Tumor-specific delivery of KRAS siRNA with iRGD-exosomes efficiently inhibits tumor growth
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Lung cancer is the major cause of cancer-related deaths globally. Mutant KRAS is a feature of 15–50% of lung cancer cases and represents one of the most prevalent oncogenic drivers in this disease. Unfortunately, although much effort has been spent on searching for small molecule inhibitors of KRAS, KRAS gene has proven extraordinarily difficult to target by current pharmacological agents. In the present study, we developed an alternative strategy to silence the so-called untargetable and undruggable KRAS gene by employing exosome-mediated siRNA delivery. Particularly, we reprogrammed HEK293T cells to simultaneously express KRAS siRNA and Lamp2b, an exosomal membrane protein, in fusion with a tumor-penetrating internalizing RGD (iRGD) peptide (CRGDKGPDC), and then purified the tumor-targeting exosomes as KRAS siRNA delivery system. In agreement with the study design, intravenously injected iRGD-exosomes specifically targeted to tumor tissues in vivo. The therapeutic potential was revealed by the strong inhibition of tumor growth in a mouse model after intravenous injection of KRAS siRNA encapsulated in iRGD-exosomes. In conclusion, our results indicate that iRGD-tagged exosomes is an ideal delivery agent to transport KRAS siRNAs for lung cancer treatment. As an extension of this finding, the vast majority of mutated genes that are difficult to target by current pharmacological agents will be targetable and druggable in the future.
KeywordsLung cancer siRNA KRAS Exosome iRGD
- HEK293T cell
human embryonic kidney 293 T cell
rabies virus glycoprotein
small interfering RNA
Globally, lung cancer is diagnosed at the highest frequency and leads to the highest mortality amongst all cancers . Lung cancers are often identified in patients at late phase due to its asymptomaticity which renders the current treatments unsensitive (e.g., surgery, radiotherapy and chemotherapy). Thus, designing new therapeutic agents for lung cancer is still crucial. The discovery of key signaling pathways involved in mediating cancer cell development and metastasis significantly expands our understanding of lung cancer biology. For example, oncogenic mutations such as EGFR and EML4-ALK have been intensively investigated as targets of personalized lung cancer therapy.
One of the commonest mutant genes in lung cancer cells is the Kirsten Rat Sarcoma viral oncogene homolog (KRAS) . KRAS gene is located at 12p12.1 and encodes a protein that is a member of the small GTPase superfamily [3, 4]. KRAS is a prominent oncogene because it can transform normal cells into malignant cells, particularly when harboring an activating mutation in codon 12 or 13 . Abnormal overexpression and somatic activating mutations in KRAS are universal in many cancer types, including majority of the pancreatic cancer (70–90%) , 30–60% of colon cancer  and 15–50% of lung cancer patients . Thus, the search for effective inhibitors for KRAS is an urgent need. Unfortunately, although much effort has been spent, KRAS has remained an untargetable and undruggable oncogene, possibly due to the absence of allosteric binding sites or activation of parallel signaling pathways that provide compensatory signals.
Short interfering RNA (siRNA) has the potential to silence the so-called untargetable and undruggable genes based on recognition and subsequent degradation of specific mRNA sequences . However, the successful implementation of siRNA in vivo is hampered by some major barriers: 1) Being target-specific based either on tissue or cell types; 2) the toxicity and immunogenicity of either siRNA or its delivery vehicle; 3) the instability of siRNA in circulation and difficulty to transfer across biological barriers. Thus, developing a new and efficient siRNA delivery system is quite essential.
Exosomes are endocytosis-relevant vesicles sized at 30–100 nm [10, 11, 12]. They are involved in intercellular interaction through mediating horizontal transfer of informative proteins, lipids and RNAs [13, 14]. Because exosomes can act as natural carriers of RNAs, they might provide a novel delivery strategy for siRNA . The concept of using exosome for siRNA delivery was first proved by Alvarez-Erviti et al., who developed modified brain-specific exosome that expresses a brain-targeting peptide derived from RVG (rabies virus glycoprotein, YTIWMPENPRPGTPCDIFTNSRGKRASNG) fused to the N-terminus of Lamp2b (lysosome-associated membrane glycoprotein 2b), a protein that is abundant in exosome outer membrane. In principle, Lamp2b can guide the RVG peptide to exosomal surface, and the modified exosomes can recognize and target neuronal cells by binding to acetylcholine receptors on the blood-brain barrier and neuronal cells [16, 17]. Intravenous injection such modified exosomes into normal mice led to efficient delivery of siRNA to the brain and consequently knockdown of BACE1 gene, highlighting the therapeutic value of this new RNAi strategy .
In this study, we developed a novel strategy for lung cancer treatment by employing exosome-mediated siRNA delivery. Tumor targeting was achieved by engineering HEK293T (human embryonic kidney 293 T) cells to express Lamp2b in fusion with the tumor-penetrating iRGD peptide (CRGDKGPDC) which has a high affinity to integrin αvβ3. Intravenously injected iRGD-exosomes delivered KRAS siRNA specifically to tumors in vivo, resulting in specific KRAS gene knockdown and tumor growth inhibition.
Characterization of the redesigned iRGD-exosomes loaded with KRAS siRNA
KRAS siRNA encapsulated in iRGD-exosomes suppressed the proliferation of lung cancer cells
iRGD-exosomes specially targeted tumor tissues in vivo
KRAS siRNA encapsulated in iRGD-exosomes inhibited tumor development in a xenograft mouse model
KRAS mutation was identified in tumor cells more than 20 years ago, but its clinical importance in cancer therapy just began to be appreciated. Abnormal activation of KRAS is extensively found in many cancer types and is often associated with drug resistance. Scientists have redoubled efforts to disable the mutated KRAS oncogene. Despite much effort spent on searching for inhibitors for activated KRAS, KRAS has proven extraordinarily difficult to target. Now scientists are trying to find another way to target KRAS gene by blocking signals from downstream genes. However, the attempts to identify druggable targets that are linked to KRAS have been challenging, since KRAS affects many signaling pathways and may function differently in different cancers. Thus, the search for an alternative approach to block KRAS activity or inhibit KRAS expression is highly required.
siRNA is highly dependent on target gene sequence and can theoretically inhibit the expression of any gene with strong specificity, including those traditionally undruggable genes. However, the transformative therapeutic potential of small RNAs has been stymied by a major obstacle—safe and efficient delivery of siRNA in vivo. Although siRNA delivery could theoretically be achieved through injection of synthetic siRNAs, it is enormously difficult to circulate siRNA at constant and high level even though the repeated injections at short intervals are given, as siRNA is rapidly cleared in vivo. Thus, several techniques using viruses or synthetic vehicles (liposomes or polymer nanoparticles) as siRNA agents have been developed. Despite being reasonably efficient in transferring siRNAs in vivo, the procedures for construction and preparation are labor-intensive, and the delivery process may induce severe immune responses and toxicity in host. Recently, direct administration of modified exosomes containing the small RNAs (siRNA and miRNA) of interest unveils a new strategy to deliver and express small RNAs in vivo. Exosomes, as the host’s own RNA shuttle service, can be employed to deliver siRNAs and miRNAs to various tissues and organs through the circulation and bio-distribution of themselves. Therefore, it is now possible to silence many different genes and/or their combinations for their therapeutic activity, even though these genes are located within the tissues and organs (e.g., pancreas and CD4+ T cells) that are currently difficult to treat and manipulate by the conventional techniques. This technique is expected to become a promising new siRNA delivery methodology for in vivo gene therapy. In consistent with this concept, we validated in this study that KRAS siRNA encapsulated in iRGD-exosomes could be delivered into tumor cells and efficiently knocked down KRAS expression both in vitro and in vivo. Targeting KRAS with such new strategy may provide a therapeutic option for lung cancer patients with KRAS mutations. As an extension of this finding, the vast majority of mutated genes that are difficult to target by current pharmacological agents will be targetable and druggable in the future.
In conclusion, the present study demonstrates that exosome-based siRNA delivery, which uses iRGD peptide as a guide, is a convenient and efficient mean to induce RNAi in vivo. Employing endogenous exosomes to deliver siRNAs has great significance in theory and application because it can overcome the barriers of delivering siRNA in vivo. Such a technological innovation may reshape the gene therapy field and enable systemic siRNA transfer for treatment of human diseases.
Materials and methods
The human embryonic kidney 293 cell line (HEK293T) and adenocarcinomic human alveolar basal epithelial cells cell line A549 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in high-glucose (4.5 g/L) DMEM (Gibco, China) supplemented with 10% foetal bovine serum (FBS, Gibco, Australia), penicillin and streptomycin in a 5% CO2, water-saturated atmosphere.
Transfection of HEK293T cells and isolation of exosomes
HEK293 cells were cultured in 225-cm2 flasks (Corning, China) and were co-transfected with plasmids encoding Lamp2b-iRGD and KRAS siRNA using Lipofectamine 2000 (Invitrogen) following manufacturer’s instructions. The cell culture medium was then collected 2 days after transfection, and the exosomes were harvested from the cultural medium with Exosome Isolation Reagent (Invitrogen). The resulting pellet was then resuspended in PBS.
Cells were washed with PBS (pH 7.4) and then lysed in RIPA Lysis buffer solution (Beyotime, Shanghai, China) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL) on ice for 30 min. The tissue samples were frozen solid in liquid nitrogen, powdered and lysed in ice-cold RIPA Lysis buffer containing a protease and phosphatase inhibitor cocktail. The samples were sonicated in an ice bath where necessary. Tissue homogenates and cell lysates were spinned down for 10 min (12,000×g at 4 °C) and the supernatant was collected. The protein levels were normalized by probing the same blots with a GAPDH antibody. KRAS antibody (F234) (SC-30) was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH antibody (FL-335) (sc-25,778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Incubation of cells with exosomes
Exosomes (100 μg) loaded with KRAS siRNA were incubated with A549 cells (106 cells). After 6 h incubation, the recipient cells were harvested for total RNA extraction. Subsequently, quantitative RT-PCR analysis of KRAS siRNA and KRAS mRNA and western blot analysis of KRAS protein were performed.
Quantitative RT-PCR assay
Total RNA was extracted from cells and tissues with TRIzol Reagent (Invitrogen, Carlsbad, CA). Assays used to quantify mature miRNAs were performed using TaqMan miRNA probes (Applied Biosystems, Foster City, CA). One μg of total RNA was reverse-transcribed to cDNA using an AMV reverse transcriptase (TaKaRa, Dalian, China) and Oligo d(T) primer (TaKaRa, Dalian, China) with the following reaction conditions: 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. Real-time PCR was performed using a TaqMan PCR kit and an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). The reactions were incubated in triplicate in a 96-well optical plate at 95 °C for 5 min followed by 40 cycles at 95 °C for 30 s, 65 °C for 30s and 72 °C for 30s. After the reactions ran to completion, the cycle threshold (CT) values were determined using fixed threshold settings, and the mean CT was determined from the triplicate PCR reactions.
Non-invasive bioluminescence imaging
Non-invasive bioluminescence imaging was used to assess tumour outgrowth because the in vivo bioluminescence imaging clearly distinguished the tumours from the surrounding tissue even without any contrast agent. Inject 10 μl/g body weight of D-luciferin firefly (15 mg/ml in PBS; Caliper Life Sciences Catalog XR-1001) into the mice abdominal cavity 5 min before anesthetizing the mice by placing it into the gas anesthesia chamber.
NanoSight particle tracking analysis (NTA) of exosomes isolated from HEK293T cell culture media
The concentration and size of exosomes were detected with the NanoSight NS300 system (Malvern Instruments, Malvern, UK) and configured with scientific CMOS camera with blue 488 nm laser. Exosomes were diluted in 1 mL of PBS. The diluted sample was collected and analysed on the NTA software (Version 3.2).
Cell proliferation assay
A549 cell was incubated with exosomes in plates. After incubation, 5-ethynyl-20-deoxyuridine (EdU) (100 mM) (Cell Light EdU DNA imaging Kit, Guangzhou RiboBio, China) was added and the cells were cultured for 2 h. The cells were then stained according to the following protocol: discard the EdU medium mixture, add 4% paraformaldehyde to fix cells at room temperature for 30 min, wash with glycine (2 mg/mL) for 5 min in a shaker, add 0.2% Trion X-100 for 10 min, wash with PBS for two times, add click reaction buffer (Tris–HCl, pH 8.5, 100 mM; CuSO4, 1 mM; Apollo 567 fluorescent azide, 100 mM; ascorbic acid, 100 mM) for 10–30 min while protecting from light, wash with 0.5% Triton X-100 for three times, stain with Hoechst (5 mg/mL) for 30 min at room temperature, wash with 0.5% Triton X-100 for five times, and, finally, add 150 ml PBS. Images were taken and analyzed using High Content Imaging Pathway 855 (BD, USA). EdU positive cell was calculated with (EdU add-in cells/Hoechst stained cells) × 100%.
All western blot results, histopathological staining and immunohistochemical staining sections shown in this manuscript are representative of at least three independent experiments. Quantitative RT-PCR assays were carried out in triplicate, then, each individual experiment was repeated for statistical accuracies. The data are presented as the mean ± SEM of at least three independent experiments. Differences were considered at significance of p < 0.05 using Student’s t-test.
This work was supported by the National Natural Science Foundation of China (No. 31871295 and 31741075) and the Natural Science Foundation of Jiangsu Province (No. BE2016737).
Availability of data and materials
All data generated or analysed during this study are included in this published article.
Xi Chen formulated the hypothesis, commented on this study and edited the manuscript. Yu Zhou and Yuan Yuan performed the experiments and Minghui Liu, Xiuting Hu offered some assistance. Yu Zhou did data analysis and Yiming Quan offered some assistance. All authors reviewed the manuscript. All authors read and approved the final the final manuscript.
Ethics approval and consent to participate
This study was approved by the Ethics Committee of Nanjing University.
All animal care and handling procedures were performed in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Review Board of Nanjing University.
Consent for publication
The authors declare that they have no competing interests.
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