.     

Long time-course monitoring of ZFP809-mediated gene silencing in transgene expression driven by promoters containing MLV-derived PBS

Expression of Moloney murine leukemia virus (MoMLV)-typed retroviral vectors is strictly suppressed in immature cells such as embryonic stem cells. The phenomenon known as gene silencing is primed by the sequence-specific binding of the zinc finger protein 809 (ZFP809) to the primer-binding site of the vectors. However, it has yet to be determined whether the ZFP809-mediated gene silencing is maintained over a long period. In this study, we established an experimental system that can monitor gene silencing during a long-term cell culture using flow cytometry technology combined with fluorescent reporters for the expression of ZFP809 and the transgene expression driven by the promoters of interest. Time-course analysis using our system revealed that ZFP809 maintains gene silencing effect even at a longtime period. Furthermore, our system was useful for the monitoring of ZFP809-mediated gene silencing regardless of the types of vectors and cell lines.     

Is it going to be SIN?

Insertional mutagenesis resulting in a leukaemia-like lymphoproliferative disease, as observed in the X-SCID (severe combined immunodeficiency) clinical trial using a gamma-retroviral vector that transferred a functional copy of the defective gene into hematopoietic precursor cells of affected children, sparked a debate about a ban on conventional gamma-retroviral vectors. This commentary summarizes the relevant data on this topic and concludes that there is no preclinical or clinical evidence as yet that SIN vectors, which self-inactivate the retroviral long terminal repeats (LTRs), will indeed show an improved safety profile. Conventional murine leukaemia virus (MLV) vectors can thus be used further in clinical gene therapy trials but require a thorough case-by-case risk-benefit analysis.     

Downstream processing of oncoretroviral and lentiviral gene therapy vectors

Retroviral vectors from both oncoretroviral and lentiviral origins have a great potential as gene delivery vehicles. A number of research groups have devoted considerable effort to the development of large-scale production strategies for retroviral vectors. However, the manufacturing of clinical-grade vectors for gene therapy, especially for in vivo applications, additionally requires scaleable purification strategies to remove the contaminants present in the harvested supernatants while preserving the functionality of the vectors. In this article, we review recent advances made in the field of downstream processing of retroviral vectors. The methods currently described in the literature for clarification, concentration and purification of retroviral vectors will be presented, with special emphasis on novel chromatography methods that open up the possibility to selectively and efficiently purify retroviruses on a large-scale. Problems associated with stability and quantification of retroviral particles will be outlined and future challenges will be discussed.     

Advances in retroviral transduction of hematopoietic stem cells for the gene therapy of atherosclerosis

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory disease that is the primary cause of morbidity and mortality in the developed world. Many studies have shown that macrophages and T-cells play critical roles in multiple aspects of the pathogenesis of the disease. Given that these cells are ultimately derived from bone marrow precursors, the concept of performing gene therapy for atherosclerosis through the retroviral transduction of hematopoietic stem cells has received much attention. This review will highlight recent advances that will help bring this goal closer. RECENT FINDINGS: The clinical application of retroviral gene transfer into hematopoietic stem cells has been hampered, in part, by the absence of vectors that can direct long-lasting, cell-type specific gene expression. In this review we will detail recent developments in the design of novel retroviral and lentiviral vectors that appear to overcome these problems, offering approaches to express therapeutic genes in specific cell-types within atherosclerotic lesions. We will also highlight advances in our understanding of the pathogenesis of atherosclerosis that may offer new gene therapeutic targets. SUMMARY: The use of retroviral transduction of hematopoietic stem cells for treatment of patients with atherosclerosis still remains a long-term goal. However, the recent development of retroviral vectors capable of directing expression to specific cell types within the lesion will allow more targeted therapeutic strategies to be devised. In addition, these vectors will provide powerful experimental tools to further our understanding of the pathogenesis of the disease.