INVITED REVIEW
Gene transfer in the kidney

¹ Renal Division, Molecular Autoimmunity, Brigham and Women's Hospital, Harvard Medical School, and ² Renal Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

	ABSTRACT

Top Abstract Introduction Conclusion References

Gene transfer approaches offer the promise of revolutionizing medicine. In this review, we focus on the current and future prospects of somatic gene transfer into the kidney. The advantages and disadvantages of current vector systems are described, and the ex vivo and in vitro approaches applicable to the kidney are reviewed. We discuss uses of gene transfer approaches to dissect the pathogenesis of kidney disease and the future directions and applications of gene transfer to combat kidney destruction.

somatic gene transfer

	INTRODUCTION

Top Abstract Introduction Conclusion References

HUMAN GENE TRANSFER, once considered a fantasy, is a clinical reality. Gene transfer approaches offer the promise of revolutionizing medicine. In this review we focus on the current and future prospects for gene transfer into the kidney. We will discuss the concepts of somatic gene transfer and the general considerations of gene transfer, and we will review the advantages and disadvantages of current vector systems. We will then address the unique features of the kidney that dictate the selection of a gene transfer strategy and review ex vivo and in vivo gene delivery approaches tailored for the kidney. Finally, we will speculate on the future directions and application of gene transfer systems to combat kidney transplant rejection and autoimmune, polycystic, malignant, and other acute glomerular and chronic interstitial kidney diseases.

	SOMATIC GENE TRANSFER

Approaches: In Vivo and Ex Vivo

Gene transfer involves the delivery of an expression cassette consisting of a single or multiple genes and the sequences regulating their expression into a target cell. Somatic gene transfer is achieved using in vivo or ex vivo approaches. In vivo gene transfer delivers the cassette directly into the cells of the recipient. The genes can be delivered regionally or systemically. Ex vivo gene delivery refers to the transfer of the expression cassette into cells removed from a donor, expanded in vitro, and then subsequently introduced into a recipient. Transfer may also be into an organ ex vivo. Gene transfer requires a vector that assists delivery of the gene cassette into an intracellular location that is dictated by the vector. The choice of an in vivo vs. ex vivo approach and the vector selected to deliver the gene is determined by the clinical target.

Disease Considerations

The vector system is determined by several broad considerations: delivery, expression, safety, and the disease target (e.g., severity, tempo, affected organs). Delivery refers to the ability to introduce the gene product at the site required to impact on disease, the minimal number of cells needed to be transduced, and the effects of unintended targets. Expression entails an assessment of the protein level: Is it sufficient for a desired biological effect? How tightly does it need to be regulated? What duration is required to alter disease? Finally, safety issues include toxicity, e.g., inflammation engendered either by the vector or gene product leading to damage to the transduced organ. For example, toxicity is a particularly important problem for gene therapy in chronic diseases.

Vectors

Retroviral, adenoviral, and nonviral vectors are popular systems for experimental gene transfer delivery that have been applied to human trials. An overview of the advantages and disadvantages of these vector systems is summarized in Table 1 and has been detailed in several reviews (2, 10, 49).

View this table: [in this window] [in a new window]   Table 1.   Advantages and disadvantages of current vectors

Retroviral vectors. Retroviruses are attractive vectors because they permanently express a foreign gene in cells. When retroviruses infect a cell, their RNA genomes are converted into a DNA by the viral enzyme reverse transcriptase. The viral DNA is efficiently integrated into the host genome where it permanently resides, replicating along with host DNA. This integrated pro-virus steadily produces viral RNA under a strong promoter located at the end of the genome in a long terminal repeat sequence. Thus the retrovirus genome or any foreign gene placed in the retrovirus becomes a permanent part of the host cell genome.

The most common retroviral vector uses the Moloney murine leukemia virus as a base (11, 49). The gene is cloned into a retroviral vector that lacks most viral genes including the gag, pol, and env sequences required for encapsidation. The recombinant plasmid is transfected into a special packaging cell line that harbors an integrated pro-virus. The pro-virus has been crippled so that, although it produces the proteins required to assemble infectious virus, its own RNA cannot be packaged. Instead, RNA transcribed from the transfer vector is packaged into infectious virus particles and released from the cell. This virus "helper-free" stock is used to infect a target cell culture.

Although murine-based retroviral vectors are well suited for ex vivo applications, the first generation vectors are limited for direct in vivo gene transfer. The major limitations include the following: 1) the inability to easily purify and concentrate the large amounts of virus often needed for direct in vivo gene transfer applications; 2) sensitivity of virus with amphotropic host range, i.e., infect a wide variety of species; and 3) inability to integrate into quiescent cells. However, second generation vectors are evolving that improve the vector and broaden its application for in vivo delivery. For example, to produce high titers of recombinant Moloney murine leukemia virus particles, a human retroviral packaging cell line (293GPG) capable of incorporating the vesicular stomatitis virus G (VSV-G) protein was generated (56). The resulting VSV-G/retroviral pseudotypes possess the wide host range of VSV and can be highly concentrated without the loss of bioactivity. However, high levels of VSV-G are toxic to cells. Therefore, a transient transfection system was constructed using tetracycline-dependent expression of VSV-G in the 293GPG producer cells controlled by the tet transactivator system. Thus it is now feasible to generate large amounts of extremely high titer (>10⁹ cfu/ml) virus critical for in vivo gene transfer. An additional advantage is that the virus produced from 293GPG cells is more resistant to serum inactivation than amphotropic virus generated from murine cells. Another second generation retroviral vector is the group of lentivirus-based vectors (54). Lentiviruses are a subclass of retroviruses that infect nondividing cells. Thus lentiviral-based vectors broaden the host range by transducing dividing and nondividing cells. In summary, new retroviral vectors and packaging cell lines are being created to circumvent the limitation of the first generation vectors.

Adenoviral vectors. These DNA viruses offer several distinct advantages. They 1) have a wide host range, 2) survive robustly in the circulation (63), 3) are produced at high titer (~10¹³ particles/ml), and 4) infect nondividing cells. However, there are some limitations. The duration of expression in vivo is largely limited by a T cell response to low levels of adenoviral proteins and transgene (if non-self) produced in the transduced cell (1). Newer versions of the virus with the nonessential E3 deleted (along with E1 portion) allow inserts up to ~8 kb in size. Moreover, an E2 mutant version is less immunogenic. Recent E4 deletion variants accept an additional 3 kb of insert DNA and are even less immunogenic (12). The ideal vector would contain only the cis elements required for packaging, namely, the so-called inverted terminal repeats and the packaging signal, and progress toward the construction of such a vector has been reported recently (6, 15, 34, 41), though the stability of gene expression in vivo is as yet uncertain (24). However, these advances will not circumvent the antibody response to adenoviral proteins that preclude secondary infection with the same serotype but not with a different serotype (44). The nonspecific inflammatory response (that can be produced even by empty virus) may also limit the amount of adenovirus that can be delivered. The reader is referred to a recent review by Anderson (2) for a summary of the use of adeno-associated and other DNA virus-based vectors.

Nonviral vectors. Nonviral vectors are relatively nontoxic and nonimmunogenic and easy to manufacture, but, in general, transduction efficiency in vivo is far less than with adenoviruses (reviewed in Ref. 37). Moreover, duration is usually transient (days), although use of replication origins and nuclear retention signals has extended this period to weeks or months.

	KIDNEY-TARGETED GENE TRANSFER

Special Considerations

Because of the structural complexity of the kidney, it is impossible to introduce genes into the multiple cell types using a single method. In principle, there are four routes for transferring genes into the kidney. They include delivery via the 1) renal artery, 2) renal subcapsule site, 3) retrograde from the ureter, or 4) parenchymal injection. These can be delivered singly or in combination, using ex vivo or in vivo approaches. It is conceivable that introduction into the peripheral circulation of cells or vectors could be targeted to the kidney. For example, Pasqualini and Ruoslahti (57) have identified (using a phase display library) sequences that selectively bind to renal vasculature in vivo.

In building a database on which kidney cells are transduced by various gene therapy vectors, it should be noted that delivery in a disease context may differ from that in a normal kidney. There are many reasons for this, e.g., multiple distinct populations of dividing cells, active inflammation, and alterations in vascular permeability. Thus it is critical to select the appropriate preclinical disease model for translating this information to design therapeutics.

Current Data on Kidney Gene Delivery

In vivo. There are many successful distinct in vivo gene transfer vectors, including HVJ liposomes, retroviruses, and adenoviruses.

HVJ LIPOSOMES. Tomita (64) has utilized HVJ, a Sendai virus. Plasmid DNA and a nucleoprotein coencapsulated in liposomes were fused to the inactivated Sendai virus. Gene transfer of an SV40 T-antigen reporter gene was conducted by inserting a catheter proximal to the right renal artery with the abdominal aorta clipped distally beneath the left renal artery. Four days after injection of the liposome suspension, SV40 T-antigen was detected immunohistochemically for several days in 15% of glomerular cells, although it was difficult to ascertain whether expression was in endothelial, epithelial, or mesangial cells. However, foreign gene expression was not detected outside of the glomerulus. The success of this gene transfer method was predicated on the ability of the HMG1 nucleoprotein, a high-mobility group 1 nonhistone nuclear protein, to enhance plasmid DNA passage into nuclei (21). The HVJ virus contains a fusogenic protein on its surface and is responsible for neutral pH-mediated cell fusion. This approach was used by Isaka et al. (19) to transfer cDNAs for transforming growth factor-1 (TGF-1) and platelet-derived growth factor (PDGF) into the rat kidney, with the expected phenotypic effects: increased matrix deposition and increased number of mesangial cells, respectively. Strategies using antisense/decoy oligodeoxynucleotides in HVJ liposomes have also been utilized (47).

CATIONIC LIPOSOMES. Lai et al. (36) have utilized a liposome formulation for -galactosidase gene transfer into the kidney via the intrapelvic route and obtained patchy and transient transfer into tubules. Importantly, this transfer strategy was used to transiently correct carbonic anhydrase deficiency (35) in a rodent model. Similar gene transfer data with a reporter gene was obtained by Boletta et al. (4), using the cationic liposome polyethylenimine.

RETROVIRUSES. Using retroviruses, Fine and co-workers (5) failed to transfer genes into a normal kidney. The very low mitotic index of the kidney was responsible for the lack of success. However, if the tubular epithelial cells (TEC) were damaged using folic acid, they subsequently proliferated. However, transduction was only noted in a few proximal TEC.

ADENOVIRUSES. Moullier (48) reported on adenoviral-mediated gene transfer into the rat kidney using two methods. The first involved selective perfusion into the renal artery of a replication-deficient adenovirus carrying the -galactosidase gene resulting in an occasional blue proximal tubular cell. Expression was not detected in vasculature. In contrast to our method (see below), venous clamping was not utilized. The use of retrograde injection into the renal pelvis was more effective, i.e., genes were transferred into numerous TEC, and expression lasted for 1-2 wk.

Thompson and colleagues (70) have utilized adenovirus as an adjunct for gene transfer purposes. Their scheme used isolated human kidneys under conditions of organ preservation. With an adenovirus polylysine DNA complex, they were able to insert a cDNA expression vector encoding -galactosidase into the intact human kidney. A pump was used to maintain pulsatile perfusion, and a solution of adenoviral particles admixed with polylysine and the expression cassette was perfused over a period of 2 days. Gene delivery and expression was localized to a large fraction of proximal TEC, as detected by immunohistochemical and in situ enzymatic analyses. The expression was patchy and largely restricted to the cortical proximal TEC. These investigators also conducted in situ hybridization analysis using a -galactosidase antisense probe, but sense probe controls were absent. Of concern, the in situ hybridization pattern was either interstitial or vascular, and not tubular as suggested by the enzymatic analysis. Moreover, the controls were pretransfection kidneys, and controls were not performed in the absence of either adenovirus or polylysine. Finally, these investigators detected -galactosidase mRNA expression in the biopsied kidney at various time points following gene transfer into the isolated perfused kidney, using RT-PCR. These findings and the antegrade data of Moullier (48) raise the intriguing question of how adenovirus infects TEC when injected into the renal artery. Is virus able to traverse the glomerular basement membranes, or does it pass through the postglomerular capillary network or vasa recta and escape from the vascular bed?

Tryggvason and colleagues (17) have also reported on the use of a perfusion system for adenoviral gene transfer into the kidney. They isolated a pig kidney in vivo and perfused it from 2 to 12 h with a -galactosidase replication-deficient adenovirus. After reconnecting the kidney, they examined expression 3 days later. Marked glomerular expression was noted in podocytes (67). In addition, they claimed that the kidney could be maintained ex vivo with continuous perfusion and genes could be transferred into glomeruli.

We have demonstrated adenovirus-mediated gene transfer into the normal rat kidney (71). Our technique utilized the following two manipulations: 1) the use of cold incubation (following antegrade injection and cross-clamping) to prolong contact time of the adenovirus while limiting ischemic injury, and 2) the use of vasodilators resulting in different localization patterns of transferred gene expression. We were able to successfully transfer a -galactosidase reporter gene into the vasculature without ischemic injury to the kidney. Transfer occurred predominantly in the capillaries in the chilled cortex, whereas combining a cold environment and vasodilators efficiently shifted transfer into the outer medulla in both the inner and outer stripe. Our data is in contrast with the results of Tryggvason et al. (17, 67) and Moullier (48). It is unclear why these studies are so remarkably different. Details of the protocol may well be responsible for the differences noted [e.g., injection vs. injection with clamping (our study) vs. perfusion (17)], and it will be particularly important to dissect these issues in the future. Very recently, we have utilized an adenovirus with a modified fiber, containing several copies of an RGD peptide (a receptor for certain integrins), to enhance endothelial cell transfer in vivo: fine peritubular capillaries in the cortex and the subcapsular area were efficiently transduced (unpublished studies).

Ex vivo. Ex vivo gene transfer approaches into the kidney are valuable for dissecting the pathogenesis of kidney disease and for the delivery of therapeutic molecules. Ex vivo approaches have provided systems with which to deliver genetically modified cells into the kidney. Selections of the cell type and route of administration for ex vivo gene transfer are based on the site targeted to receive the "carrier cell" product. Gene engineered TEC and mesangial cells offer distinct features making them suitable for delivery of molecules into the interstitium and glomerulus of syngeneic rodents. We will review the ex vivo gene transfer experimental systems that have applied renal parenchymal cells as vehicles to deliver molecules to a prescribed site in the kidney. Because these renal parenchymal cells are genetically modified to generate a selected molecule and these cells are then reintroduced into the kidney with the intention of delivering the selected molecule, we use the term "carrier cells." We will review the use of renal TEC and mesangial cells as carrier cells to transfer a gene product into the kidney. We will review ex vivo gene transfer using renal parenchymal cells and review the strategies for reintroducing these carrier cells into the kidney.

KIDNEY TUBULAR EPITHELIAL CELLS: GENE TRANSFER PROBING FOR THE PATHOGENESIS OF AUTOIMMUNE KIDNEY DISEASE. Cytokines and chemokines are ubiquitously expressed during kidney injury. To define the importance of each of these individual molecules in the pathogenesis of kidney disease, we have tailored an ex vivo approach to deliver a selected individual or multiple molecules into the kidney. This system offers a pragmatic approach for sustained delivery of a test molecule into the kidney and/or circulation. Our strategy has been to identify a molecule (growth factor, cytokine, chemokine) expressed in the kidney either in advance of renal injury or during the initial phases of autoimmune destruction and determine the impact of delivery of this molecule on promoting or thwarting renal injury. This gene transfer approach is a potent tool with which to tease apart the distinct role of molecules whose expression is increased in the kidney and circulation prior to and during the progression of autoimmune renal injury. We review this strategy using several specific examples.

1) Single gene transfer. Single gene transfer into the kidney has provided an approach to determine the importance of selected molecules, such as colony-stimulating factor-1 (CSF-1), on the pathogenesis of kidney disease. For example, although we had gathered a wealth of data linking CSF-1, a cytokine which is increased in advance of kidney disease in autoimmune MRL-Fas^lpr mice, kidney-infiltrating macrophages, and renal disease, the challenge remained to test the hypothesis that CSF-1 expression could elicit autoimmune renal injury. Therefore, we constructed an ex vivo gene transfer system to deliver CSF-1 into the kidney (53). We selected a retroviral vector approach. DNA sequences encoding CSF-1 were subcloned into the MFG vector. This vector was transfected into a CRIP packaging cell line, thereby converting this packaging cell into a producer cell shedding recombinant retroviruses (11). We infected primary cultures of TEC with the recombinant retroviruses encoding CSF-1. CSF-1 transcripts were reverse transcribed into the TEC genome. These genetically modified TEC, which we call CSF-1 carrier cells, offer a pragmatic approach for local, stable delivery of CSF-1 into the kidney. We implanted CSF-1 carrier cells under the renal capsule of MRL strains (Fas^lpr and ++) prior to any evidence of kidney disease (53). CSF-1 was detected in the carrier cells, and we established that sufficient CSF-1 was released to increase the circulating CSF-1 levels for at least 28 days. To track the CSF-1 carrier cells, TEC were co-infected with the -galactosidase (-gal) gene, LacZ. The CSF-1, -gal carrier cells, identified by the -gal reaction product, remained confined to the implant site and did not migrate into the renal cortex. Therefore, local and systemic delivery of CSF-1 released from carrier cells in the kidney capsule is enduring.

CSF-1 carrier cells, but not control uninfected TEC or -gal carrier cells, elicited renal injury in MRL-Fas^lpr. The tempo of CSF-1-elicited kidney injury was rapid and the lesion circumscribed. Several days after the CSF carrier cells were implanted into the kidney, there was an influx of macrophages into the implant site, followed by T cells. Although the renal lesion expanded for at least 3 mo, it remained localized to the area proximal to the implant site. Thus CSF-1-incited injury is discrete and restricted to the segment of the kidney adjacent to the CSF-1 carrier cells.

Using our gene transfer approach, we tested the impact of several other cytokines on the pathogenesis of renal disease. For example, the -chemokine, RANTES, is expressed in MRL-Fas^lpr mice in advance of renal injury (46). Although RANTES carrier cells elicited autoimmune kidney damage in MRL-Fas^lpr mice, CSF-1 and RANTES recruited different T cell populations into the kidney. Since kidney disease in MRL-Fas^lpr mice is composed of multiple T cell populations, this suggests that both CSF-1 and RANTES contribute to fostering T cell accumulation in MRL-Fas^lpr kidneys.

2) Dual gene transfer. Although several cytokines are increased prior to kidney injury in MRL-Fas^lpr mice, we used our gene transfer approach to establish whether the interaction of multiple cytokines was more damaging than either alone. Therefore, we delivered multiple cytokines into the kidney via carrier cells. We implanted tumor necrosis factor- (TNF-) carrier cells alone and together with CSF-1 carrier cells under the renal capsule of MRL-Fas^lpr and MRL +/+ strains prior to renal injury. TNF- alone did not incite renal injury. However, TNF- plus CSF-1 proved to be more potent in promoting renal disease than CSF-1 alone (46). We concluded that the simultaneous exposure of the kidney and/or the macrophages to CSF-1 and TNF- is instrumental in promoting autoimmune glomerular and interstitial injury in MRL-Fas^lpr mice. Taken together, this ex vivo gene transfer approach provides a potent strategy to define the impact of intrarenal cytokines and other molecules on kidney injury.

3) Regional vs. systemic impact of gene product. Ex vivo gene transfer has enabled us to determine the impact of molecules released regionally versus systemically on renal injury. To establish whether systemic delivery of cytokines promoted kidney damage, we compared renal pathology in the kidneys receiving a select cytokine regionally with kidneys exposed to the same cytokine in the circulation. Therefore, we implanted carrier cells releasing a select cytokine into a single kidney. The select cytokine is increased locally in the kidney and systemically. Therefore, we compared the implanted kidney with the unmanipulated contralateral kidney. Systemic exposure alone to cytokines [e.g., CSF-1, granulocyte-macrophage-CSF (GM-CSF), TNF-, interleukin-6 (IL-6), IL-2, RANTES, IL-12] increased renal pathology in the contralateral kidney (45, 46, 52, 53). The most plausible explanation for the differential impact of regional and systemic exposure of cytokines on the kidney is related to the concentration of the cytokine. The local regional concentration of cytokines into the kidney is substantially greater than the more dilute titers delivered to the kidney via the circulation.

MESANGIAL CELLS. Mesangial cells have been genetically modified to serve as gene carrier cells, or as Dr. Kitamura terms them, vectors to deliver genes into the glomerulus. Thus, similar to TEC, mesangial cells were propagated from the autologous kidney and genetically modified in culture to confer stable expression of a selected gene (28, 30). However, rather than being infused under the kidney capsule, mesangial cells were transferred back into the glomeruli via the renal circulation. These reintroduced mesangial cells lodged in the glomerular capillary and, perhaps, in the mesangium. By use of a -galactosidase gene (LacZ) as a marker, gene transfer after 4 wk was reported to be ~50%, albeit in limited amounts. Of note, expression of -galactosidase was amplified by injecting anti-mesangial cell antibody (anti-Thy-1). Mesangial cells expressing LacZ proliferated in situ, thus amplifying the expression of -galactosidase. More recently, Kitamura and co-workers (29, 69) have used this approach to deliver TGF- and an IL-1 receptor antagonist protein into the glomerulus. Both of these proteins blunted the glomerular response to IL-1. Kitamura (27) tested regulatory controls to switch on and off -galactosidase expression in mesangial cells using the tetracycline-responsive promoter. In addition, he has tested the concept of an in vivo cytosenor by introducing LacZ into mesangial cells under the control of the CArG box element (32). This is based on the concept that -smooth muscle actin is expressed in mesangial cells during pathological states. The CArG box element, the crucial regulatory sequence for the -smooth muscle promoter, was used as a sensor for glomerular inflammation. Testing this in vivo, Kitamura and Kqwachi (32) transferred serum-stimulated and unstimulated cells into normal and anti-Thy-1 nephritic rats. Transferring stimulated cells into normal rats switched off -galactosidase expression, whereas unstimulated cells transferred into nephritic rats enhanced expression. This novel approach provides a potential strategy for gene transfer by automatic regulation of local gene expression when the gene is required to dampen inflammation and then cease when inflammation subsides.

	FUTURE APPLICATIONS FOR KIDNEY DISEASE

Below are possible applications of gene therapy for treating renal disease. The reader is also referred to several recent reviews (14, 31, 39, 42).

Transplantation

Somatic gene transfer offers a powerful approach for the delivery of a therapeutic molecule into the kidney, or other tissues, to abort transplant rejection. Gene transfer provides distinct advantages over conventional, systemic administration of therapeutics by delivering a therapeutic locally within an organ. Local delivery avoids systemic complications and is economical since lesser amounts of therapeutics are required. The general barriers of gene transfer in medicine include 1) imprecise site-targeted expression, 2) transient expression, and 3) ethical pitfalls. However, these are not barriers for gene transfer in transplantation. Imprecise site (organ) targeting and issues concerning exclusively targeting the kidney do not apply to transplantation, since gene transfer into harvested organs is readily achievable. Furthermore, the second barrier, transient expression, may not necessarily be a problem in transplantation, since short doses of biologics may tolerize, and transplant therapies are initially intense and then are tapered to low doses. Finally, some ethical pitfalls may be dismissed. Problems of vector toxicity to the host are largely avoided by gene transfer into harvested organs.

Gene transfer approaches to block kidney transplant rejection remain in the planning stages. Nevertheless, studies in other organ transplants have provided clues to combat rejection applicable to the kidney. Strategies that block T cell activation or destroy T cells are particularly appealing approaches to combat allograft rejection. Since the interaction of antigen-presenting cells and T cells requires adhesion, recognition, and costimulation to activate T cells, gene transfer interfering with any of these steps offers attractive therapeutic approaches. Let us review several examples.

It was initially thought that local delivery of Fas ligand in genetically engineered syngeneic carrier cells would protect against rejection. This was based on the concept that upon T cell activation, a signal induces Fas and Fas ligand on the T cells (51). Since the interaction of Fas with its ligand induces apoptosis and thus eliminates T cells, it was reasoned that provision of Fas ligand into an allograft would introduce a barrier to dampen or eliminate allograft rejection (3). Although this strategy was claimed to combat islet allograft rejection (38), this data has been challenged (23). In fact, recent evidence determined that -cells in the pancreatic islet in diabetic mice express Fas and Fas ligand and induce apoptosis of islets (22). In addition, Fas ligand expression on pancreatic -cells results in massive neutrophilic infiltration and islet destruction (23). Furthermore, evidence suggests that engagement of these molecules is required for insulin-dependent diabetes at least in mice (20). Thus provision of Fas ligand is harmful and not protective for islet transplants. However, this is not necessarily the case for other tissues. For example, although activated kidney parenchymal cells express Fas and Fas ligand, we have recently provided evidence that, unlike islets, the expression of Fas and Fas ligand on renal parenchymal cells destroys T cells but does not induce apoptosis of renal parenchymal cells in vivo (68). Thus provision of FasL may be suitable for deleting activated T cells in the kidney transplants but may be lethal for some other organs. Another approach has been to block the costimulatory pathway required for T cell activation. Without costimulation, T cells are paralyzed and may enter a state of anergy. The interaction of CTLA4 expressed by T cells and B7 proteins displayed on activated macrophages is a potent costimulation pathway. Masking B7 antigens using a soluble CTLA4-Ig fusion protein prolonged allogenic and xenogenic engraftment (40). Based on this concept, adenovirus-mediated transduction of CTLA4-Ig into the cold preserved liver allograft protected the liver from allograft rejection (55). Thus this gene transfer approach to block T cell activation offers promise for preserving kidney transplant engraftment.

There are many other promising gene delivery approaches to combat rejection. Gene delivery of sequences encoding allogenic major histocompatibility complex (MHC) antigens prior to transplantation may modify foreign determinants and induce tolerance. For example, fibroblasts transfected with donor-specific class I or II MHC genes injected into recipients prior to mouse cardiac allogenic transplantation induced immune unresponsiveness (43). In addition, intrathymic administration of autologous plasmid-transfected myoblasts and myotubes bearing donor MHC class I antigens induced donor-specific unresponsiveness to liver grafts in allogeneic responder rats (33). In addition, cytokines that dampen immune events offer potential gene transfer opportunities to combat transplant rejection. Gene transfer with plasmid DNA encoding TGF-1 in vivo prolonged cardiac mouse allografts from 12 to 25 days without altering systemic immunity (58). However, transient expression of TGF-1 may have been responsible for rejection, since the duration of expression was unclear.

Although the studies highlighted in this section are encouraging, there remain issues concerning the design of better vectors to deliver more consistent and enduring gene transfer to block transplant rejection in the absence of harmful immune events or toxicity.

Autoimmune Kidney Disease

Successful engraftment and protection from autoimmune kidney destruction share a common theme, i.e., tolerance. In either case, the loss of self or tolerance and perception of an invading foreign component sets in motion a series of tissue self-destructive events. Thus the strategies that combat tissue rejection should also offer therapeutic promise to blocking autoimmune kidney diseases. Several in vivo and ex vivo approaches have successfully delivered genes into the kidney; however, gene transfer approaches targeting autoimmune kidney diseases are as yet unrealized. The challenge will be to have sufficient local gene delivery to preserve the entire kidney, although subtotal preservation may also be clinically useful.

Polycystic Kidney Disease

The identification of the PKD1 and PKD2 cDNAs (7, 8) might suggest that transduction of the appropriate cDNAs could provide a therapeutic treatment of autosomal dominant polycystic kidney disease (ADPKD). However, this approach is fraught with problems; the genetics of ADPKD, that is, haplotype insufficiency vs. a dominant-negative mechanism, the requirement for highly efficient tubular cyst transduction, and problems of vector capacity and stability, e.g., the open-reading frame for the PKD1 cDNA of 13 kb, all make this a daunting proposition. On the other hand, the idea of antagonizing downstream events, for example, the inflammatory process, epithelial cell proliferation, or the fibrotic process, is more realistic. Moreover, with agents working in a paracrine manner, gene transfer into a small number of cells, vascular and/or TEC, might be therapeutic. For example, it is conceivable that delivery of a cytotoxic gene into cyst epithelium would result in destruction of nonlethal cysts somewhat akin to the unroofing procedure (surgical cyst ablation) still used occasionally for large cysts. Safety concerns will, however, be paramount, since ADPKD is a chronic nonlethal disorder. Although similar considerations apply to autosomal recessive PKD, the disease phenotype, genetics, and population affected are clearly different, and even gene transfer to a few cysts in the kidney (and liver) could be beneficial once the appropriate disease gene(s) are identified.

Our data in the Han:SPRD rat model (71) are a first step in gene therapy approaches for cystic disease. The Han:SPRD rat is an excellent model for ADPKD, with genetics and histological features resembling that of the human disease (9, 25, 26). A replication-deficient adenovirus carrying a -galactosidase reporter was introduced into the renal artery. Of note, some of the cysts stained blue and were entirely blue, suggesting that adenovirus had reached the cyst lumen. Moreover, some interstitial cells, in addition to vascular staining noted in normal animals, were also positive. One explanation for these findings is that vascular permeability/integrity in cystic kidneys is compromised compared with normal kidneys. In very recent studies, we have shown that pelvic (retrograde) injection into cystic kidneys leads to substantial interstitial expression, with the highest density of -galactosidase cells in areas of greatest disease activity (63). An occasional blue cyst was also noted. This procedure is remarkably straightforward, in contrast to the intra-arterial injection method. These studies underscore the importance of assessing gene delivery in the context of pathology.

Renal Cancer

The clinically challenging problem in renal cancer is largely that of metastatic disease. Nevertheless, it is worth reviewing all the gene therapy modalities that might impact on the treatment of cancer. Several options exist: 1) a gene correction strategy (e.g., replacement of an intracellularly acting tumor suppressor gene would likely require 100% of tumor cells transduced), unless there were a bystander effect, as recently suggested for p53; 2) introduction of an agent that would cause cell differentiation, acting intracellularly or extracellularly; 3) cytotoxic therapy, e.g., with HSV-TK, the herpes simplex virus-thymidine kinase gene, which has a bystander effect, i.e., the ability to kill neighboring nontransduced cells; 4) anti-angiogenic therapies; and 5) immunotherapy. All of these options are useful for the treatment of local disease. For localized renal cell carcinoma, nephrectomy is a reasonable choice; therefore, gene therapy would have to be even more beneficial, which is a difficult goal. For the treatment of metastatic disease by gene therapy, "immunotherapy" and anti-angiogenics are rational approaches, perhaps in combination with other modalities. In addition, since renal cancer is sensitive to systemic IL-2 and to natural and recombinant interferon, this approach merits consideration. Recent work has focused on the reintroduction of renal cells transduced ex vivo with the GM-CSF gene to elicit an immune response to tumor antigens (60). Other treatment modalities would include anti-angiogenic therapy or cytoprotective gene therapy. Studies from our laboratory in a xenograft renal cancer model point to the efficacy of endostatin, an anti-angiogenic protein, for renal cancer (M. Dhanabal, R. Ramchandran, and V. P. Sukhatme, unpublished data). Such a molecule could, in principle, be delivered to the vascular bed of a tumor via a suitably targeted vector. Regarding cytoprotective gene therapy, a multidrug resistance gene could be transferred into bone marrow or gut epithelial cells, allowing more vigorous chemotherapy or reducing toxicity of current regimes. Thus various gene therapy-based options exist for the treatment of renal and other cancers.

Acute Glomerular Disease

The notion of utilizing gene therapy (perhaps delivered as antisense oligonucleotides) to counter the action of various inflammatory, proliferative, or profibrotic cytokines in acute glomerulonephritides is not unreasonable. The rapidly progressive glomerulonephritides may provide a suitable first setting since disease progression is rapid, thereby making the efficacy of therapy easy to judge. It is possible that retroviral vectors may be particularly advantageous in this context, since they selectively transduce dividing cells. Moreover, gene therapy to muscle or liver (72), with the aim of creating a transient source of circulating protein, could also be useful in glomerular disease. Border and colleagues (18) have utilized the HVJ system in skeletal muscle to deliver a cDNA for decorin, a proteoglycan known to bind TGF-1, -2, and -3. They have found decorin gene therapy ameliorates disease in the anti-Thy-1-induced model of glomerulonephritis (18).

Chronic Interstitial Disease

Glomerular and/or interstitial fibrosis is the hallmark of chronic renal failure, with the degree of interstitial fibrosis correlating with long-term renal prognosis irrespective of initiating insult. Approaches, including gene transfer, for blocking fibrosis have been recently reviewed (61). Long-term therapy would be needed. There are many patients (500,000 in the United States) and therapies could be tested in a single kidney with clearances measured from each kidney over a 2- to 3-yr period. However, before therapies could be initiated in humans, preclinical studies are urgently needed on vectors for sustained delivery, delivery modalities, and cell types transduced, and reagents for antagonizing the action of target molecules such as TGF-1, basic fibroblast growth factor, and PDGF. Since fibrosis is a key component of chronic rejection, the transplant kidney would also be a suitable target for such interventions. Our previously cited retrograde studies (63) with adenovirus injected into the pelvis of a cystic (and fibrotic) kidney highlight the relative ease of transducing interstitial cells in such an organ, and this method might be used advantageously in this setting.

Miscellaneous

Applications "outside of the kidney" of interest to nephrologists include vascular access, and in particular, the problem of graft stenosis (62), and genes such as EPO expressed from liver, e.g., utilizing adenovirus or muscle (13, 16, 50, 59, 65, 66) as a "depot" source. Even without the ability to regulate gene expression quantitatively, gene therapy for the anemia of chronic disease could be used to raise the baseline hematocrit in an end-stage renal disease patient to a level in the high twenties, with fine tuning accomplished by EPO protein injections. Primary hyperoxaluria is another kidney disease in which therapy would be directed to the liver. Cloning of the relevant genes would clearly be a first step. Moreover, techniques to obtain long-term expression from the liver would also need to be perfected, unless repeated transduction were contemplated.

	CONCLUSION

Top Abstract Introduction Conclusion References

There are numerous hurdles to tackle before gene transfer for the treatment of kidney disease is a clinical reality. Constructing databases for vectors and gene transfer methods for the normal and diseased kidney are critical first steps.

	FOOTNOTES

Address for reprint requests: V. R. Kelley, Harvard Institutes of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115.

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