Immunosuppression Strategies

Since preventive strategies are not always sufficient to avoid immune responses, immunosuppressive drugs will be required. Current treatment for immunological disorders are nearly all empirical in origin, using immunosuppressive drugs identified by screening large numbers of natural and synthetic compounds.

Therapeutic immunosuppression (IS) can be achieved by depleting lymphocytes, blocking lymphocyte response pathways, or diverting lymphocyte traffic. The most commonly used drugs in clinical studies include glucocorticoids, small-molecule drugs, depleting and nondepleting protein drugs (polyclonal and monoclonal antibodies), fusion proteins, and intravenous immune globulin (Table 11.1). In the majority of IS protocols for organ transplants, IS drugs are given in combination because many of the classes of IS drugs are able to act synergistically. This allows for greater efficacy from lower doses of drugs, an important consideration when trying to avoid unwanted dose-dependent side effects (Halloran 2004). The regimen and the duration of the IS required to prevent or ameliorate undesirable immune responses following gene therapy is not yet defined. There is evidence in several large animal models of disease, suggesting that transient (short-duration) immune modulation would allow sustained transgene expression and correction of the disease phenotype.

Early studies in DMD patients using IS drugs alone or in combination with myo-blast implantation revealed a beneficial role of IS in the clinical management of the disease. The use of anti-inflammatory and IS drugs can reduce the severity of muscular dystrophy by producing short-term functional improvement or slow disease progression. Clinical trials with cyclosporine in DMD subjects revealed a significant increase in muscle force after 8 weeks of treatment (Sharma et al. 1993). The beneficial effect of IS was also observed with prednisone in boys with DMD, data similar to those obtained in the GRMD model (Liu et al. 2004). Ongoing clinical studies of cyclosporine and prednisone in DMD patients will define the potential role of IS in the disease phenotype. A series of small-scale clinical trials testing the feasibility of myoblast transplantation were performed in the last decades. In some of these studies, humoral responses to the cell and/or the dystrophin protein were detected (Wells et al. 2002). Early phase studies of allogeneic myoblast transplantation in patients with DMD were typically of limited efficacy due to immune responses, rapid cell death and poor migration of the therapeutic cell. Long-term studies using cyclosporine combined with myoblast therapy resulted in increased muscle strength after 7 months, probably due to the use of IS (Miller et al. 1997). Successful cell transplants were also obtained by sustained administration of tacrolimus (FK506) or cyclophosphamide. The addition of IS in this setting was beneficial, but resulted in low efficacy due to non-immunological toxicity of the transplanted myoblasts

Table 11.1 Classification of immunosuppression drugs and their effect on T regulatory (Treg)

General class


In vivo effects on Tregs








Calcineurin inhibitor

Tacrolimus (FK-506)


Cyclosporine (CsA)



Azathioprine (AZA)




Mycophenolate mofetil (MMF)

No effect

Target of Rapamycin

Sirolimus (rapamycin)





Polyclonal antibodies

Rabbit Antithymocyte globulin


Horse Antithymocyte globulin


Monoclonal antibodies

Muromonab-CD3 (anti-CD3)


Alemtuzumab (anti-CD52)


Basiliximab (anti-IL-2 receptor)


Daclizumab (anti-IL-2 receptor)


Rituximab (anti-CD20)







Intravenous immune globulin (IVIG)


(Peault et al. 2007). Nevertheless, these studies provide evidence on the safety of some IS drugs on the DMD phenotype that could be adapted for preclinical and clinical studies based on gene- and/or cell-based therapies. Route of Vector Administration

Proof of concept studies of intramuscular (IM) or intravascular (IV) injection of vectors encoding a series of therapeutic genes in murine models for DMD significantly improve muscle membrane integrity and muscle function (Odom et al. 2007; Wang et al. 2009). Before translating these discoveries into clinical therapy, a major limitation is the formidable scale-up from mouse to dog to human. Skeletal muscle contains one of the highest capillary densities in the body that can be chemically and/ or mechanically modified to ensure vascular leakage of fluid containing vectors. The capillaries, consisting of a single layer of endothelial cells, permit rapid exchange with the interstitial fluid. To achieve scale-independent dosing, one needs to take advantage of intravascular delivery techniques aimed at widespread vector dissemination. Regional perfusion utilizing the vascular tree of the isolated limb as the delivery network affords such a solution to the problem of scaleable delivery. It is important to take into consideration that the vascular permeability of large vertebrates is substantially less permeable than their homologues in mice (Williamson et al. 1971).

Therefore, the delivery of vector to the muscle in the large adult animal has relied upon transient alteration in endothelial permeability by using vasoactive drugs (Arruda et al. 2005; Greelish et al. 1999), or by increasing the hydrostatic pressure (Hagstrom et al. 2004; Su et al. 2005). Thus, based on the existing knowledge, it is clear that the combination of vector system, route of delivery, vascular-modulating drugs and the animal model will define the clinical applicability of the many distinct approaches for the treatment of DMD. Non Viral Vectors

Direct intramuscular injection of naked plasmid DNA into the GRMD dog muscles was also attempted with profoundly low efficiency (Howell et al. 1997; Howell et al. 1998). Newly developed hydrodynamic delivery through peripheral vein injection of plasmid DNA into nonhuman primate and canine muscles has shown much improved efficiencies (Hagstrom et al. 2004) with no major local or systemic toxicity (Toumi et al. 2006). However, the possibility of using nonviral vectors for transduction of widespread areas of the skeletal muscle and heart has not been tested in large animals. Adenoviral Vectors

Initial studies on gene transfer show that adenoviral (Ad) vectors were among the most commonly used vectors for gene therapy due to their large cloning capacity, ability of transducing post-mitotic cells, and the low risk for insertional mutagenesis and germline transmission. (Verma and Weitzman 2005). However, the ability of adenoviral vectors to induce long-term transgene expression has been hampered by both the host immune response and the nonimmune loss of vector genomes. The use of IS drugs have been attempted to circumvent the immunological barriers. In mdx mice (Jiang et al. 2004), coblockade of both CD28/B7 and CD40L/ CD40 costimulatory pathways was required for effective inhibition of the Ad vector-induced humoral immune response in DMD mice, whereas blockade of CD28/B7 alone by murine CTLA4Ig would be sufficient for prolonged dystrophin expression in treated muscles. Transient IS with tacrolimus (FK506) efficiently suppressed both cellular and immune responses in Ad-injected mdx mice (Lochmuller et al. 1996). In GRMD dogs, expression of human dystrophin by adenoviral vectors was sustained by continuous use of cyclosporine, whereas in dogs without IS both humoral responses and robust infiltrates of CD4+ and CD8+ T cells prevented transgene expression (Howell et al. 1998). Efforts from several investigators resulted in the development of helper-dependent (HDAd) or gutless adenoviral vectors (Verma and Weitzman 2005). These engineered vectors are deleted of all viral genes from the parental vector backbone, whereas maintaining the inverted terminal repeats to keep them intact. Encouraging data obtained in mice without IS (Dellorusso et al. 2002) motivates further studies of HDAd in large animal models. However, short-term gene transfer studies using the HDAd in the muscle of GRMD newborn dogs showed that this vector has limited efficacy (Gilbert et al. 2001). Systemic administration of HDAd vector is further complicated by the potential liver toxicity and transient thrombocytopenia as observed in canine models of hemophilia (Arruda 2006). Thus, therapies based on repeated doses of HDAd vectors for widespread sustained transduction of the skeletal muscle for the treatment of DMD at this point are not supported by the safety profile of these preclinical studies. Retroviral Vectors

Oncoretroviral vectors are attractive for the treatment of genetic disease when stable long-term integration in the genome is required. These vectors are efficient in gene transfer for dividing cells and they can accommodate up to 11 Kd transgene cassettes (Verma and Weitzman 2005). However their efficacy in DMD models is limited and the results in mdx mice showed poor transduction of skeletal muscle. Moreover, safety concerns of the risk of leukemia development by insertional muta-genesis in patients enrolled in a phase I clinical study for severe combined immune deficiency further diminished the use of this vector system for genetic diseases (Hacein-Bey-Abina et al. 2003).

In contrast, lentiviral vectors based on the human immunodeficiency virus has the ability of transducing dividing and non-dividing cells; the insert capacity is 7.5-9 Kb, and early data showed stable expression of transgenes in muscle cells, muscle stem cells or early precursors. Data from the use of lentiviral for ex vivo transduction of autologous mesangioblast stem cell with m-dystrophin gene or heterologous mesangioblast stem cells harvested from normal dogs and injected into the GRMD model are encouraging (Sampaolesi et al. 2006). Following the delivery of the mesangioblasts by intra-arterial (femoral) injection, dystrophin expression was associated with remarkable improvement of both muscle morphology and function. There was improvement of the disease phenotype in both groups of treated dogs. The group of dogs receiving heterolo-gous cells required transient IS with cyclosporine or cyclosporine with rapamy-cin to avoid immune responses to the donor cells (containing the wild-type canine dystrophin derived from normal young donor dogs). These animals presented improved strength and gait of skeletal muscles and reduced serum creatine kinase levels, compared to lentiviral-transduced mesangioblasts. One potential explanation is that the IS was playing a co-adjuvant role in the improvement of the disease phenotype, and further studies will be required to confirm the potential of this strategy. Nevertheless, this study provides evidence that intravascular delivery of stem cell and/or gene therapy coupled with IS has potential therapeutic use in DMD. In a nonhuman primate model, autologous or allogenic (with IS with FK-506) transplantation of myoblast modified by lenti-viral-mediated gene transfer was also associated with sustained, yet low, local gene expression (Quenneville et al. 2007). Adeno-Associated Viral (AAV) Vector

AAV is single-stranded DNA virus belonging to the Parvovirus family. Recombinant AAV vectors are advantageous for human gene therapy because of their ability to transduce dividing and nondividing cells, including skeletal muscle and cardiac muscle fibers. To date there are 12 serotypes characterized and more derivatives identified. The use of AAV vectors as a gene delivery vehicle to skeletal muscle has shown promise both in preclinical studies and early-phase clinical trials (Brantly et al. 2006; Kay et al. 2000; Stroes et al. 2008). Overall the safety profile was excellent with no serious sustained adverse effects. Moreover, the therapeutic potential of AAV serotype 2 (AAV-2) following local delivery to skeletal muscle is attested to by documented long-term local transgene expression (Jiang et al. 2006b; Manno et al. 2003), which is an attractive property for the treatment of genetic diseases.

However, two major drawbacks associated with IM injection of AAV vectors are the risk of immune responses and the restricted transduction of muscle area. Studies in mice and dogs had previously shown that local immune responses in skeletal muscle could cause activation of T and B lymphocytes in draining lymph nodes following IM injection of AAV-2 (High 2005). In the case of a coagulation factor IX (FIX) transgene, the underlying mutation in the dog FIX gene predicts the risk of immune responses to the transgene. Dogs with missense mutation had a lower risk than dogs with null mutation. In the later model, a relatively mild transient suppression protocol using cyclophosphamide was often sufficient to prevent this response in hemophilia B dogs (Herzog et al. 2001). The identification of novel potent AAV vectors offers the opportunity to further increase AAV transduction efficiency (Gao et al. 2002). In the hemophilia B dog model, IM injection of AAV-1 resulted in > tenfold higher levels of FIX compared to AAV-2 vector, but immune response to FIX limited the duration of expression and required IS for long periods (Arruda et al. 2004). Thus, by increasing the amount of local FIX synthesis, the risk for antibody formation increased. In other large animal models, IM AAV vectors of alternate serotypes (-1, -5, -7, -8) encoding erythropoietin in nonhuman primates resulted in an anemia due to inadvertent immune response, following supraphysi-ological levels of transgene expression (Chenuaud et al. 2004; Gao et al. 2004). In a model of limb-girdle muscular dystrophy due to deficiency of a-sarcoglycan (SGCA), overexpression of the therapeutic transgene by AAV under the control of the CMV promoter resulted in cellular toxicity (Dressman et al. 2002). Thus, immune responses following IM injection of AAV prevent the clinical translation of the full potential of these vectors.

In the GRMD model, Storb and colleagues demonstrated T cell-mediated immune responses following IM injection of AAV-2 or AAV-6. This prompted the authors to use short-term IS (up to 3 months post vector injection) to prevent immune responses (Wang et al. 2007). The regimen, containing cyclosporine, MMF and antithymocyte globulin was effective in sustaining expression of canine m-dystrophin after discontinuation of the drugs without local T cell infiltrates (Wang et al. 2007). Some lymphocytic infiltrates were still observed at later timepoints (4 months after immune suppression was discontinued). Recently, studies in mice demonstrated that antigen-specific CD8+ T cells in AAV-transduced skeletal muscle were associated with programmed death of effector cells; the cellular infiltrates were considered immunologically silent (Lin et al. 2007; Velazquez et al. 2009). Moreover, strong CD8+ cytotoxic T cell response against a b-galactosidase transgene with severely limited expression in AAV-2-transduced canine muscle (normal dog) and IS with mycophenolate mofetil (MMF) and cyclosporine partially blocked this immune response, resulting in improved transgene expression (Yuasa et al. 2007). Interestingly, in this study no cellular immune response to the vector capsid was observed. The use of AAV-8 in this dog model was also associated with both cellular and humoral immune responses in the absence of IS (Ohshima et al. 2009). Thus, further characterization of these cells are required to assess whether in GRMD dogs the residual cellular infiltrates at the injection site are directed to the AAV capsid (see below) and/or the transgene and notably, whether these cells are immunologically functional or not. These data in dogs and nonhuman primates demonstrate that immune responses following IM injection of early or novel AAV remain the main obstacle in translating these studies to humans. It is possible that AAV vectors more readily yield undesired gene transfer to antigen presenting cells in large immunocompetent animals than in mice, or that antigen presenting cells and/or T cells respond more strongly to inflammatory signals. Therefore, optimized immune suppression protocols will be required in such gene therapies in humans, if the canine model is a good predictor of the response.

Although access to the skeletal muscle is easily performed by direct IM injections, achievement of AAV therapeutic target doses in humans has proved impractical because of the large number of injections required. Moreover, it is an ineffective strategy for the treatment of DMD that requires widespread transduction of the skeletal muscle, diaphragm, and heart. Thus, the development of intravascular delivery of vector for regional or systemic tissue transduction is critical for translation to humans. In early trials on intravascular delivery of AAV vectors, immune responses to the vector capsid and the risk of germline transmission were recognized as critical challenges to the safety of this strategy. These observations underlie the importance of translational studies in large animals, which have proven a more stringent screen and more accurate predictor of success in humans. Preclinical studies in the GRMD model are critical in terms of defining the feasibility and safety of a given strategy.

Greelish et al. showed that regional intra-arterial delivery of rAAV to the skeletal muscle, with either adenovirus or AAV vectors, resulted in extensive transduction of skeletal muscle in rats and hamsters (Greelish et al. 1999). This procedure required the use of histamine and papaverine to enhance vector extravascular dissemination to the target muscle. Studies in hemophilia B dogs demonstrate that this technique was also successful in widespread transgene expression in a large animal. Regional vascular delivery of AAV-2 canine FIX to the muscle tissue in a single limb perfusion model resulted in long-term expression (> 4 years) of the transgene (Factor IX) at therapeutic levels (ranging from 4 to 15% of normal). In this study, transient IS with weekly injections of cyclosphosphamide (total 6 doses) prevented the formation of antibody to the transgene. In the hind limb that received the direct intramuscular injection, cF.IX expression was confined to the sites of injection, with a radius of diffusion of ~0.5 mm, whereas the hind limb that received vector by the intravascular infusion process showed transduction throughout the muscle groups supplied by the injected vessel (Arruda et al. 2005). In nonhuman primates, intra-femoral delivery of an isolated limb of AAV-8-GFP vector resulted in transduction of > 65% of the muscle area perfused three weeks after injection with no acute toxicity (Rodino-Klapac et al. 2007). Although it will be necessary to address details of the immune response to the transgene product in a GRMD model, and to identify an approved pharmacologic agent that can induce a vascular leak (i.e., a drug other than histamine), these studies have established the efficacy of the strategy.

Stedman and colleagues developed an alternative noninvasive delivery method aimed at ensuring widespread gene transfer to the skeletal muscle named afferent transvenular retrograde extravasation (ATVRX). This delivery method is achieved by an intravenous injection of vector through a peripheral vein of an isolated limb under elevated hydrostatic pressure to ensure vascular leakage in large animals (Su et al. 2005). The increase in venous pressure induces passive capillary dilation and changes in transmural pressure, which facilitates tissue perfusion (Williams 1999). Thus, a sudden increase in the venous pressure by injecting enough volume of a solution-containing vector will forcibly deliver vector to large areas of the skeletal muscle. This strategy has been successful in AAV-mediated gene transfer to the skeletal muscle of normal dogs or nonhuman primates through the superficial saphenous vein under elevated hydrostatic pressure. ATRVX delivery of AAV-1 or AAV-8 vectors encoding lacZ gene in the normal dog resulted in widespread trans-duction of the injected limb, but the expression is short-lived due to immune responses to the transgene (Su et al. 2005; Ohshima et al. 2009). ATRVX delivery of AAV-1 or AAV-8 was well tolerated in nonhuman primates with sustained transgene expression for the duration of the experiment with transient IS with MMF and prednisone (Toromanoff et al. 2008). Transgene expression was detected in most of the muscle of the injected limb, but the local gene copy number is lower when compared to the IM injection of a similar vector. Vector biodistribution revealed that vector sequences were found outside the ATRVX-treated limb and these findings raise safety concerns. ATRVX-mediated human m-dystrophin in two GRMD dogs was effective in ensuring transgene expression in muscle biopsies collected at week 4, but the number of m-dystrophin positive cells decreased by week 8, probably, in part due to the use of non-species specific transgene (Ohshima et al. 2009). In normal young dogs, ATRVX delivery of AAV-8 of canine myostatin propeptide gene (MPRO) resulted in muscle growth, increased muscle myofiber size in multiple muscles of the injected limb, and muscle volume (Qiao et al. 2008b). Notably, no T cell infiltrates were observed for the duration of the experiment (3 months). The data on ATRVX delivery to skeletal muscle in large animals are encouraging, and a comprehensive study on the rates of cellular and humoral immune responses to the vector and/or transgene need to be addressed in adult GRMD dogs using species-specific m-dystrophin transgene. Moreover, vector dissemination in this model will require further refinement since the use of appropriate tourniquets are critical to restrict regional delivery of the vector.

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