Gene therapy in polycystic kidney disease: A promising future

Introduction

Polycystic kidney disease (PKD) is a genetic condition primarily marked by the development of numerous cysts within the kidneys, leading to progressive renal impairment over time.^[1] This disease manifests in two key forms: autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD), distinguished by their patterns of inheritance. ADPKD, the more prevalent variant, affects approximately 1 in 1000 to 2500 individuals, with symptoms typically emerging in adulthood.^[2] Conversely, ARPKD is less common, occurring in about 1 in 20,000 births, and often presents in infancy or childhood. Beyond its impact on kidney function, PKD may also adversely affect other organs, including the liver, pancreas, and cardiovascular system, underscoring the systemic nature of the disorder.^[3] Current treatment strategies primarily aim to mitigate symptoms and decelerate the disease’s progression, with Tolvaptan being the only medication approved for slowing kidney volume growth and renal function decline in ADPKD patients.^[4] However, its use is tempered by side effects such as polyuria and elevated liver enzymes, necessitating careful medical supervision.^[3] The advent of gene editing technologies and advancements in molecular biology in recent years has sparked new optimism for PKD treatment, particularly for ADPKD.^[5] The potential of gene therapy to directly alter or regulate the genes responsible for PKD offers a groundbreaking approach to tackling the disease’s underlying pathogenesis. This review will explore the latest developments in gene therapy research and treatment for both ADPKD and ARPKD, examining the promising avenues, inherent challenges, and the prospective future of these innovative treatments.

PKD genes, pathogenesis, clinical manifestations, and prognosis

The genetic underpinnings of PKD, particularly ADPKD, outline a sophisticated pathophysiological landscape. At the heart of ADPKD are two primary genes, Pkd1 and Pkd2. Pkd1 resides on the short arm of chromosome 16 (16p13.3), where the presence of six pseudogenes with high G-C content complicates mutation detection efforts.^[6] Conversely, Pkd2 is found on the long arm of chromosome 4 (4q22-23). Together, mutations in Pkd1 and Pkd2 account for 78% and 15% of ADPKD cases, respectively, leaving 7% of cases undetected by these two genes.^[6] Pkd1’s expansive gene length, featuring 46 exons, encodes polycystin-1 (PC1)—a protein integral to calcium signaling and essential for maintaining tubular epithelial cell polarity and function, with its 4303 amino acid residues. Meanwhile, Pkd2 gene produces polycystin-2, which collaborates with polycystin-1 to regulate cellular calcium levels. Disruptions in these genes impair the functions of their protein products, promoting abnormal cell proliferation, differentiation, inflammation, epigenetics, and fluid secretion, culminating in cyst development and progressive renal decline.^[7,8] The widespread distribution of Pkd1 mutations across its coding region further complicates the genetic landscape of ADPKD.

Mayo PKD gene database has documented the genetic diversity within the PKD community, reporting mutations in 22,077 families—322 in Pkd1 and 278 in Pkd2. Our analysis^[9] of 98 Chinese ADPKD families revealed 93 pathogenic mutations, 85 in Pkd1 and 8 in Pkd2, with 55 (59.1%) being newly reported mutations and 9 de novo mutations. Then, our subsequent search among 44 ADPKD families in China identified 37 PKD2 gene variants, including 18 nonsense mutations, 10 frameshift mutations, 4 missense mutations, and 5 splice-site mutations, 11 of which were reported for the first time.^[10]

The advent of high-throughput gene sequencing has unveiled additional genes implicated in ADPKD and ARPKD, such as GANAB and DNAJB11, enhancing our comprehension of PKD’s genetic diversity. GANAB mutations are associated with atypical ADPKD presentations, while DNAJB11 has unveiled the significance of endoplasmic reticulum responses in PKD’s progression. These discoveries not only deepen our understanding of PKD’s pathogenesis but also spotlight crucial targets for developing focused gene therapy approaches, offering new avenues for personalized and precision medicine in combating PKD.

Symptoms of ADPKD usually appear in adulthood, including hypertension, hematuria, flank pain, and progressive renal impairment. Patients may also develop cysts in other organs such as the liver, pancreas, and spleen, and face an increased risk of cardiovascular abnormalities, including aneurysms.^[2] PKD1 mutations are typically associated with a more severe form of the disease, with symptoms such as hypertension, hematuria, flank pain, and kidney enlargement generally presenting earlier in life. Patients with PKD1 mutations tend to progress to endstage renal disease (ESRD) faster.^[11] In contrast, PKD2 mutations usually result in a milder form of the disease with a later onset of symptoms. Renal function declines more slowly in these patients. Extra-renal manifestations, such as liver cysts, are less common compared to PKD1 mutations.^[11,12]

The severity and rate of progression in ADPKD can vary significantly, influenced by the specific mutation type and location, as well as other genetic and environmental factors. Patients with PKD1 mutations especially truncation mutations generally have a poorer prognosis due to the earlier onset and rapid progression of the disease, with most developing ESRD by their 50s, necessitating dialysis or kidney transplantation.^[11] On the other hand, patients with PKD2 mutations tend to have a better prognosis, with slower disease progression. ESRD typically occurs later, often in the 60s or beyond, allowing for a longer period of preserved kidney function.^[11]

ARPKD primarily stems from mutations in the PKHD1 gene, located at 6p12.2, which codes for the protein fibrocystin. Despite differing mechanisms from Pkd1 and Pkd2 mutations in ADPKD, PKHD1 mutations similarly disrupt renal cell signaling and structural integrity, leading to cyst formation primarily in the collecting ducts of the kidneys and often affecting the liver. A notable study identified 77 PKHD1 variants in 78 ARPKD patients, indicating the gene’s extensive mutational spectrum.^[13] Symptoms often present in infancy or early childhood and include enlarged kidneys, hypertension, and varying degrees of renal insufficiency. Liver fibrosis is a common extrarenal manifestation, and affected children may suffer from significant morbidity and mortality related to renal and hepatic complications. ARPKD prognosis is generally poorer, with many affected individuals developing significant renal impairment early in life. Advances in supportive care and potential gene therapies offer hope for improved outcomes, but the disease often leads to ESRD or severe liver disease in childhood or adolescence.

The relationship between genetic mutations and PKD encompasses the disruption of normal protein function, leading to cyst formation and progressive kidney damage. The clinical manifestations and prognosis of the disease depend on the specific genetic mutations involved, with PKD1 mutations generally resulting in more severe and earlier-onset symptoms compared to PKD2 mutations in ADPKD, and ARPKD presenting severe symptoms early in life. Advances in gene therapy and other treatments hold promise for altering the course of the disease and improving patient outcomes.

Classification of gene therapy

Gene therapy has emerged as a transformative approach in modern medical research, leveraging advancements in science and technology to treat or prevent diseases by modifying genes.^[14] This innovative field encompasses a variety of strategies based on their unique mechanisms and objectives (Table 1): (1) replacement therapy: This foundational method involves inserting healthy genes into a patient’s cells to replace or augment defective or missing ones, thus restoring normal cellular functions. It’s primarily employed for genetic diseases attributable to single-gene defects; (2) knockout therapy: Utilizing technology to disable or “knock out” specific genes that cause disease, this approach is effective in managing conditions resulting from gene overexpression or mutation, such as certain cancers;^[15] (3) repair therapy: Concentrating on the precise correction of gene mutations to reinstate their normal function, this technique represents a direct intervention to rectify genetic abnormalities; (4) enhancement therapy: By introducing specific genes into cells, this method boosts particular cellular functions without replacing or repairing the mutated genes. It is especially useful for treating metabolic disorders by supplementing genes to increase the production of missing or insufficient enzymes or proteins; (5) RNA interference therapy: This strategy employs small RNA molecules to suppress the expression of targeted genes, offering a promising avenue for the treatment of diseases characterized by abnormal gene expression, including cancer and genetic disorders;^[16,17] and (6) immunomodulatory therapy: While not a conventional form of gene therapy, genetically engineering patients’ immune cells (e.g., through CAR-T cell therapy) to combat cancer cells is also recognized as a gene therapy variant.

The advent of CRISPR-Cas9 technology has notably advanced the capabilities of gene editing, providing a mechanism for precise gene modification through the use of guide RNA (gRNA) that directs the Cas9 enzyme to specific DNA sequences for cutting, thereby triggering the cell’s inherent DNA repair mechanisms.^[18] This breakthrough has not only revolutionized gene manipulation techniques but also expanded the potential applications of gene editing.

Adeno-associated virus (AAV) vectors have gained prominence as preferred carriers for gene therapy due to their low toxicity, minimal immunogenicity, stable gene expression, and straightforward production process, mitigating the risks associated with gene integration seen in retroviral vectors.^[19]

In summary, the field of gene therapy offers diverse strategies, each with its specific applications and challenges, including considerations of safety, delivery efficiency, and treatment longevity. The development and application of technologies like viral vectors and CRISPR-Cas9 are pivotal in advancing gene therapy, opening new possibilities for treating a wide range of diseases.

Advances in hereditary clinical research on gene therapy

The inaugural clinical trials spearheaded by Anderson at the National Institutes of Health (NIH) during the 1990s signified a pivotal moment for gene therapy, transitioning from theoretical constructs to tangible applications in human health. A landmark trial involved infusing genetically modified autologous T cells via retroviral vectors into two young girls suffering from severe combined immunodeficiency due to adenosine dehydrogenase deficiency (ADA-SCID).^[20] Despite facing substantial challenges, including the refinement of delivery mechanisms, managing immune responses, and enhancing gene transfer efficiency, this trial established a foundational pillar for gene therapy’s potential. Since these early days, significant technological advancements have enabled the approval and commercialization of various gene therapies, offering new avenues of hope for patients with conditions once deemed intractable. Notably, Luxturna’s 2017 approval marked a breakthrough in treating inherited blindness,^[21] followed by the 2019 sanctioning of Zolgensma for spinal muscular atrophy, each representing monumental success stories within the gene therapy realm.^[22]

Recently, a pioneering clinical trial targeting genetic deafness showcased the efficacy and safety of the AAV1-hOTOF vector, delivering the OTOF gene to treat autosomal recessive deafness type 9.^[23] Furthermore, gene therapy strategies employing AAV vectors have shown promise in addressing glomerulonephritis by targeting the NPHS2 gene mutation.^[24] In vitro experiments on human podocytes and subsequent in vivo treatments in model mice with an AAV serotype 2/9 vector have demonstrated significant reductions in albuminuria levels and disease progression, underscoring gene therapy’s versatile potential across a spectrum of diseases.^[24]

In the context of Fabry disease, a clinical trial named FACTs explored the in vitro recombinant lentivirus-mediated transfer of alpha-galactosidase A cDNA to treat the condition (NCT02800070). This approach involved reintroducing genetically modified autologous hematopoietic stem/progenitor cells into patients, with the first treatment administered in January 2017.^[25] Impressively, no serious adverse events were reported among trial participants, and a two-year study revealed significant enhancements in alpha-galactosidase A activity and reductions in enzyme substrates and metabolites in plasma and urine. These findings suggest that genetically modified hematopoietic stem/progenitor cells hold the capacity for sustained alpha-galactosidase A production, highlighting the innovative strides gene therapy continues to make in treating a broad range of medical conditions.

Gene therapy for ADPKD

Antisense oligonucleotides therapy

Abnormally expressed noncoding RNAs (ncRNAs), especially microRNAs (miRNAs), play a key role in the complex pathogenesis of PKD. miRNAs affect cellular functions by regulating the expression of specific protein-coding genes. Recent studies have emphasized the critical role of miRNAs in inhibiting cyst formation, and have proposed the use of miRNAs as a therapeutic new strategy for ADPKD. For example, miR-17-5p can affect the PKD process by specifically targeting the Pkd1 and Pkd2 genes, as well as regulating the transcription factor hepatocyte nuclear factor-1 (HNF1), leading to an increase in renal cysts as well as down-regulation of miR-200 family expression.^[26,27] In the ADPKD model, decreasing the expression of RNA deconjugated p68 reduced the maturation of specific miRNAs, which in turn inhibits cyst formation.^[28] In addition, studies targeting transcription factor-regulated miRNAs overexpressed in PKD have demonstrated the possibility of preventing cyst formation by adjusting miRNA expression.^[29]

RGLS4326, an ASO inhibitor targeting miR-17, demonstrated favorable efficacy and safety by subcutaneous administration in an animal model of ADPKD (Figure 1).^[30] The drug is preferentially distributed in the kidney. The drug was able to preferentially distribute in cysts in the kidney and collecting ducts, inhibiting miR-17 targets including Pkd1 and Pkd2 and effectively controlling cyst growth (Table 2). However, the potential toxicity of RGLS4326 to the central nervous system at high doses prompted researchers to develop RGLS8429 with similar efficacy,^[31] but does not affect AMPA receptors, and has been approved by the U.S. Food and Drug Administration (FDA) to initiate a Phase I clinical trial in the second quarter of 2022 (clinicaltrials.gov#NCT05429073), which is currently ongoing.

Figure 1

Gene therapies for autosomal dominant polycystic kidney disease.

Table 2

Applications, advantages, and disadvantages of each gene therapy technique in polycystic kidney disease

Disease	Treatments	Methods	Target	Status	Advantages	Disadvantages
ADPKD	ASO (RGLS4326)	Subcutaneous injection by lipid nanoparticles	microRNA-17	Clinical trial	Specific targeting of microRNA-17	Potential injection site reactions
	Genetic blockade	PGT in combination with in vitro fertilization	PKD1/2	Clinical trial	Prevention of disease transmission	Ethical and technical challenges
	shRNA	Adenovirus vector	RAGE	Animal study	Effective gene silencing	Potential for immune response
	Transgenic mice	Inducible gene enhancement in embryo	PKD1	Animal study	Long-term study of gene function	Ethical considerations, complexity
	Transgenic mice	Inducible gene enhancement in embryo	PC1-CTT	Animal study	Long-term study of gene function	Ethical considerations, complexity
	Gene editing	Elimination of miR-17 binding element in 3’-UTR by CRISPR/Cas9	PKD1Δ17/PKD2Δ17	Animal study	Enhances mRNA stability and protein levels, retards cyst growth	Off-target effects, potential for unintended gene regulation changes
ARPKD	ASO (AVI-4126, Eteplirsen)	Intravenous infusion by lipid nanoparticles	c-myc	Clinical trial	Potential for systemic delivery	Potential for immune response, cost
	Gene editing	CRISPR/Cas9	P2rx7	Cell experiment	High-precision gene editing	Off-target effects, technical challenges

1. ASO: antisense oligonucleotides; RAGE: receptor of advanced glycation end product; PGT: preimplantation genetic testing.

Gene therapy for ARPKD

Challenges and prospects

Although gene editing and RNA interference technologies have shown great potential in laboratory research, there are still significant challenges in translating these technologies into safe and effective clinical treatments. The technical challenges mainly include improving editing efficiency, ensuring editing precision to minimize off-target effects, and developing efficient delivery systems to ensure that therapeutic molecules can reach and enter target cells. For example, the development of miRNA therapeutics faces technical limitations in improving drug delivery mechanisms and molecular stability. Effective delivery of therapeutic genes to the kidney is a major challenge in current gene therapy research, and existing gene therapy vectors such as AAVs and lipid nanoparticles (LNPs) have limited efficacy when targeting the kidney. The complex structure of the kidney and the glomerular filtration barrier pose challenges for targeting specific renal cell types. In PKD, this means targeting the renal tubular epithelial cells where cysts form. Ensuring specificity, achieving efficient delivery, and minimizing off-target effects are crucial for successful targeting.

The choice of vector is critical in gene therapy, as it influences the efficiency, safety, and duration of gene expression. Commonly used vectors in PKD gene therapy research include AAV vectors, which have low immunogenicity and the ability to infect both dividing and non-dividing cells but face challenges like limited cargo capacity and pre-existing immunity. Perhaps using two or more AAV vectors could successfully deliver the entire Pkd1 gene. Lentiviral vectors, known for their high efficiency of gene transfer, pose risks of insertional mutagenesis. The CRISPR-Cas9 system offers high precision in gene editing but requires efficient delivery mechanisms and strategies to minimize immune responses.

To this end, scientists are exploring new ways to deliver therapeutic genes directly to the kidney, including retrograde ureteral injections, subcapsular injections, renal artery injections, and retrograde renal vein injections, to achieve precision therapies in an in situ setting. The route of administration is crucial for ensuring that therapeutic agents reach the kidneys in sufficient concentrations. Intravenous (IV) injection offers a non-invasive option but may struggle to achieve high concentrations in the kidneys. Direct delivery methods like retrograde ureteral injection, subcapsular injection, and renal artery injection provide higher local concentrations but are more invasive and technically challenging.

Moreover, a potential immune response is an issue to overcome for CRISPR/Cas9 technology.CRISPR/Cas9 gene editing may trigger p53-mediated DNA damage responses in some cases.^[49] Safety issues of gene therapy, especially potential long-term side effects, are key factors that must be considered when moving to clinical applications.

The application of gene-editing technologies raises ethical issues, particularly concerning the boundaries of genetic modification, how to ensure that these technologies are not used for non-medical purposes given the current tentative absence of a regulatory system, and so on. Social acceptance of these technologies and the establishment of relevant legal and ethical guidelines will be an important part of the development of gene therapy. For the time being, researchers need to be extra vigilant on how to safeguard the rights of subjects from being violated, while avoiding the subsequent incalculable problems associated with gene therapy. In addition, the high cost of gene therapy makes these potential treatments potentially difficult for a wide range of patients to accept. How to ensure that these treatments are available to patients globally, especially in resource-limited regions, is therefore another major challenge.

As gene editing and RNA interference technologies continue to advance, the future holds the promise of solving the technical challenges currently faced. For example, the precision and efficiency of gene editing can be improved by improving the design of CRISPR systems and developing new delivery methods. Bioinformaticians have invented a series of computational tools to optimize CRISPR systems, helping researchers design more efficient gRNAs to reduce off-target effects.^[50] Next-generation gene editing tools, such as variants of CRISPR and entirely new editing systems, may provide safer and more effective therapeutic options. The development of gene therapy will accelerate the realization of personalized medicine. By analyzing an individual’s genetic information, therapeutic strategies can be designed to target specific genetic variants, thus providing more precise treatment. This will not only improve the effectiveness of treatment but also help reduce unwanted side effects. Addressing the challenges facing gene therapy requires multidisciplinary collaboration. Experts in the fields of biology, medicine, engineering, ethics, and law need to work together to advance the technology and ensure safe and ethical treatments, as well as to pave the way for the commercialization and diffusion of gene therapy. As global awareness of the importance of treating genetic diseases increases, the field of gene therapy will receive more support and investment in research and applications. The establishment of international collaborative programs and global research networks will help accelerate the development and application of gene therapy technology to benefit patients worldwide.

In conclusion, although gene therapy for PKD faces many challenges on the road to achieving clinical application, it offers unprecedented opportunities for the treatment of genetic diseases as science and technology advance and global collaboration deepens. In the future, gene therapy has the potential to revolutionize the way we treat diseases and bring new hope to patients with PKD as well as other genetic diseases.