Strategies to Modulate Immune Responses: A New Frontier for Gene Therapy
Authors: Valder R Arruda, Patricia Favaro, Jonathan D Finn
Journal: Molecular Therapy (2009)

The success of gene therapy strategies to cure disease relies on the control of unwanted immune responses to transgene products, genetically modified cells and/or to the vector. Effective treatment of an established immune response is much harder to achieve than prevention of a response before it has had a chance to develop. However, preventive strategies are not always effective in avoiding immune responses, thus the use of drugs to induce immunosuppression (IS) is required. The growing discovery of novel drugs provides a conceptual shift from using generalized, moderately intensive immunosuppressive regimens towards a refined approach to attain the optimal balance of naive cells, effector cells, memory cells, and regulatory cells, harnessing the natural tolerance mechanisms of the body. We review several strategies based on transient IS coupled with gene therapy for sustained immune tolerance induction to the therapeutic transgene. The success of gene therapy strategies to cure disease relies on the control of unwanted immune responses to transgene products, genetically modified cells and/or to the vector. Effective treatment of an established immune response is much harder to achieve than prevention of a response before it has had a chance to develop. However, preventive strategies are not always effective in avoiding immune responses, thus the use of drugs to induce immunosuppression (IS) is required. The growing discovery of novel drugs provides a conceptual shift from using generalized, moderately intensive immunosuppressive regimens towards a refined approach to attain the optimal balance of naive cells, effector cells, memory cells, and regulatory cells, harnessing the natural tolerance mechanisms of the body. We review several strategies based on transient IS coupled with gene therapy for sustained immune tolerance induction to the therapeutic transgene. IntroductionOver the past decades the gene therapy field has rapidly evolved from an initial focus on the efficacy of several viral and nonviral gene-transfer systems to the safety of these strategies, and this has culminated in the initiation of large numbers of early-phase clinical trials. The major safety issues identified from these preclinical and clinical studies include the risk of insertional mutagenesis, inadvertent germline transmission of vector sequences, and unwanted immune responses to the vector and to the therapeutic transgene.Two of the central safety issues in using gene-based strategies to treat disease are tolerance induction to the transgene and avoiding any unwanted immune responses to the vector. Most gene therapy trials for genetic diseases are aimed at sustained expression of therapeutic genes by introducing the vector into the target tissue with minimal or no tissue damage. Transduced cells and/or the expression of the therapeutic transgene following delivery of vectors are potentially able to trigger alloimmune responses involving both naive and memory lymphocytes, including lymphocytes specific for viral antigens.1Halloran PF Immunosuppressive drugs for kidney transplantation.N Engl J Med. 2004; 351: 2715-2729Crossref PubMed Scopus (782) Google Scholar This scenario creates, to a certain extent, a clinical parallel to the immune responses following organ transplantation in which neoantigens in the graft are presented to the host-immune system. To avoid allograft rejection, immunosuppression (IS) is required during the induction phase followed by a long-term maintenance regimen. There are major differences between gene therapy and organ transplantation, such as the amounts of antigen presented, nature of antigen and number of antigen-specific T cells. Thus, the intense IS that is required for organ transplantation is unlikely needed for gene-transfer based strategies. It is well known that avoiding immune responses such as allograft rejection is more successful than attempting to eradicate an already established antiallograft B- or T-cell–mediated response. Similarly, in gene therapy every effort should be made to avoid immune responses prophylactically.In this review, we will focus on drug-based strategies to avoid immune responses to the vector and/or the transgene following in vivo delivery of recombinant vectors. Most of immune suppression strategies described in this review directed at avoiding adaptive immune response will also have an affect on the innate response to the gene delivery vector (viral proteins or CpG DNA, etc.) by decreasing inflammatory responses. The use of vector-modified hematopoietic stem cell therapy in which myelocytotoxic and IS drugs are given to the host to create space in the bone marrow for the homing and expansion of gene-corrected cells will not be reviewed.Mechanism of Immune Responses and Tolerance InductionThe immune systems reaction to antigen depends on (i) the relative frequencies of responding T and B cells and on the thresholds of binding affinity that their receptors display, (ii) the levels of antigen present, and (iii) the period during which the antigen remains in secondary lymphoid tissue, where primary immune responses are initiated.Tolerance induction is the process by which the immune system is able to adapt to exogenous antigens and is characterized by an antigen-specific nonreactivity (Figure 1). T- and B-cell tolerance can be established or disrupted either centrally, at the site of primary lymphocyte development in the thymus or bone marrow; or peripherally in the lymphoid tissue where antigen recognition and processing occur. In the peripheral immune system the key mechanisms that induce and maintain tolerance include clonal deletion, anergy, ignorance, and suppression. Ignorance describes the situation whereby T cells fail to respond to a specific antigen. This can be due to (i) low levels of antigen that are insufficient to activate T cells, (ii) antigens that are physically separated from T cells (such as blood-brain barrier). Antigens that are presented in the absence of co-stimulation signaling can induce anergy, characterized by state of T-cell unresponsiveness. Deletion of T cells can occur when the cell is activated in the absence of co-stimulation, or due to a lack of growth factors. Tolerance induction by suppression is an active process by which a regulatory subset of T cells (Tregs, Tr1, etc.) specifically suppresses the activity of T cells.2Kamradt T Mitchison NA Tolerance and autoimmunity.N Engl J Med. 2001; 344: 655-664Crossref PubMed Scopus (0) Google Scholar,3Vignali DA Collison LW Workman CJ How regulatory T cells work.Nat Rev Immunol. 2008; 8: 523-532Crossref PubMed Scopus (1112) Google ScholarStrategies to Prevent Immune Responses in the Context of Gene TransferIn an effort to avoid immune responses during gene transfer, viral gene therapy vectors have been designed to contain few or no viral coding genes and avoid expression of pathogenic genes.4Thomas CE Ehrhardt A Kay MA Progress and problems with the use of viral vectors for gene therapy.Nat Rev Genet. 2003; 4: 346-358Crossref PubMed Scopus (1161) Google Scholar Factors influencing the host-immune response against the vector, such as route of vector administration, dose of vector, choice of promoter/enhancer, alterations to vector genome sequence and/or structure, the status and the nature of the target tissue (e.g., underlying disease or immune-privileged sites), and patient-related factors (age, gender, immune status, drug intake, comorbid pathology and others) are all critical to the development of a clinically relevant gene-based strategy to treat human diseases.5Bessis N GarciaCozar FJ Boissier MC Immune responses to gene therapy vectors: influence on vector function and effector mechanisms.Gene Ther. 2004; 11 Suppl 1: S10-S17Crossref PubMed Scopus (263) Google Scholar For some clinical conditions, fetal or neonatal therapy are critical for the treatment of the disease and in these strategies the immune responses to the vector and/or transgene may be minimized.6Ponder KP Immunology of neonatal gene transfer.Curr Gene Ther. 2007; 7: 403-410Crossref PubMed Scopus (22) Google ScholarTransgene expression restricted to the target tissue by using tissue-specific promoters has been extensively exploited to avoid immune responses to the transgene. One important strategy to avoid an immune response is to prevent transgene expression within antigen-presenting cells (APCs), such as dendritic cells, B cells, or macrophages. However, the uptake of exogenous protein by APC and presentation in the context of major histocompatibility complex class I or class II does not require direct transduction of APCs by the recombinant vectors. For muscle-restricted expression, plasmid DNA appears to generate cytotoxic CD8+ lymphocytes using a cross-priming mechanism whereby APCs take up, process and present exogenous antigen and present it on major histocompatibility complex class I molecules.7Ulmer JB Deck RR Dewitt CM Donnhly JI Liu MA Generation of MHC class I-restricted cytotoxic T lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells.Immunology. 1996; 89: 59-67Crossref PubMed Google Scholar Therefore the use of muscle-specific promoters would not prevent immune responses if cross priming is involved, even if the vectors do not transduce APCs. That being said, it is still preferable to avoid expressing in APCs as direct transduction of APCs can exacerbate immune responses.8Xin KQ Mizukami H Urabe M Toda Y Shinoda K Yoshida A et al.Induction of robust immune responses against human immunodeficiency virus is supported by the inherent tropism of adeno-associated virus type 5 for dendritic cells.J Virol. 2006; 80: 11899-11910Crossref PubMed Scopus (51) Google Scholar,9Vandenberghe LH Wang L Somanathan S Zhi Y Figueredo J Calcedo R et al.Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.Nat Med. 2006; 12: 967-971Crossref PubMed Scopus (137) Google Scholar It should be noted that there have been some examples of tolerance induction by expressing peptide-immunoglobulin fusion proteins in B cells. The exact mechanism of this tolerance induction is unclear, however it appears to involve T-regulatory epitopes encoded in the immunoglobulin (Ig) G molecule.10Su Y Carey G Maric M Scott DW B cells induce tolerance by presenting endogenous peptide-IgG on MHC class II molecules via an IFN-gamma-inducible lysosomal thiol reductase-dependent pathway.J Immunol. 2008; 181: 1153-1160Crossref PubMed Google Scholar,11De Groot AS Moise L McMurry JA Wambre E Van Overtvelt L Moingeon P et al.Activation of natural regulatory T cells by IgG Fc-derived peptide "Tregitopes".Blood. 2008; 112: 3303-3311Crossref PubMed Scopus (203) Google ScholarThe liver is an attractive target for gene transfer as it has long been known as tolerogenic organ.12Benseler V McCaughan GW Schlitt HJ Bishop GA Bowen DG Bertolino P The liver: a special case in transplantation tolerance.Semin Liver Dis. 2007; 27: 194-213Crossref PubMed Scopus (0) Google Scholar Studies in mice have shown that tolerance induction by liver-specific expression of the transgene is an active suppresive mechanism involving the induction of Treg cells (for detailed review see LoDuca et al.13Loduca PA Hoffman BE Herzog RW Hepatic gene transfer as a means of tolerance induction to transgene products.Curr Gene Ther. 2009; 9: 104-114Crossref PubMed Scopus (53) Google Scholar). Liver-specific promoters are successful in inducing long-term, sustained expression of the therapeutic transgene in large animal models following delivery of adeno-associated virus (AAV) vectors to adult animals14Sarkar R Mucci M Addya S Tetreault R Bellinger DA Nichols TC et al.Long-term efficacy of adeno-associated virus serotypes 8 and 9 in hemophilia a dogs and mice.Hum Gene Ther. 2006; 17: 427-439Crossref PubMed Scopus (64) Google Scholar,15Niemeyer GP Herzog RW Mount J Arruda VR Tillson DM Hathcock J et al.Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy.Blood. 2009; 113: 797-806Crossref PubMed Scopus (122) Google Scholar or murine Moloney leukemia virus-based retroviral vectors to neonatal dogs.16Xu L Nichols TC Sarkar R McCorquodale S Bellinger DA Ponder KP Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy.Proc Natl Acad Sci USA. 2005; 102: 6080-6085Crossref PubMed Scopus (0) Google Scholar Interestingly, the use of a liver-specific promoter was not sufficient to completely prevent an immune response in the context of lentiviral vectors delivered to liver of adult mice,17Follenzi A Battaglia M Lombardo A Annoni A Roncarolo MG Naldini L Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice.Blood. 2004; 103: 3700-3709Crossref PubMed Scopus (149) Google Scholar nor to prevent the generation of inhibitory antibodies using nonviral vectors encoding human factor VIII.18Ye P Thompson AR Sarkar R Shen Z Lillicrap DP Kaufman RJ et al.Naked DNA transfer of Factor VIII induced transgene-specific, species-independent immune response in hemophilia A mice.Mol Ther. 2004; 10: 117-126Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar In order to overcome these limitations, Brown et al. described a gene-transfer system that exploits the endogenous microRNA machinery for transgene regulation. They have shown the incorporation of the microRNA mir-142-3p target sequence suppresses the expression of the transgene in hematopoietic lineages, thus avoiding neutralizing antibodies against the transgene product.19Brown BD Cantore A Annoni A Sergi LS Lombardo A Della Valle P et al.A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice.Blood. 2007; 110: 4144-4152Crossref PubMed Scopus (158) Google Scholar Similar studies have been carried out using hydrodynamic delivery of plasmid under the control of tissue-specific (liver or skeletal muscle) promoters and mir-142-3p. Although incorporation of the microRNA sequence did decrease antitransgene antibody titers, transgene-specific immune tolerance was not achieved.20Wolff LJ Wolff JA Sebestyén MG Effect of tissue-specific promoters and microRNA recognition elements on stability of transgene expression after hydrodynamic naked plasmid DNA delivery.Hum Gene Ther. 2009; 20: 374-388Crossref PubMed Scopus (26) Google Scholar Therefore, in some systems the use of tissue-specific promoters will be enough to avoid immune responses, whereas in a different context (vector type, tissue target, route of administration, etc.) additional strategies may be required.Regulated expression of the transgene is another strategy that can be used to minimize the risk of unwanted immune responses. In this approach a regulated promoter is used to delay transgene expression until the tissue has recovered from underlying inflammation and/or trauma that can be associated with vector administration. This prevents the immune system from first encountering the transgene in the context of a "danger signal," one that is likely to prompt an immune response. Several systems have been exploited for such an immunoevasion strategy, such as Tet-On tetracycline regulatable system. However, nonhuman primate (NHP) studies have shown humoral and cytotoxic immune response against the nonspecies specific transactivator. Novel regulated expression systems based on human transcription factors are in development and probably are likely less immunogenic.21Ye X Rivera VM Zoltick P Cerasoli F Schnell MA Gao G et al.Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer.Science. 1999; 283: 88-91Crossref PubMed Scopus (253) Google Scholar,22Yang W Keenan TP Rozamus LW Wang X Rivera VM Rollins CT et al.Regulation of gene expression by synthetic dimerizers with novel specificity.Bioorg Med Chem Lett. 2003; 13: 3181-3184Crossref PubMed Scopus (13) Google ScholarDelivering vector to tissue and/or a space considered to be immune-privileged is a logical option to evade unwanted immune responses in gene therapy. These areas include the brain, eye, testis, and uterus among others. Therefore, gene transfer at these tissues may avoid or minimize immune responses to both vector and transgene. Lowenstein et al. reviewed a series of studies on viral vector delivery into the brain of naive and previously vector-immunized animal models demonstrate that the immunologic protection of the naive brain could be hampered by the local of the injection, vector dose and vector type.23Lowenstein PR Mandel RJ Xiong WD Kroeger K Castro MG Immune responses to adenovirus and adeno-associated vectors used for gene therapy of brain diseases: the role of immunological synapses in understanding the cell biology of neuroimmune interactions.Curr Gene Ther. 2007; 7: 347-360Crossref PubMed Scopus (95) Google Scholar Thus, it is likely that perturbations of the "immune-privileged sites" may compromise the anatomical integrity of these natural barriers and change local immune responses.24Doi K Kong J Hargitai J Goff SP Gouras P Transient immunosuppression stops rejection of virus-transduced enhanced green fluorescent protein in rabbit retina.J Virol. 2004; 78: 11327-11333Crossref PubMed Scopus (0) Google ScholarImmune Suppression StrategiesPreventive strategies are not always sufficient to avoid immune responses to transgenes and/or vectors, thus the use of more potent alternatives is necessary. One of these alternatives is the use of drug-induced IS, a very well established strategy for organ transplantation that has been recently translated to the gene therapy field.Tolerance induction or IS are possible strategies to enhance the efficacy and the duration of gene expression without major safety concerns. Some factors need to be taken into consideration for IS drug therapy coupled with gene therapy. The safety aspects of this combination need to be addressed in preclinical studies and from epidemiological clinical studies in other settings requiring long-term IS. The main considerations for the use of IS therapy are described below: 1. Interference with vector internalization, stability, and transduction efficiency. 2. Modification of the biodistribution of vector to target and nontarget tissues. 3. Compromising host-defense mechanisms required to prevent infection by opportunistic pathogens and potentially altering vector transduction efficacy and/or immunological profile. 4. Higher susceptibility of tissue damage by the combination of vector and/or drug, especially in strategies targeting tissues with underlying diseases. Examples include primary or iatrogenic liver disease, renal diseases, and inflammatory status of skeletal muscle in patients with muscular dystrophy, inflammatory joint diseases, and bone marrow failure. 5. Drug side effects that can exacerbate underlying metabolic complications such as lipid disorders, glucose metabolism and others. 6. Long-term complications such as increased risk of malignancies, impairing gonadal function and infertility.Immune Suppression Versus Tolerance InductionIS involves blocking the activity or efficacy of the immune system. Since the introduction of IS therapy in the 1950s, IS has been an integral part of organ transplant protocols. Much progress has been made in the prevention of acute immune responses to organ transplants; however, chronic allograft rejection is still a major problem. This demands the re-evaluation of early concepts focused mainly on aggressive IS rather than balanced IS and tolerance induction. IS protocols involve the use of a wide range of drugs (see Table 1), each having side effects, and most protocols require the patient to stay on IS agents for many years. The combination of different classes of drugs have allowed a more sophisticated application of IS. There has been a shift from high intensity ablative therapy to less intense, more refined use of IS that can tip the balance from total immune suppression to a setting more prone to induce tolerance.Table 1Immunosuppression drugs: mechanism of action and side effectsClassDrugMechanism of actionEffect on TregsSide effectsNotesGlucocorticoidsMethyprednisoloneNon specific anti-inflammatoryPositive effect: increase FoxP3 expression and increases suppressive activity and cell survivalFluid retention, weight gain, diabetes, bone mineral loss, dyslipidemia, irritabitilyUsed in combination with other IS agents in transplant recipients and to treat inflammatory autoimmune diseaseAntiproliferative agentsAzathioprineInterferes with nucleotide synthesis and results in inhibition of T-cell activationN/AUsually well tolerated. Side effects include bone marrow suppression, hepatoxicityCyclophosphamideInterferes with DNA synthesis. Major effect on dividing cellsNegative effect: decreases Tregs in miceMyelosuppression, vomiting and nausea, gastrointestinal discomfort, hemorrhagic cystitis, and hair lossHas been successfully used in multiple preclinical gene therapy modelsMycophenolate mofetilSelective inhibitor of de novo pathway of purine biosynthesis. Inhibits T- and B-cell proliferationNo effectDiarrhea, anemia, leukopenia, may increase cytomegalovirus diseaseHas been successfully used in multiple preclinical gene therapy modelsCalcineurin inhibitorsCyclosporine, tacrolimus (FK506)Block production of interleukin-2 and inhibits T-cell proliferation and differentiationNegative effect: inhibits FoxP3 expression and Treg functionNephrotoxicity, hypertension, hyperlipidemia, hirsutism, gingival hyperplasia, tremorSafe in patients with a high risk of diabetesTarget-of-rapamycin inhibitorsSirolimus (Rapamycin), EverolimusPrevents cytokine receptors from activating cell cyclePositive effect: can increase Treg numbers and/or functionHyperlipidemia, thrombocytopenia, diarrhea, poor wound healingSynergistic with calcineurin inhibitors. May have antineoplastic and arterial protective effectsDepleting antibodiesAntithymocyte globulin (ATG)Polyclonal antithymocyte globulin depletes all lymphoid cellsRabbit ATG has a positive effect (expands Tregs). Horse ATG has a negative effect (depletes Tregs)Lymphopenia, increased risk of infection, thrombocytopenia, cytokine release syndrome, serum sicknessRabbit ATG is documented to increase Tregs and promote tolerance in human cells in vitroAnti-CD3 mAb First Generation: Muromonab-CD3 (OKT3) Second Generation: hOKTγl (Ala-Ala) Hu291 ChAglyCD3Depletes CD3+ T cellsPositive effect: expands TregsCytokine release syndromeFirst generation mAbs have been used for decades, however humans develop neutralizing antibodies and side effects that limits its clinical use. Second generation mAbs show promising efficacy in autoimmune disease (diabetes, arthritis)AlemtuzumabmAb against CD52. Depletes B cells, T cells, NK and monocytesPositive effect: expands TregsNeutropenia, anemia, autoimmunityApproved for treatment of refractory B-cell chronic lymphoid leukemia. Currently being evaluated for organ transplantRituximabMonoclonal anti-CD20 antibody. Depletes most B cellsN/ASome infusion related reactions. Possible reactivation of hepatitis B (rare). Impaired B-cell functionUsed in combination with other IS drugs to control alloantibody responses in transplantation and for the for treatment of autoimmune diseasesNondepleting antibodies or proteinsDaclizumab and basilixumabMonoclonal anti-CD25 antibody. Binds to activated T cellsNegative effect: depletes Tregs (in nonhuman primates)Hypersensitivity reactions, abdominal discomfort and myalgiasDaclizumab is a humanized antibody (half-life 11 days), Basilizumab is a chimeric antibody (half-life 6.5 days). Often used in combination with calcineurin inhibitorsCTLA4-IgBlocks co-stimulation of T cellsPositive effect: increases Tregs in mice, however human data is unclearIncreased risk of thromboembolism and upper respiratory tract infectionHas been used successfully in humans with rheumatoid arthritis and in animal models of transplantation. LEA29Y is a second generation fusion protein with enhanced potencyIntravenous immunoglobulinPooled human plasma from thousands of volunteers. Modulation of Fc receptors, interference with activity of complement, cytokine and idiotype network. Regulation of cell growth and effects on activation and function of dendritic cells, T cells and B cellsPositive effect: increases TregsAcute renal toxicity, hypersensitivity reactions, hemolytic anemia, urticarial rashes, hemolysis, cytopeniaContains high levels of neutralizing antibodies to most commonly used recombinant viral vectors such as AAV and adenovirus. This may hamper the efficiency of gene transfer by these vectorsmAb, monoclonal antibody; N/A, data not available. Open table in a new tab In gene therapy applications, the ultimate goal is to achieve long-term antigen-specific tolerance to the transgene product. There is a delicate balance between immune suppression and tolerance induction. The identification and characterization of T-regulatory cells has enabled the design of effective strategies to control immune responsiveness. The mechanisms by which Tregs control immune responses are complex and variable, but there is a consensus that Treg-mediated immune regulation plays crucial roles in both the induction and maintenance of tolerance.25Sakaguchi S Yamaguchi T Nomura T Ono M Regulatory T cells and immune tolerance.Cell. 2008; 133: 775-787Abstract Full Text Full Text PDF PubMed Scopus (1860) Google Scholar IS strategies that block activation/proliferation of Tregs or completely deplete them from circulation are predicted to hamper tolerance induction, necessitating the long-term use of IS. Thus, intensive IS may prevent the achievement of the ultimate goal of IS regimens, which is induction of tolerance to the foreign antigens.Immunosuppressive DrugsCurrent treatment for immunological disorders are nearly all empirical in origin, using immunosuppressive drugs identified by screening large numbers of natural and synthetic compounds. In the majority of IS protocols for organ transplants, IS drugs are given in combination because many of the classes of IS drugs act synergistically. This allows greater efficacy from lower doses of drug, an important consideration when trying to avoid unwanted dose-dependent side effects.1Halloran PF Immunosuppressive drugs for kidney transplantation.N Engl J Med. 2004; 351: 2715-2729Crossref PubMed Scopus (782) Google Scholar IS can be achieved by depleting lymphocytes, blocking lymphocyte response pathways, or diverting lymphocyte traffic. IS drugs include glucocorticoids, small-molecule drugs, depleting and nondepleting protein drugs (polyclonal and monoclonal antibodies), fusion proteins, and intravenous IgG (Table 1 and Figure 2). Table 1 summarizes the different classes of immunomodulatory drugs and includes information as to the mechanism of action, possible side effects, and other pertinent information on the use of these drugs in IS regimens. Of note, drugs are also classified according with their ability to interfere with Treg cell population and/or function.Figure 2T- and B-cell activation. Overview of the major proteins and pathways involved in (a) T-cell activation by antigen-presenting cells (APCs) and (b) B-cell activation by CD4 T helper cells. CTL, cytotoxic T lymphocyte; IFN, interferon; IL, interleukin; TOR, target-of-rapamycin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)There is not a single IS regimen that is largely used in organ transplant even within an organ-specific group. Ongoing and planned trials consist of heterogeneous drug combinations. Therefore, it is prudent to consider all major characteristics of the underlying disease to be treated by gene therapy in the light of the organ transplantation experience to evaluate both efficacy and side effects of all available drugs. In organ transplantation models, the unusually large number of T cells that are responsive to transplant tissues (5 in 20 T cells) as compared with the response to a foreign protein (one in one million) is remarkable.26Strom TB 2006 Homer W. Smith Lecture: taming T cells.J Am Soc Nephrol. 2007; 18: 2824-2832Crossref PubMed Scopus (6) Google Scholar Thus, the pharmacological IS regimens to induce successful immune modulation most likely required in gene-transfer protocols may be less intense than for those to control organ transplant rejection.27Koyama K Hatsushika K Ando T Sakuma M Wako M Kato R et al.Imatinib mesylate both prevents and treats the arthritis induced by type II collagen antibody in mice.Mod Rheumatol. 2007; 17: 306-310Crossref PubMed Google Scholar This may argue against the need for intensive induction therapy with monoclonal or polyclonal antibodies in a gene therapy setting.Notably, most of these IS drugs have been used in the context of other alloimmune-mediated, primary autoimmune and benign diseases. For example, the efficacy of mycophenolate mofetil (MMF), tacrolimus and cyclosporine in various regimens has been extensively tested in solid organ transplantation including liver, kidney, lung, heart among adults1Halloran PF Immunosuppressive drugs for kidney transplantation.N Engl J Med. 2004; 351: 2715-2729Crossref PubMed Scopus (782) Google Scholar,28Mueller XM Drug immunosuppression therapy for adult heart transplantation. Part 2: clinical applications and results.Ann Thorac Surg. 2004; 77: 363-371Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,29Perry I Neuberger J Immunosuppression: