Gene therapy and solid-organ transplantation
ENVER
AKALIN
and JONATHAN
S. BROMBERG
Division of Nephrology and Institute of Molecular Medicine and Gene Therapy, and Recanati-Miller Transplant Institute, Mount Sinai School of Medicine, New York, New York, USA
Gene therapy and solid-organ transplantation.Recent develop- absence of immunosuppressive treatment, (2) the preven-ments in transplantation medicine improved the short- and tion of nonimmunologic causes of graft loss, such as ische-long-term survival of solid-organ transplantation. However,
mia-reperfusion injury and arteriosclerosis, and (3) xeno-chronic allograft rejection, the side effects of the long-term
transplantation as a potential solution for organ shortage. immunosuppressive treatment, and organ shortage are still the
Gene therapy has the potential to meet these chal-major obstacles to achieving long-term survival. Gene therapy
has the potential to meet these challenges and has unique lenges and has unique advantages in transplantation. advantages in transplantation. In this review we summarize Allotransplantation requires the removal of tissues from the studies using gene therapy in solid-organ transplantation.
one individual and offers the possibility of treating cells or organs ex vivo with gene transfer vectors prior to implantation, thus avoiding many of the hurdles encoun-The introduction of new immunosuppressive
medica-tered with in vivo gene transfer. This gives the advantage tions significantly increased the short-term and long-term
of local production of immunosuppressive molecules, survival of kidney grafts from both living and cadaver
without exposing the recipient to the systemic side-donors. The projected half-life of cadaver
transplanta-effects of the drugs and may potentially achieve antigen tion was increased to 13.8 years, and living-related
trans-specific immunosuppression. Higher concentrations of plantation was increased to 21.6 years in 1995. Despite
vector can be administered to organs ex vivo or cells in the reduction of the rate of acute rejection, it is still one
vitro, and excess vector washed away, decreasing chances of the major obstacles to achieving long-term survival.
for toxicity, immunogenecity, and systemic transduction The projected half-life of cadaver transplants with an
or expression of the vector. One of the major limitations episode of acute rejection was only increased to 8.8 years
to the gene therapy is that gene expression and protein from 7.0 years [1]. Chronic rejection remains the most
production are transient, which significantly limits the important cause of graft loss in long-term studies, and
ability of gene transfer techniques for the treatment of acute rejection is the major predictor of chronic
rejec-inborn errors of metabolism, genetic deficiencies, or tion. Death with graft function is increasingly becoming
chronic disease. However, in the setting of transplanta-an importtransplanta-ant cause of graft loss, occurring in 10 to 40%
tion, transient gene expression may be sufficient for im-of transplanted patients, due to long-term
immunosup-mune modulation or even desirable because the poten-pressive treatment in particular the increased risk of
in-tial toxicities and adverse effects of prolonged vector fection, malignancy, and cardiovascular disease [2].
De-persistence and expression or immunosuppressive mole-layed graft function is another independent risk factor
cule production could be avoided [4]. for long-term survival [3]. Another hurdle in
transplanta-Gene therapy can be used for the transfer of gene that tion is the increasing number of patients on a waiting
induces secreted proteins [such as interleukin-10 (IL-10), list because of organ shortage, limiting the opportunities
transforming growth factor-(TGF-)], overexpression for transplantation. To overcome these obstacles, three
of membrane-bound molecules (such as Fas ligand) or main research areas in the field of transplantation have
in-intracellular proteins (such as antiapoptotic molecules). creased interest: (1) the prevention of acute and chronic
In this review, we summarize the studies using gene rejection with new immunotherapies with less systemic
therapy in solid-organ transplantation. immunosuppression and preferably that induce
trans-plantation tolerance, as defined by graft acceptance in the
IMMUNOMODULATION
The alloimmune response is a T-cell-dependent pro-Key words: transplantation, rejection, allograft, gene therapy,
xeno-transplantation. cess that involves the orchestration of different amplifi-cation and effector mechanisms, including natural killer 2002 by the International Society of Nephrology
cells, B cells, macrophages, cytokines, and chemokines. studied in the context of the Th1/Th2 paradigm. This paradigm proposes that Th1-derived cytokines (IL-2, T cell recognizes the antigenic peptides in the groove of
major histocompatibility complex (MHC) molecules on interferon␥) promote allograft rejection by mediating delayed-type hypersensitivity reactions, cytotoxic T-lym-the surface of antigen-presenting cells (APCs). This
so-called signal 1 delivers intracellular signals and confers phocyte generation, and macrophage activation and that Th2-derived cytokines (IL-4 and IL-10) protect rejection antigen specificity to the immune response but alone is
insufficient for full T-cell activation. The second signal, by down-regulating immune responses. Previous studies in rat transplantation models showed that the induction or “costimulatory signal,” is provided by interactions
be-tween specific receptors on the T cells, CD28, CTLA4 of tolerance in some studies is associated with the inhibi-tion of Th1 cytokines and the sparing Th2 cytokines [15]. (CD152), CD40 ligand, and their respective ligands on
APCs, B7-1 (CD80), B7-2 (CD86), and CD40. Block- TGF-can also suppress T-cell, B-cell, neutrophil, and inflammatory cytokine functions. Many studies have in-ing the costimulatory signals with the fusion protein
CTLA4Ig and/or anti-CD40 ligand antibodies results in vestigated the role of these immunomodulatory cyto-kines by using gene therapy. IL-10 gene therapy by using long-term allograft acceptance [5].
Although allo-MHC is the main target for alloimmune different vectors (such as, naked plasmid DNA, retrovi-ral, herpes simplex viral and adenoviral vector or lipid response, exposure of these donor MHC antigens through
the thymus or hematopoietic stem cells could serve as a mediated) significantly prolonged murine and rabbit car-diac allograft survival [16–19]. In addition, TGF-gene strategy for inducing antigen-specific tolerance [6]. The
involution of the adult thymus makes the thymic ap- therapy was shown to prolong the murine cardiac and liver allograft survival [16, 20, 21]. Along with the immu-proach not feasible for human gene therapy. However,
DNA-mediated gene transfer of MHC classes I and II nosuppressive properties of TGF-, it induces fibrosis and may play a role in chronic rejection. This issue has molecules to recipient bone marrow cells prolonged
sur-vival of murine cardiac allografts [7]. By using a similar not been addressed in these studies. One recent study investigated the effects of IL-4 gene therapy by using approach, it was shown that pretransplant administration
of a single donor class I MHC molecule was sufficient for recombinant adenovirus vector in rat heart transplanta-tion model. Although IL-10 gene therapy significantly the indefinite survival of cardiac allografts [8]. Retroviral
transduction of bone marrow with the donor MHC gene prolonged the cardiac allograft survival, IL-4 gene ther-apy had no effect [22].
significantly prolonged murine skin allograft survival and
miniature swine kidney allograft survival [9, 10]. A recent Chemokines are members of a family of small, ducible, and secreted proteins that are produced in in-study, using liposomes to transfect cultured recipient
hepatocytes with plasmid DNA encoding the soluble flammation and regulate leukocyte and lymphocyte re-cruitment. Increasing number studies have shown that donor MHC class I Ag, abrogated HAR and prolonged
allograft survival in presensitized rat cardiac allograft chemokines play an important role in allograft rejection [23]. vMIP-II and MC148 are two recently identified model [11]. These studies demonstrate that the
applica-tion of gene therapy of donor MHC molecules through chemokine homologues encoded by human herpes virus 8 and molluscum contagiosum that have antagonistic bone marrow cells provides a new strategy for inducing
transplantation tolerance. activities against multiple different CC and CXC
chemo-kine receptors. Plasmid-mediated gene transfer of either Systemic administration of CTLA4Ig and anti-CD40
ligand has been tested in numerous allograft and xeno- chemokine antagonists markedly prolonged vascularized and nonvascularized cardiac allograft survival in mice graft models, and it has been shown in some studies that
they significantly prolong survival and induce antigen- [24]. This approach resulted in a marked decrease of donor-specific cytotoxic T lymphocytes infiltrating the specific tolerance [5]. The effect of local expression of
CTLA4Ig has been studied by using replication-defec- grafts and inhibited alloantibody production. Targeting the chemokine functions by gene therapy can be another tive adenoviral vector in cold-preserved murine liver
al-lograft model. This method induced indefinite survival novel approach for inhibition of alloimmune response. Following the activation of effector mechanisms, allo-of the recipient with donor-specific unresponsiveness
[12]. Ex vivo perfusion of the donor hearts with recombi- immune responses need to be down-regulated. This is carried out by several mechanisms that inhibit T cells at nant adenovirus encoding CTLA4Ig cDNA induced
in-definite graft survival in rat cardiac allotransplantation the later stages of the alloimmune response. CTLA4 molecules expressed on activated T cells interact with [13]. Transplantation of allogenic mouse islets expressing
the human CTLA4Ig cDNA following gene gun delivery B7 molecules on APCs and deliver inhibitory signals to T cells. The Fas/Fas ligand (FasL) system plays an also prolonged allograft survival significantly [14]. Gene
transfer of anti-CD40 ligand has not yet been studied. important role in the induction of lymphoid apoptosis and can turn off immune responses. Two immune-privi-The role of cytokines in the induction of allograft
FasL and protect cells from immune destruction. Gene prevent antibody-induced transplant arteriosclerosis in mouse cardiac allografts [32].
therapy, using FasL, has been studied by several groups
Endothelium-derived nitric oxide is an endogenous in different transplantation models, but the results are
inhibitor of vascular lesion formation. Gene transfer of controversial. Gene transfer of murine FasL by
replica-endothelial cell nitric oxide synthase by the Sendai virus/ tion defective adenovirus significantly prolonged rat
re-liposome in vivo gene transfer technique to the endothe-nal allograft survival [25]. Similar results were obtained
lium of rat carotid arteries significantly decreased the in the rat liver transplantation model by using rat pFasL
balloon injury-induced damage and neointima formation conjugated to liposome vesicles (hemagglutinating virus
[33]. Adenoviral-mediated inducible nitric oxide syn-of Japan liposome) [26]. However, heart allografts from
thase gene transfer completely suppressed the develop-FasL transgenic mice were more rapidly rejected than
ment of arteriosclerosis in both untreated or cyclosporine nontransgenic control allografts [27].
A-treated rat aortic allografts [34]. A recent interesting study demonstrated the
impor-Intercellular adhesion molecule-1 (ICAM-1) is an tance of secondary lymphoid tissue in alloimmune
re-important mediator of cell adhesion and T-cell costimu-sponse. In this study, cardiac allografts were accepted
in-lation. The use of ex vivo hyperbaric transfection of anti-definitely in recipient mice that lack secondary lymphoid
sense oligodeoxynucleotides that blocked ICAM-1 re-tissue, indicating that the alloimmune response to a
vas-sulted in the reduction of chronic graft vascular disease cularized organ cannot be initiated in the graft itself and
and reperfusion injury in rat cardiac allografts [35, 36]. requires the presence of secondary lymphoid tissue [28].
Cyclin-dependent kinase 2 is an important enzyme in This study may challenge the idea of using gene therapy
the transition from the G1-S phase of the cell cycle and methods locally at the transplanted organ and may
sug-mediates smooth muscle cell proliferation. The use of gene gest that secondary lymphoid organs could be potential
transfer of antisense phosphorothioate oligodeoxynucle-targets. Dendritic cells are highly specialized APCs that
otide against this enzyme inhibited intimal hyperplasia initiate and modulate immune responses and play an
and expression of vascular cell adhesion molecule-1 [37]. essential role in T-cell activation and central and
periph-eral tolerance. A recent study used dendritic cells for
XENOTRANSPLANTATION
adenoviral delivery of CTLA4Ig and induced
alloanti-gen-specific T-cell hyporesponsiveness [29]. Organ shortage is one of the major limitations in trans-plantation, and this has stimulated a great deal of re-search into the possibility of using nonhuman donors for
MODULATION OF NONIMMUNOLOGIC
transplantation. The preferred donor animal species for
CAUSES OF GRAFT LOSS
xenotransplantation is the pig. The initial barrier to xeno-Ischemia-reperfusion injury is an important cause of transplantation is hyperacute rejection (HAR), which early nonimmune loss of the allograft. It induces the results in the loss of graft within a few minutes to hours formation of reactive oxygen species by endothelial dam- because of antibody-mediated complement activation of age, which may cause cell death within the graft or may the graft endothelium. This xenoreactive natural anti-induce acute or chronic rejection by activating cytokines body predominantly recognizes the unique carbohydrate and adhesion molecules and recruiting leukocytes. This structure, Gal␣1-3Gal, which is found as the terminal subject has also been of interest in gene therapy. Mito- sugar on glycolipids and glycoproteins present on the chondrial superoxide dismutase gene was transferred to donor pig endothelium. Humans do not express this anti-the mouse liver prior to anti-the lobar ischemia-reperfusion gen and have antibody to this antigen, in a similar fashion injury. It was shown to reduce acute liver damage signifi- to blood group antigens. This epitope is synthesized by cantly by inhibiting the redox-mediated activation of nu- an enzyme called␣-galactosyltransferase. It was shown clear factor-B and activator protein-1, which are the that introducing a functioning gene that encodes this immediate early transcription factors that represent com- enzyme by retroviral gene transfer into autologous bone mon pathways by which cells respond to environmental marrow cells overcame the HAR in a mouse model [38].
stress [30]. The effector mechanism of HAR is the activation of
com-Apoptosis plays an important role in organ preserva- plement cascade. There are complement-regulatory pro-tion and rejecpro-tion. Overexpression of antiapoptotic genes teins (CRPs) present on the donor endothelium that in the allograft may prevent ischemia/reperfusion-induced regulate the activation of complement. These proteins apoptosis. Bcl-2 gene transfer to the mouse liver by re- include decay-accelerating factor (DAF;CD55), mem-combinant adenovirus before procurement significantly brane cofactor protein (MCP;CD46), and CD59. Xeno-decreased hepatocyte injury and the number of apoptotic graft endothelium is severely damaged because of a lack cells [31]. It was also demonstrated that the expression of these CRPs. Transgenic pigs that express human CRPs have been developed to limit complement-induced dam-of genes that encode heme oxygenase-1, bcl-xl, and A20
9. Sykes M, Sachs DH, Nienhuis AW,et al: Specific prolongation age. Heart and kidneys derived from transgenic pigs that
of skin graft survival following retroviral transduction of bone express DAF did not develop HAR if transplanted into marrow with an allogenic major histocompatibilty complex gene.
Transplantation55:197–202, 1993 nonhuman primates [39–42]. Furthermore, the use of
10. Emery DW, Sablinski T, Shimada H,et al: Expression of an alloge-transgenic pigs that express DAF with CD46 or CD59 as
nic MHC DRB transgene, through retroviral transduction of bone donors for kidney or heart transplantation into baboons marrow, induces specific reduction of alloreactivity.
Transplanta-tion27:1414–1423, 1997 show to prolong graft survival without
immunopatholog-11. Geissler EK, Graeb C, Tange S,et al: Effective use of donor ical findings of HAR [43].
MHC class I gene therapy in organ transplantation: Prevention of If HAR can be avoided, the xenograft is subject to antibody-mediated hyperacute heart allograft rejection in highly
sensitized rat recipients.Hum Gene Ther11:459–469, 2000 acute vascular rejection (delayed xenograft rejection)
12. Olthoff KM, Judge TA, Gelman AE,et al: Adenovirus-mediated within a few days to weeks post-transplantation, which
gene transfer into cold-preserved liver allografts: Survival pattern is probably T-cell independent and is caused by antibod- and unresponsiveness following transduction with CTLA4Ig.Nat
Med4:194–200, 1998 ies, macrophages, cytokines, and chemokines. If these
13. Yang Z, Rostami S, Koeberlein B,et al: Cardiac allograft toler-problems are overcome, which is very difficult, a third
ance induced by intra-arterial infusion of recombinant adenoviral hurdle to the final success of the xenotransplantation is CTLA4Ig.Transplantation67:1517–1523, 1999
14. Gainer AL, Suarez-Pinzon WL, Min WP,et al: Improved sur-T-cell-mediated rejection. Similar approaches as in
al-vival of biolistically transfected mouse islet allografts expressing lotransplantation (using CTLA4Ig, IL-10, TGF-gene
CTLA4Ig or soluble fas ligand.Transplantation66:194–199, 1998 transfer) can be used in xenotransplantation to prevent 15. Sayegh MH, Akalin E, Hancock WW,et al: CD28–B7 blockade after alloantigen challenge in vivo inhibits Th1 cytokines but spares T-cell-mediated rejection.
Th2.J Exp Med181:1869–1874, 1995
16. Qin L, Chavin KD, Ding Y,et al: Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model:
CONCLUSION Immunosuppression with TGF1 or vIL-10.Transplantation59:
809–816, 1995 Gene therapy, which is in the early stages of
develop-17. Qin L, Chavin KD, Ding Y,et al: Retrovirus-mediated transfer ment, holds great promise for allotransplantation and
of viral IL-10 gene prolongs murine cardiac allograft survival. xenotransplantation. Improvements in transfer vectors J Immunol156:2316–2323, 1996
18. Brauner R, Nonoyama M, Laks H,et al: Intracoronary adeno-and delivery protocols to obtain efficient transfection
virus-mediated transfer of immunosuppressive cytokine genes with minimal toxicity, along with a better understanding prolongs allograft survival. J Thorac Cardiovasc Surg 114:923– of alloimmune responses to identify the most efficient 933, 1997
19. DeBruyne LA, Li K, Chan SY,et al: Lipid-mediated gene transfer gene or gene combinations, would make gene therapy
of viral IL-10 prolongs vascularized cardiac allograft survival by an important field of transplantation in this century. inhibiting donor-specific cellular and humoral immune responses.
Gene Ther5:1079–1087, 1998
Reprint requests to Enver Akalin, M.D., Mount Sinai Medical Center, 20. Qin L, Ding Y, Bromberg JS: Gene transfer of transforming Recanati-Miller Transplant Institute, One Gustave L. Levy Place, Box growth factor–beta 1 prolongs murine cardiac allograft survival by 1104, New York, New York 10029-6574, USA. inhibiting cell-mediated immunity.Hum Gene Ther7:1981–1988,
1996
21. Drazan KE, Olthoff KM, Wu L,et al: Adenovirus-mediated
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