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Many pediatric diseases have reached a therapeutic plateau using currently available surgical and pharmacological approaches. Gene therapy has emerged as an exciting new technology to manipulate cells in the mammalian system, and in some cases, this method has achieved amazing therapeutic benefits. Compared to other organs, such as the brain, liver and lung, methods to genetically modify renal cells have received relatively little attention. The current review will discuss the challenges and important developments regarding gene therapy to the kidney, and relate the recent successes and failures to the future potential of gene therapy as a treatment modality in the context of pediatric disease.
The developing kidney is known to be subject to numerous inherited and/or acquired diseases with many of them being developmentally debilitating and in some cases, lethal (1, 2). Conventional surgical and pharmacological methods to ameliorate or even provide a cure for many of these diseases have yet to be developed due to a lack of information regarding the etiology for many of them. For this reason, therapeutics for various pediatric renal diseases have reached a plateau and alternative strategies are required, including the development of gene therapy vectors derived from viruses.
Gene therapy vectors based on viruses have been studied over the past 30 years, but so far, limited information has been obtained regarding the use of various gene therapy vectors for genetic modification of renal cells, particularly for pediatric diseases. The biology of developing cells can vary greatly in comparison to terminally differentiated, adult cells, and so the requirements in the design and development of viral vectors in the treatment of pediatric diseases can be quite challenging. For genetic and environmental diseases that require long-term gene expression to ameliorate their aberrant phenotypes, viral vectors will need to be capable of maintaining their presence within the cell, since most of the normal and pathological cells in pediatric patients will be in a continual and random state of mitotic flux. Other proliferative diseases, such as renal cell carcinoma or nephroblastoma (Wilms’ tumor), may not require long-term persistence by the vector as long as the duration of the therapeutic effect is sufficient to eliminate the oncogenic cells prior to the loss of the vectors from the targeted cells.
It is important to note that another complicating issue in the development of a treatment regimen for the kidney using viral vectors is the complex anatomical structure of this organ. The kidney is comprised of various types of distinct vascular, interstitial and tubular cells, and the access to some of these cells can be limited due to their location within the kidney and lack of adequate blood flow. The route of delivery into the kidney will prove to be an important factor regardless of the vector being considered for use as a gene therapy vehicle.
In this review, we will discuss the pros and cons of the well-known viral vector systems in the context of the kidney, and discuss other gene therapy applications that may provide therapeutic potential in the future.
Adenoviral (Ad) vectors have been one of the most well characterized and extensively studied gene therapy systems over the past 20 years. Characteristically, Ad vectors are predominantly non-integrating episomal, non-enveloped, and double-stranded DNA viruses that have demonstrated broad tropism to numerous cell types in vivo, including the kidney (3, 4, 6). Early-generation Ad vectors had moderate cloning capacities (~8 kb), but more recently developed helper-dependent (also known as “gutted”) Ad vectors have extremely large cloning capacities (~37 kb) (5). The moderate-to-large cloning capacities constitute a clear advantage for these vectors to insert large promoter and/or transgene fragments for gene expression studies. A generic schematic of viral vector production is shown in Figure 1.
Through Ad vectorology, this virus has been modified to generate both replication-defective and -competent systems to treat various types of genetic- and environmentally-based pathologies in many different organs (4), including the kidney. However, the majority of the studies to date have focused on the replication-defective vectors for gene therapy applications in the kidney (6). In general, most of the pre-clinical experiments using Ad vectors have investigated the transduction efficiency in the kidneys of adult small and large animal models, but the efficiencies have been relatively poor. One reason for this is related to the complexity of the renal vascular and tubular architecture, which has been found to make the route of administration (as shown in Figure 2) into the kidney an important factor for transduction of distinct cell types (7–21). Numerous studies over the years have shown a fairly poor transduction into various renal tubular, vascular, glomerular and interstitial cells regardless of whether the Ad vector was administered anterograde through the renal artery (7, 8), retrograde into the ureter (7), or directly into the renal interstitial parenchyma (9). Even prolonged infusion of the Ad vector in vivo and ex vivo into pig (10, 11), rat (21) or isolated human kidneys (12) has not markedly increased the transduction efficiency regardless of the species. In general, most of the intravenous and intrarenal infusion studies have shown a propensity for the Ad vector to be more effective in transducing renal epithelial cells than other cell types in the kidney.
Another reason for the lack of efficient transduction may be attributed to the low vector titer that reaches the target cells. It has been found that the liver and spleen remove a large percentage of the vector from circulation following a single pass (22, 23). By blocking the portal circulation, Ye et al. (24, 25) achieved ~85±10% glomerular transduction efficiency by intravenous administration of Ad vector. Moreover, prolonged infusion of high-titer Ad vector (>7.5 × 1011) into young Sprague Dawley rats resulted in predominant expression of lacZ transgene in glomeruli (30–70%) (15, 25), whereas lower vector titers were unable to transduce the glomerular cells efficiently. Similar high levels of glomerular transduction was also observed in pigs (10, 11). Finally, there may be differences in the species and cell-type localization of the receptors needed to promote Ad vector internalization resulting in the varying degrees of transduction efficiency (26).
In order to optimize transduction of renal cells in vivo, a molecular strategy to re-target the Ad vector is needed. Ad vector binding to target cells and its subsequent internalization require particular receptors and co-receptors (27, 28). Until recently, the tropism of the Ad5 vector has been attributed largely to the fiber region on the capsid (29). Manipulations of the fiber region by pseudotyping the Ad5 capsid with either wild-type Ad-19p fiber (30) or Ad-19p fiber modified to include various kidney specific peptides (31) results in a striking lack of hepatic tropism concomitant with successful transduction into renal glomeruli and/or tubular structures, which was shown to be effective even with systemic administration of the vector without the requirement of direct renal infusion. Recent work demonstrated that coagulation factor X (FX) can be vital to Ad vector targeting to the liver (32, 33), and that blockade of the Ad-FX interaction resulted in the re-targeting of the vector to extra-hepatic organs. In a seminal work, Waddington et al. (34) has demonstrated that the binding of the Ad vector to FX is not attributed to the fiber, but rather to the hexon protein found in the capsid. This new revelation has opened the door to a whole new area of Ad vectorology which may produce a new method to more efficiently re-target these vectors to the kidney.
While the high tropism and the large cloning capacity are the two major advantages of this vector system, the hallmark complication associated with Ad vectors is its transient gene expression (generally <3 weeks) (3, 4) due to the episomal nature of the viral genome following infection and the robust immune response against the Ad vector-transduced cells (35). Lipshutz et al. (36) showed that the transgene expression following Ad vector administration in utero was barely detectable 2-weeks after birth due to loss of the Ad vector in the rapidly growing fetus. Investigators have attempted to overcome the episomal loss of the Ad vector by developing hybrid Ad vectors in which components from retroviruses (37), retrotransposons (38), transposons (39) and AAV (40) have been incorporated in the Ad genome. These newer hybrid vectors have not been tested for renal-based gene therapy approaches in vivo, but they should be able to maintain long-term transgene expression similar to the results obtained in other organs.
The development of innate and adaptive immune responses as well as activation of other inflammatory pathways promoting adverse effects due to Ad vector administration (41, 42) remain difficult problems that gene therapy researchers have been working feverishly to overcome. One solution may be the application of the hybrid Ad vectors during embryogenesis, at which stage the immune system may not have had the time to mature (43). For children with more mature immune systems, investigators may be able to use helper-dependent Ad vectors, which are devoid of the viral genes associated with activating the immune system, and have been shown to persist over a long period of time even in immune-competent animals (5). However, even for these “gutted” Ad vectors, modifications in the capsid will be necessary to minimize the acute toxic effects that are associated with the delivery of high-titer Ad vector (41, 42), due to the composition of the capsid proteins.
The transient persistence of the non-hybrid Ad vectors may limit their use for gene therapy applications requiring chronic gene expression in vivo, but this is not likely to be an issue in the treatment of prevalent renal cancers in pediatrics, such as nephroblastoma (Wilms’ tumor) or renal cell carcinoma (RCC) (44). Replication-defective Ad vectors have been shown to reduce RCC tumor size following expression of suicide genes, such as herpes simplex virus thymidine kinase (HSV-tk) gene (44). However, there has been shown to be a lack of the Coxsackie and Adenovirus Receptor (CAR) on many types of renal cancer cells (45), so the efficiency for manipulating oncogenic cells may be limited. To circumvent this problem, an alternative approach could be to use replication-competent or oncolytic Ad vectors, which aim to kill the tumor cells as a result of Ad vector replication, even though only a few cells may initially be infected. Development of re-targeting strategies for the Ad vector using tumor-specific antigens (46), CAR-independent uptake of Ad vectors (45), or capsid modifications (45, 47), will lead to increased specificity and transduction efficiency in various cancer cells isolated from animal models and human kidney patients (47).
Compilation of currently available studies has shown significant promise in the application of Ad vectors for renal gene therapy as shown by the successful expression of numerous gene products, including FasL (18), Bcl-2 (19), CTLA4Ig (20) and heme oxygenase (13). By combining the previous studies with the latest technology related to the re-targeting of the Ad vectors, these vectors may still prove to have an important role in gene therapy applications in pediatric kidneys. At the present time, however, there are clear barriers as to which cell types can be transduced within distinct regions of the kidney, and the transduction efficiency appears to be dependent on the route of administration. It may be concluded that the slow perfusion of a high-titer Ad vector at a dose greater than 1 × 109 I.U. via the renal artery or ureter, which allows for the maximization of the contact time between the vector and target cell, provides the best possible method to modify renal cells in vivo.
AAV was originally discovered as a “contaminant” of the adenovirus, and has become a popular virus in the development of gene therapy applications for a number of reasons: First, AAV has been found to be non-pathogenic in humans (48). Second, AAV genome can persist following infection into host cells either as multimeric concatemers or as an integrant into the host genome. However, the latter event is extremely rare for AAV vectors (49), and unlike retroviral vectors (see next section on Integrating viral vectors), the lack of integration minimizes the likelihood for insertional mutagenesis. It is interesting that wild-type AAV can integrate into a specific region of chromosome 19, but the replication-defective AAV vectors that are used for gene therapy applications have been found to integrate randomly into the genome (49). One of the main limitations to the use of AAV vectors for gene therapy is its relatively small packaging capacity (~4.5 kb), and so extremely large cDNAs cannot be cloned into the vector and packaged efficiently (50). Similar to Ad vectors, an innate and/or humoral response against the vector has been shown to be developed (48), although alterations in the viral vector could help weaken the anti-vector immune response by removing the antigenic proteins in the AAV capsids. The replacement of the capsid proteins from alternative serotypes, such as AAV7 and AAV8, would minimize the humoral response in most patients (51, 52). A generic schematic of viral vector production is shown in Figure 1.
Although many gene therapy applications have been performed over the past decade in different organ systems, the number of studies targeted to the kidney with various serotypes of AAV has been limited. Similar to the adenoviral vector, the efficiency of transduction was dependent on the route of administration (see Figure 2). Retrograde infusion of AAV serotype 2 (AAV2) by Ito et. al. (53) resulted in moderate expression exclusively in the tubular cells in the renal medulla. Renal arterial infusion into rat kidneys showed restricted transduction into the S3 segment of the proximal tubules and intercalated cells of the renal medulla with no expression detected in the glomeruli (54). To circumvent the complex architecture of the kidney, Lipkowitz et al. (55) attempted to genetically modify renal cells by direct intraparenchymal injection of AAV2 into mouse kidneys which resulted in only low expression of tubular structures near the needle track with no expression of glomeruli or vasculature indicating that the AAV vector did not efficiently spread following direct infusion into the kidney. In a comparative study using serotypes of AAV1 to AAV5, it was found that AAV2 was the best transducing system in the kidney among the various serotypes (56).
Recent investigations by numerous researchers resulted in isolation of novel serotypes to potentially enhance the transduction efficiency into various organs in vivo, including the kidney. Nakai and his associates have shown that AAV8 and AAV9 could infect mouse kidneys as determined by the relatively high AAV genome copy numbers using Southern blot analysis (57, 58). Transgene expression in the kidney as a result of AAV infection was not determined in these studies. More recently, Bostick et al. (59) demonstrated that AAV9 transduction into the kidney was age-dependent, and that only adult kidneys, but not the newborn kidneys, were capable of being infected by this particular serotype when administered intravenously. This relates to early studies by Lipshutz et al. (60, 61), which showed decreasing expression of transgene during the first month of life, followed by AAV2-mediated in utero administration. Even though long-term expression was maintained throughout the life of the animal at detectable levels, the initial loss in transgene expression stands as a potential problem in the use of this vector to treat chronic, debilitating diseases in the developing kidney. Considerable work is needed to elucidate the importance of the alternative serotypes of AAV, and their potential role for therapeutic applications for the kidney.
Biologically, simple and complex retroviruses contain single-stranded RNA genomes that are converted into double stranded DNA through a reverse transcription phase prior to integrating into the host genome. For this reason, these viral vectors have been widely studied as gene therapy tools for a number of different diseases. More detailed vectorology of the retroviral and lentiviral vectors can be read elsewhere (3) and a generic schematic of vector production is shown in Figure 1. Because active proliferation occurs during embryonic and adolescent development, retroviruses would be ideal genetic modifiers to treat pediatric diseases, since copies of the integrated transgene would be able to be propagated into progeny cells unlike the non-integrating vector systems.
The first clinical gene therapy trial in SCID-ADA children was performed using ex vivo genetically manipulated cells using a simple retrovirus derived from Moloney leukemia virus (MLV) (62). Subsequent studies attempted to investigate the efficiency of retroviral vector transduction into the kidney, but the absolute number of genetically modified cells was extremely low. One of the reasons that in vivo gene therapy using retroviruses has been limited is the need for nuclear membrane breakdown for the vector to be capable of genomic DNA integration (63, 64). Even studies in which the vector was introduced into actively proliferating mouse kidneys in vivo following an injury response to a toxic chemical (65) or ex vivo manipulation of proliferating metanephric tissue (66) transplanted into adolescent kidneys resulted in extremely low numbers of genetically modified cells. In addition to the poor transduction, serious adverse effects in children with X-linked SCID (67–70) following treatment with MLV-transduced hematopoietic stem cells ex vivo has led to renewed effort in the development of alternative complex retroviruses (also known as lentiviruses) isolated from either humans (71) or other species (72–74).
Few studies have documented the efficiency of lentiviral vectors pseudotyped with vesicular stomatitis virus G protein (VSV-G) into the kidneys in vivo (75, 76). Compared to retroviral vectors, lentiviral vectors have been shown to be more efficient in transducing terminally differentiated cells, yet integration efficiency can significantly increase in actively cycling cells (77, 78). Details regarding the vector design has been detailed elsewhere (3, 79). In adult mouse kidneys, Gusella et al. (75, 76) found that renal artery perfusion resulted in tubular cell transduction in the outer medullary region of the kidney, whereas retrograde infusion into the ureter led to exclusive collecting duct transduction in the medullary region. Our lab has infused higher titers of VSV-G pseudotyped lentiviral vectors (>109 T.U./kidney) into rodent renal arteries, and we found similar results as Gusella et al. (75, 76) except we did find random glomeruli expressing EGFP marker gene (unpublished observations). Due to the relatively moderate transduction in low mitotic renal cells, it is likely that increased efficiencies would be obtained if the lentiviral vector were applied in younger, actively proliferating kidneys, as previously found in mouse livers and other organs (9, 80) or even in early embryogenesis (73, 81).
With increased genetic screening for kidney-based diseases (82), such as autosomal dominant or recessive polycystic kidney disease, there may come a time when genetic modification of early progenitor cells may become a viable method to circumvent the need to abort the fetus or have a child with a lethal or severely debilitating disorder. Recent studies have manipulated fertilized zygotes using lentiviral vectors to produce transgenic animals with global transgene expression in the kidneys (81). To minimize the off-target effects of expressing transferred genes, vectors would need to be designed with organ- or even cell-type specific promoters to produce exclusive expression in the kidney. Another possibility may be to manipulate renal cells during fetal development as shown by Waddington et al. (73), although only few renal cells were found to express the marker gene.
One of the limitations of lentiviral vector transduction to the kidney is likely due to the currently used envelopes for coating the vector system. In general, the VSV-G protein that was isolated from the rhabdovirus has been widely used to study lentiviral vector transduction. However, but the native virus is neurotropic and so the swapping with renal-tropic envelope proteins (also known as pseudotyping) may provide significant enhancements for renal cell transduction. Another benefit of pseudotyping is that alternative envelopes may circumvent the activation of the immune system whereby the VSV-G envelope has been shown to stimulate a humoral responses against the vector (83). Advances in lentiviral vectorology will inevitably demonstrate the viability of this vector system for renal-based diseases.
To date, human clinical trials have been conducted using lentiviral vectors in which hematopoietic stem cells were transduced to conditionally express antisense RNA sequences against HIV genes (84, 85). The first human trial using lentiviral vectors documented several biosafety features of this system to help promote its future use for other gene therapy applications (85). Although there are no current trials including children using lentiviral vectors, the findings from the adult trials may prove to be an important treatment for pediatric AIDS, and this should open the door to many new trials for lentiviral vector applications to the kidney.
Alternative methods consisting of naked DNA transfection with liposomes (86, 87) or inactivated Sendai virus (or hemagglutinating virus of Japan, HJV) (88–90) have been examined as potential vehicles to transfect the kidney in vivo, but in general, the transgene expression and transfection efficiency remains poor and transient. In many cases, they still lag behind the currently discussed viral vector systems. However, the HVJ-liposome vector approach remains one of the few methods to target the glomeruli (88, 89), and even with the relatively low transfection efficiencies, it has shown to mediate biological changes. Moreover, retrograde infusion of a modified HJV-liposome complex using an artificial viral envelope (AVE) has also been found to transfect interstitial cells (90), which may be important as a potential tool to study therapies for interstitial fibrosis.
It is likely that alternative viruses will need to be studied and developed as gene therapy vectors to treat renal diseases, since a viral vector that can be effectively used for renal gene therapy has yet to be found. Candidate viruses that may become important players could be human foamy virus vectors (91) or other as-yet-to-be-developed viruses from the polyoma family, such as BK (92, 93), or bunyavirus family, such as hantavirus (94). The latter two viruses are both known to have renal cell tropism. It will be interesting to observe what new vector systems will become available for use in renal gene therapy as the field of vectorology continues to evolve. However, in the near future, vectors may need to be used to genetically modify extra-renal sites to secrete proteins into the circulation for targeting to the kidney in order to mediate its therapeutic effect until more efficient vector systems are designed and developed for direct renal gene therapy (74).
Although the effectiveness of viral vectors to manipulate renal cells has not been extremely successful unlike other organ systems, there is a definite need to continue to investigate vectors to effectively modify this important organ. There are too many diseases that originate in the kidney, particularly in newborns and young infants, whereby the evolution of viral vector technology must continue to find successful vectors to treat renal diseases. The field of viral vectorology is still in its infancy with respect to the kidney, so there is great optimism for success especially with the expanding information regarding genomics and gene therapy trials. Continued research into viral vectorology will ultimately produce a vector system, likely derived in some form from those already discussed in the current review to treat renal-based pediatric diseases in the future.