Development and evaluation of siRNA-mediated gene silencing strategies for ADO2 therapy utilizing iPSCs model and DMPC-SPIONs delivery system

Abstract

Background

Autosomal dominant osteodystrophy type II (ADO2) is an inherited disease characterized by an abnormal increase in bone mineral density, and CLCN7 (R286W) is its most common causative mutation. The aim of this study was to explore the new idea of siRNA technology applied to the in vitro treatment of ADO2.

Methods

Urinary-derived cells from ADO2 patients were collected to establish induced pluripotent stem cells (iPSCs) model. The siRNA targeting CLCN7 (R286W) mutant mRNA was designed. the cytotoxicity of the delivery vector DMPC-SPIONs was comprehensively evaluated by CCK-8 assay, flow cytometry and scratch assay. Finally, qPCR was utilized to verify the post-transcriptional silencing effect of siRNAs.

Results

We found that DMPC-SPIONs had low cytotoxicity and were able to effectively deliver siRNAs into ADO2-iPSCs. qPCR confirmed that siRNA-DMPC-SPIONs were able to significantly reduce the expression level of mutant CLCN7 (66%), while there was no significant effect on the expression of wild-type CLCN7.

Conclusions

This study developed a gene silencing strategy based on siRNAs and DMPC-SPIONs, which provides a potential new approach for the treatment of ADO2 and demonstrates the potential application of siRNA technology in the treatment of autosomal dominant genetic diseases.

Innovative statements

In this study, we used the established ADO2-iPSCs using patient's urine-derived cells to explore the safety and efficacy of siRNA technology based on the principle of RNA interference for ADO2 treatment for the first time. In addition, we chose DMPC-SPIONs as the delivery vehicle for siRNA, which cleverly exploits the advantages of nanoparticles such as superparamagnetism, low cytotoxicity, and good bio-histocompatibility.

Introduction

Osteopetrosis is a human genetic disorder characterized by an abnormal increase in bone mineral density [1]. The disease can be categorized based on its inheritance patterns: autosomal recessive inheritance, autosomal dominant inheritance, and X-linked inheritance. Specifically, ADO2 is a commonly occurring subtype with an incidence rate of approximately 1 in 20,000 individuals [2]. Patients with ADO2 frequently experience diffuse sclerosis in the skull base, pelvis, and vertebrae, which often lead to complications such as fractures, osteomyelitis, anemia, extramedullary hematopoiesis, cranial nerve impairment, and developmental stunting with deformities. These conditions collectively impact the patients’ quality of life significantly. The most prevalent pathogenic mutation linked to ADO2 is the CLCN7 (R286W) variant, which encodes for the ClC-7 [3, 4]. This gene mutation can result in the dysregulated expression of crucial signaling pathways, including Wnt, nuclear factor-kB (NF-kB), and transforming growth factor-beta (TGF-beta), within bone marrow cells. This disruption may alter the dynamic equilibrium of bone metabolism by affecting the activities of osteoblasts and osteoclasts, ultimately manifesting as clinical symptoms [5]. In clinical practice, hematopoietic stem cell transplantation emerges as a frequently utilized approach for treating malignant osteopetrosis, predominantly advanced cases of autosomal recessive osteopetrosis. However, ADO2 often manifests with a delayed onset, and the success and survival rates of this therapy are contingent on factors like patient age and donor compatibility. Consequently, it is typically not employed for the treatment of ADO2 [6]. Besides providing temporary pain relief and managing symptomatic complications, there remains a conspicuous lack of an effective targeted treatment for ADO2. Hence, there lies a significant research value in exploring innovative cellular-level therapeutic strategies for the treatment of ADO2.

Current treatments for monogenic genetic diseases primarily rely on targeted gene editing, yet their widespread clinical adoption is hindered by the substantial costs associated with research, development, and treatment [7]. However, advancements in modern biomedical technology, particularly in the realms of gene editing and cell therapy, have yielded remarkable breakthroughs. Notably, the integration of siRNA, iPSCs, and nanomaterials has opened up unprecedented avenues for disease treatment. As a potent gene-silencing tool, siRNA precisely regulates gene expression by selectively binding and degrading target mRNA [8]. With the continuous refinement of RNA interference technology, the application of siRNA in gene therapy has expanded significantly, particularly in the management of complex diseases like cancer and hereditary disorders. For instance, Jun Jiang et al. devised a fusion protein consisting of a single-chain variable fragment (scFv), which targets the estimated glomerular filtration rate (eGFR), and lysosomal associated membrane protein 2b (Lamp2b), an exosomal membrane protein genetically engineered to reside on exosomes derived from HEK293 cells. By loading LPCAT1 siRNA into these engineered exosomes, they successfully traversed the blood-brain barrier in an animal model, inhibiting the progression of brain metastases from non-small cell lung cancer [9]. Furthermore, the FDA’s approval of five siRNA-based drugs—patisiran [10], givosiran [11], lumasiran [12], inclisiran [13], and vutrisiran [14]—underscores the maturing of siRNA therapeutic technologies and their clinical potential. Concurrently, breakthroughs in iPSCs technology have introduced new horizons for gene therapy. iPSCs, akin to embryonic stem cells in their pluripotency, circumvent ethical concerns and immune rejection, thereby transcending cell source limitations [15]. This enables the utilization of patients’ own cells for disease modeling and personalized therapies [16]. Recently, the combination of siRNAs and iPSCs has emerged as a research focus, aiming to specifically modify disease-causing genes in iPSCs using siRNAs, thereby restoring normal function or inducing differentiation into therapeutically valuable cell types [17].

Nonetheless, the efficient intracellular delivery of siRNAs remains a pivotal challenge that necessitates resolution. Traditional delivery methods are often plagued by low efficiency and high cytotoxicity, impeding the clinical translation of siRNA technology [18]. Nanomaterials, as an emerging technology platform, present themselves as promising carriers for siRNA due to their unique physicochemical properties, including low immunogenicity, facile surface modification, and multifunctionality [19]. By strategically designing nanomaterials, efficient encapsulation, protection, and targeted delivery of siRNAs can be achieved, enhancing their stability and efficacy within cells. Notably, biocompatible nanomaterials like liposomes, polymer nanoparticles, and inorganic nanoparticles have demonstrated remarkable outcomes in siRNA delivery studies [20]. For example, Manfang Zhu et al. encapsulated lactobacillus lysates within DMP nanoparticles, which were self-assembled from 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and monomethoxy poly(ethylene glycol)-poly(epsilon-caprolactone) (mPEG-PCL), to create DMPLAC. This DMPLAC was then complexed with siSTAT3 to target signal transducer and activator of transcription 3 (STAT3). This DMPLAC/siSTAT3 complex exhibited potent anti-tumor activity and safety in multiple tumor models, showcasing the potential of immunogenic gene vectors for tumor immunogene therapy [21]. Inspired by these advancements, we posit that siRNAs may offer novel therapeutic avenues for ADO2. In this study, we endeavored to establish ADO2-iPSCs using somatic cells derived from ADO2 patients, based on preliminary screening and identification of disease-causing gene mutations within the lithiasis family lineage. Subsequently, we selected superparamagnetic iron oxide nanoparticles (SPIONs) modified with dimethyl phosphatidylcholine (DMPC) as the delivery vehicle for siRNAs. SPIONs, renowned for their tunable size and shape, superparamagnetism, straightforward synthesis, high specific surface area, ease of modification, low toxicity, and biodegradability, have found widespread applications in magnetic resonance contrast enhancement, magnetic particle imaging, and drug/gene delivery [22, 23]. Notably, DMPC-modified SPIONs facilitate cellular endocytosis, evade recognition by the reticuloendothelial system, and minimize immune responses, making them suitable carriers for coupling and delivering siRNAs [24].

Methods and results

Preparation of ADO2-iPSCs, siRNA and DMPC-SPIONs

Disease-specific cells are an indispensable tool for evaluating the efficacy and safety of siRNA. To this end, we commenced by obtaining blood samples from ADO2 patients and their parents. These samples were subsequently genotyped using whole exome sequencing (WES) to confirm the presence of the heterozygous mutation in CLCN7 (R286W). Following this verification, urine-derived cells of the patient were reprogrammed and induced by Sendai virus vectors containing key transcription factors including kruppel-like factor 4 (KLF4), octamer-binding transcription factor 4 (OCT4), v-myc myelocytomatosis viral oncogene homolog (c-MYC), and sry-box transcription factor 2 (SOX2) [3]. As a result, the initial ADO2-iPSCs were successfully established, as illustrated in Fig. 1A. siRNA, a double-stranded RNA molecule with a length of 20–25 nucleotides, can specifically recognize and bind to a specific sequence of target mRNA. This interaction can trigger the degradation of the mRNA or hinder its translation process, ultimately resulting in the silencing of the target gene’s expression. To design siRNA, we employed siDirect software, considering parameters like thermodynamic stability and G/C content of the double-stranded RNA. As a result, we determined the sequence to be 5’-GAUACAGAGAAGUGGGAUUTT-3’. In the subsequent stages of our study, we proceed with the synthesis of DMPC-SPIONs. Briefly, we weigh and combine 0.3 g of polyethyleneimine (PEI) and 15 g of polyethylene glycol (PEG) in a three-necked flask, which is then heated to 80 °C. Subsequently, we introduce 0.7 g of iron acetylacetonate and continue heating until a temperature of 260 °C is reached, resulting in the formation of SPIONs. Once the mixture has cooled down, the SPIONs are dispersed in toluene, purified through magnetic column adsorption, and then dispersed in water to produce PEG/PEI-SPIONs. These are then combined with DMPC and subjected to dialysis to generate DMPC-SPIONs. Finally, through electrostatic adsorption, siRNA is complexed with the previously formed DMPC-SPIONs. The resulting siRNA-DMPC-SPIONs complexes are subsequently internalized into ADO2-iPSCs via endocytosis (Fig. 1A).

Fig. 1

(A) The construction of ADO2-iPSCs model, the preparation of DMPC-SPIONs, and the post-transcriptional targeted silence effect of siRNA-DMPC-SPIONs through endocytosis in ADO2-iPSCs. (B) CCK-8 assay to detect the survival rate of ADO2-iPSCs. (C) Flow cytometry to detect the apoptosis rate of ADO2-iPSCs. (D) Scratch assay to detect the healing migration status of ADO2-iPSCs. (E) qPCR to verify the post-transcriptional silencing effect of siRNA on CLCN7(R286W) pathogenic gene. ***P<0.001

Validation of the safety and efficacy of siRNA-DMPC-SPIONs

Biosafety considerations are paramount for the utilization of siRNA-DMPC-SPIONs in ADO2 treatment. We have meticulously assessed the cytotoxic potential of DMPC-SPIONs using the cell counting kit-8 (CCK-8) assay, flow cytometry, and scratch tests. Following the adherence of ADO2-iPSCs to 80% confluence, we introduced culture media containing concentrations of 0, 20, 40, 60, 80, and 100 µg/mL of DMPC-SPIONs, respectively. The co-culture process was maintained at 37 °C and 5% CO₂ for a duration of 48 h, strictly adhering to the operational instructions outlined for the subsequent experimental procedures. The CCK-8 assay results indicate that the cell viability of each experimental group exceeded 97% (Fig. 1B), suggesting that DMPC-SPIONs had minimal impact on the proliferation of ADO2-iPSCs. Flow cytometry data revealed that the apoptosis rates of cells exposed to various concentrations of DMPC-SPIONs (0, 20, 40, 60, 80, and 100 µg/mL) were 2.74%, 3.40%, 4.47%, 4.32%, 3.35%, and 3.03%, respectively (Fig. 1C). The collective apoptosis rate remained below 5%, indicating that DMPC-SPIONs did not significantly enhance the apoptosis of ADO2-iPSCs. The scratch test outcomes indicate that, within a duration of 0 to 72 h, there was no discernible difference in scratch repair among the blank control group and the experimental groups treated with various concentration gradients of DMPC-SPIONs intervention (Fig. 1D). This suggests that DMPC-SPIONs do not influence the healing and migration of ADO2-iPSCs. Through comprehensive analysis of both qualitative and quantitative experiments, it can be inferred that DMPC-SPIONs possess low cytotoxicity and remain within an acceptable biosafety range. To assess the post-transcriptional silencing impact of siRNA on the pathogenic ADO2 gene, we conducted quantitative polymerase chain reaction (qPCR) verifications. Following 24 h of adhesive growth of ADO2-iPSCs, the culture medium was supplemented with siRNA-loaded DMPC-SPION nanoparticles and allowed to incubate for an additional 48 h. A blank control was established to serve as a baseline. Subsequently, total RNA was extracted from the cells, followed by the synthesis of complementary DNA (cDNA) through reverse transcription. qPCR was then performed using specific primers targeting CLCN7 (R286W) with the following sequences: forward primer (5’-GCAGAGCCTCCCACAACACC-3’) and reverse primer (5’-CTCCAAATGCAGCAGATACACCAG-3’). The results of qPCR, depicted in Fig. 1.

E, indicate that while the expression of wild-type CLCN7 did not decrease in the experimental group, the expression of mutant CLCN7 decreased by 66%. This finding confirms the delivery capability of DMPC-SPIONs and the intracellular therapeutic effect of siRNA, thereby validating its potential in gene silencing therapies.

Statistical analysis

All statistical analyses in this study were performed using GraphPad Prism software (version 9.5.0 for Windows, GraphPad Software, La Jolla, CA, USA). Data are presented as the mean ± standard deviation (SD) for normally distributed continuous variables, and as the median (interquartile range) for non-normally distributed continuous variables or categorical variables. For the CCK-8 assay, cell viability data at different concentrations of DMPC-SPIONs were compared using one-way ANOVA with Tukey’s post hoc test to assess the impact of DMPC-SPIONs on cell proliferation. For the flow cytometry data, the apoptosis rates at various DMPC-SPIONs concentrations were analyzed using the Kruskal-Wallis test with Dunn’s post hoc test, given the non-normal distribution of apoptosis rates. For the qPCR data depicted in Figure E, the expression levels of mutant CLCN7 were compared between the siRNA-DMPC-SPIONs treated group and the control group using the unpaired two-tailed Student’s t-test. The gene expression level was determined by the 2^−∆∆Ct method, with GAPDH serving as the internal control. All tests were two-tailed, and a p-value of less than 0.05 was considered statistically significant.

Discussion

ADO2, a rare genetic disease characterized by imbalance of bone metabolism, lacks effective targeted therapies. Based on the concept of precision medicine, this study innovatively proposed a strategy to specifically silence mutant CLCN7 (R286W) gene expression using siRNA technology, aiming at restoring the dynamic balance between osteoblasts and osteoclasts, thereby alleviating or reversing the disease process. It avoids permanent alterations to the genome, reducing potential side effects and ethical issues. By utilizing iPSCs as a disease model in combination with DMPC-SPIONs as a delivery vehicle for siRNA, we successfully validated the feasibility and effectiveness of this approach at the stem cell level. Compared with traditional gene editing technology, siRNA technology has higher specificity and lower off-target effect, and the technical principle is relatively simple and easy for clinical translation [25]. This strategy not only provides a new idea for the treatment of ADO2, but also provides a new reference for the treatment of other autosomal dominant genetic diseases.

Chemical modification refers to the process of changing the properties and functions of a molecule by altering the composition or structure of the chemical bonds in the molecule [26]. In the biomedical field, chemical modifications are commonly used to improve the stability and efficiency of drugs, nucleic acids and other biomolecules [27]. It is noteworthy that this time we did not increase the stability of siRNAs by chemical modifications, which have been reported to have important effects on the biotoxicity, immunostimulation and off-target effects of siRNAs. For example, 2’-O-methylation (2’OMe) [28], fluorouracil (FU/FC) modification [17], phosphorothioate (PS) modification of the phosphodiester bond [29], and ribose five-membered ring modification [30] can increase the stability of siRNA to a certain extent by altering the charge of siRNAs, increasing their hydrophilicity, or decreasing their interactions with cellular membranes, and thus prolong their half-life. Unmodified siRNAs are more likely to be degraded by nuclease in vivo, resulting in low bioavailability [31]. Certain chemical modifications are also capable of reducing the non-specific binding of siRNA to non-target molecules. For example, PS modifications may reduce non-specific interactions of siRNAs with certain proteins [18], thereby reducing cytotoxicity. Unmodified siRNAs are unstable in the in vivo circulation and may not reach the target tissues or cells efficiently, which also increases unnecessary exposure and potential toxicity. Of course, in addition to chemical modification, the delivery system is also an important factor affecting siRNA biotoxicity. Sophisticated delivery systems, such as lipid nanoparticles (LNPs) and N-acetylgalactosamine (GalNAc) conjugates, efficiently transport siRNAs to target cells while minimizing their accumulation in non-target tissues, thereby mitigating potential biotoxicity [32]. siRNAs, as exogenous short chains of nucleic acids, can activate pattern recognition receptors such as Toll-like receptors, trigger immune responses, and promote the expression and release of inflammatory factors [33]. Chemical modification can block the activation of pattern recognition receptors (e.g., TLR3, TLR7, and TLR8) by altering features such as nucleotide chain length, base sequence, and ribose structure of siRNAs, thereby inhibiting immune response and reducing immunotoxicity [34]. siRNAs can also activate intracellular pattern recognition receptors such as double-stranded RNA-dependent protein kinase R (PKR), leading to fever, cytokine storm, and other adverse reactions, causing dysfunction in the body [30]. Appropriate chemical modifications (e.g., methoxyl modification and fluorine modification) can inhibit this intrinsic immune activation and reduce the immunostimulatory properties of siRNAs [35]. Off-target effect is one of the major challenges in siRNA drug discovery and development, which refers to the non-specific binding of siRNAs to non-target mRNA strands, resulting in unintended gene silencing [36]. This may be due to mismatch pairing due to high sequence homology, or due to the wide-ranging effects of the siRNA-induced RNAi pathway on the entire transcriptome. Chemical modifications, especially those targeting the siRNA seed region (a seven-nucleotide region critical for target recognition), can reduce off-target effects by altering the thermodynamic stability and base-pairing properties of siRNAs [37]. The research group at Nagoya University succeeded in reducing the risk of off-target effects and improving the specificity and safety of siRNA drugs by introducing formamide modifications to the seed region of siRNAs, inhibiting the formation of hydrogen bonds, and interfering with the stability of the helical structure of mRNAs, which resulted in strand denaturation or separation [38]. In addition, by binding a specific ligand (e.g., an antibody, peptide, or small molecule) to the siRNA, it can be more easily recognized and bound to the target gene. This approach is known as “ligand-mediated targeting” and improves the specificity and affinity of the siRNA. Adding markers such as fluorescein or radioisotopes to siRNAs enables real-time monitoring and quantitative analysis of siRNAs. Recent studies have shown that it is also possible to design bifunctional siRNAs with pro-inflammatory and specific silencing activities by chemical modification [17]. this design not only enhances the therapeutic effect of siRNAs, but may also have potential applications in antiviral therapy. For example, uridine bump-designed siRNAs enhance immune stimulation while maintaining a conserved silencing efficiency [39]. In the next phase of our research program, we may map out different chemical modifications to optimize the targeted silencing effect of siRNAs.

In recent years, remarkable progress has been made in the field of nanomaterials and gene therapy. On the one hand, the continuous emergence of new nanomaterials has provided more choices of tools for gene therapy; on the other hand, the continuous improvement of gene editing technology and siRNA technology has opened up new avenues for the treatment of hereditary diseases. In the future, with the continuous integration and innovation of technology, we have reason to believe that gene therapy based on nanomaterials and siRNA technology will become an important tool for the treatment of hereditary diseases. Despite the initial success of this study at the cellular level, it is still far from clinical application. Future studies should further optimize the preparation process of nanomaterials to improve their stability and bioavailability; meanwhile, validation in more advanced animal models is needed to assess the effectiveness and safety of this therapeutic strategy in vivo. In addition, the distribution, metabolism and pharmacokinetics of siRNA in vivo should be thoroughly explored to develop a more rational dosing regimen. Due to the genetic heterogeneity of ADO2, different patients may carry different mutations, and thus siRNA design for specific mutations may need to be individually tailored. With the continuous maturation of gene editing technologies, such as CRISPR/Cas9, the combination of siRNA technology and gene editing technology may also be considered in the future to further improve the therapeutic efficacy.

Future directions

In the future, we plan to employ CRISPR-Cas9 technology in constructing a Clcn7 (r284w) mutant ADO2 mouse model. For this purpose, we will administer intraperitoneal injections of siRNA-DMPC-SPIONs to eight 10-day-old sex-matched WT mice and ADO2 mice, three times per week for a duration of four weeks. Additionally, an identical group of eight 10-day-old sex-matched WT mice and ADO2 mice will receive the same volume of normal saline as a control measure. Post-treatment, comprehensive Computed Tomography (CT) examinations and precise bone mineral density measurements will be conducted to assess the effectiveness of the treatments. The mice will undergo euthanasia, and their internal organs including liver, kidney, spleen, brain, heart, and lungs will be procured for examination. This process will involve assessing their size, weight, and pathological morphology, along with analyzing any nanoparticle residues. Additionally, the femurs will be prepared for qPCR experiments to determine whether there is a decrease in the expression of Clcn7 (r284w) gene. Concurrently, the bone marrow cells from the femoral region will be extracted and induced to transform into osteoclasts. Following this, Tartrate-Resistant Acid Phosphatase (TRAP) staining, Calcitonin Receptor (CTR) staining, and bone resorption experiments will be conducted to determine whether there is an increase in the number and functionality of osteoclasts. Ultimately, the in vivo therapeutic efficacy of siRNA-DMPC-SPIONs on Clcn7 (r284w) mutant ADO2 mice will be evaluated based on the experimental outcomes mentioned above.

Conclusion

In this study, we successfully achieved effective silencing of the mutant mRNA of the CLCN7 (R286W) variant within the ADO2 pathogenic gene at the level of iPSCs by meticulously designing specific siRNAs and employing DMPC-SPIONs as a delivery vehicle. Notably, DMPC-SPIONs exhibited favorable biocompatibility and minimal cytotoxicity, ensuring the safe delivery of siRNAs. Nevertheless, further investigations are warranted to thoroughly validate their efficacy and safety in animal models, thereby assessing their potential as viable therapeutic agents. Additionally, despite the impressive delivery efficiency demonstrated by DMPC-SPIONs, there is scope for enhancing the cellular uptake and endosomal escape of siRNAs through additional chemical modifications or structural optimizations, ultimately amplifying the therapeutic outcome. In conclusion, the targeted delivery of siRNAs utilizing nanoparticles, as established in our study, presents a novel and comparable therapeutic avenue for researchers working in the realm of monogenic genetic diseases. This approach is anticipated to propel the translation and application of siRNA technology further.