Gene Therapy for Autoimmune Disorders

Although many autoimmune disorders do not have a strong genetic basis, their treatment may nevertheless be improved by gene therapies. Most strategies seek to transfer genes encoding immunomodulatory products that will alter host immune responses in a beneficial manner. Used in this fashion, genes serve as biological delivery vehicles for the products they encode. By this means gene therapy overcomes obstacles to the targeted delivery of proteins and RNA, and improves their efficacy while providing a longer duration of effect, and, potentially, greater safety. Additional genetic strategies include DNA vaccination and the ablation of selected tissues and cell populations. There is considerable evidence from animal studies that gene therapies work: examples include the treatment of experimental models of rheumatoid arthritis, multiple sclerosis, diabetes, and lupus. Pre-clinical success in treating animal models of rheumatoid arthritis has led to the first clinical trial of gene therapy for an autoimmune disease. In this Phase I study, a cDNA encoding the interleukin-1 receptor antagonist was transferred to the knuckle joints of patients with advanced rheumatoid arthritis. Two additional clinical trials are in progress. It is likely that gene therapy will provide effective new treatments for a wide range of autoimmune disorders.     

Optimal modes and targets of gene therapy in transplantation

Summary:  Genetic modification strategies have the potential to improve outcome following cell/organ transplantation. A unique opportunity in transplantation is that gene therapies need not be restricted to in vivo approaches and that ex vivo genetic modification of cell and/or organs can be of value. Improvements in vector design, production, and delivery should enhance transfection efficiency and optimize gene expression. Herein, we discuss potential modes of gene therapy, focusing on viral, liposome, or naked DNA-based systems for gene delivery. We suggest gene therapy targets taking into consideration the essential constituents of anti-allograft repertory. In addition to strategies that may have salutary effects in mitigating the threat of acute rejection, we suggest genetic strategies for minimizing ischemia/reperfusion injury as well as for the perennial problem of progressive functional loss of the transplanted organ. Data from pre-clinical transplant models support the idea that gene therapy may improve allograft function and survival. We are optimistic that gene therapy will be of clinical value in the near future in the management of recipients of allografts; we believe that genetic strategies would be essential for successful breaching of the formidable challenge of xenotransplantation.     

Gene therapy in peripheral nerve reconstruction approaches

Gene transfer to a transected peripheral nerve or avulsed nerve root is discussed to be helpful where neurosurgical peripheral nerve reconstruction alone will not result in full recovery of function. Axonal regeneration is supposed to be facilitated by this new therapeutic approach via delivery of specific regeneration promoting molecules as well as survival proteins for the injured sensory and motor neurons. Therefore gene therapy aims in long-term and site-specific delivery of those neurotrophic factors. This paper reviews methods and perspectives for gene therapy to promote functional recovery of severely injured and thereafter reconstructed peripheral nerves. Experimental in vivo and ex vivo gene therapy approaches are reported by different groups. In vivo gene therapy generally uses direct injection of cDNA vectors to injured peripheral nerves. Ex vivo gene therapy is based on the isolation of autologous cells followed by genetic modification of these cells in vitro and re-transplantation of the modified cells to the patient as part of tissue engineered nerve transplants. Vectors of different origin are published to be suitable for peripheral nerve gene therapy and this review discusses the different strategies with regard to their efficiency in gene transfer, their risks and their potential relevance for clinical application.     

非HLA 抗体在肾脏移植中作用的研究进展

肾移植术后产生的抗体与移植肾排斥反应密切相关,并影响移植肾长期存活。近年来非HLA 抗体在肾移植 领域越来越受到关注。尽管对非HLA 抗体的研究并不全面,然而实验及临床研究均发现针对某些非HLA 抗原诸如血管紧张 素1 类受体、基底膜聚糖、自身抗原等产生的抗体能够影响移植肾急性及慢性排斥反应的进程。非HLA 抗体影响移植肾排斥 反应及存活的潜在机制是目前研究的重要领域之一。本文针对肾移植领域主要的非HLA 抗体做一综述,并阐述其对移植物 的致病机制。鉴别出非HLA 介导的移植肾抗体性排斥反应的免疫表型,有利于为治疗移植肾抗体性排斥反应提供新的靶点, 并为提高移植物的长期存活提供有效策略。     

肾移植后急性排异反应12例

背景：同种异体肾移植后发生的急性排斥反应是移植肾功能减退和最终移植肾丧失的最主要原因之一。有效预防和早期发现与治疗急性排异反应是关系到肾脏移植患者能否长期存活的重要问题。 目的：总结肾移植后1个月内急性排异反应患者治疗过程中免疫抑制剂的应用体会。 方法：选择首次肾移植患者12例,移植后采用霉酚酸酯+环孢素A+甲泼尼龙三联预防排异反应。当肾移植后3~30 d内出现尿量减少、移植肾区胀痛不适、血肌酐升高、尿蛋白增加等不同临床表现,确诊为肾移植后急性排斥反应时,先选用甲强龙500 mg/d静脉滴注,连续3 d。然后改甲泼尼龙24 mg口服1次/d,每5~7 d递减4 mg,至8 mg/d维持。 结果与结论：12例患者成功逆转,其中6例甲强龙冲击疗法成功;不能逆转者选用抗胸腺细胞球蛋白或CD3治疗。4例经抗胸腺细胞球蛋白治疗患者中1例8h内尿量迅速增加,2例24 h内尿量迅速增加,1例72 h后尿量迅速增加;1例选用CD3治疗48 h内尿量迅速增加;1例将环孢素转换为他克莫司治疗,同时服用霉酚酸酯胶囊和甲泼尼龙片。经以上治疗12例患者肾功能逐渐恢复。提示肾移植后早期发现、早期诊断、及时治疗是急性排异反应成功逆转的关键。     

Mycophenolate Mofetil

Mycophenolate mofetil is the morpholinoethyl ester prodrug of mycophenolic acid, an uncompetitive reversible inhibitor of the rate-limiting enzyme in de novo purine synthesis, inosine monophosphate dehydrogenase. As an antimetabolite immunosuppressant, mycophenolate mofetil has been evaluated for the prevention and treatment of acute rejection of a variety of solid organ allografts. It is generally added to post-transplant therapy regimens in place of azathioprine, and in conjunction with cyclosporin and corticosteroids. In large, randomised controlled trials in renal and cardiac transplant recipients, mycophenolate mofetil has shown significant efficacy in reducing the incidence of acute rejection compared with azathioprine in the first year after transplantation. Whereas patient and graft survival rates are improved with mycophenolate mofetil in cardiac transplantation, significant benefits for patient or graft survival have not been demonstrated in clinical trials in renal transplant patients. Mycophenolate mofetil does, however, appear to reduce the incidence of renal graft loss due to rejection. Mycophenolate mofetil has been shown to reverse ongoing acute rejection episodes in heart, kidney and liver transplant patients, and to improve graft function when used to treat chronic lung or heart graft vasculopathy. The efficacy of mycophenolate mofetil immunosuppression appears to allow sparing of other immunosuppressive agents, particularly cyclosporin and corticosteroids, in selected patients. The main adverse effects associated with oral mycophenolate mofetil are GI events, haematological toxicity (especially leucocytopenia) and an increased incidence of some types of infections relative to placebo. Lower dosages (2 g/day) are generally better tolerated than higher dosages (3 g/day). Although mycophenolate mofetil carries a high acquisition cost relative to azathioprine, when other direct costs of treatment of organ transplant patients are considered, it appears to be a cost-effective alternative, at least during the first post-transplant year. CONCLUSIONS: The benefits in terms of a reduction in the morbidity associated with acute organ allograft rejection indicate that mycophenolate mofetil should be considered as part of a primary therapy regimen in renal and cardiac transplant recipients, and as a treatment for reversal of acute refractory rejection in these patients. Further study is required to confirm its benefits in the transplantation of other solid organs.     

Genetic engineering in allotransplantation of vascularized organs

Transplantation offers a unique opportunity for gene transfer into allografts before grafting. After organ retrieval, the cold ischemic period renders organs available for manipulation and gene transfer. Local expression of protective or immunomodulatory molecules within the graft environment offers a better local bioavailability of bioreagents and potentially less systemic side effects. Protection against ischemia-reperfusion injury, acute and/or chronic rejection without significant side effects would be a major breakthrough in transplant research. However, protocols of transfection adapted to the transplant setting and control of gene expression must be clearly evaluated before going to clinical trials. The first part of this review deals with gene transfer techniques into the allograft, emphasizing particular transplant conditions that are encountered and that must be respected when designing protocols for gene transfer experiments. The second part deals with specific therapeutic strategies to protect and prolong allograft survival.     

Association between a TGFBR2 Gene Polymorphism (rs2228048, Asn389Asn) and Acute Rejection in Korean Kidney Transplantation Recipients

Transforming growth factor-β (TGF-β) signaling transduction initiates TGF-β activation, resulting in activation of TGF-β receptor II (TGFBR2). Any quantitative and qualitative changes in TGFBR2 are expected to affect the TGF-β signaling pathway, which occupies a central position with respect to the regulation of cell growth, differentiation, apoptosis, immune reaction, angiogenesis and extracellular matrix formation. Recent studies have shown that TGF-β1 gene polymorphisms may confer susceptibility to early acute and chronic allograft rejection in kidney transplantation recipients. In this study, we assessed whether polymorphisms of the TGFBR2 gene were associated with susceptibility to kidney transplantation rejection. A total of 347 renal allograft recipients transplanted at three centers in Korea were analyzed. Three SNPs (rs764522, rs3087465, rs2228048) of the TGFBR2 gene were genotyped from genomic DNA with direct sequencing. Multiple logistic regression models (codominant, dominant, recessive, and log-additive) were performed to evaluate odds ratios (ORs), 95% confidence intervals (CIs), and p-values. A total of 63 patients (18%) developed acute rejection (AR). There were no significant differences in age, sex, number of HLA mismatches, cause of renal failure, or immunosuppressant regimen between the AR and non-AR group. The synonymous SNP rs2228048 was significantly associated with AR (p = 0.020 in recessive model, and p = 0.036 in log-additive model. The allele frequencies of rs2228048 were different between the AR and non-AR group (p = 0.026). These results suggest that the synonymous TGFBR2 gene SNP rs2228048 may be associated with the development of AR in Korean kidney transplantation recipients.     

Gene Therapy: The Potential Applicability of Gene Transfer Technology to the Human Germline

The theoretical possibility of applying gene transfer methodologies to the human germline is explored. Transgenic methods for genetically manipulating embryos may in principle be applied to humans. In particular, microinjection of retroviral vector appears to hold the greatest promise, with transgenic primates already obtained from this approach. Sperm-mediated gene transfer offers potentially the easiest route to the human germline, however the requisite methodology is presently underdeveloped. Nuclear transfer (cloning) offers an alternative approach to germline genetic modification, however there are major health concerns associated with current nuclear transfer methods. It is concluded that human germline gene therapy remains for all practical purposes a future possibility that must await significant and important advances in gene transfer technology.     

Nonviral gene therapy

The last 10 years have seen substantial progress in the development and application of nonviral vectors in gene therapy. Several novel nonviral methods have been developed that approach viruses with respect to transfection efficiency. A variety of nonviral delivery systems that can be used for gene therapy in different clinical settings are also available. In this review article, we will detail all of the major nonviral vectors that are currently used in gene therapy while highlighting some recent developments, particularly the progress towards the understanding of the cellular and in vivo barriers in gene transfer. Recent advancement in achieving sustained and regulated gene expression will also be addressed. Finally, this review will briefly cover targeted gene repair using nonviral delivery systems. Their impact in gene therapy will also be discussed.     

Deficiency of C4 from donor or recipient mouse fails to prevent renal allograft rejection

Complement effector products generated in the transplanted kidney are known to mediate transplant rejection, but which of the three main activation pathways of complement trigger this response is unclear. Here we assessed the role of the classical and lectin pathways by studying the common component C4 in mouse kidney transplant rejection. We transplanted wild-type or C4-null H-2(b) donor kidneys into H-2(k) or H-2(d) recipients, or vice-versa, to assess the roles of donor kidney and recipient expression of complement. Intragraft C4 gene expression rose substantially during rejection. However, we found no significant association between graft acceptance and the presence of C4 in either the donor kidney or recipient mouse. At the time of rejection, we found no significant differences in alloantibody response in the different groups. Tubular deposition of C3 to C9 occurred regardless of the absence or presence of C4 in either the donor or recipient mouse, indicating that C4 was dispensable for complement activation at this site. These data suggest that complement activation and renal allograft rejection are independent of the classical and lectin pathways in these models, implying that in the absence of these pathways the alternative pathway is the main trigger for complement-mediated rejection.     

趋化因子受体多态性与肾移植临床结果的关系

目的：探讨趋化因子受体CCR5-59029、CCR2-64I多态性与肾移植临床结果的关系。方法：应用PCR限制性片段长度多态性分析方法（PCR-RFLP）检测72例同种异体肾移植病人CCR5-59029、CCR2-64I基因多态性，并分析两个等位基因多态性与急性排斥反应和存活时间的关系。结果：本组16例发生急性排斥反应。25例CCR5-59029A／A基因型受者，9例发生急性排斥反应，排斥反应发生率明显增高。具有CCR2-64I基因型受者，急性排斥反应发生率无明显变化。CCR2-64I基因型受者平均存活时间长于非此基因型受者。结论：趋化因子CCR5-59029A／A基因型是急性排斥反应的高危因素。CCR2-64I与肾移植术后长期存活相关。     

Developments in Inhaled Immunosuppressive Therapy for the Prevention of Pulmonary Graft Rejection

Cyclosporin is the main immunosuppressive treatment for lung and heart-lung transplantation. When combined with azathioprine and oral corticosteroids, repeated episodes of acute rejection are limited to a minority of transplant patients. Despite early successful transplantation, many patients developed a disabling and fatal condition called obliterative bronchiolitis. This is currently thought to be a result of chronic rejection. The principal risk factor for the development of obliterative bronchiolitis is undercontrolled acute rejection in the first 3 months after transplantation. Frequent early acute rejection increases the later risk of death and disability. A new approach to immunosuppressive therapy is needed to prevent this complication. Simply increasing the dosage of cyclosporin or oral corticosteroids results in the major complications of opportunistic infection and renal failure. Targeted immunosuppressive treatment delivered to the transplanted organ may offer certain advantages, since a high topical inhaled dosage should be relatively free from systemic complications. The lung as a transplanted organ is easily accessible to targeted therapy by means of inhalation. Inhaled nebulised corticosteroids have been shown to be effective in preventing obliterative bronchiolitis in patients at risk after heart-lung transplantation. Similarly, inhaled cyclosporin has also been reported to be more effective than oral administration, with substantially lower blood concentrations. Such new approaches to targeting immunosuppressive treatment could have specific advantages in long term therapy of lung and heart-lung transplant recipients. They might also be of use in other types of solid organ transplantation.     

Plasmid-mediated muscle-targeted gene therapy for circulating therapeutic protein replacement: a tale of the tortoise and the hare?

There is now conclusive evidence that gene therapy can lead to real clinical benefit. Initial enthusiasm has been muted by set-backs related to viral vectors including retroviral oncogenesis and adenoviral inflammatory response. Plasmid-mediated muscle-targeted gene transfer offers the potential of a cost-effective pharmaceutical grade therapy delivered by simple intramuscular injection without the need for anaesthetic, cell culture, transplantation or immunosuppression. This approach is particularly appropriate for long-term circulating therapeutic protein replacement currently requiring repeated injection therapy. Wide-ranging clinical applications include haemophilia, chronic anaemia, growth hormone deficiency and diabetes. Inadequate transgene expression, unregulated protein delivery and immune response have been major limiting factors. Recent innovations including in situ electroporation enabling sustained systemic protein delivery within the therapeutic range are reviewed. Pharmacological and physiological approaches to regulation are discussed in addition to the role of innate and humoral immunity. Translation of advances in all of these areas to clinical success will enable muscle-targeted gene therapy to capitalise on its inherent strengths and realise its long-standing promise.     

Mycophenolate Mofetil

Mycophenolate mofetil (MMF) is an immunosuppressive drug designed to inhibit inosine monophosphate dehydrogenase (IMPDH). IMPDH is a key enzyme in the de novo purine synthesis of lymphocytes. It is crucially important for proliferative responses of human T and B lymphocytes. The inhibition of IMPDH thus leads to selective lymphocyte suppression. After successful use in various in vitro and animal models, MMF was brought to clinical trial in patients undergoing transplantation. The drug is rapidly and completely absorbed following oral administration. Pilot studies of administration with cyclosporin and corticosteroids suggested a significant reduction in the incidence of organ rejection at dosages of 1 to 3 g/day As a result of these studies, 3 pivotal randomised double-blind multicentre trials, involving nearly 1500 patients, were designed to investigate the effects of addition of MMF to different standard immunosuppressive protocols on the prevention of acute renal allograft rejection. After 6 months, the rates of biopsy-proven rejection were significantly reduced in patients receiving MMF. In combination with cyclosporin and corticosteroids, the adverse effect profile resembled that of azathioprine. Most adverse effects were associated with the gastrointestinal tract, the blood system and opportunistic infections. MMF offers improved immunosuppressive therapy following renal and probably other solid organ transplantation. MMF has been licensed since 1995 for the prevention of acute renal allograft rejection in most countries. It has been used in different combinations of immunosuppressive drugs and in various dosages and regimens.     

Immune responses to lentiviral vectors

Efficient delivery and sustained expression of a therapeutic gene into human tissues are the requisite to accomplish the high expectations of gene therapy. A major challenge has concerned development of gene transfer systems capable of efficient cell transduction and transgene expression without harm to the recipient. A lot of work has been done to demonstrate the efficacy of gene therapy in animal models that mimic situations in humans. Use of lentiviral vectors (LVs) offers multiple advantages for gene replacement therapy, because they combine efficient delivery, ability to transduce proliferating and resting cells, capacity to integrate into the host chromatin to provide stable long-term expression of the transgene, absence of any viral genes in the vector and absence of interference from preexisting viral immunity. However, one of the major barriers to stable gene transfer by LVs and other viral vectors is the development of innate and adaptive immune responses to the delivery vector and the transferred therapeutic transgene. Since this greatly hinders the therapeutical benefits of gene therapy by LVs, developing strategies to overcome the host immune response to the transfer vector and the transgene is a matter of current investigation.     

Fetal somatic gene therapy

Fetal somatic gene therapy is emerging as a new experimentalapproach, in particular to prevent irreversible perinatal diseasemanifestation for many inherited conditions. Early therapeuticgene application may also allow targeting of still expandingstem cell populations of organ or cell systems inaccessiblelater in life and help to avoid immune sensitization againstthe therapeutic vector system or transgene protein product.The progress in development of ultrasound scanning and embryofetoscopyover the last decade has made minimally invasive administrationof therapeutic gene transfer vectors to the fetus in utero possiblein principle. We review here the different considerations inchoosing candidate diseases, the possible routes of administrationand times in fetal development for application of a therapeuticgene and discuss the benefits and problems of present vectorsystems in this context. Given the many unknown aspects of fetalgene transfer, it is essential to extensively investigate thisnew approach to gene therapy in animal models for specific diseases,to improve on the technology of delivery and to assess efficacyof expression as well as the possible side effects before applicationto humans can be considered. animal models/DNA delivery system/fetal surgery/gene therapy/immunity     

A comprehensive non‐invasive framework for automated evaluation of acute renal transplant rejection using DCE‐MRI

The objective was to develop a novel and automated comprehensive framework for the non‐invasive identification and classification of kidney non‐rejection and acute rejection transplants using 2D dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI). The proposed approach consists of four steps. First, kidney objects are segmented from the surrounding structures with a geometric deformable model. Second, a non‐rigid registration approach is employed to account for any local kidney deformation. In the third step, the cortex of the kidney is extracted in order to determine dynamic agent delivery, since it is the cortex that is primarily affected by the perfusion deficits that underlie the pathophysiology of acute rejection. Finally, we use an analytical function‐based model to fit the dynamic contrast agent kinetic curves in order to determine possible rejection candidates. Five features that map the data from the original data space to the feature space are chosen with a k‐nearest‐neighbor (KNN) classifier to distinguish between acute rejection and non‐rejection transplants. Our study includes 50 transplant patients divided into two groups: 27 patients with stable kidney function and the remainder with impaired kidney function. All of the patients underwent DCE‐MRI, while the patients in the impaired group also underwent ultrasound‐guided fine needle biopsy. We extracted the kidney objects and the renal cortex from DCE‐MRI for accurate medical evaluation with an accuracy of 0.97 ± 0.02 and 0.90 ± 0.03, respectively, using the Dice similarity metric. In a cohort of 50 participants, our framework classified all cases correctly (100%) as rejection or non‐rejection transplant candidates, which is comparable to the gold standard of biopsy but without the associated deleterious side‐effects. Both the 95% confidence interval (CI) statistic and the receiver operating characteristic (ROC) analysis document the ability to separate rejection and non‐rejection groups. The average plateau (AP) signal magnitude and the gamma‐variate model functional parameter α have the best individual discriminating characteristics. Copyright © 2013 John Wiley & Sons, Ltd.     

Current status of genetic modulation of growth factors in wound repair

Growth factors are members of a large functional group of polypeptide regulatory molecules secreted by different cells. They are important players in orchestrating all stages of wound healing exerting their influence through autocrine and paracrine fashions within sites of injury and repair. They are mitogen, chemotactic, they regulate cell-cell interactions and influence synthesis and composition of extracellular matrix components. The use of growth factors to stimulate wound healing is a promising therapeutic approach to repair chronic tissue defects. The delivery of genetic material offers an attractive treatment modality to produce an appropriate amount of growth factor proteins within the wound site. Gene therapy might become a significant treatment modality for those wound healing pathologies refractory to other wound management approaches. This review discusses several methods of growth factor gene transfer into wound tissue.