Developing protocols for recombinant adeno-associated virus-mediated gene therapy in space

With the advent of the era of International Space Station (ISS) and Mars exploration, it is important more than ever to develop means to cure genetic and acquired diseases, which include cancer and AIDS, for these diseases hamper human activities. Thus, our ultimate goal is to develop protocols for gene therapy, which are suitable to humans on the earth as well as in space. Specifically, we are trying to cure the hemoglobinopathies, beta-thalassemia (Cooley's anemia) and sickle cell anemia, by gene therapy. These well-characterized molecular diseases serve as models for developing ex vivo gene therapy, which would apply to other disorders as well. For example, the procedure may become directly relevant to treating astronauts for space-anemia, immune suppression and bone marrow derived tumors, e.g. leukemia. The adeno-associated virus serotype 2 (AAV2) is a non-pathogenic human parvovirus with broad host-range and tissue specificity. Exploiting these characteristics we have been developing protocols for recombinant AAV2 (rAAV)-based gene therapy. With the rAAV constructs and hematopoietic stem cell (HSC) culture systems in hand, we are currently attempting to cure the mouse model of beta-thalassemia [C57BL/6- Hbbth/Hbbth, Hb(d-minor)] by HSC transplantation (HST) as well as by gene therapy. This paper describes the current status of our rAAV-gene therapy research.     

Kinetics of recombinant adeno-associated virus-mediated gene transfer

Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IX (hFIX) gene transfer into human primary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduction, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold higher expression than that with a muscle-specific creatine kinase enhancer linked to beta-actin promoter. Factor IX expression from transduced myoblasts as well as myotubes reached levels as high as approximately 2 microgram of hFIX/10(6) cells/day. Southern blot analyses of high-molecular-weight (HMW) cellular genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells showed that the conversion of single-stranded vector genomes to double-stranded DNA forms, but not the level of the integrated forms in HMW DNA, correlated with increasing expression of the transgene. Together, these results indicate that rAAV can transduce both proliferating and terminally differentiated muscle cells at about the same efficiency, that expression of transgenes increases linearly over their lifetime with no initial lag phase, and that increasing expression correlates with the appearance of double-stranded episomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained.     

Efficient adeno-associated virus-mediated gene expression in human placenta-derived mesenchymal cells

Mesenchymal cells from various sources are pluripotent and are attractive sources for cell transplantation. In this study, we analyzed recombinant adeno-associated virus (rAAV)-mediated gene expression in human placenta-derived mesenchymal cells (hPDMCs), which reside in placental villi. After transduction of AV-CAG-EGFP, a rAAV expressing enhanced green fluorescence protein (EGFP), hPDMCs showed much higher level of EGFP expression than human umbilical vein endothelial cells or rat aortic smooth muscle cells. The number of EGFP-positive hPDMCs infected by AV-CAG-EGFP alone did not increase significantly by coinfection of adenovirus, which enhanced expression level of the rAAV vector. Moreover, flow cytometric analysis showed discrete positive fraction of EGFP-expressing hPDMCs, which is about 15-20% of the cells infected with AV-CAG-EGFP. Therefore, some cell population in hPDMCs might be highly susceptible to rAAV-mediated gene transduction. In addition, stable EGFP expressions were observed in about 1% of hPDMCs infected with AV-CAG-EGFP at 4 weeks post-infection. Collectively, hPDMCs have characters favorable for rAAV-mediated gene expression.     

Suppression of autoimmune diabetes by viral IL-10 gene transfer

Th1 cell activation and cytokine production shift the balance between Th1 and Th2, favoring the up-regulation of proinflammatory activity that leads to destruction of insulin-producing pancreatic beta cells in type 1 diabetes. Th2-type cytokines, such as IL-10, have immune regulatory function. Administration of IL-10, or IL-10 gene transfer, prevents autoimmune diabetes in nonobese diabetic (NOD) mice. However, constant administration of purified rIL-10 is not practical for long-term therapy to prevent diabetes. In this study, we transferred the BCRF-1 gene, an open reading frame in the Epstein-Barr viral genome with remarkable homology to mouse IL-10 (viral IL-10 or vIL-10), by an adeno-associated viral (AAV) vector to NOD mice to attain sustained vIL-10 gene expression. Like endogenous mouse IL-10, vIL-10 has potent immunoregulatory and immunosuppressive functions, but can be specifically distinguished from endogenous mouse IL-10 for monitoring of the transgene expression. A single systemic administration of AAV vIL-10 significantly reduced insulitis and prevented diabetes development in NOD mice. This protective effect correlated with sustained transgene expression and protein production. Moreover, splenocytes from the treated mice blocked diabetes transfer to NOD recipients, suggesting that vIL-10 induces an active suppression of autoimmunity. This study provides evidence to support the possibility of using vIL-10 gene therapy to prevent type 1 diabetes.     

降钙素基因相关肽转基因预防小鼠自身免疫性糖尿病发病及其抗氧化应激机制

有证据表明,活性氧(reactive oxygens pecies,ROS)参与了胰岛β细胞自身免疫损伤,是自身免疫性糖尿病发病的重要原因之一。一氧化氮(NO)和过氧化氢(H2O2)介导了致炎性细胞因子对胰岛B细胞的损害,引起脂质过氧化反应,进而损伤胰岛细胞。降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在心肌细胞中可抑制ROS生成而具有细胞保护作用。通过CGRP裸质粒肌肉注射体细胞电针强化转基因方法,使骨骼肌持续表达CGRP,观察其对小剂量多次注射链脲佐菌素(streptozotocin,STZ)造成的小鼠自身免疫性糖尿病发病的影响,进一步测定胰腺局部活性氧含量以及抗氧化酶的改变,探讨其抗氧化应激机制。结果发现,CGRP裸质粒直接注射入小鼠双后肢胫前肌。继以程控电针刺激导入(体内电穿孔法),可使血浆和骨骼肌组织CGRP表达水平显著增高,且持续4周以上;注射STZ同时给予CGRP转基因治疗可减轻胰岛β细胞损伤,显著降低自身免疫性糖尿病的发病率及血糖水平;CGRP转基因可显著抑制自身免疫性糖尿病小鼠胰腺局部活性氧和丙二醛的生成,增加过氧化氢酶(CAT)及超氧化物歧化酶(superoxide dismutase,SOD)的活性。结果提示,CGRP裸质粒直接注射、电针辅助导人转基因可获得CGRF持续高水平的表达,能够预防小鼠自身免疫性糖尿病的发病,其机制之一可能为CGRP抑制了活性氧对胰岛β细胞的损伤。     

Adeno-associated virus-mediated antiapoptotic gene delivery: in vivo gene therapy for neurological disorders

Apoptosis is an important mechanism of physiological and pathological cell death and is known to occur in various neurological disorders. Apoptosis is associated with activation of genetic programs in which apoptosis-effector genes promote cell death, thereby opposing repressor genes that enhance cell survival. In this review, we describe various apoptotic pathways, with a special reference to the caspase cascade and discuss the role of individual antiapoptotic factors in various target diseases. Apoptosis could be suppressed by in vivo gene delivery of antiapoptotic factors directly into the central nervous system. The adeno-associated virus (AAV) vector is a good candidate for such gene therapy because it can infect postmitotic neurons. We also describe our in vivo system for overexpression of apoptotic protease activating factor-1 (Apaf-1) caspase recruitment domain as an Apaf1-dominant negative inhibitor (Apaf-1-DN) to regulate the mitochondrial caspase cascade. Apaf-1-DN delivery using an AAV vector system inhibited mitochondrial apoptotic signaling pathway and prevented dopaminergic cell death in a mouse model of Parkinson's disease. Our results suggest that AAV-Apaf-1-DN is potentially useful as an antimitochondrial apoptotic gene therapy for neurodegenerative disorders such as Parkinson's disease.     

Evaluation of cytokine toxicity induced by vaccinia virus-mediated IL-2 and IL-12 antitumour immunotherapy

Single intratumoural treatment of nude mice with a vaccinia virus (VV)-expressing interleukin-1 (IL-2) or IL-12 induced significant tumour growth inhibition associated with clear signs of toxicity. At a low virus dose, only some treated animals showed signs of toxicity. We characterized and compared the activity of NK and B cells and major pro-inflammatory factors (IFN-gamma, TNF-alpha) in treated animals with and without toxicity. One week after treatment animals exhibiting signs of cytokine-related toxicity showed dramatic increases in several measured parameters. High leukocyte and lymphocyte counts in blood and marked increases in NK and CD25(+)cells in both blood and spleen were associated with IL-2-induced toxicity, while IL-12-induced toxicity was related to a great elevation of CD25(+)cells in blood and CD71(+)cells in the spleen. In contrast, immune activation in animals free of toxicity was observed on day 2 after the treatment, which drastically declined by day 7. Thus, immune responses induced by IL-2 and IL-12 therapy appear to play important roles in both tumour inhibition and the accompanying toxicity. Short-term effects induced by IL-2 and IL-12 could be critical for antitumour therapy that prolongs survival and protects from adverse side effects.     

Long-term rescue of a lethal inherited disease by adeno-associated virus-mediated gene transfer in a mouse model of molybdenum-cofactor deficiency

Molybdenum cofactor (MoCo) deficiency is a progressive neurological disorder that inevitably leads to early childhood death because of the lack of any effective therapy. In a mouse model of MoCo deficiency type A, the most frequent form of this autosomal recessively inherited disease, the affected animals show the biochemical characteristics of sulphite and xanthine intoxication and do not survive >2 wk after birth. We have constructed a recombinant-expression cassette for the gene MOCS1, which, via alternative splicing, facilitates the expression of the proteins MOCS1A and MOCS1B, both of which are necessary for the formation of a first intermediate, cyclic pyranopterin monophosphate (cPMP), within the biosynthetic pathway leading to active MoCo. A recombinant adeno-associated virus (AAV) vector was used to express the artificial MOCS1 minigene, in an attempt to cure the lethal MOCS1-deficient phenotype. The vector was used to transduce Mocs1-deficient mice at both 1 and 4 d after birth or, after a pretreatment with purified cPMP, at 40 d after birth. We report here that all Mocs1-deficient animals injected with a control AAV-enhanced green fluorescent protein vector died approximately 8 d after birth or after withdrawal of cPMP supplementation, whereas AAV-MOCS1-transduced animals show significantly increased longevity. A single intrahepatic injection of AAV-MOCS1 resulted in fertile adult animals without any pathological phenotypes.     

Plasmid vaccination with insulin B chain prevents autoimmune diabetes in nonobese diabetic mice

The insulin B (InsB) chain bears major type 1 diabetes-associated epitopes of significance for disease in humans and nonobese diabetic (NOD) mice. Somatic expression of InsB chain initiated early in life by plasmid inoculation resulted in substantial protection of female NOD mice against disease. This was associated with a T2 shift in spleen, expansion of IL-4-producing and, to a lesser extent, of IFN-gamma-secreting T cells in pancreatic lymph nodes, as well as intermolecular Th2 epitope spreading to glutamic acid decarboxylase determinants. A critical role of IL-4 for the Ag-specific protective effect triggered by plasmid administration was revealed in female IL-4(-/-) NOD mice that developed diabetes and higher Th1 responses. Coadministration of IL-4-expressing plasmid or extension of the vaccination schedule corrected the unfavorable response of male NOD mice to DNA vaccination with InsB chain. Thus, plasmid-mediated expression of the InsB chain early in diabetes-prone mice has the potential to prevent transition to full-blown disease depending on the presence of IL-4.     

The natural killer T-cell ligand alpha-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice

Diabetes in non-obese diabetic (NOD) mice is mediated by pathogenic T-helper type 1 (Th1) cells that arise because of a deficiency in regulatory or suppressor T cells. V alpha 14-J alpha 15 natural killer T (NKT) cells recognize lipid antigens presented by the major histocompatibility complex class I-like protein CD1d (refs. 3,4). We have previously shown that in vivo activation of V alpha 14 NKT cells by alpha-galactosylceramide (alpha-GalCer) and CD1d potentiates Th2-mediated adaptive immune responses. Here we show that alpha-GalCer prevents development of diabetes in wild-type but not CD1d-deficient NOD mice. Disease prevention correlated with the ability of alpha-GalCer to suppress interferon-gamma but not interleukin-4 production by NKT cells, to increase serum immunoglobulin E levels, and to promote the generation of islet autoantigen-specific Th2 cells. Because alpha-GalCer recognition by NKT cells is conserved among mice and humans, these findings indicate that alpha-GalCer might be useful for therapeutic intervention in human diseases characterized by Th1-mediated pathology such as Type 1 diabetes.     

Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation

Adeno-associated virus (AAV) is so far the most valuable vehicle for gene therapy because it has no association with immune response and human disease. The present study was conducted to investigate the feasibility of AAV-mediated BMP4 gene transfer for bone formation. In vitro study suggested that AAV-BMP4 vectors could transduce myoblast C2C12 cells and produce osteogenic BMP4. In vivo study demonstrated that new bone formation could be induced by direct injection of AAV-BMP4 into the skeletal muscle of immunocompetent rats. Histological analysis revealed that the newly formed bone was induced through endochondral mechanism. Immunohistochemical staining further demonstrated that AAV-BMP4 gene delivery could mediate long-term transduction, and the involvement of BMP4 expression was responsible for the endochondral ossification. This study is, to our knowledge, the first report in the field of AAV-based BMP gene transfer and should be promising for clinical orthopaedic applications.     

IL-17 silencing does not protect nonobese diabetic mice from autoimmune diabetes

The long-held view that many autoimmune disorders are primarily driven by a Th1 response has been challenged by the discovery of Th17 cells. Since the identification of this distinct T cell subset, Th17 cells have been implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. Type 1 diabetes has also long been considered a Th1-dependent disease. In light of the emerging role for Th17 cells in autoimmunity, several recent studies investigated the potential of this subset to initiate autoimmune diabetes. However, direct evidence supporting the involvement of Th17 cells in actual pathogenesis, particularly during spontaneous onset, is lacking. In this study, we sought to directly address the role of IL-17, the cytokine by which Th17 cells are primarily characterized, in the pathogenesis of autoimmune diabetes. We used lentiviral transgenesis to generate NOD mice in which IL-17 is silenced by RNA interference. The loss of IL-17 had no effect on the frequency of spontaneous or cyclophosphamide-induced diabetes. In contrast, IL-17 silencing in transgenic NOD mice was sufficient to reduce the severity of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, consistent with reports that IL-17 deficiency is protective in this experimental model of multiple sclerosis. We concluded that IL-17 is dispensable, at least in large part, in the pathogenesis of autoimmune diabetes.     

Bronchoalveolar fluid is not a major hindrance to virus-mediated gene therapy in cystic fibrosis

Successfully targeting the airway epithelium is essential for gene therapy of some pulmonary diseases. However, the airway epithelium is resistant to virus-mediated gene transfer with commonly used vectors. Vectors that interact with endogenously expressed receptors on the apical surface significantly increase gene transfer efficiency. However, other endogenous components involved in host immunity may hinder virus-mediated gene transfer. We tested the effect of bronchoalveolar lavage liquid (BAL) from patients with cystic fibrosis (CF), BAL from subjects without CF (non-CF BAL), Pseudomonas aeruginosa-derived proteins, and an array of inflammatory proteins on gene transfer mediated by adeno-associated virus type 5 (AAV5) and adenovirus targeted to an apically expressed glycosylphosphatidylinositol-modified coxsackie-adenovirus receptor. We found that neither CF BAL nor its components had a significant effect on gene transfer to human airway epithelium by these vectors. Non-CF BAL significantly impaired adenovirus-mediated gene transfer. Removal of immunoglobulins in non-CF BAL restored gene transfer efficiency. As virus vectors are improved and mechanisms of humoral immunity are elucidated, barriers to successful gene therapy found in the complex environment of the human lung can be circumvented.     

Structure of adeno-associated virus serotype 8, a gene therapy vector

Adeno-associated viruses (AAVs) are being developed as gene therapy vectors, and their efficacy could be improved by a detailed understanding of their viral capsid structures. AAV serotype 8 (AAV8) shows a significantly greater liver transduction efficiency than those of other serotypes, which has resulted in efforts to develop this virus as a gene therapy vector for hemophilia A and familial hypercholesterolemia. Pseudotyping studies show that the differential tissue tropism and transduction efficiencies exhibited by the AAVs result from differences in their capsid viral protein (VP) amino acids. Towards identifying the structural features underpinning these disparities, we report the crystal structure of the AAV8 viral capsid determined to 2.6-A resolution. The overall topology of its common overlapping VP is similar to that previously reported for the crystal structures of AAV2 and AAV4, with an eight-stranded beta-barrel and long loops between the beta-strands. The most significant structural differences between AAV8 and AAV2 (the best-characterized serotype) are located on the capsid surface at protrusions surrounding the two-, three-, and fivefold axes at residues reported to control transduction efficiency and antibody recognition for AAV2. In addition, a comparison of the AAV8 and AAV2 capsid surface amino acids showed a reduced distribution of basic charge for AAV8 at the mapped AAV2 heparin sulfate receptor binding region, consistent with an observed non-heparin-binding phenotype for AAV8. Thus, this AAV8 structure provides an additional platform for mutagenesis efforts to characterize AAV capsid regions responsible for differential cellular tropism, transduction, and antigenicity for these promising gene therapy vectors.     

Absence of IL-4, and not suppression of the Th2 response,prevents development of experimental autoimmune Graves' disease

In autoimmune Graves' disease (GD), autoantibodies bind to the thyrotropin receptor (TSHR) and cause hyperthyroidism. We studied the effects of fms-like tyrosine kinase receptor 3 ligand (Flt3-L) or GM-CSF treatment on the development of experimental autoimmune GD (EAGD) in mice, a slowly progressing Ab-mediated organ-specific autoimmune disease of the thyroid induced by immunization with syngeneic cells expressing TSHR. Flt3-L and GM-CSF treatment resulted in up-regulation of CD8a(+) and CD8a(-) dendritic cells, and skewing of cytokine and immune responses to TSHR in favor of Th1 and Th2, respectively. However, this skewing did not persist until the later stages, and thus failed to affect the course or severity of the disease. To determine whether the total absence of either IL-4 or IFN-gamma could affect the development of EAGD, we immunized wild-type, IFN-gamma(-/-) and IL-4(-/-) BALB/c mice with TSHR. Nearly 100% of the wild-type and IFN-gamma(-/-) mice developed EAGD with optimal TSHR-specific immune responses, while IL-4(-/-) mice completely resisted disease and showed delayed and suboptimal pathogenic Ab response. These data demonstrated that skewing immune responses to TSHR, using either Flt3-L or GM-CSF, in favor of Th1 or Th2, respectively, may not be sufficient to alter the course of the disease, while the complete absence of IL-4, but not IFN-gamma, can prevent the development of EAGD.     

Cutting edge: interactions through the IL-10 receptor regulate autoimmune diabetes.

BDC2.5/nonobese diabetic (NOD) transgenic mice express a TCR from a diabetogenic T cell clone yet do not spontaneously develop diabetes at high incidence. Evidence exists showing that in the absence of endogenous TCR alpha-chain rearrangements this transgenic mouse spontaneously develops diabetes and that CTLA-4 negatively regulates diabetes onset. This strongly suggests that onset of diabetes in BDC2.5/NOD mice is governed by T cell regulation. We addressed the mechanism of immune regulation in BDC2.5/NOD mice. We find that activated spleen cells from young, but not old, BDC2.5/NOD mice are able to transfer diabetes to NOD-scid recipients. We have used anti-IL-10R to show that the failure of splenocytes from older mice to transfer diabetes is due to dominant regulation. We furthermore found that diabetes developed following anti-IL-10R treatment of 6-wk old BDC2.5/NOD mice indicating that endogenous IL-10 plays a key role in the regulation of diabetes onset in this transgenic mouse.     

The state of the art of adeno-associated virus-based vectors in gene therapy

Background

Genotyping of HBV is generally used for determining the epidemiological relationship between various virus strains and origin of infection mostly in research studies. The utility of genotyping for clinical applications is only beginning to gain importance. Whether HBV genotyping will constitute part of the clinical evaluation of Hepatitis B patients depends largely on the availability of the relevance of the evidence based information. Since Pakistan has a HBV genotype distribution which has been considered less virulent as investigated by earlier studies from south East Asian countries, a study on correlation between HBV genotypes and risk of progression to further complex hepatic infection was much needed

Methods

A total of 295 patients with HBsAg positive were selected from the Pakistan Medical Research Council's (PMRC) out patient clinics. Two hundred and twenty six (77%) were males, sixty nine (23%) were females (M to F ratio 3.3:1).

Results

Out of 295 patients, 156 (53.2%) had Acute(CAH), 71 (24.2%) were HBV Carriers, 54 (18.4%) had Chronic liver disease (CLD) Hepatitis. 14 (4.7%) were Cirrhosis and HCC patients. Genotype D was the most prevalent genotype in all categories of HBV patients, Acute (108), Chronic (39), and Carrier (53). Cirrhosis/HCC (7) were HBV/D positive. Genotype A was the second most prevalent with 28 (13%) in acute cases, 12 (22.2%) in chronics, 14 (19.7%) in carriers and 5 (41.7) in Cirrhosis/HCC patients. Mixed genotype (A/D) was found in 20 (12.8%) of Acute patients, 3 (5.6%) of Chronic and 4 (5.6%) of carriers, none in case of severe liver conditions.

Conclusion

Mixed HBV genotypes A, D and A/D combination were present in all categories of patients except that no A/D combination was detected in severe conditions. Genotype D was the dominant genotype. However, genotype A was found to be more strongly associated with severe liver disease. Mixed genotype (A/D) did not significantly appear to influence the clinical outcome.     

Inhibition of autoimmune diabetes by TLR2 tolerance

We have reported that apoptotic β cells undergoing secondary necrosis, called "late apoptotic (LA) β cells," stimulated APCs and induced diabetogenic T cell priming through TLR2, which might be one of the initial events in autoimmune diabetes. Indeed, diabetogenic T cell priming and the development of autoimmune diabetes were significantly inhibited in TLR2-null NOD mice, suggesting the possibility that TLR2 blockade could be used to inhibit autoimmune diabetes. Because prolonged TLR stimulation can induce TLR tolerance, we investigated whether repeated TLR2 administration affects responses to LA β cells and inhibits autoimmune diabetes in NOD mice by inducing TLR2 tolerance. Treatment of primary peritoneal macrophages with a TLR2 agonist, Pam3CSK(4), suppressed cytokine release in response to LA insulinoma cells or further TLR2 stimulation. The expression of signal transducer IRAK-1 and -4 proteins was decreased by repeated TLR2 stimulation, whereas expression of IRAK-M, an inhibitory signal transducer, was enhanced. Chronic Pam3CSK(4) administration inhibited the development of diabetes in NOD mice. Diabetogenic T cell priming by dendritic cells and upregulation of costimulatory molecules on dendritic cells by in vitro stimulation were attenuated by Pam3CSK(4) administration in vivo. Pam3CSK(4) inhibited diabetes after adoptive transfer of diabetogenic T cells or recurrence of diabetes after islet transplantation by pre-existing sensitized T cells. These results showed that TLR2 tolerance can be achieved by prolonged treatment with TLR2 agonists, which could inhibit priming of naive T cells, as well as the activity of sensitized T cells. TLR2 modulation could be used as a novel therapeutic modality against autoimmune diabetes.     

Haematopoietic stem cell gene therapy to treat autoimmune disease

Autoimmune diseases affect approximately 6% of the population and are characterised by a pathogenic immune response that targets self-antigens. Well known diseases of this nature include type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. Treatment is often restricted to replacement therapy or immunosuppressive regimes and to date there are no cures. The strategy of utilising autologous or allogeneic haematopoietic stem cell transplantation to treat autoimmunity and induce immunological tolerance has been trailed with various levels of success. A major issue is disease relapse as the autoimmune response is reinitiated. Cells of the immune system originate from bone marrow and have a central role in the induction of immunological tolerance. The ability to isolate and genetically manipulate bone marrow haematopoietic stem cells therefore makes these cells a suitable vehicle for driving ectopic expression of defined autoantigens and induction of immunological tolerance.