Assembly PCR synthesis of optimally designed, compact, multi-responsive promoters suited to gene therapy application.

Gene therapy has the potential to provide innovative treatments for genetic and non-genetic diseases, with the ability to auto-regulate expression levels of therapeutic molecules so that they are produced locally and in direct response to disease activity. Generating disease responsive gene therapy vectors requires knowledge of the activation profile of transcription factors (TFs) during active disease, in order to assemble binding sites for these TFs into synthetic promoters, which can be appropriately activated by the disease process. In this study, we optimised a PCR random assembly approach to generate promoters with optimal spacing between TF binding sites (TFBSs) and their distance from the TATA box. In promoters with optimal spacing, it was possible to demonstrate activation by individual transcription pathways and either additive or synergistic promoter activation when transfected cells were treated with combined stimuli. The kinetics and sensitivity of promoter activation was further explored in transduced cells and when lentivirus was directly delivered to mouse paws a synthetic promoter demonstrated excellent activation by real-time imaging in response to local inflammation.

Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1ß and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme.

Historical development of monoclonal antibody therapeutics.

Since the first publication by Kohler and Milstein on the production of mouse monoclonal antibodies (mAbs) by hybridoma technology, mAbs have had a profound impact on medicine by providing an almost limitless source of therapeutic and diagnostic reagents. Therapeutic use of mAbs has become a major part of treatments in various diseases including transplantation, oncology, autoimmune, cardiovascular, and infectious diseases. The limitation of murine mAbs due to immunogenicity was overcome by replacement of the murine sequences with their human counterpart leading to the development of chimeric, humanized, and human therapeutic antibodies. Remarkable progress has also been made following the development of the display technologies, enabling of engineering antibodies with modified properties such as molecular size, affinity, specificity, and valency. Moreover, antibody engineering technologies are constantly advancing to enable further tuning of the effector function and serum half life. Optimal delivery to the target tissue still remains to be addressed to avoid unwanted side effects as a result of systemic treatment while achieving meaningful therapeutic effect.

Inflammation-inducible anti-TNF gene expression mediated by intra-articular injection of serotype 5 adeno-associated virus reduces arthritis.

BACKGROUND: The tumor necrosis factor (TNF)-alpha plays a central role in rheumatoid arthritis (RA) and current biotherapies targeting TNF-alpha have a major impact on RA treatment. The long-term safety concerns associated with the repetitive TNF blockade prompt optimization of therapeutic anti-TNF approaches. Since we recently demonstrated that intra-articular gene transfer using a recombinant adeno-associated virus serotype 5 (rAAV5) efficiently transduces arthritic joints, we evaluate its effect on collagen-induced arthritis (CIA) when encoding TNF antagonists. METHODS: Recombinant AAV5 vectors encoding the human TNFRp55 extracellular domain fused to the Fc region of mice IgG1 (TR1) or a small molecular weight dimeric human TNFRp75 extracellular domain (TR2), under two different promoters, the CMV or a chimeric NF-kappaB-based promoter inducible by inflammation, were injected into mouse CIA joints. RESULTS: Best protection against arthritis was obtained with the rAAV5 encoding the TR1, as reflected by delayed disease onset, decreased incidence and severity of joint damage. This effect was associated with a transient expression of the anti-TNF agent when expressed under a NF-kappaB-responsive promoter, only detectable during disease flare, while the antagonist expression was rapidly increased and stable when expressed from a CMV promoter. Importantly, using the intra-articular administration of the rAAV5-NF-kappaB-TR1 vector, we observed a striking correlation between local TR1 expression and inflammation. CONCLUSIONS: These findings strongly support the feasibility of improving the safety of anti-TNF approaches for the treatment of arthritis by local rAAV5-mediated gene expression under an inflammation-responsive promoter, able to provide a limited, transient and therapeutically relevant expression of anti-TNF compounds.

Reduction of arthritis following intra-articular administration of an adeno-associated virus serotype 5 expressing a disease-inducible TNF-blocking agent.

BACKGROUND: In the context of preclinical development, we studied the potential of intra-articular gene delivery using a recombinant adeno-associated virus 5 (rAAV5) encoding a chimeric human tumour necrosis factoralpha (TNFalpha) soluble receptor I linked to a mouse immunoglobulin heavy chain Fc portion (TNF receptor I; TNFRI-Ig). METHODS: Expression was under control of a nuclear factor kappa B (NFkappaB)-responsive promoter and compared with a cytomegalovirus (CMV) promoter (rAAV5.NFkappaB-TNFRI-Ig and rAAV5.CMV-TNFRI-Ig, respectively). RESULTS: Fibroblast-like synoviocytes transduced in vitro with rAAV5.NFkappaB-TNFRI-Ig were able to produce TNFRI-Ig protein in response to several stimuli, and this was inhibited upon treatment with a specific NFkappaB blocking agent. A bioassay revealed that the synthesised TNFRI-Ig was bioactive, showing a higher affinity for human than for rat TNFalpha. Transcription of the transgene and protein production were detectable in joints injected with both constructs. No dissemination of the vector was observed outside the joints. A significant reduction in paw swelling was seen in rats treated with rAAV5.NFkappaB-TNFRI-Ig. This clinical effect was accompanied by a decrease in pro-inflammatory cytokine levels and an increase in IL10 expression in the synovium. CONCLUSION: These results provide evidence that intra-articular gene therapy using rAAV5 encoding TNFRI-Ig may be a safe and feasible approach for the treatment of rheumatoid arthritis. The higher affinity for human TNFalpha suggests that in patients with rheumatoid arthritis the therapeutic effect might be even more pronounced than in rat adjuvant arthritis.

The differential expression of corticosteroid receptor isoforms in corticosteroid-resistant and -sensitive patients with rheumatoid arthritis.

OBJECTIVE: A proportion of patients with rheumatoid arthritis (RA) fail to respond adequately to corticosteroid (CS) therapy. Using an in vitro CS sensitivity bioassay, we have subdivided RA patients into steroid-sensitive (SS) and -resistant (SR) subgroups and this correlates with clinical responses to CS therapy. CSs exert their effects via the CS receptor (CR), which exists as two main isoforms, CRalpha and CRbeta. CRbeta can function as a negative inhibitor of CRalpha. We have hypothesized that steroid resistance in RA patients is due in part to a relative over-expression of the CRbeta. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from SS and SR RA patients. CRalpha and CRbeta mRNA expression was determined by quantitative real time polymerase chain reaction (qRT-PCR). The ratio of CRbeta/CRalpha mRNA expression was determined. CRalpha and CRbeta protein expression by PBMCs was analysed by flow cytometry. RESULTS: qRT-PCR analysis showed a trend towards higher expression of both CRbeta and basal CRbeta/CRalpha ratio in SR RA patients. Stimulation of PBMCs in vitro with concanavalin-A induced a significantly higher CRbeta mRNA expression, and CRbeta/CRalpha ratio in SR RA patients compared with SS patients, which was not inhibited by hydrocortisone. Flow cytometry showed that the percentage of PBMCs staining for CRbeta protein was significantly lower in the SS RA group (SS 43.3 +/- 14.8% vs SR 88.6 +/- 8.6%; P < 0.0010). The mean intensity of fluorescence CRbeta staining was higher in the SR RA patients (P < 0.001). CONCLUSION: We show for the first time that CRbeta is over-expressed in SR RA patients and that hydrocortisone fails to inhibit concanavalin-A stimulated increase in CRbeta mRNA in SR RA patients. This mechanism may contribute in part to the CS hyporesponsiveness seen in some RA patients.

Prevention of experimental autoimmune encephalomyelitis in DA rats by grafting primary skin fibroblasts engineered to express transforming growth factor-beta1.

To determine whether primary fibroblasts producing latent transforming growth factor beta1 (TGF-beta1) are capable of down-regulating experimental autoimmune encephalomyelitis (EAE), a retroviral vector TGF-beta1-pBabe-neo (-5'UTR) was used for efficient gene transfer into primary skin fibroblasts of DA rats. After heat activation, conditioned medium from the transduced fibroblasts was found to inhibit significantly in vitro proliferation of lymphocytes from lymph nodes of DA rats with EAE. Intraperitoneal administration of TGF-beta1-transduced fibroblasts into DA rats during the priming phase of EAE resulted in a significant reduction in mortality and in the mean clinical and EAE scores versus the control immunized animals treated with non-transduced fibroblasts.

The molecular and cellular basis of corticosteroid resistance.

Corticosteroids (CS) can modulate gene expression and are often used to treat a range of immunological and inflammatory diseases such as asthma, inflammatory bowel disease and rheumatoid arthritis. However, a proportion of patients fail to show an adequate response. On this basis patients have been subdivided into CS-sensitive (SS) and -resistant (SR) subgroups. The ability of CS to inhibit peripheral blood T cell proliferation in vitro has also been used similarly. In rheumatoid arthritis (RA), the in vitro-defined SS and SR subgroups correlate with the clinical responses to CS therapy. The mechanisms responsible for this observation are unknown but they appear to involve a number of known molecular events related to the described mechanisms of action of CS. These include alterations in the functional status of CS receptor-alpha, perturbations of the cytokine and hormonal milieu and intracellular signalling pathways. Peripheral blood mononuclear cells (MNCs) from SR significantly overexpress activated NF-kappaB. In vitro, CS fail to significantly inhibit concanavalin A (conA)-induced NF-kappaB activation in MNCs from SR RA patients. The alterations in the intracellular signalling pathways may explain in part our observations seen in SR RA subjects, CS fail to significantly inhibit conA-induced interleukin (IL)-2 and IL-4 secretion and lipopolysaccharide-induced IL-8 and IL-1beta secretion in vitro. CS therapy fails to reduce the circulating levels of IL-8 and IL-1beta in RA patients. In asthma, CS fail to induce L10 in SR asthma patients. Other molecular mechanisms such as enhanced AP-1 expression and alterations in the MAP kinase pathway are most likely to be involved too and we are currently investigating such possibilities. A full understanding of the molecular basis of SR will lead to the development of more rational therapeutic strategies.

Gene therapy for arthritis.

Rheumatoid arthritis is an autoimmune disease with intra-articular inflammation and synovial hyperplasia that results in progressive degradation of cartilage and bone, in severe cases it causes systemic complications. Recently, biological agents that suppress the activities of proinflammatory cytokines have shown efficacy as antiarthritic drugs, but require frequent administration. Thus, gene transfer approaches are being developed as an alternative approach for targeted, more efficient and sustained delivery of inhibitors of inflammatory cytokines as well as other therapeutic agents. Indeed, the efficacy of gene transfer for the treatment of arthritis has been demonstrated in mouse, rat, rabbit, and horse models of disease whereas the feasibility of the approach has been demonstrated in Phase I clinical trials. In this review, the current status of both preclinical and clinical arthritis gene therapy is presented. In addition, the advantages and disadvantages of different types of vectors, target cells and therapeutic genes being developed for the treatment of arthritis are summarized. Finally, the future directions of the rapidly developed field of arthritis gene therapy are outlined.

Amelioration of antigen-induced arthritis in rats by transfer of extracellular superoxide dismutase and catalase genes.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of rheumatoid arthritis (RA), while antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD) and catalase, block radical-induced events. The present study tested if the ex vivo transfer of EC-SOD and catalase genes alone or in combination in the knee joint of rats with monoarticular antigen-induced arthritis (AIA) was anti-inflammatory, and examined the potential mechanisms involved. Synoviocytes isolated from female Wistar rats were immortalized with a retroviral vector SUV19.5. These cells were permanently transfected with an EC-SOD expression plasmid (pEC-SODZeo) or a catalase expression plasmid (pCatalaseZeo) to create cells overexpressing EC-SOD or catalase, as measured by RT-PCR and Western blots. The cells were engrafted in knee joints of animals at the time of the induction of AIA. Three gene transfer groups, an EC-SOD group, a catalase group and a combined therapy group (EC-SOD and catalase) were included in these experiments. Animals in the control group were engrafted with synoviocytes transfected with the plasmid pZeoSV2 without an insert. Clinical and histological assessments were performed, as well as tissue measurements of SOD, catalase and gelatinase activities. Ex vivo gene transfer of EC-SOD and catalase into rat knee joints produced about a six- to seven-fold increase in EC-SOD activity and a two- to three-fold increase in catalase activity compared with the control animals. Rats treated with cells overexpressing EC-SOD, catalase or a combination of EC-SOD and catalase showed significant suppression of knee joint swelling, decreased infiltration of inflammatory cells within the synovial membrane and reduced gelatinase activity in knee joints, compared with animals receiving cells transfected with the plasmid alone. No statistically significant difference was found between the groups treated with cells overexpressing EC-SOD, catalase or a combination of both. Gene therapy involving the local intra-articular overexpression of two antioxidant enzymes, EC-SOD and catalase, was anti-inflammatory in AIA. One mechanism appears to be the suppression of gelatinase activities by both EC-SOD and catalase.

Prolonged exposure of T cells to TNF down-regulates TCR zeta and expression of the TCR/CD3 complex at the cell surface.

A role for TNF-alpha in the pathogenesis of chronic inflammatory disease is now firmly established. Paradoxically, TNF also has potent immunomodulatory effects on CD4(+) T lymphocytes, because Ag-specific proliferative and cytokine responses are suppressed following prolonged exposure to TNF. We explored whether TNF attenuated T cell activation by uncoupling proximal TCR signal transduction pathways using a mouse T cell hybridoma model. Chronic TNF exposure induced profound, but reversible, T cell hyporesponsiveness, with TNF-treated T cells requiring TCR engagement with higher peptide concentrations for longer periods of time for commitment to IL-2 production. Subsequent experiments revealed that chronic TNF exposure led to a reversible loss of TCRzeta chain expression, in part through a reduction in gene transcription. Down-regulation of TCRzeta expression impaired TCR/CD3 assembly and expression at the cell surface and uncoupled membrane-proximal tyrosine phosphorylation events, including phosphorylation of the TCRzeta chain itself, CD3epsilon, ZAP-70 protein tyrosine kinase, and linker for activation of T cells (LAT). Intracellular Ca(2+) mobilization was also suppressed in TNF-treated T cells. We propose that TNF may contribute to T cell hyporesponsiveness in chronic inflammatory and infectious diseases by mechanisms that include down-regulation of TCRzeta expression. We speculate that by uncoupling proximal TCR signals TNF could also interrupt mechanisms of peripheral tolerance that are dependent upon intact TCR signal transduction pathways.

The activity of immunoregulatory T cells mediating active tolerance is potentiated in nonobese diabetic mice by an IL-4-based retroviral gene therapy.

Splenocytes from nonobese diabetic mice overexpressing murine IL (mIL)-4 upon recombinant retrovirus infection lose their capacity to transfer diabetes to nonobese diabetic-scid recipients. Diabetes appeared in 0-20% of mice injected with mIL-4-transduced cells vs 80-100% of controls injected with beta-galactosidase-transduced cells. Protected mice showed a majority of islets (60%) presenting with noninvasive peri-insulitis at variance with beta-galactosidase controls that exhibited invasive/destructive insulitis. Importantly, in all recipients, the transduced proteins were detected within islet infiltrates. Infiltrating lymphocytes from recipients of mIL-4-transduced cells produced high levels of mIL-4, as assessed by ELISA. In recipients of beta-galactosidase-transduced cells, approximately 60% of TCRalphabeta(+) islet-infiltrating cells expressed beta-galactosidase, as assessed by flow cytometry. The protection from disease transfer is due to a direct effect of mIL-4 gene therapy on immunoregulatory T cells rather than on diabetogenic cells. mIL-4-transduced purified CD62L(-) effector cells or transgenic BDC2.5 diabetogenic T cells still transferred disease efficiently. Conversely, mIL-4 transduction up-regulated the capacity of purified immunoregulatory CD62L(+) cells to inhibit disease transfer. These data open new perspectives for gene therapy in insulin-dependent diabetes using T cells devoid of any intrinsic diabetogenic potential.

Different therapeutic outcomes in experimental allergic encephalomyelitis dependent upon the mode of delivery of IL-10: a comparison of the effects of protein, adenoviral or retroviral IL-10 delivery into the central nervous system.

Experimental allergic encephalomyelitis (EAE) is a CNS autoimmune disease mediated by the action of CD4(+) T cells, macrophages, and proinflammatory cytokines. IL-10 is a cytokine shown to have many anti-inflammatory properties. Studies have shown both inhibition and exacerbation of EAE after systemic IL-10 protein administration. We have compared the inhibitory effect in EAE of Il10 gene delivery in the CNS. Fibroblasts transduced with retroviral vectors expressing IL-10 could inhibit EAE. This was not associated with a prevention of cellular recruitment but an alteration in their phenotype, notably an increase in the numbers of CD8(+) T and B cells. In marked contrast, CNS delivery of adenovirus coding for mouse IL-10 or IL-10 protein performed over a wide dose range failed to inhibit disease, despite producing similar or greater amounts of IL-10 protein. Thus the action of IL-10 may differ depending on the local cytokine microenvironment produced by the gene-secreting cell types.

Mice vaccination with interleukin 12-transduced colon cancer cells potentiates rejection of syngeneic non-organ-related tumor cells.

Cell-based gene therapy after cytokine gene transfer is being investigated for autologous and allogeneic vaccination in cancer therapy. Here we show that mice vaccinated with 3-5 x 10(6) interleukin 12 (IL-12) gene-transduced CT26 colon cancer cells developed a long-lasting antitumor immune memory able to reject not only parental cells but also syngeneic, LM3 mammary, and MCE fibrosarcoma tumorigenic cells. In contrast, mice vaccinated with 0.5-1 x 10(6) CT26 cells transduced with pBabe neo IL-12 retrovirus cells (CT26-IL12) were only able to reject parental cells. An increase in the total circulating levels of IgG2a and a clear shift toward a systemic Th1 response developed, regardless of the amount of injected CT26-IL12 cells. On the contrary, a strong increase in anti-CT26-specific IgG2a levels was observed only when 3-5 x 10(6) CT26-IL12 cells were injected. Immunocompetent mice vaccinated with 3-5 x 10(6) CT26-IL12 cells developed local nodules for a few days, which then ceased growing. These nodules comprised mainly blood vessels, suggesting that an angiogenic process was taking place. CD8+ T cells were responsible for the anti-LM3 tumor cell memory, whereas CD4+ T cells were not involved. Splenocytes and lymphocytes obtained from mice immunized against CT26 cells were able to kill LM3 cells in vitro. Adoptive transfer of lymphocytes obtained from animals immunized against CT26 colon cancer cells suppressed LM3 mammary tumor growth in tumor-bearing mice. The present studies raised the possibility of isolating CTL clones and identifying CTL epitopes shared by different tumor cell types, which can be a target for cancer therapy.

Soluble complement receptor 1 (CD35) delivered by retrovirally infected syngeneic cells or by naked DNA injection prevents the progression of collagen-induced arthritis.

OBJECTIVE: The complement system is important in the development of autoimmune inflammation, including rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). Complement receptor 1 (CR1) is involved in regulation of complement activity. Studies on models of autoimmunity have demonstrated that soluble CR1 (sCR1) is a potent therapeutic agent. The present study was thus undertaken to investigate the feasibility of antiinflammatory gene therapy to prevent CIA by delivery of genes encoding truncated sCR1 (tsCR1) and dimeric tsCR1-Ig. METHODS: Syngeneic fibroblasts or arthritogenic splenocytes, engineered to express tsCR1 using retrovirus-mediated gene transfer, were injected into DBA/1 recipients that had been immunized with bovine type II collagen (CII). In separate experiments, naked DNA containing tsCR1 and tsCR1-Ig genes was injected intramuscularly into the immunized animals. The clinical development of arthritis was monitored, anti-CII levels measured, and antigenic T cell response studied. Affinity-purified tsCR1-Ig was assayed for its inhibitory effect on the alternative complement pathway in mouse serum. RESULTS: Treatment of CII-immunized mice with the tsCR1-expressing cells inhibited development of CIA, reduced anti-CII antibody levels, and inhibited T cell response to CII in vitro. Intramuscular injections of DNA encoding the CR1 genes prevented the progression of disease. Furthermore, compared with full-length sCR1, purified tsCR1-Ig was more active in inhibiting the murine alternative complement pathway. CONCLUSION: Our findings demonstrated that tsCR1 and tsCR1-Ig, when delivered via gene therapy, had a beneficial effect on autoimmune inflammation. These results indicate that targeting the complement system in RA patients may be of clinical importance.

Engineering mouse T lymphocytes specific to type II collagen by transduction with a chimeric receptor consisting of a single chain Fv and TCR zeta.

The chimeric cell surface receptor scC2Fv/CD8/zeta was constructed to engineer primary mouse T lymphocytes with antibody-type specificity to type II collagen (CII). Such cells could be used as gene carriers in the anti-inflammatory gene therapy of an autoimmune arthritis. This receptor includes the single chain Fv domain (scFv) of the anti-CII monoclonal antibody (mAb) C2, hinge region of CD8alpha and the transmembrane and cytoplasmic domains of TCRzeta. The scC2Fv/CD8/zeta gene was transduced into T cell hybridomas and primary mouse lymphocytes using retrovirus-mediated gene transfer. The chimeric receptor scC2Fv/CD8/zeta forms covalently bound homodimers, as demonstrated in T cell hybridomas and packaging fibroblasts. It does not associate with endogenous signalling subunits of the TCR complex. When scC2Fv/CD8/zeta-expressing clones of T cell hybridomas MD.45 and HCQ6 were stimulated with CII they produced IL-2. The level of their IL-2 response correlated with the expression level of the chimeric receptor on the cell surface. Splenocytes isolated from DBA/1 mice were stimulated with Con A in vitro to facilitate retrovirus-mediated transfer of the scC2Fv/CD8/zeta gene. As a result of transduction, approximately 4% of the Con A-activated splenocytes expressed the chimeric receptor scC2Fv/CD8/zeta on the cell surface. These cells proliferated in response to stimulation with CII.

Gene therapy for chronic relapsing experimental allergic encephalomyelitis using cells expressing a novel soluble p75 dimeric TNF receptor.

In a murine relapsing experimental allergic encephalomyelitis (EAE) model, gene therapy to block TNF was investigated with the use of a retroviral dimeric p75 TNF receptor (dTNFR) construct. To effectively produce these TNF inhibitors in vivo, a conditionally immortalized syngeneic fibroblast line was established, using a temperature-sensitive SV40 large T Ag-expressing retrovirus. These cells were subsequently infected with a retrovirus expressing soluble dTNFR. CNS-injected cells could be detected 3 mo after transplantation and were shown to produce the transgene product by immunocytochemistry and ELISA of tissue fluids. These levels of dTNFR protein were biologically active and could significantly ameliorate both acute and relapsing EAE. This cell-based gene-vector approach is ideal for delivering proteins to the CNS and has particular relevance to the control of inflammatory CNS disease.

Immuno- and genetic therapy in autoimmune diseases.

Animal models of autoimmune disease have been developed that mimic some aspects of the pathophysiology of human disease. These models have increased our understanding of possible mechanisms of pathogenesis at the molecular and cellular level and have been important in the testing, development and validation of new immunotherapies. The susceptibility to develop disease in the majority of these models is polygenic as is the case in humans. The exceptions to this rule are gene knock outs and transgenic models of particular genes which, in particular genetic backgrounds, have also contributed to the understanding of single gene function and their possible contribution to pathogenesis. Gene therapy approaches that target immune functions are being developed with encouraging results, despite the polygenic nature of these diseases. Basically this novel immuno-genetic therapy harnesses the knowledge of immunology with the myriad of biotechnological breakthroughs in vector design and delivery. Autoimmune disease is the result of genetic dysregulation which could be controlled by gene therapy. Here we summarize the genetic basis of these human diseases as well as some of the best characterized murine models. We discuss the strategies for their treatment using immuno- and gene therapy.