Gene therapy in transplantation

Transplant Immunology 9 (2002) 301–314

Review

Gene therapy in transplantation D. Chen, R. Sung, J.S. Bromberg* Carl C. Icahn Institute for Gene Therapy and Molecular Medicine and the RecanatiyMiller Transplantation Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1496, New York, NY 10029-6574, USA

Abstract Gene transfer and gene therapy represent a relatively new field that has grown and expanded enormously in the last 5–10 years. The application of gene transfer and gene medicines to transplantation is currently in its infancy. Consideration for gene medicines in transplantation requires delivery of vectors, either to the graft or to the immune system. Delivery of vectors to the graft provides a choice of potential immunologic targets including: costimulatory signals; inhibitory cytokines; adhesion molecules; and molecules relating to apoptosis. In addition, non-immunologic targets, that increase graft protective mechanisms by reducing ischemic and immunologic damage, represent significant targets for gene transfer. Delivery of vectors to the immune system includes potential targets to modify the immune system, and results in tolerance. Other considerations for gene therapy include the development of additional technologies, such as gene conversion or transgenesis coupled with xenotransplantation, which may provide genetically modified organs. Another important aspect of gene transfer relates to regulation of the transgene expression. A variety of issues concerning innate immunity, adaptive immunity, response to vector components, response to transgene products, and entry of vectors into the antigen presentation and processing pathway require further investigation and refinement of approaches. Lastly, regulatable promoters and the understanding of their interaction with individual cells, tissues and organs, and their interaction with innate and adaptive immunity, are of paramount importance to improving the efficacy and utility of gene transfer. There is no doubt that there is much exciting basic and translational science to be accomplished in the next decade in order to solve these potential barriers and advance gene medicines into the clinical realm in transplantation. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Transplantation; Gene therapy; Tolerance

1. Introduction Organ transplantation has become a conventional treatment for failure of various organs, but chronic allograft rejection, the side effects of chronic immunosuppressive therapy and severe donor organ shortages continue to limit its success. Gene therapy has the potential to prevent graft rejection by manipulating the immune response in the microenvironment of the graft, by facilitating the induction of tolerance, or by generating transgenic animals for xenotransplantation. 2. Modification of the graft One of the most important advantages of gene therapy in transplantation is that it can be targeted to a specific *Corresponding author. Tel.: q1-212-241-8938; fax: q1-212-8492437. E-mail address: [email protected] (J.S. Bromberg).

organ or tissue through ex vivo manipulation. Therefore, local immunosuppression, confined to the transplanted organ or tissue, can be achieved. Almost every transplantable organ or tissue has been shown to be receptive to gene transfer by one or more of the currently available gene transfer vectors. The susceptibility of different organs, tissues, or cells to gene transduction may vary depending on their biological characteristics. For example, when considering the application of gene therapy to tissue transplantation, the cornea stands out as having unique properties. The tissue is small; can be maintained in culture for several weeks at 32–37 8C, instead of a few hours at 0 8C; and the key target cell, the corneal endothelial cell, forms a single sheet of cells on the inner surface of the cornea and is, therefore, perfectly accessible to vectors. Another area of transplantation that is likely to benefit most from advances in gene therapy techniques is that of cell transplantation, in

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Table 1 Transgenes used in the modification of the graft Transgenes

Mechanism of suppression

Allograft

Reference

CTLA4Ig

Inhibit CD28-CD80y86 interaction, interrupt costimulatory pathway Inhibit cytokine synthesis, negative regulation of antigen presenting cell function Suppress Th1-mediated immune response Block cell adhesion

Rat kidney, liver, islets

w4–6x

Mouse heart, rat heart, sheep cornea

w7–12x

Mouse heart

w13x

Rat heart, mouse islets

w15,16x

Chemokine antagonist

Mouse heart

w14x

Induce apoptosis of immune cells Inhibit inflammatory signals, attenuate ischemia-reperfusion injury Anti-oxidative stress Protect tissue cells from apoptosis

Mouse heart, islets Mouse heart, rat heart

w17,18x w19x

Rat aorta Mouse liver, endothelium Rat heart

w21x w22,23x

vIL-10, IL-10, TGF-b IL-12 p40 ICAM-1 antisense vMIP-II, MC148 FasL NF–kB decoy iNOS Bcl-2 HSP 70

Enhance myocardial tolerance after normothermic ischemia-reperfusion

particular, islet transplantation. Because islets can be placed in culture before transplantation, they have the potential for efficient and targeted transfection. Furthermore, culture allows for selection of transduced cells. Although efficient gene transfer into primary cultures of human or pig vascular endothelial cells has been reported, uptake by vascular endothelial cells in pieces of aorta was found to be much less efficient w1x. Delivery to the myocardium was found to be more efficient than to the artery, with the former resulting in reporter expression in a localized area of the heart which peaked between days 3 and 6 after injection and returned to control levels within a month w2x. As an alternative to directly modifying the graft, genetically engineered cells have been co-transplanted with cellular grafts (for example islets) to produce locally active immunosuppressive bioreagents w3x. Many different biologically active molecules have been delivered into grafts to prolong survival, as outlined in Table 1. Two major approaches can be defined: (1) inhibiting anti-graft immune responses; and (2) increasing graft protective mechanisms against the host immune response. 2.1. Inhibition of anti-graft immune responses 2.1.1. Blockade of costimulatory signals Costimulatory signaling between CD28-CD80y86, or CD40-CD40L, plays a pivotal role in alloimmune responses and transplant allograft rejection. Failure of this receptor–ligand engagement at the time of antigen recognition induces a state of anergy. Administration of anti-CD40L antibody or CTLA4Ig effectively prolongs allograft survival in heart, kidney, liver and islet cell transplantation in rodents and subhuman primates. Gene

w20x

transfer to modify the grafts has been described for CTLA4Ig. Adenovirus-mediated CTLA4Ig gene therapy has resulted in prolonged survival in rat liver, kidney and islet transplantation w4–6x. 2.1.2. Inhibitory cytokines Expression of type 2 cytokines or TGFb has the potential to promote graft survival by inhibiting the production of type 1 cytokines and blocking some antigen presenting cell and macrophage functions. Viral IL-10 (vIL-10) is a viral form of interleukin-10 (IL-10) that is encoded by the Epstein–Barr virus, but lacks some of the T cell immunostimulatory activities of IL10. Gene transfer of the sequence encoding vIL-10 or TGFb have resulted in prolonged allograft survival in various models w7–9x. Cellular IL-10 has also been reported to promote permanent liver graft acceptance w10x and prolong cardiac allograft survival in rats w11x. A recent study reported that in sheep, ex vivo transfer of the gene encoding IL-10 to cornea can prevent rejection after allotransplantation. Five of nine genetically manipulated corneas showed prolonged survival, and another two showed indefinite survival, without any other treatment w12x. Retrovirus-mediated local production of the p40 subunit of IL-12 suppressed Th1mediated immune responses and prevented allogeneic myoblast rejection in the mouse w13x. 2.1.3. Inhibition of leukocyte infiltration or adhesion The recruitment and activation of recipient leukocytes in response to a transplanted organ constitute the initial steps of a complex immune response, which ultimately culminates in rejection of the graft. Chemokines are the primary molecules involved in the leukocyte recruitment. Plasmid-mediated gene transfer of virally encoded

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chemokine antagonists vMIP-ll and MC148 have been reported can block donor-specific lymphocyte immunity within cardiac allografts and prolong graft survival w14x. Adhesion molecule interactions including LFA-1y ICAM-1, LFA-3-CD2 and VLA-4yVCAM-1 promote interactions between leukocytes and other cells. Transfection of rat hearts with antisense oligonucleotides, for blockade of ICAM-1, demonstrated specific reduction of chronic graft vascular disease w15x. 2.1.4. Inducing apoptosis of immune cells Theoretically, expression of FasL by grafted tissue may protect allogeneic grafts against immune cell infiltration and destruction by inducing apoptosis of graftreactive T cells expressing Fas. But, contradictory results have been obtained. The survival of islet allografts cotransplanted with myoblasts overexpressing FasL was significantly prolonged w17x, whereas mice transplanted with FasL expressing islet allografts were rejected in an accelerated fashion due to massive granulocyte infiltration w18x. These results indicate that regulation of inflammation and immune response by Fas and FasL is complex. 2.2. Increasing graft protective mechanisms It has been postulated that chronic allograft dysfunction is mediated by both alloantigen-dependent (MHC incompatibility, immune mechanism) and alloantigenindependent factors, such as anoxia and ischemia-reperfusion injury that result in inflammatory reactions. Some molecules which have protective actions in non-immune processes, may also help graft survival. 2.2.1. Preventing endothelial activation NF–kB, is an inducible activator of transcription of a large array of genes, some of which are required for inflammatory and immune response. Inhibition of NF– kB may block the activation of endothelial cells, and subsequently protect the graft from inflammatory injury. In one such approach double-stranded oligodeoxynucleotides with a specific affinity for NF–kB (NF–kB decoy), were transfected into rat hearts. Gene transfection of the NF–kB decoy attenuated ischemia-reperfusion injury after prolonged heart preservation w19x. Heat shock protein 70 (HSP70) gene transfection has been shown to enhance myocardial tolerance after normothermic ischemia-reperfusion. In a clinically relevant donor heart preservation protocol, HSP70 gene transfection protects both mechanical and endothelial function w20x. 2.2.2. Anti-oxidative stress Reactive oxygen species are responsible for cell death within the graft, induce cytokine production and recruit leukocytes. Nitric oxide has been shown to suppress T

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cell proliferation, inhibit leukocyte chemotaxis and prevent vascular smooth muscle cell proliferation. Transduction of rat heart allografts with an inducible nitric oxide synthase gene suppressed the development of allograft arteriosclerosis for 4 weeks w21x. 2.2.3. Anti-apoptotic genes In vitro expression of anti-apoptotic genes (Bcl-2, Bcl-xL and A20) by endothelial cells has been shown not only to protect hepatocyte or endothelial cells from apoptosis (as a consequence of ischemia-reperfusion injury), but also to inhibit their activation through an NF–kB-dependent mechanism w22,23x. 2.2.4. Immunomodulation using dendritic cells Dendritic cells (DC), highly specialized antigen-presenting cells (APC), are now regarded not only as the initiators and regulators of immune responses, but also as potentially powerful tools for the therapeutic manipulation of immune reactivity in allograft rejection as well as cancer, autoimmunity and infectious disease w24x. A role for DC in tolerance induction was recognized initially in the context of intra-thymic self-tolerance. In addition to having a role in central tolerance induction, DC are now regarded as potential modulators of peripheral immune responses w25x. Compared with strategies that aim at directly manipulating the transplanted organ, the induction of T-cell non-reactivity or tolerance via DC manipulation can be fulfilled outside the transplanted tissue and before transplantation. Direct damage to the organ that might be caused by the vectors or immunosuppressive genes used for manipulation could thus be avoided. Another advantage of DC treatment is that they can be manipulated much more easily than solid organs under controlled laboratory conditions. Since DC constitutively express MHC antigens, home to T-dependent areas of recipient lymphoid tissue, and interact selectively with host T cells, they are an attractive target for genetic manipulation to improve the outcome of allograft survival. Studies have reported that immunosuppressive genes such as CTLA4Ig w26x, TGFb w27x and vIL-10 w28x can be successfully delivered into DC and promote T cell hyporesponsiveness. 3. Modification of the host Tolerance remains the primary goal in transplantation immunology. There are several methods that can be used to create a state of acceptance, such as micro- and macrochimerism, co-stimulatory blockade, deletion of donor-specific immune cells, and induction of active, persistent regulatory mechanism w29x. When genetically engineering the host’s immune system, researchers also aim at these mechanisms.

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3.1. DST type protocol—MHC gene transfer and tolerance induction Although MHC molecules are the main targets for allorejection, exposure of the recipient to donor MHC antigens (such as donor blood or bone marrow, MHC molecules, or MHC-derived peptides) in a non-immunogeneic antigen presentation context has been shown to promote graft acceptance. Donor-specific blood transfusion (DST) provided to prospective kidney transplant recipients has resulted in significant improvement in graft survival rates in livingrelated kidney transplant recipients w30x. A state of permanent chimerism may result from the transfusion of stem cells contained in DST w31x, which is considered one of the important tolerance-inducing mechanisms. However, bone marrow may be more effective at establishing chimerism than peripheral blood, because bone marrow has 10–100 times more stem cells than peripheral blood. Indeed, the infusion donor-specific bone marrow (DBM) has been found to be effective in inducing donor-specific graft acceptance w32x. The potential mechanism by which DST and DBT immunomodulate the immune system are likely to be different only in degree rather than kind. These mechanisms include anergy, development of anti-idiotypic antibodies, provision of HLA antigen, alteration in T cell subsets, stimulation of regulatory cells, clonal deletion, regulation of cytokines, andyor chimerism w33x. Among these mechanisms, microchimerism with the concomitant persistence of soluble donor HLA antigen is felt by many to be the most important. With the concern that DST may sensitize patients, while DBM infusion may predispose the recipient to graft-vs.-host disease and is not easily accomplished in living donor organ transplantation, DNA-mediated gene transfer may, represent a safer approach to molecular chimerism. Gene delivery of sequences encoding alloMHC class I antigens to autologous BM cells facilitated the development of tolerance to a mismatched skin graft in mice, by inducing molecular, rather than cellular, chimerism w34x. This strategy might also facilitate the development of unresponsiveness to a fully mismatched vascularized organ, and similar results were reported in a class I transgenic model of heart transplantation w35x. The hyporesponsiveness was likely the result of T cell anergy rather than clonal deletion w36x. Gene delivery of sequences encoding donor MHC class II antigen has also induced prolonged donor-specific organ survival w37x. The high level of MHC polymorphism may not be an obstacle to this approach, since it has been shown that the presentation of a single allelic product can result in inhibition of recipient response to the entire donor haplotype. It is well known that tolerance to self-MHC antigen develops via presentation of this antigen on the thymic

epithelium. Thus, the thymus seems to be a good target for tolerance induction by MHC gene transfer. Delivery of donor cells to the thymus in combination with modification of the peripheral immune system has been shown to induce tolerance to donor tissue subsequently transplanted at peripheral sites w38x. Indirect gene transfer to thymus using myoblasts, myotubes and fibroblasts as cellular intermediates has also been accomplished successfully. This approach, using donor MHC class I presentation to thymus, led to long-term specific tolerance to subsequent liver transplants w39x. Another study used direct gene injection to the thymus to express a single foreign MHC class I Ag in the thymus, prolonging liver allograft survival over 100 days in the majority of recipients following this manipulation w40x. 3.2. Systemic immunosuppression It was reported recently, in a rat lung allograft model, that significant reduction in rejection grade (from grade 3 to 2) occurred after systemic but not intrabronchial adenoviral vector CTLA4Ig gene transduction. The authors concluded that local expression of immunomodulatory proteins could be achieved within lung allograft by intrabronchial delivery of adenoviral vector, but that systemic immunomodulation is required to modify acute rejection w41x. In contrast to protein administration, in vivo gene transfer may be able to produce persistent, biologically controlled long-term expression of gene product. Therefore, gene therapy may have a role in providing long-term systemic immunosuppression. Some studies show that systemically administered adenovirusmediated gene transfer with CTLA4Ig is effective for prolonging both transgene expression and allo- and xenograft survival w42x. Another group reported that a single treatment with AdCD40Ig produced a substantial amount of CD40Ig protein in serum and allowed indefinite graft survival in a stringent rat liver allograft model w43x. These approaches, however, suffer from the same problem of conventional immunosuppression in that chronic systemic administration is associated with a number of adverse events and side effects. Recently, a new type of adenovirus vector, Adex1CALoxCTLA4IgGLox was constructed w44x. It could be manipulated to terminate in vivo CTLA4IgG expression at a desired time point by using Adex1CACre virus. This termination system may minimize the adverse effects of CTLA4Ig gene therapy. It was demonstrated that: CTLA4IgG expression from Adex1CALox-CTL4IgGlox was persistent and efficient; in vivo gene expression was completely terminated after administration of Adex1CACre virus; long-term acceptance of allografts (pancreatic islet and skin) was achieved even after the termination of CTLA4IgG expression; and immune responses against adenovirus were preserved. Thus, the

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principle of systemic gene transfer and stringent regulation of expression may achieve tolerogenic effects. 3.3. Selective T cell suicide gene therapy Control of anti-donor activated T cells involved in allograft rejection, while preserving immunocompetence, is a challenging goal in transplantation. Engineered T cells expressing a viral thymidine kinase (TK) suicide gene metabolize the non-toxic prodrug ganciclovir (GCV) into a metabolite toxic only to dividing cells. This system is particularly well suited to developing an immunomodulation strategy based on elimination of the dividing T cells engaged in pathological immune responses, while sparing quiescent T cells. In transgenic mice expressing TK in T cells, it has been shown that rejection of fully allogeneic skin graft and non-vascularized heart graft can be significantly delayed by GCV treatment w45x. In addition, a short course of GCV induces long lasting acceptance of vascularized abdominal heart allograft, and the tolerance generated in this system is robust, as shown by the acceptance of a second abdominal allograft, without any additional treatment. These mice remain immunocompetent since they are capable of rejecting third-party allografts with normal kinetics w46x.

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DNA, and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics and gene therapy w48x. 3.5. Gene therapy in xenotransplantation Shortages of human organ for transplantation have made it necessary to examine the possibility of using non-human organs for xenotransplantation. The pig is presently considered the most likely source of organs for human xenotransplantation because it is easy to breed, has compatibly sized organs and offers the possibility of genetic manipulation. The major immediate immunological hurdle that a discordant vascularized xenograft will most probably face is a complementmediated hyperacute rejection (HRA) following the binding of preformed, xenoreactive natural antibodies (XNAs) against xenoantigens. In fact almost all of the human Abs bind to an oligosaccharide pig xenoepitope defined as Gal-a(1,3)-Gal-b(1,4)-GlcNAc-protein, also known as aGal. Attempts to use gene therapy to prevent xenograft rejection also involve the modification of donor organ and the host. 4. Modification of donor organs

3.4. Gene conversion Gene therapy is usually taken to mean the delivery of a gene(s) of interest in an expression vector to a target cell, however, there are alternative approaches to gene therapy, such as targeted DNA or RNA conversion. Although germ line mutations can be incorporated by means of embryonic stem cell technology, little progress has been made toward introducing mutations in somatic cells of living organisms. Triplex-forming oligonucleotides (TFOs) recognize and bind to specific duplex DNA sequences and have been used extensively to modify gene function in cells. It has been reported that TFOs can induce mutations at specific genomic sites in somatic cells of adult mice, providing the initial evidence that sequence-specific, DNA-modifying reagents may prove useful in intact animals w47x. Group II introns are catalytic RNAs that function as mobile genetic elements by inserting directly into target sites in doublestranded DNA, and then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a )14-nucleotide region of the DNA target site, as well as fixed interactions between the intron-encoded protein and flanking regions. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, it has been shown that group II introns can be retargeted to insert efficiently into virtually any target

Eliminating the aGal epitope from porcine tissue through genetic engineering of pigs would be the most direct way to prevent HAR or delayed xenograft rejection (DXR). It is not yet possible to conduct genetargeting in pigs to knock out the aGal gene because porcine embryonic stem cell lines capable of achieving germ line transmission of mutations induced by homologous recombination are not available. Preventing HAR by genetic transfer of human complement regulators has been another major focus. Several groups have successfully introduced genes that encode human complement regulators, such as human decayaccelerating factor (DAF), membrane cofactor protein (MCP) or CD59, into the pronuclei of fertilized pig oocytes w49x. Organs from DAF-expressing pigs survived for up to 8 days when transplanted into baboons, in the continued absence of any visible HAR w50x. In these studies, there was no attempt to reduce the titers of xenoreactive natural antibodies or to provide other immunosuppression, clearly indicating that HAR can be surmounted by the local inhibition of complement activation by genetically manipulating the donor organ. Both HAR and DXR involve the activation of endothelial cells (ECs), so modification of the graft by transferring protective genes is another important approach to xenograft survival. Transfer of genes encoding CTLA4Ig w51x, IL-10 and TGF-b w52x has been reported to result in significant prolongation of islet

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xenograft survival. The anti-inflammatory properties of hemoxygenase-1 (HO-1) have been shown to promote xenograft survival, and rapid synthesis of HO-1 in cardiac xenografts is essential for ensuring long-term xenograft survival w53x. Another approach is to eliminate or reduce aGal expression by replacing aGal epitopes with other carbohydrate moieties to which humans are immunologically tolerant. This method involves the genetic engineering of xenogenetic cells to express a-(1,2)fucosyltransferase (a-FT), which adds fucose residues to an oligosaccharide. In both pigs and humans, the substrate for the fucosyltransferase is identical. It has been shown that expression of a-FT in vascular endothelium can eliminate approximately 90% of cell surface aGal epitopes w54x, although the remaining 105;106 aGal epitopes is sufficient to permit DXR mediated by XNAs without further immunologic manipulation w55x. 5. Modification of the host Lasting mixed chimerism was achieved in alpha1, 3galactosyltransferase knockout (GalT KO) mice by cotransplantation of GalT KO and GalTqyq wild-type (WT) marrow after lethal irradiation. Mixed chimerism efficiently induces anti-Gal-specific B cell tolerance in addition to T cell tolerance, providing a single approach to overcoming both the humoral and the cellular immune barriers to discordant xenotransplantation w56x. Currently, this approach is not yet practical in humans. In another report, a functioning gene that encodes agalactosyltransferase (aGT) was introduced by retroviral gene transfer into autologous murine bone marrow cells, preventing aGal XNA production w57x. Thus, genetic engineering of bone marrow may overcome humoral rejection of discordant xenografts and may be useful for inducing B cell tolerance. MHC gene transfer into BM cells have also shown promising results in controlling cellular responses to xenoantigens. Transfer and expression of xenogeneic MHC class II DR transgenes can be achieved in baboons. This therapy may prevent late T cell-dependent responses to porcine xenografts, which include induced non-aGal IgG antibody responses w58x. 6. Limitations to gene transfer and expression A variety of barriers exist to efficient, targeted expression by gene therapy vectors. To achieve this goal, vectors must selectively reach and be retained by the target tissue, enter cells and negotiate endosomal compartments. Vector-derived nucleic acid must be transported to the nucleus, and must be efficiently transcribed. At each of these levels, conditions particular to the vector and the intended application may ultimately impair effective expression of transgene. In addition, host innate and adaptive immune responses to vector

components and transgene products limit vector expression through apoptosis, T-cell mediated cytotoxicity, vector clearance and inhibition of transcription. 6.1. Adaptive immune responses to vector antigens Adenoviral vectors, although highly efficient, are unfortunately also highly immunogenic. These vectors are attractive for gene transfer because of their broad host range, and capacity for large DNA inserts. However, administration of first-generation adenovirus vectors (those with viral genes E1 and E3 deleted) generates well-characterized adaptive immune responses against viral capsid proteins, which appear to be the primary mechanism by which expression is extinguished w59,60x. Both the cellular and humoral arms of the adaptive immune response are triggered, the former effecting viral clearance, and the latter preventing effective readministration of vector. These responses have been attributed to low levels of viral gene expression, or may reflect contamination by wild-type virus. The importance of viral gene expression is supported by a diminution of both Th2 responses and neutralizing antibodies when E4 is deleted from first-generation vectors w61x, and by the absence of late inflammation in muscle when vectors lacking all viral genes are employed w62x. However, cellular responses may not necessarily require viral gene expression, as vectors rendered biologically inactive generate similar infiltration of CD4q and CD8q lymphocytes as do untreated vectors, and antivector CTL responses can be generated in the absence of vector transcription w63,64x. Adeno-associated virus (AAV) vectors, although less efficient than adenovectors, incite much less of an immune response. Consequently, expression by these vectors results in greater vector persistence and transgene expression w65x. Transfection of dendritic cells (DC) by adenovirus vectors is an important component in the development of the anti-vector immune response. Adenovirus vectors efficiently transfect both mature and immature DC without altering maturation or function w66x. DC transfected by adenovirus can be adoptively transferred to effect immune mediated elimination of adenovirus or AAV infected cells, but AAV-transfected DCs cannot. This is likely due to the inability of AAV to transduce or to effectively activate DCs, and may account for the decreased immunogenicity of AAV vectors w67,68x. Dendritic cells infected in vitro with adenovirus encoding an antigenic peptide can subsequently induce strong specific CTL responses that can be augmented with repeated administration of transfected DC w69x. Low titers of neutralizing antibody are generated with this method; in contrast, direct immunization generates high titers of neutralizing antibody, which limits the efficacy of repeated administration. Such an approach may have

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advantages in designing vaccination therapies or in tumor immunization. 6.2. Adaptive immune responses to transgenes Immune responses to encoded transgenes also limit expression by gene therapy vectors. Much of the literature describing immunity to adenovirus vectors has been generated using vectors expressing the immunogenic reporter b–galactosidase. Human a1-antitrypsin and human factor IX are two examples of less immunogenic reporter proteins whose duration of expression by adenovirus vectors exceeds that of b–galactosidase w70,71x. Long-term expression has also been demonstrated in mice transgenic for hAAT and b–galactosidase w70,72x. Vectors encoding murine erythropoietin demonstrate superior long-term expression in mice compared with identical vectors encoding the human homologue w73x. Thus, while immune responses to adenovirus proteins per se may be less important in limiting expression than originally believed, the immune response to the vector may potentiate the antitransgene response; immune responses to Factor IX, while activated by adenoviral vectors, are absent following administration of AAV vectors expressing Factor IX w74x. The magnitude of immune responses to transgene products may be more strain-dependent than responses to viral antigens, which may account for the strain variation in expression by adenoviral vectors w75x. Regardless of the relative contributions of responses to vector antigens and transgene products, it is clear that any gene therapy strategy must account for both in order to achieve long-term expression. The mechanisms underlying adaptive immune responses to transgenes also include a prominent role for DC not only in indirect presentation of soluble antigen, but also directly via vector uptake. Inoculation of plasmid DNA into muscle or skin results in activation of plasmid-containing dendritic cells at the injection site, in draining lymph nodes and in the systemic circulation w76,77x. These cells function as antigen presenting cells (APC), can induce specific proliferation of CD4q T cells and generation of CD8q CTLs, and can induce both primary and secondary adaptive immune responses w78,79x. Much of the transgene product can be presented directly by transfected DC, since antigen-bearing DC in draining lymph nodes can be depleted by antibodies to a cell surface protein encoded by cotransfected DNA w79x. This direct pathway appears to be the primary mode of antigen presentation in this model, rather than cross- or indirect presentation of antigen by untransfected DC that have taken up soluble antigen. 6.3. Immune responses to nucleic acids Immune responses to nucleic acids in gene therapy vectors also contribute to limitation of expression. Bac-

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terial DNA is a powerful stimulator of polyclonal proliferation in B cells and cytokine production by monocytes and lymphocytes w80,81x The immunostimulatory properties of bacterial DNA are attributed to unmethylated CpG motifs, which may be present in plasmid DNA or synthetic oligonucleotides. These motifs are much less frequent in mammalian DNA, which is not immunostimulatory w82x. These responses are sensitive to inhibitors of endosomal acidification such as bafilomycin A or chloroquine w83,84x. This acidification is coupled to the generation of intracellular reactive oxygen species, which in turn is linked to the degradation of IkB w83x, and inhibition of IkB degradation suppresses the immunostimulatory effects of CpG DNA w85x. CpG DNA also induces the activation of cJun N-terminal kinase and p38 which leads ultimately to activation of the transcription factor AP-1 w86,87x. The actions of CpG motifs stimulate innate immune responses via TNFa and IL-6 secretion, monocyte activation and increased NK cell activity w83,88,89x. In addition, type I interferons, IFNg, IL-12 and IL-18 are also induced, and subsequently stimulate Th1 responses and enhance adaptive immunity w90–93x. Although CpG motifs are generally immunostimulatory to most cell types, evidence exists that different sequences possess different cellular profiles of activity w82x. While the ability of these motifs to function as immune adjuvants to coadministered antigens are advantageous for immunization, they are potentially deleterious to gene therapy applications, which require sustained expression of transgene product. Efforts to minimize CpG immunostimulation by methylation of stimulatory CpG sequences, while effective in reducing cytokine induction, have resulted in dramatic decreases in gene expression due to methylation of CpG sequences in the promoter w82x. 6.4. Viral components Vector efficiency is also limited by the ability to enter host cells via defined receptors. The tissue distribution of cellular receptors for viral vectors may influence the distribution of vector and can result in either inefficient transduction of target tissue or in transduction of undesirable cell types. Adenoviral vector infection can be mediated by the cocksackie and adenovirus receptor (CAR), av-integrins or MHC class I heavy chain w94x. Tissue distribution of CAR expression varies in a fashion that is similar among species w95x. CAR expression is greatest in liver, which is also the organ with the greatest transgene expression following systemic administration. More efficient vector transduction and transgene expression has been achieved in typically inaccessible tissues such as lung and brain in CAR transgenic mice, which express higher levels of CAR on these tissues, compared with wild-type mice w96x. Enhanced expression and the ability to express transgene following repetitive admin-

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istration of vector has also been reported with adenoviral vectors that encode the CAR receptor in addition to reporter gene w97x. In this model, enhanced CAR expression on the target tissue presumably permits greater cell entry of vector following secondary administration. However, despite the important role of CAR binding, other barriers to effective expression exist. Many tissues expressing levels of CAR and av –integrins, which would make them potential targets for adenoviral vectors, do not express well in vivo; indeed, receptor expression and in vivo expression patterns correlate poorly w95x. Presumably significant anatomic barriers to vector accessibility to target tissues exist. Mutations of the adenoviral knob domain of the fiber protein, which binds CAR, do not alter the in vivo biodistribution of vector compared to wild-type vector w98x. However, replacing the CAR-binding domain with the integrinbinding motif RGD results in an expanded vector tropism in vivo w99x. Other barriers to efficient, targeted adenovirus infection include endothelial cells of solid organs, antiadherence layers of glycosaminoglycans in urothelium and diffusion of vector to distant sites in applications where direct injection of vector is employed. Targeting of adenoviral vectors to tissues such as endothelial cells which have low transduction efficiency has been achieved by utilization of recombinant bispecific antibodies which serve as a bridge between the knob domain and tissue-specific cell surface proteins w100x. Administering vectors in specific polyamide vehicles, which abrogate the GAG barrier w101x, has enhanced transfection to urothelial cell types such as bladder. Enhanced local expression of vector and decreased peripheral transduction of non-target tissues has been achieved by complexing adenoviral vectors in a collagen-avidin gel using a specific anti-adenoviral IgG w102x. Expression by retroviral vectors is also restricted by tissue distribution of cell surface receptors. Retroviruses such as amphotrophic murine leukemia virus (MuLV) and gibbon ape leukemia virus (GALV) possess a broad tissue tropism due to the ubiquitous expression of their cell surface receptors, the phosphate transporter molecules Pit1 and Pit2, respectively. Pseudotyping of other retroviral vectors such as feline leukemia virus with chimeric envelope proteins has produced vectors capable of binding either of these receptors, thereby enhancing the potential infectivity of these vectors w103x. Envelope pseudotyping may also effect tissue selectivity of retroviral vectors; vectors have been successfully pseudotyped to target melanoma cells w104x and human lymphocytes w105x. However, others directed against a folate receptor, EGF receptor and IGF receptor have resulted in impairment of virus transduction, implying that other barriers to vector entry may exist w106–108x.

6.5. Antigen processingypresentation pathways By their nature gene transfer vectors must effect the transfer of nucleic acid through endosomal compartments, and cytoplasm to the nucleus for transcriptional activation. The type of gene transfer vehicle influences the rate of transfer of DNA to acidic lysosomes, the degree of intracellular recycling and exocytosis of DNA, and thus influence expression w109x. These vectors are invariably introduced into antigen processing and presentation pathways, which engender antivector immune responses. Antigen presentation may ensue due to direct transduction of APC by vectors, or by transfer of proteins from target tissue to APC w110–113x. Both viral and non-viral vectors can efficiently direct antigen to MHC class I and class II pathways w114–116x. Retroviral nucleic acid sequences have also been shown to upregulate MHC class I expression in target tissue w117x. Other investigators have shown that double stranded polynucleotides in the cytoplasm of target cells perturb MHC expression and increase the expression of proteasome proteins, antigen peptide transporters and costimulatory molecules, thereby enhancing antigen presentation and recognition w118x. Viral vector antigens also interfere with antigen presentation pathways through changes in the expression or function of MHC molecules. Adenovirus E1 region proteins decrease transcriptional activation of class I genes w119x. The adenovirus E3-19K product binds to class I MHC in the endoplasmic reticulum, resulting in decreased cell surface expression of class I MHC and decrease cytotoxic T cell responses to adenoviral antigens w120–123x. Herpesviruses express genes which selectively interfere with viral antigen presentation by MHC class I, and retroviral vectors encoding HSV ICP47, HCMV US3, or HCMV US11 proteins all decrease target tissue MHC class I expression and evade CTL recognition w124x. These virally encoded proteins may be potentially utilized to potentiate vector persistence and expression. 6.6. Innate immunity While the major mechanism for the attenuation of gene expression is the specific cellular immune response mediated by cytotoxic T lymphocytes and immunoglobulins to either viral proteins and to the encoded transgene product, loss of transgene expression frequently precedes the development of specific immune responses. Thus, non-specific, innate immune mechanisms must also contribute to limiting vector efficiency and persistence w125,126x. Resident macrophages clear a vast majority of adenovirus vector genomes within 24 h of administration w127,128x, and Kupffer cell depletion by gadolinium chloride treatment substantially reduces this vector clearance in the liver w129x. Innate immune responses

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are characterized by pattern recognition receptors such as mannan-binding lectin, which focus on highly conserved sequences common to many pathogens. Effector responses are rapid and do not require effector cell proliferation. Signaling receptors, such as those of the toll family, induce expression of a variety of cytokines and chemokines involved in innate and adaptive immune responses w130x. A broad range of cytokines (TNFa, IL-6, IFNg, IL-1b) and chemokines (MCP-1, RANTES, IP-10, MIP-1a) are induced following vector administration. While some of these cytokines such as TNFa and IFNg possess intrinsic antiviral properties, they also induce a profound inflammatory response mediated by neutrophils, NK cells and monocytes, which effect vector clearance and apoptosis of transduced cells w125,126,129,131x. In addition, so-called ‘natural interferon producing cells’, recently shown to be dendritic cell precursors, are the chief producers of IFNa and – b upon viral challenge, and are critical components of the antiviral immune response w132–134x. While innate immunity to gene therapy vectors has primarily been characterized in response to viral vectors in mice, similar cytokine and inflammatory responses have also been demonstrated to non-viral vectors w135x, and to adenoviral vectors in non-human primates w136x. As with anti-vector adaptive immunity, this inflammatory response is often not dependent on viral or transgene expression, as such responses occur very early or immediately after infection, and adenoviral vectors progressively deleted of viral genes incite similar inflammatory responses and interference with vector persistence w137,138x. Furthermore, specific antigen recognition may not be required w139x, suggesting that infection of parenchymal cells, that are not part of the leukocytic immune system, induces an innate stereotyped response in these cells that inhibit vector function and gene expression w140–142x. This may occur by apoptosis of vector-transduced cells or by inhibition of vector promoter expression by cytokines w143x. The cytokines induced following virus or viral vector administration are potent inducers of proinflammatory chemokines such as RANTES, MCP-1, MIP-1a, MIP1b, and MIP-2 w131,137,144,145x. Specific inhibition of TNFa or MIP-2 can attenuate virus-induced inflammation w137,146x. Many of these effects are mediated through NF–kB activation, as inhibition of NF–kB activation either by overexpression of its inhibitor IkB or by expression of a mutant of the p65yRelA subunit of NF–kB leads to inhibition of cytokine expression, chemokine expression, and leukocyte infiltration w145,147,148x. However, NF–kB also appears to exert a protective effect on vector-induced hepatic injury; NF–kB inhibition by introduction of an expression vector encoding IkB results in greater susceptibility to TNFa-mediated apoptosis, although antagonism of p65y RelA binding does not. Such protection may be due to

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feedback inhibition of TNFa production by the p50 subunit of NF–kB w149x. Vector persistence during NF– kB inhibition by IkB overexpression is enhanced only by simultaneous overexpression of the anti-apoptotic gene bcl-2 w147x. 6.7. Promoter extinctionyattenuation The type of promoter directing transgene expression influences expression by gene transfer vectors. A wide variety of promoter types have been studied, and the most commonly used are viral promoters such as MMLV, RSV, SV40, or CMV, but cellular promoters such as the b–actin promoter have also been utilized. Other promoter characteristics may have dramatic influences on transgene expression. The efficiency of the CMVie promoter differs depending on whether the promoter is derived from human or murine CMV, on the size of the promoter fragment used in the vector, and on the cell type transfected in vitro w150x. The nature of the promoter may determine interactions with a number of transcription factors or viral gene products, which may ultimately determine vector efficacy. For example, the adenoviral E4 region gene products can activate gene expression in cis or in trans directed by an RSV but not a CMV promoter w151x. Interactions of promoters with components of the immune system represent an important means by which transgene expression is attenuated. Interferons and TNFa are known to have a variety of antiviral activities. Interferon–g regulates MCMV infection and inhibits reactivation of MCMV from latency w152x. Such inhibition may occur by blockade of replication w153x, but there is evidence that interferons inhibit the onset of MCMVie gene transcription as well, implying a more direct effect on promoter function w141,154,155x. Inhibition of transgene expression by interferons at a posttranscriptional level has been demonstrated in HBV infection, and in MMLV-based retroviral vectors w156,157x. In contrast, CMV promoter activity, which increases following adenovirus vector administration, is stimulated by the transcription factor NF–kB w158x. This may explain the early efficiency of CMV promoter based vectors, as NF–kB activation can be induced by TNFa, IL-1 and IL-6, all of which are stimulated by vector administration. Cytokines induced by immune responses to gene transfer vectors may also exert competitive or synergistic effects on transgene expression at the promoter level. We have shown that IFNg and TNFa in combination are synergistic in inhibition of b–gal expression by adenoviral, retroviral and plasmid vectors in vitro, and by adenoviral vectors in vivo w141x. In contrast, NF–kB-dependent HIV gene expression is stimulated by TNFa but inhibited by IFNa by competitive binding of transcription factors induced by these cytokines to the NF–kB coactivator p300 w159x. Wheth-

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er an individual cytokine inhibits or augments transgene expression may also be determined by vector promoter structure; for example, the deletion or mutation of a transcription factor binding site may lead to loss of inhibition by a given cytokine. Precise characterization of the interactions between cytokines and promoter elements may ultimately lead to gene therapy vectors with promoters designed to capitalize on the cytokine milieu of the target tissue, resulting in augmentation of expression rather than attenuation. References w1x Merrick AF, Shewring LD, Sawyer GJ, Gustafsson KT, Fabre JW. Comparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture and in vivo. Transplantation 1996;62(8):1085 –1089. w2x Muhlhauser J, Jones M, Yamada I, et al. Safety and efficacy of in vivo gene transfer into the porcine heart with replicationdeficient, recombinant adenovirus vectors. Gene Ther 1996;3(2):145 –153. w3x Chahine AA, Yu M, McKernan MM, Stoeckert C, Lau HT. Immunomodulation of pancreatic islet allografts in mice with CTLA4Ig secreting muscle cells. Transplantation 1995;59(9):1313 –1318. w4x Olthoff KM, Judge TA, Gelman AE, et al. Adenovirusmediated gene transfer into cold-preserved liver allografts: survival pattern and unresponsiveness following transduction with CTLA4Ig. Nat Med 1998;4(2):194 –200. w5x Feng S, Quickel RR, Hollister-Lock J, et al. Prolonged xenograft survival of islets infected with small doses of adenovirus expressing CTLA4Ig. Transplantation 1999;67(12):1607 – 1613. w6x Tomasoni S, Azzollini N, Casiraghi F, Capogrossi MC, Remuzzi G, Benigni A. CTLA4Ig gene transfer prolongs survival and induces donor-specific tolerance in a rat renal allograft. J Am Soc Nephrol 2000;11(4):747 –752. w7x Qin L, Chavin KD, Ding Y, et al. Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immunosuppression with TGF-beta 1 or vIL-10. Transplantation 1995;59(6):809 –816. w8x Qin L, Chavin KD, Ding Y, et al. Retrovirus-mediated transfer of viral IL-10 gene prolongs murine cardiac allograft survival. J Immunol 1996;156(6):2316 –2323. w9x Brauner R, Nonoyama M, Laks H, et al. Intracoronary adenovirus-mediated transfer of immunosuppressive cytokine genes prolongs allograft survival. J Thorac Cardiovasc Surg 1997;114(6):923 –933. w10x Shinozaki K, Yahata H, Tanji H, Sakaguchi T, Ito H, Dohi K. Allograft transduction of IL-10 prolongs survival following orthotopic liver transplantation. Gene Ther 1999;6(5):816 – 822. w11x David A, Chetritt J, Guillot C, et al. Interleukin-10 produced by recombinant adenovirus prolongs survival of cardiac allografts in rats. Gene Ther 2000;7(6):505 –510. w12x Klebe S, Sykes P, Coster D, Krishnan And R, Williams K. Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10. Transplantation 2001;71(9):1207 –1209. w13x Kato K, Shimozato O, Hoshi K, et al. Local production of the p40 subunit of interleukin 12 suppresses T-helper 1-mediated immune responses and prevents allogeneic myoblast rejection. Proc Natl Acad Sci USA 1996;93(17):9085 –9089.

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