RNA repair for haemophilia A

The mainstay of gene transfer studies is the use of wild-type cDNAs to effect phenotypic correction of diseases. However, this strategy is not feasible for genetic diseases caused either by mutations of large genes or by dominant-negative mutations, or where the regulation of the gene is critical. In this review, we will discuss a novel RNA reprogramming strategy - spliceosome-mediated RNA trans-splicing - where the pre-messenger RNA is modified by the splicing of two independent RNA species. The use of trans-splicing to effect phenotypic change in the hereditary bleeding disorder haemophilia A will be discussed.     

Recent advances in gene therapy and future prospects

Gene therapy, which is treatment of diseases by introducing normal genes into the body, is becoming feasible as the result of advances in genetic engineering. The hematopoietic stem cells have been considered as the appropriate target for gene transfer in many genetic diseases for which allogeneic bone marrow transplantation has been employed successfully. However, there are still many problems to be solved. In particular, expression from retrovirally transduced genes in bone marrow cells has been transient and unstable. On the other hand, an alternative approach to somatic cell gene therapy using nonhematopoietic cells, including skin fibroblasts, endothelial cells, keratinocytes, and lymphocytes, has been shown to possess several advantages. This kind of approach is usually applied to supplementation therapy in not only hereditary disorders but also various acquired diseases, such as cancer or infectious diseases. Recently, clinical application of gene transfer into lymphocytes to treat cancer and immunodeficiency have been approved at NIH (USA). The trial could represent the start of a new era in molecular medicine.     

Protection and selection for gene therapy in the hematopoietic system

Hematopoietic stem cell gene therapy is potentially curative for a number of inherited and acquired disorders. However, poor gene transfer and expression in repopulating hematopoietic stem cells attenuate this potential. Here we review potential means of conferring a selective advantage to hematopoietic stem cells and their progeny, and discuss the issues that surround the use of selective advantages in vivo.     

Bone marrow transplantation beyond treatment of aplasia and neoplasia.

Marrow transplantation has proved to be an important modality in the treatment of aplastic anemia, acute leukemia, and some other malignant diseases of hemopoietic cells. Much less attention has been paid to the role of marrow transplantation in the treatment of stem cell disorders such as sickle cell disease, chronic granulomatous disease, or Gaucher''s disease. Allogeneic transplantation is successful in these disorders, but carries a considerable risk to the recipient. Autologous transplantation becomes a reality when efficient gene transfer with sustained expression is developed. As methods are developed to harvest hemopoietic stem cells from the peripheral blood and to amplify these cells without causing them to differentiate, transplantation will become increasingly valuable in the treatment of stem cell disorders.     

In pursuit of new developments for gene therapy of human diseases

Gene therapy aims at transferring a therapeutic gene into human somatic cells in order to treat a disease. Originally addressed to hereditary genetic disorders, gene therapy has found therapeutic applications in cancer, infectious diseases and degenerative disorders, particularly those of the nervous system. Although gene transfer into humans has been demonstrated in several clinical trials, with more than 300 currently underway worldwide, there is still no single outcome that undoubtedly showed a consistent benefit for the patient. Nevertheless, the expectations for gene therapy are still high, and the prospects of future clinical success are increasing together with the growing of the field. The development of better delivery systems specifically tailored to individual diseases, with sustained expression of the therapeutic gene in the appropriate cells, will in the end make possible true therapeutic applications of human gene transfer.     

Lentiviral Vector-Mediated Gene Transfer and RNA Silencing Technology in Neuronal Dysfunctions

Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson’s disease, or in investigating gene function in Huntington’s disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition.     

Gene Transfer Approaches to the Lysosomal Storage Disorders

The work summarized in this paper used animal and cell culture models systems to develop gene therapy approaches for the lysosomal storage disorders. The results have provided the scientific basis for a clinical trial of gene transfer to hematopoietic stem cells (HSC) in Gaucher disease which is now in progress. The clinical experiment is providing evidence of HSC transduction, competitive engraftment of genetically corrected HSC, expression of the GC transgene, and the suggestion of a clinical response. In this paper we will review the progress made in Gaucher disease and include how gene transfer might be studied in other lysosomal storage disorders.     

Myosin-Va-Dependent Cell-To-Cell Transfer of RNA from Schwann Cells to Axons

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.     

牛雄性生殖系干细胞的体外诱导分化

雄性生殖系干细胞(Male germ-line stem cells, mGSCs)是一群具有高度自我更新能力和分化潜能的细胞, 是雄性成体内唯一可复制的二倍体永生细胞。转基因技术与雄性生殖系干细胞异体及异种移植技术相结合, 将会为克隆动物、转基因动物生产及一些人类遗传性疾病的基因治疗提供新的机遇与途径。本试验采用组合酶消化和选择贴壁法, 对5月龄、6月龄牛胎儿及新生牛雄性生殖系干细胞体外培养及分化进行了研究。试验结果显示, 睾丸支持细胞对雄性生殖系干细胞体外增殖、分化所必需的, 同时对数期睾丸支持细胞对雄性生殖系干细胞贴壁、增殖与分化效果明显; 共培养16 d后, 牛雄性生殖系干细胞分化为长形精子细胞, 试验建立了牛雄性生殖系干细胞体外诱导培养分化体系。     

慢病毒载体介导RNAi的研究进展

RNAi通过双链RNA的介导,特异性阻抑相关序列的表达,从而导致转录后水平的基因沉默.广泛存在于真菌、植物和动物等真核生物中.慢病毒载体是理想的真核细胞基因转移工具,被广泛应用于相关的RNAi研究领域,例如抗病毒研究、癌症及其治疗、遗传性疾病的治疗、基因治疗.现已发现,慢病毒载体能够介导组织特异、时间特异的RNAi,在疾病的基因靶向性治疗上必有广阔的前景.     

Messenger RNA electroporation: an efficient tool in immunotherapy and stem cell research

Over the last decades medicine has developed tremendously, but still many diseases are incurable. The last years, cellular (gene) therapy has become a hot topic in biomedical research for the potential treatment of cancer, AIDS and diseases involving cell loss or degeneration. Here, we will focus on two major areas within cellular therapy, cellular immunotherapy and stem cell therapy, that could benefit from the introduction of neo-expressed genes through mRNA electroporation for basic research as well as for clinical applications. For cellular immunotherapy, we will provide a state-of-the-art on loading antigen-presenting cells with antigens in the mRNA format for manipulation of T cell immunity. In the area of stem cell research, we will highlight current gene transfer methods into adult and embryonic stem cells and discuss the use of mRNA electroporation for controlling guided differentiation of stem cells into specialized cell lineages.     

Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscle-derived stem cells: potential for muscle regeneration

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disease characterized by progressive muscle weakness caused by the lack of dystrophin expression at the sarcolemma of muscle fibers. Although various approaches to delivering dystrophin in dystrophic muscle have been investigated extensively (e.g., cell and gene therapy), there is still no treatment that alleviates the muscle weakness in this common inherited muscle disease. The transplantation of myoblasts can enable transient delivery of dystrophin and improve the strength of injected dystrophic muscle, but this approach has various limitations, including immune rejection, poor cellular survival rates, and the limited spread of the injected cells. The isolation of muscle cells that can overcome these limitations would enhance the success of myoblast transplantation significantly. The efficiency of cell transplantation might be improved through the use of stem cells, which display unique features, including (1) self-renewal with production of progeny, (2) appearance early in development and persistence throughout life, and (3) long-term proliferation and multipotency. For these reasons, the development of muscle stem cells for use in transplantation or gene transfer (ex vivo approach) as treatment for patients with muscle disorders has become more attractive in the past few years. In this paper, we review the current knowledge regarding the isolation and characterization of stem cells isolated from skeletal muscle by highlighting their biological features and their relationship to satellite cells as well as other populations of stem cells derived from other tissues. We also describe the remarkable ability of stem cells to regenerate skeletal muscle and their potential use to alleviate the muscle weakness associated with DMD.     

Hematopoietic stem cell gene therapy for Niemann-Pick disease and other lysosomal storage diseases

Of the various gene therapy approaches under investigation for the treatment of genetic diseases, hematopoietic stem cell-mediated gene therapy has attracted the most interest. Enriched populations of hematopoietic stem cells can be obtained from diseased individuals, genetically modified to express normal gene products, and then transplanted back into these individuals without the risk of graft versus host disease. Following transplantation and engraftment, hematopoietically-derived cells can repopulate various sites of pathology and express the normal gene product in vivo. Such a procedure has been accomplished in several mouse models of human genetics diseases, leading to partial or complete correction of the disease phenotype, and current efforts are now focused on adapting the success of murine systems to larger animals, including man. This review will focus on the use of hematopoietic stem cell-mediated gene therapy for the treatment of lysosomal storage disorders, and discuss recent data obtained in the laboratory using a murine knock-out mouse model of Types A and B Niemann-Pick disease (NPD).     

转基因动物新技术研究进展

转基因技术经过数十年的发展日渐成熟,并推动转基因技术研究进入一个新的发展阶段。文章综述了体细胞核移植技术、慢病毒载体法、转座子介导的基因转移法、RNA干扰介导的基因敲除法和锌指核酸酶-基因打靶技术等近年发展起来的方法。而近来诱导多能干细胞(iPS细胞)的成功为尚未获得ES细胞的大动物建立多能干细胞系提供了一种新的方案,为转基因动物研究开创一片更广阔的天地。文章在前人研究的基础上重点总结了以上各种转基因技术的最新研究动态并对各种转基因技术的特点进行了探讨。     

遗传物质的横向传递与转基因植物的安全性

横向传递是在同种或异种生物不同个体之间沿水平方向进行遗传物质的单方向转移,有多种不同的转移方式。在生物界中,遗传物质的横向传递通常是借助某种载体如病毒来完成,高等生物还可以通过有性生殖在同种生物不同个体之间或异种生物不同个体之间传递遗传物质。基因的横向传递是普遍存在的,是生物进化的重要动力之一。转基因植物是人工遗传物质横向转移的结果,人工遗传物质横向转移正在越来越明显地影响着生物的生存状态。     

The use of patient-specific stem cells in different autoimmune diseases

Autoimmune diseases are developed when the immune system mistakenly attacks the body’s cells. These inflammatory disorders can be inherited or triggered by external forces, such as type 1 diabetes, which is caused by the immune system's destruction of pancreatic beta cells. So far, stem cells such as hESC and iPSC have been used to treat autoimmune disorders such as type 1 diabetes, rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE), although these procedures have certain ethical concerns. On the other hand, bone marrow-derived mesenchymal stem cells (BM-MSC) are thought to be the best source of stem cells. Later, it was shown that mesenchymal stem cells produced from autologous adipose tissues have a great potential for producing huge volumes of stem cells. In-vitro and in-vivo investigations using autologous hematopoietic stem cells and autologous mesenchymal stem cells have been carried out on various rodent and human models, while clinical trials for inflammatory diseases such as multiple sclerosis and diabetes mellitus have yielded promising results. We attempted to summarise the usage of diverse stem cells in the therapy of various autoimmune disorders in this review. Shortly, we expect that the use of autologous stem cells will provide a new perspective on the treatment of autoimmune disorders.     

Cutaneous gene transfer and therapy: the present and the future

The easy accessibility of the skin as a therapeutic target provides an exciting potential for this organ for the development of gene therapy protocols for cutaneous diseases and a variety of metabolic disorders. Thus far, full phenotypic reversion of a diseased phenotype has been achieved in vivo for junctional epidermolysis bullosa and X-linked or lamellar ichthyosis and in vitro for xeroderma pigmentosum. These recessive skin diseases are characterized by skin blistering, abnormalities in epidermal differentiation and increased development of skin cancers, respectively. Corrective gene delivery at both molecular and functional levels was achieved by transduction of cultured skin cells using retroviral vectors carrying the specific curative cDNA. These positive results should prompt clinical trials based on transplantation of artificial epithelia reconstructed ex vivo using genetically modified keratinocytes. Promising results have also been obtained in phenotypic reversion of cells isolated from patients suffering from a number of metabolic diseases such as gyrate atrophy, familial hypercholesterolemia or phenylketonuria. In these diseases transplantation of autologous artificial epithelia expressing the transgenes of interest or direct transfer of the DNA to the skin represents a potential therapeutic approach for the systemic delivery of active molecules. Successful cutaneous gene therapy trials, however, require development of protocols for efficient gene transfer to epidermal stem cells, and information about the host immune response to the recombinant polypeptides produced by the implanted keratinocytes. The availability of spontaneous animal models for genodermatoses will validate the gene therapy approach in preclinical trials.     

Evaluation of optimal concentration and exposure duration of valproic acid alone or in combination with ViraDuctin to augment adenovirus transduction in human adipose stem cells

Background aimsRecombinant adenoviruses have tremendous potential in both gene therapy research and therapeutic applications. Mesenchymal stromal cells have a set of several properties that make them ideally suited for both regenerative medicine and gene and drug delivery. A limitation of adenoviral-mediated gene transfer is indeed the poor transduction rate of cells with low or no levels of the specific adenoviral cell surface receptor coxsackie virus and adenovirus receptor (CAR), such as human mesenchymal stromal cells. In the present work, we tried to increase the adenovirus transduction level and mediated gene delivery of human adipose stem cells with the use of valproic acid (VPA) and determined the proper concentration and duration of treatment alone or in combination with ViraDuctin adenovirus transduction reagent.MethodsGreen fluorescent protein–expressing recombinant adenovirus was propagated. The effects of various doses and exposure periods of VPA on CAR expression in human adipose stem cells were speculated by quantitative real-time polymerase chain reaction and adenoviral transduction rate by flow cytometry in different doses and time intervals of VPA and in combination with ViraDuctin transduction reagent.ResultsCAR messenger RNA upregulation through VPA was observed in human adipose stem cells; it was a dependent factor of dose and exposure time. Consequently, adenoviral transduction level of human adipose stem cells treated with VPA was increased, and co-administration of VPA and ViraDuctin further enhanced the transduction rate.ConclusionsThese results confirm that addition of VPA to hASCs alone or in combination with ViraDuctin has enhancing effects on adenoviral transduction rate, which can be auspicious in adenoviral-mediated gene therapy.