Gene therapy for kidney disease: targeting cystinuria

Abstract

Purpose of review

The aim of this study was to summarize recent findings in kidney gene therapy while proposing cystinuria as a model kidney disease target for genome engineering therapeutics.

Recent findings

Despite the advances of gene therapy for treating diseases of other organs, the kidney lags behind. Kidney-targeted gene delivery remains an obstacle to gene therapy of kidney disease. Nanoparticle and adeno-associated viral vector technologies offer emerging hope for kidney gene therapy. Cystinuria represents a model potential target for kidney gene therapy due to its known genetic and molecular basis, targetability, and capacity for phenotypic rescue.

Summary

Although gene therapy for kidney disease remains a major challenge, new and evolving technologies may actualize treatment for cystinuria and other kidney diseases.

INTRODUCTION

Gene therapy involves the change, removal, or introduction of genetic material in cells with the goal of treating disease. The revolutionary advances in gene therapy over the past few decades have led to effective therapies for a wide range of diseases that were previously difficult to treat and have expanded potential therapies for previously undruggable targets [1]. Gene therapy targets somatic cells to prevent heritability of changes that would be introduced in germline cells. Current methodologies edit or correct gene mutations, insert or delete entire genes, or create genetic knock outs [2]. There is also diversity in the tools used for gene delivery including viral and non-viral methods. Delivered genetic material can remain episomal, integrate into the genome, or edit the genome. Programable nucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, zinc-finger nucleases, and transcription-activator-like effector nucleases can induce targeted double stranded breaks that can be repaired through template-dependent homologous directed repair or the error-prone nonhomologous end-joining pathway [2]. With this broad diversity in the delivery and editing technologies, many researchers have identified new strategies for creating targeted therapeutic options. Currently, somatic cell gene therapy has been developed for several hematopoietic and metabolic disorders including lymphoma, thalassemia, leukemia, and lysosomal storage disorders [1, 3, 4]. In the U.S., gene therapy has been FDA approved for chimeric antigen receptor (CAR) T cell products treating leukemias, lymphomas and multiple myeloma, and adeno-associated viral (AAV) vectors treating retinal dystrophy and spinal muscular atrophy [5].

KIDNEY GENE THERAPY LAGS BEHIND

Notably, while gene therapy has been achieved within perhaps more easily targetable tissues, other organs such as the kidney have lagged behind. Kidney diseases are rarely if at all mentioned as an indication for gene therapy clinical trials [1]. Kidney targeted gene therapy would offer new hope for preventing chronic kidney disease (CKD), as recent data has shown that 20–30% of CKD is due to monogenic disease-causing variants [6]. The kidney is potentially accessible through multiple routes of delivery including systemic administration, injection into the renal artery, retrograde administration into the ureter, and intraparenchymal injection, which could allow for targeted administration based on the desired effect [7]. Despite the potential advantages for kidney gene therapy, the gap in the gene therapy field is primarily due to challenges with delivery. The complex kidney architecture makes targeting specific kidney cell types difficult, as the glomerulus actively excludes proteins above 50 kDa in size and slit diaphragms within podocytes are only 10 nm [8]. In contrast, viral vectors often exceed those limits, as one of the smallest vectors, AAV, is 25 nm and has a mass of over 200 kDa [9–11]. Additionally, there are at least 26 unique cell types within the kidney [12]. Compared to tissues where gene therapy has been accomplished such as the liver and retina which have 4 and 5 cell types respectively [13, 14], the diversity and complexity of cell types in the kidney adds to the delivery challenges. Therefore, the unique tissue architecture and number and diversity of cell types contribute to the ongoing challenges of kidney gene delivery.

RECENT ADVANCES IN NANOPARTICLES AND NON-VIRAL DELIVERY

To attempt to overcome the kidney delivery challenges as described above, many have pivoted to the use of nanoparticles. These constructs have several advantages over viral delivery, such as low immunogenicity, flexibility, size, and cost effectiveness [15, 16]. Delivery of naked plasmid DNA to tubules has been achieved in mice and rats, though phenotypic correction of an inherited disease has not been demonstrated [17, 18]. Recent studies have developed nanocarriers that localize to proximal tubule epithelial cells using a targeted transmembrane protein, megalin [19]. A similar study designed mesoscale nanoparticles that targeted proximal tubular NF-kB essential modulator (NEMO) in a model of AKI [20]. While these models were used for renal cell carcinoma and AKI therapeutics respectively, the efficiency of the nanoparticles demonstrates the potential for nucleic acid delivery and their use in other disorders of the proximal tubule. Both studies also showed minimal localization to other commonly off-target tissues such as liver and lung. Therefore, the use of a highly specific proximal tubule nanoparticle would reduce therapeutic dosing and off-target effects for gene therapy in vivo.

Another recent study that created a non-viral tubular-targeted system utilized KIM-1 specific extracellular vesicles [21]. Kidney injury molecule-1 or KIM-1 has shown to be a marker for tubular injury during AKI and CKD [22]. By selecting for particles that bound Kim-1 with high affinity and fusing them with red blood cell derived extracellular vesicles, they created a system that targeted tubular epithelial cells, which could also have applications for gene therapy.

Finally, several recent studies have designed non-specific nanoparticles that have shown high affinity for the kidney. These studies all exhibit significant off-target delivery, notably in the liver, lungs, and heart, but the authors note that the nanoparticles could be modified to induce further specificity to a single tissue. The first of these studies modified a chitosan polymer with an anionic coating to deliver anti-fibrotic therapy in a model of CKD [16]. Another group assessed lipid nanoparticle delivery of α-galactosidase A in a model of Fabry disease and found expression in the kidney [23]. Finally, a group modified an existing polyamidoamine (PAMAM) dendrimer polymer to reduce toxicity and found their novel nanoparticle also showed significant localization to the kidney [15]. While these studies are not as specific to a single cell type within the kidney, they are promising for global kidney expression or diseases such as lysosomal storage disorders where multi-organ targeting is beneficial.

RECENT ADVANCES IN VIRAL DELIVERY

Viral delivery is a common approach in the gene therapy field due to its transduction efficiency, longevity, and current use in FDA-approved therapies [5]. However, there is a current gap in the field for creating kidney cell specific viral vectors. Viral targeting of the kidney remains a challenge. A recent study has used recombinant AAV9 (rAAV9) by both systemic delivery and renal vein injection to deliver a therapeutic protein in a model of acrodysostosis [24]. They demonstrated up to 70% transduction in kidney cortex tubular cells and subsequent phenotypic restoration. However, such success with AAV9 in the kidney has not been reported by others. For instance, in a separate AAV delivery study testing six AAV serotypes, researchers demonstrated transduction of kidney mesenchymal cells including pericytes, fibroblasts, and mesangial cells with a novel AAV serotype; however, they observed no kidney transduction with AAV9 [25]. Gene editing or delivery of Cre-recombinase to the kidney in reporter mice has been achieved [9, 26]. However, these studies do not necessarily translate to therapeutic transgene delivery and expression. Delivery of a recombinase to cleave genomic DNA is a different challenge than integrating new sequences into the genome to treat a genetic disease. Similar to the non-viral delivery approaches above, viral vectors often lead to transduction of off-target tissues, so specificity may likely need to be further refined before translation to human health. Cell-type specific promoters may obviate such issues.

Some researchers have shown effects on kidney disease or its complications without targeting the kidney directly. Antigen specific T lymphocytes have been gene-modified to serve as micro-pharmacies to deliver erythropoietin (EPO) to correct anemia of CKD [27]. In order to better control timing of transgene expression, a recent study has used a reversible RNA on-switch through AAV with the goal of treating anemia of CKD [28]. They showed a 200-fold induction of their AAV-RNA on-switch system compared to native transcription factor AAV regulation for EPO production. This temporal control allows for new flexibility and feasibility for disorders where constant transgene production may not be optimal. Recent advancements in lentivirus delivery have also shown implications for kidney gene therapy. A clinical study isolated CD34+ stem cells from five adult male patients with Fabry disease and transduced them to express α-galactosidase A [29]. All five patients demonstrated almost baseline rescue of α-galactosidase A levels within a week, and all but one patient showed stabilization of their CKD up to 33 months post infusion. Finally, new work on renal ex vivo organ gene therapy has created new avenues for treatment [30]. This recent study also used lentiviral transduction to perfuse a donor ex vivo kidney to silence MHC transcripts before transplanting into an allogenic recipient. While only applicable to gene therapy for organ transplant, this technique allows for specificity and control over viral transduction which could be utilized for other applications.

CYSTINURIA AS A MODEL TARGET FOR KIDNEY GENE THERAPY

Compared to other monogenic kidney diseases, cystinuria offers several advantages as a target for gene therapy. Cystinuria is an autosomal recessive disorder that occurs in 1 in 7,000 globally [31]. Type A cystinuria results from mutation of SLC3A1 (rBAT protein) while type B results from mutation of SLC7A9 (b^0,+AT protein), and the two protein products come together to transport cystine as well as dibasic amino acids (ornithine, lysine, and arginine) [32, 33]. Cystinuria results from defective reabsorption of cystine in the proximal tubule of the kidney and leads to cystine stones in the urinary tract. Cystine stone formation typically begins between ages 10 to 30 years and patients continue to form multiple stones per year [33]. The resultant urinary obstruction leads to repeated kidney injury, with up to 70% of patients progressing to CKD [34]. Despite sufficient characterization of the genetics and molecular basis of cystinuria, effective treatments are lacking. α-lipoic acid has been shown to improve cystine solubility in mice [35], but studies will reveal if it works in humans. It doesn’t correct the transporter defect which can also affect amino acid levels in the blood and other tissues [36].

The proximal tubule is potentially targetable within the overall kidney architecture. The highly endocytic proximal tubule is accessible from the urinary apical side via glomerular filtration, or via retrograde administration from further down the nephron or the urinary tract. The proximal tubule is also accessible via fenestrated endothelium on the basolateral side. As cystinuria is autosomal recessive, it is presumably more easily treatable with gene therapy than other diseases like autosomal dominant polycystic kidney disease. The SLC3A1 and SLC7A9 cDNAs are 2.3kb and 1.8kb respectively such that they could fit in AAV vectors for instance. Off-target expression of transgenes should limit toxicity as functional transporter protein complexes would only be expected to be expressed in the target tissue where the complementary SLC3A1 or SLC7A9 is expressed. Albino cystinuria mice have been generated with CRISPR/Cas9 that can be tested with in vivo imaging technologies to visualize delivery to the kidney in live animals, as well as gene editing to rescue cystine transporter activity [37]. Potential gene therapy for cystinuria patients would not need to completely reduce urinary cystine levels to baseline levels. If urinary cystine levels are fractionally reduced below the crystallization threshold of 1mM [38], that would be sufficient for stone prevention. A low level of cystine transport reconstitution in the proximal tubule may be enough to rescue the phenotype. This could occur via high expression in a low number of cells. Additionally, many monogenic kidney diseases affect development and are hard to reverse at older ages, so gene therapy would be required at the neonatal stage. However, cystinuria could be targeted in adulthood especially if gene therapy was before the development of CKD. As described above, there are several current advances in the nanoparticle and viral delivery field that target the proximal tubule. Other structures in the nephron, such as the glomeruli and collecting ducts, lack current specific targeting strategies compared to the proximal tubular epithelium. Overall, cystinuria represents a model target for kidney gene therapy.

CONCLUSIONS

Kidney gene therapy remains a difficult challenge, namely due to obstacles with delivery. With recent advances in non-viral and viral delivery methods, there is emerging hope for gene therapy for monogenic kidney diseases. Cystinuria represents a model target within the kidney gene therapy field due to the understanding of its genetic and molecular basis, its unique targetable location along the nephron, and the possiblity that high transduction efficiency may not be needed for disease correction.

KEY POINTS.

• Kidney gene therapy lags behind gene therapy targeting other organs.

• Kidney-targeted gene delivery remains a major challenge.

• Recent advances in nanoparticle and adeno-associated viral vector technologies have improved gene delivery, particularly to the proximal tubule segment of the nephron.

• Cystinuria represents a model monogenic disease target for kidney gene therapy.