Targeting GDF15 to enhance immunotherapy efficacy in glioblastoma through tumor microenvironment-responsive CRISPR-Cas9 nanoparticles | Journal of Nanobiotechnology

In vivo CRISPR screens identify GDF15 as a critical driver of immune escape

To systematically identify gene targets whose loss enhances antitumor immunity, we used a murine lentiviral CRISPR-Cas9 knockout (MusCK) library. This library includes 5 sgRNAs for each of the more than 4,900 genes implicated in tumor immune modulation. Once we validated the MusCK library, our subsequent step was to transduce the lentiviral MusCK library into Luc-expressing GL261 cells. Following in vitro passage to enable gene editing, we proceeded to transplant the tumor cells into designated regions of the brains of mice to establish orthotopic GBM models. The mouse types used for the orthotopic GBM models included: C57BL/6 mice, immunodeficient Rag1^−/− mice—lacking both T cells and B cells and C57BL/6J mice subjected to PD-1 blockade treatment with a monoclonal antibody. (Fig. 1A). These treatments were used to generate an adaptive immune response strong enough to exert immune selective pressure on tumor cells. After 14 days, the mice were euthanized, and the tumors were harvested for high-throughput sgRNA library sequencing, after which significantly different levels of tumor growth were observed among the different model mice. T-cell-deficient Rag1^−/− mice had the largest tumors, and immune-competent mice treated with an anti-PD-1 antibody had the smallest tumors (Fig. 1B). While the library representation of primary lentiviral and pretransplanted tumor cells (day 0) followed a log-normal distribution, the library representation of posttransplanted cells obtained from tumor masses from C57BL/6 and Rag1^−/− mice showed a distinct shift (Fig. 1C).

Next, the CRISPR/Cas9 knockout screening readout from the C57BL/6 group was compared to those of the α-PD-1 and Rag1^−/− groups. The analysis revealed that three genes (GDF15, SIK2, and CPLX2) overlapped and were found to be associated with the immune response in glioma (Fig. 1D-E and Supplementary Fig. 1). We sought to further investigate crucial immune-related genes in the complex TME that are involved in antitumor activities as possible targets for immunotherapy in GBM. Next, validation of the three genes was conducted using The Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA), and a Gene Expression Omnibus (GEO) dataset uploaded by Gravendeel, LA. Strikingly, the analysis of the datasets from all three public databases revealed a notable correlation between increased levels of GDF15 and unfavorable prognosis (Fig. 1F). Conversely, no discernible effect on patient prognosis was observed in relation to SIK2 and CPLX2 (Supplementary Fig. 2). Furthermore, we examined the correlations among the three genes and CD8⁺ T cells using the Tumor Immune Estimation Resource (TIMER) database. The results revealed that only GDF15 is negatively correlated with infiltration of CD8⁺ T cells (Supplementary Fig. 3). We primarily examined gliomas classified as World Health Organization (WHO) grades II, III, or IV from the TCGA, CGGA, and GEO datasets. The findings indicated a rise in GDF15 expression in high-grade glioma (Fig. 1G). Our clinical specimens further supported the finding that GDF15 expression was significantly higher in high-grade glioma compared to low-grade glioma (Supplementary Fig. 4). To investigate the potential association between GDF15 and prognosis in glioma patients, we performed immunohistochemistry (IHC) analysis of GDF15 expression on a human tissue microarray comprising 180 glioma tumors. Among the 180 samples, a rise in GDF15 levels was observed among glioma patients who later experienced relapse. The fraction of individuals displaying elevated GDF15 expression was notably decreased within the nonrelapsed patient cohort in contrast to the relapsed cohort upon segregating patients into high and low GDF15 expression categories (Fig. 1H). These data suggest a potential association between GDF15 and relapse in glioma patients. Meanwhile, elevated GDF15 expression was shown to significantly influence the outcomes of both nonrelapsed and relapsed glioma patients (Fig. 1I). These findings indicate a strong correlation between GDF15 overexpression in tumor cells, an immunosuppressive TME and unfavorable patient survival outcomes.

In Vivo CRISPR screens identify GDF15 as a critical driver of immune evasion. (A) Workflow of in vivo CRISPR screens to identify potential therapeutic targets involved in GBM immune evasion. (B) Time course luminescence images of mice bearing orthotopic GL261-Luc tumors following different treatments (n = 8 mice in each group). (C) Cumulative distribution function plots of MusCK library sgRNAs in cells before transplantation, tumors in C57/B6 mice, and tumors in Rag1 mice and C57BL/6 mice subjected to PD-1 blockade treatment. (D) Venn diagram of the two criteria used to identify candidate gene hits. (E) Dynamic distribution of sgRNA read counts of enriched genes. (F) Prognostic value of GDF15 in the TCGA, CGGA, and Grvaendeel databases. (G) The expression level of GDF15 was correlated with the pathological stage of glioma in the TCGA, CGGA, and Gravendeel databases. (H) Representative images of IHC staining of GDF15 in samples from nonrelapsed and relapsed patients. (I) Kaplan‒Meier estimate of survival time for glioma patients with low versus high expression of GDF15

GDF15 ablation promotes M1-like macrophage polarization and T-cell activation

To further investigate the role of GDF15 in GBM, we individually knocked out GDF15 in GL261 cell lines using three sgRNAs obtained from the CRISPR-Cas9 knockout (MusCK) library (Supplementary Fig. 5). Surprisingly, our study revealed that GDF15 did not exhibit an intrinsic role in tumor proliferation and apoptosis in vitro (Supplementary Fig. 6A-C). Furthermore, we inoculated murine glioma cells into immunodeficient mice (Rag1^−/−), and no obvious differences were detected between the sgGDF15 group and the corresponding control (Fig. 2A). However, when GL261 cells were inoculated into the brains of normal syngeneic mice (C57BL/6), the knockout of GDF15 significantly suppressed tumor growth, extended the lifespan of the mice, and reduced GDF15 concentrations in both the circulation and tumor microenvironment back to a physiological level (Fig. 2B and Supplementary Fig. 7). Thus, GDF15 failed to impede tumor cell proliferation in immunodeficient mice but impaired tumor progression in immunocompetent mice, indicating that the anti-tumor effect mediated by GDF15 may rely on the TME. These results prompted further investigation into how GDF15 shapes the TME in vivo.

We further used single-cell mass cytometry (CyTOF) to analyze immune cell infiltration in GDF15-deficient GL261 or control tumors derived from C57BL/6 mice. After being stained with 42 heavy metal-labeled antibodies, immune cells were categorized using a validated, data-driven, unsupervised clustering method (Fig. 2C). By employing the t-distributed stochastic neighbor embedding (tSNE) algorithm, CD45⁺ immune cells were visually represented. The results of these assessments revealed that the CD45⁺ cell population could be divided into 24 unique clusters (Fig. 2D-E). Further investigation was conducted on the variances within these clusters between the sgGDF15 and sgNC groups. The sgGDF15 group exhibited a marked increase in the numbers of CD8⁺ effector T cells (cluster 4) and M1 macrophages (cluster 21). Conversely, the sgGDF15 group presented significant decreases in the numbers of CD8⁺ exhausted T cells (cluster 9) and M2 macrophages (cluster 20) (Fig. 2F-G).

The expression of major immune cell markers in tumor-infiltrating immunocytes (TILs) was analyzed across different groups. The CyTOF results showed a notable increase in the proportion of CD8⁺CD69⁺ T cells after GDF15 knockout (Fig. 2H). CD69 expression is known to be rapidly upregulated upon activation in various leukocytes, making it a commonly used marker for activated T cells and NK cells [17]. Furthermore, the expression of T-cell exhaustion markers, such as PD1 and TIM3, was significantly reduced in the GDF15 knockout group. Moreover, analysis of the expression of markers associated with macrophages revealed that the levels of iNOS, CD86 and MHC II, which are markers of M1 macrophages, substantially increased following GDF15 knockout. In contrast, the expression levels of CD206 and CD163, which are markers of suppressive TAMs, decreased in the sgGDF15 group (Fig. 2H). The tumor tissues were stained for DAPI, PanCK, CD86, CD206, CD8, and GZMB using multiplex immunofluorescence. Our research indicated that tumors in the sgGDF15 group harbored a greater number of CD8⁺ CTLs than sgNC group. Similarly, there was a notable reduction in the proportion of CD206⁺ M2 macrophages within the tumor tissue (Fig. 2I). Moreover, the flow cytometry findings also indicated that, compared with the sgNC group, the sgGDF15 group presented the greatest quantity of CD3⁺CD8⁺GZMB⁺ CTLs. Furthermore, the mice in the sgGDF15 group displayed low CD86^–CD206⁺ TAM (M2-like TAM) infiltration into tumors and high CD86⁺CD206– TAM (M1-like TAM) infiltration within the tumors (Fig. 2J and Supplementary Fig. 8). These findings indicate that disrupting GDF15 has the potential to reshape the immunosuppressive TME by increasing M1-like infiltration and activating CD8⁺ T cells.

GDF15 modulates the immune profile and impairs the antitumor T-cell response. (A) Luminescence images of mice bearing orthotopic GL261-Luc tumors in different groups (n = 8 mice in each group); Proliferation curves of tumors orthotopically transplanted into GL261-bearing Rag1^−/− mice; Kaplan–Meier survival curves of GL261-bearing Rag1^−/− mice. (B) Luminescence images of GL261 tumor-bearing C57BL/6 mice (n = 8 mice in each group); Proliferation curves of orthotopically transplanted tumors in GL261-bearing C57BL/6 mice; Kaplan–Meier survival curves of GL261-bearing C57BL/6 mice. (C) Schematic illustration of the CyTOF analysis of the immune response landscape in different groups. (D) Heatmap displaying the normalized expression of selected markers in each group. (E) t-SNE plots of immune cells in tumor tissues from each group. (F) The cell type corresponding to each cluster and the proportion of each cell type in the sgNC group and sgGDF15 group. (G) Relative abundance of tumor-infiltrating immune cell subpopulations based on CyTOF analysis. (H) tSNE visualization of CD206, iNOS, CD86, TIM3, CD69, PD1, MHC II, and CD163 expression. (I) Multiplex immunofluorescence analysis of PanCK, CD8, GZMB, CD86, and CD206 expression. Scale bar, 50 μm. (J) Flow cytometric quantification of CD206⁺ TAMs, CD86⁺ TAMs, and CD3⁺CD8⁺GZMB⁺ T cells in tumors

Synthesis and characterization of TME-responsive nanoparticles for GDF15 gene editing therapy

Our above research indicated that GDF15 has the potential to alter the tumor immune microenvironment and facilitate GBM progression, identifying it as a key target for GBM immunotherapy. Compared with current immunotherapies such as adoptive immune cells or ICB, directly inhibiting the expression of GDF15 in tumor cells via genome editing offers superior advantages in the restoration of antitumor immunity. This includes enhanced specificity and prolonged therapeutic effects. However, the use of viral vectors to deliver the CRISPR-Cas9 system into organisms for effective editing of tumor sites is impeded by issues of specificity and biosecurity. To overcome this issue, we constructed nanoparticles cross-linked with a disulfide bond loaded with Cas9 and sgRNA targeting GDF15 [NP_SS(Cas9/sgGDF15)]. These nanoparticles were fabricated through an effective in situ polymerization technique employing free radicals. The Cas9/sgRNA complex was encapsulated with positively charged acrylate guanidine through electrostatic interactions, followed by polymerization and cross-linking with N, N’-bis(acryloyl) cystamine and polyethylene glycol (PEG) with acrylate- or succinate-decorated end-groups. Then the resulted nanoparticles were decorated with Angiopep-2 on their surface by an amidation reaction. (Fig. 3A). Angiopep-2 specifically binds to the low-density lipoprotein receptor (LRP-1), which is highly expressed on the surfaces of blood-brain barrier (BBB) endothelial cells and glioblastoma (GBM) tumor cells, thereby facilitating BBB penetration and the active targeting of tumor cells [18, 19]. Dynamic light scattering (DLS) was performed to determine the size of the nanoparticles, and the results revealed that the size and polydispersity index (PDI) of ANP_SS(Cas9/sgGDF15) were 124 nm and 0.12, respectively (Fig. 3B). The zeta potential of ANP_SS(Cas9/sgGDF15) was ≈ + 24.65 ± 3.79 Mv (Fig. 3C). Moreover, we employed transmission electron microscopy (TEM) to view the morphology of ANP_SS(Cas9/sgGDF15), validating their spherical structure. Notably, the nanoparticles exhibited rapid degradation and released Cas9/sgGDF15 in an intracellular reducing environment simulating high GSH levels. Interestingly, this phenomenon was not seen in the nonreducible control environment (Fig. 3D). Next, we investigated the potential of nanoparticle-mediated Cas9/sgGDF15 delivery for gene editing in vitro. We introduced the nanoparticles into GL261 cells, and the Cas9/sgRNA complex was used to identify target DNA sequences. Upon identifying a match, the Cas9 protein cleaved the DNA at the precise location, resulting in a double-stranded break (DSB) within the DNA. Subsequently, the cell activated its repair mechanism, primarily through nonhomologous end joining (NHEJ) (Fig. 3E). To evaluate the efficacy of our CRISPR/Cas9 nanoparticles in GDF15 gene editing and their ability to protect sgRNA, we performed T7 endonuclease I (T7E1) cleavage assays. Notably, the presence of RNase did not significantly affect the efficiency of ANP_SS(Cas9/sgGDF15)-mediated gene editing, which displayed an efficacy similar to that of free Cas9/sgGDF15 in an environment devoid of RNase. However, the introduction of RNase hindered the ability of free Cas9/sgGDF15 to cleave DNA (Fig. 3F). The efficiency and specificity of GDF15 gene disruption by ANP_SS(Cas9/sgGDF15) in GL261 cells were further evaluated through sanger sequencing. The results revealed that the editing site of the CRISPR/Cas9 system was located 3–5 bases ahead of the adjacent motif (PAM) sequence of the protospacer (Fig. 3G). Consistently, the ELISA results revealed that GDF15 protein secretion was reduced to 23.1% in the supernatant when treated with ANP_SS(Cas9/sgGDF15) nanoparticles, while ANP_SS(Cas9/sgNC) and saline did not cause a significant alteration in GDF15 secretion (Supplementary Fig. 9). These findings collectively show that ANP_SS(Cas9/sgGDF15) nanoparticles are capable of achieving precise GDF15 gene editing within GL261 cells. The disulfide cross-linking within the nanoparticles is essential for the specific release of Cas9/sgRNA, thus enhancing the safety of gene editing.

The escape of the nanoparticle content from endosomal confinement is necessary for its functionality, and we examined the endosomal escape capability of ANP_SS(Cas9/sgGDF15). ANP_SS(Cas9/sgGDF15) strongly colocalized with endosomes after 3 h of incubation. Moreover, ANP_SS(Cas9/sgGDF15) also highly colocalized with lysosomes after 3 h of incubation, suggesting their trafficking and accumulation to the lysosome. Interestingly, after 6 h of incubation, most of the ANP_SS(Cas9/sgGDF15) and endosomes/lysosomes did not overlap, suggesting that ANP_SS(Cas9/sgGDF15) escaped from endosomes/lysosomes over time (Supplementary Fig. 10A-B). Moreover, after long-term storage at room temperature and in medium containing 10% FBS, the ANP_SS(Cas9/sgGDF15) remained relatively constant in size, indicating satisfactory stability (Supplementary Fig. 11).

To assess the nanoparticles’ ability to target GBM cells, we labeled the Cas9 protein with FITC and the sgRNA with sulfo-cyanine5.5 (Cy5.5) before encapsulating them in the nanoparticles. Subsequently, we developed an in vitro BBB model by culturing a monolayer of bEnd.3 cells. Confocal microscopy imaging demonstrated that ANP_SS(Cas9/sgGDF15) possessed the highest targeting efficiency compared with NP_SS(Cas9/sgGDF15) (Fig. 3H). We then investigate the biodistribution of the nanoparticles in vivo, fluorescence images were captured at various time points using an IVIS Spectrum system following the intravenous administration of ANP_SS(Cas9/sgGDF15) or NP_SS(Cas9/sgGDF15). Compared with that of NP_SS(Cas9/sgGDF15), the fluorescence intensity of ANP_SS(Cas9/sgGDF15) was greater in the brain (Fig. 3I, upper panel). Given that LDL receptor-related protein 1 (LRP1) is overexpressed by both endothelial cells of the BBB and GL261 glioma cells, LRP-1–targeting angiopep-2–functionalized ANP_SS(Cas9/sgGDF15) are expected to increase BBB permeability via receptor-mediated transcytosis. The fluorescent signal of Cy5.5 emitted by the ANP_SS(Cas9/sgGDF15) group appeared brighter than that of the NP_SS(Cas9/sgGDF15) group during ex vivo imaging of mouse brains (Fig. 3I, lower panel). More importantly, the predominant localization of ANP_SS(Cas9/sgGDF15) occurred within the confines of the tumor border, indicating its exceptional ability to target tumor cells (Fig. 3J). Considering that the microenvironment of a tumor produced by a cancer cell line varies from that of a tumor that arises naturally, we also constructed a spontaneous GBM model by using RCAS viruses carrying oncogenes to specifically infect tv-a-expressing cells on N/tv-a; Ink4a/Arf^−/− mice [20]. Similar results were observed in the spontaneous GBM mouse model, indicating that ANP_SS(Cas9/sgGDF15) exhibited a higher targeting ability towards GBM compared to NP_SS(Cas9/sgGDF15) in vivo (Supplementary Fig. 12A-C). These results show that ANP_SS (Cas9/sgRNA) have excellent BBB penetration ability and successfully accumulate in tumors in a GL261-bearing mouse model and a spontaneous GBM mouse model.

Design and construction of ANP_SS(Cas9/sgGDF15). (A) Disulfide cross-linked nanoparticles containing Cas9/sgRNA were synthesized through in situ free-radical polymerization and functionalized with the Ang glioma-targeting peptide. (B) DLS image showing the particle size of ANP_SS(Cas9/sgGDF15). (C) Zeta potential of ANP_SS(Cas9/sgGDF15). (D) TEM images were taken to compare the spherical shape of the ANP_SS(Cas9/sgGDF15) in saline with or without GSH. (E) Schematic representation of genome editing by ANP_SS(Cas9/sgGDF15). (F) Agarose gel electrophoresis analysis was performed to observe insertions and deletions (indels) in the GDF15 gene following treatment with ANP_SS(Cas9/sgGDF15) or other specified treatments, with or without RNase treatment at a concentration of 2 mg/ml for 20 min. (G) GDF15 gene editing in GL261 cells treated with ANP_SS(Cas9/sgGDF15) was confirmed through DNA sequencing. (H) Immunofluorescence images showing NP_SS(Cas9/sgGDF15) and ANP_SS(Cas9/sgGDF15) uptake into GL261 cells. Scale bar, 10 μm (I) Fluorescence images of mice with orthotopic GL261 tumors were captured following the injection of NP_SS(Cas9/sgGDF15) or ANP_SS(Cas9/sgGDF15). (J) Confocal microscopy revealed the tumor penetration of NP_SS(Cas9/sgGDF15) and ANP_SS(Cas9/sgGDF15). Nuclei were counterstained with DAPI (blue), and Cy5.5-Cas9 fluorescence revealed a violet color. Dotted lines were used to outline the tumor boundary, with brain tissue labeled N and the tumor labeled T. Scale bar, 100 μm

Assessment of the effect of ANPSS(Cas9/sgGDF15) in the orthotopic GBM model

To evaluate the therapeutic potential of ANP_SS(Cas9/sgGDF15), the orthotopic GBM mouse models were established. The mice were randomly assigned to different treatment groups and received intravenous injections of saline, ANP_SS(Cas9/sgNC), or ANP_SS(Cas9/sgGDF15) every 7 days (Fig. 4A). Remarkably, mice treated with ANP_SS(Cas9/sgGDF15) exhibited a substantial decrease in tumor growth, as indicated by a noticeable reduction in bioluminescence signal intensity (Fig. 4B-C). Conversely, mice treated with saline or ANP_SS(Cas9/sgNC) presented an increase in bioluminescence signal intensity, indicating a lack of efficacy in suppressing tumor growth (Fig. 4D). Survival curve analysis demonstrated a significant improvement in median survival time, exceeding 41 days with ANP_SS(Cas9/sgGDF15) treatment compared to 31 and 32.5 days with saline and ANP_SS(Cas9/sgNC), respectively (Fig. 4E).

To confirm that the inhibition of tumor growth was attributed to the disruption of the GDF15 gene and the subsequent decrease in GDF15 protein expression, excised tumor tissues from mice treated with ANP_SS(Cas9/sgGDF15), ANP_SS(Cas9/sgNC), or saline were analyzed on day 28. The evaluation of gene editing efficiency, as indicated by the indel frequency, showed a notable 67.3% efficiency for ANP_SS(Cas9/sgGDF15) treatment (Fig. 4F). Furthermore, a significant reduction in GDF15 protein expression was noted in the group treated with ANP_SS(Cas9/sgGDF15) in comparison to the control group (Supplementary Fig. 13). Moreover, disruption of the GDF15 gene was verified through next-generation sequencing (NGS), which revealed a mutation rate of 59.4% (Fig. 4G). Immunohistochemical analysis showed a significant decrease in the presence of GDF15-positive tumor cells, and Ki67 and cleaved caspase-3 IHC staining confirmed the potent tumor inhibitory effect of ANP_SS(Cas9/sgGDF15) (Fig. 4H). Flow cytometry analysis demonstrated that the number of cytotoxic T lymphocytes (CD3⁺CD8⁺GZMB⁺) was significantly greater in the ANP_SS(Cas9/sgGDF15) group than in the other treatment groups. Additionally, ANP_SS(Cas9/sgGDF15) treatment resulted in the lowest infiltration of M2-like TAMs (CD86^–CD206⁺), with a greater number of M1-like TAMs (CD86⁺CD206^–) within the tumors (Fig. 4I-K). Multiplex immunofluorescence analysis revealed a substantial increase in CD3⁺CD8⁺GZMB⁺ T cells and M1-like TAMs after ANP_SS(Cas9/sgGDF15) treatment (Fig. 4L). These results indicate that targeting GDF15 with ANP_SS(Cas9/sgGDF15) in orthotopic GBM xenografts can remodel the TME and effectively suppress GBM progression.

Gene editing therapy of ANP_SS(Cas9/sgGDF15) in the GL261 orthotopic GBM mouse model. (A) Diagram illustrating the timeline of the study conducted using the GL261 orthotopic tumor model. The intravenous injection of normal saline, ANP_SS (Cas9/sgNC), or ANP_SS(Cas9/sgGDF15) (a 1.5 mg dose of Cas9 equivalent per kilogram) was performed on days 7, 14, 21, and 28 after tumor implantation. (B) Quantification of tumor volume in mice after the indicated treatments. (C) Images displaying luminescence in orthotopic GL261-bearing C57BL/6 mice following the indicated treatments. (D) Individual tumor growth curves of tumor-bearing mice subjected to various treatments. (E) Mouse survival after the indicated treatments was evaluated in another three groups of mice (n = 8). (F) Frequencies of indel mutations in the GDF15 gene observed in tumor tissues from mice subjected to various treatments. (G) The results of DNA sequencing showing GDF15 gene editing in GBM tumors excised from mice treated with ANP_SS(Cas9/sgGDF15). (H) Immunohistochemical analysis of GDF15, Caspase-3 and Ki67 expression in tumor tissues excised from mice subjected to the indicated treatments. Scale bar, 50 μm I-K. Flow cytometric quantification of CD206⁺ TAMs, CD86⁺ TAMs, and CD3⁺CD8⁺GZMB⁺ T cells in tumors. L. Representative multiplex immunofluorescence staining of tumor tissues from mice treated with the indicated drugs on day 28 after tumor implantation. The stained markers included DAPI (blue), PanCK (pink), CD8 (red), GZMB (yellow), CD86 (orange), and CD206 (green). Scale bar, 50 μm

In vivo antitumor activity of ANPSS(Cas9/sgGDF15) in the spontaneous GBM model

The above results indicate that ANP_SS(Cas9/sgGDF15) can exert significant antitumor effects in the orthotopic GBM models. To further confirm the antitumor effects of ANP_SS(Cas9/sgGDF15), we also conducted therapeutic evaluation experiments in the spontaneous GBM mouse model. We first analyzed GDF15 protein levels in glioma tissues and observed significantly higher GDF15 expression in the brains of spontaneous glioma model than in those of normal controls (Supplementary Fig. 14). The results were consistent with those observed in GL261-bearing mice, where ANP_SS(Cas9/sgGDF15) treatment successfully inhibited tumor growth, as evidenced by the reduction in tumor volume in treated mice (Fig. 5A-D). Additionally, mice treated with ANP_SS(Cas9/sgGDF15) showed a significant improvement in median survival (41 days), surpassing the median survival time (29 days) of mice treated with saline (Fig. 5E). T7E1 assays revealed a significant indel frequency of 61.3% in mice treated with ANP_SS(Cas9/sgGDF15) (Fig. 5F). Subsequent NGS analysis confirmed efficient editing of the GDF15 gene, with an indel frequency of 56.8%, which was consistent with the T7E1 assay findings (Fig. 5G). Additionally, a reduction in GDF15 protein expression was noted in spontaneous glioma tissues, indicating a possible disruption of the GDF15 gene (Supplementary Fig. 15). Immunohistochemical analysis of cleaved caspase-3 and Ki67 signals in tumor tissues revealed a gradual increase in apoptosis in mice treated with ANP_SS(Cas9/sgGDF15), along with a subsequent decrease in cell proliferation (Fig. 5H). Furthermore, ANP_SS(Cas9/sgGDF15) therapy led to a decrease in the presence of CD86^–CD206⁺ M2-like TAMs, while boosting the population of CD86⁺CD206– M1-like TAMs in the tumor microenvironment (Fig. 5I). The tumors of the mice treated with ANP_SS(Cas9/sgGDF15) presented a significant increase in T-cell infiltration. Subsequent analysis revealed elevated levels of GZMB in CD8⁺ T cells, suggesting increased cell lysis ability in the ANP_SS(Cas9/sgGDF15)-treated mice (Fig. 5J-K). Multiplex immunofluorescence examination also demonstrated a notable rise in CD3⁺CD8⁺GZMB⁺ T cells and M1-like TAMs following ANP_SS(Cas9/sgGDF15) administration (Fig. 5L). These findings validated the immunostimulatory effects of ANP_SS(Cas9/sgGDF15) in mice with tumors, resulting in enhanced therapeutic outcomes in a spontaneous GBM model.

Gene editing therapy of ANP_SS(Cas9/sgGDF15) in the spontaneous GBM mouse model. A. Schematic of spontaneous GBM model establishment. The intravenous injection of normal saline, ANP_SS (Cas9/sgNC), or ANP_SS(Cas9/sgGDF15) (a 1.5 mg dose of Cas9 equivalent per kilogram) was performed on days 7, 14, 21, and 28 after tumor implantation. B. H&E staining images of whole brains excised from mice treated as described above on day 20 and the tumor volume of each mouse. C-D. Individual tumor growth curves of tumor-bearing mice subjected to various treatments. E. Survival rates of the mice in the different groups (n = 5). F. Frequencies of indel mutations in the GDF15 gene observed in tumor tissues from mice subjected to the indicated treatments. G. Sequencing results of GDF15 gene editing in the spontaneous GBM model treated with ANP_SS(Cas9/sgGDF15) are presented. H. Immunohistochemistry analysis was conducted to assess the expression of GDF15, Caspase-3, and Ki67 in tumor tissues. Scale bar, 50 μm. I-K. Flow cytometric quantification of CD206⁺ TAM, CD86⁺ TAM, and CD3⁺CD8⁺GZMB⁺ T cells in tumors. L. Multiplex immunofluorescence analysis of PanCK, CD8, GZMB, CD86, and CD206 expression

ANPSS(Cas9/sgGDF15) potentiates the efficacy of PD-1 blockade therapy

Immunotherapy with antibodies that target like PD-1 and CLTA-4 has shown different levels of effectiveness in the treatment of different types of cancers, such as hepatocellular carcinoma, melanoma and non-small cell lung cancer [21]. Nevertheless, research has suggested that the use of an α-PD-1 antibody does not result in improved overall survival rates in patients with GBM [22]. Crucially, the functional screening readouts in this study revealed that the sgRNAs targeting GDF15 were significantly depleted in the α-PD-1 group compared with the C57BL/6 group (Fig. 1E). This finding suggests that the loss of GDF15 may increase sensitivity to α-PD-1 therapy in GBM. To further investigate the efficacy of our constructed nanoparticles, GL261-bearing mice were randomly allocated to different groups and were subsequently administered intravenous tail vein injections of various substances, including saline, ANP_SS(Cas9/sgGDF15), α-PD-1, and a combination of ANP_SS(Cas9/sgGDF15) and α-PD-1 every 7 days. Notably, combination therapy demonstrated superior efficacy compared with the other treatments as evidenced by the intensity of the bioluminescence signal (Fig. 6A). In contrast, the mice that received saline presented increased bioluminescence intensity, indicating a faster rate of tumor growth. After the treatment period ended, the results revealed a notable reduction in tumor volume across all three intervention groups compared with the saline-treated group, among them, the combination therapy-treated group exhibited the smallest tumor volume (Fig. 6B). Survival curve analysis demonstrated that combination therapy significantly prolonged the median survival time to over 46 days and resulted in complete tumor eradication in 25% (2/8) of the mice. In contrast, the mice treated with saline exhibited a substantially shorter median survival time of 32 days (Fig. 6C). Moreover, NGS revealed that the gene editing efficiencies of ANP_SS(Cas9/sgGDF15) and the combination therapy were 55.9% and 57.4%, respectively (Fig. 6D). Furthermore, the examination of Ki67 and cleaved caspase-3 IHC staining evidenced the remarkable tumor inhibitory efficacy of ANP_SS(Cas9/sgGDF15) + α-PD-1 therapy (Fig. 6E). The synergistic effect of combining α-PD-1 with ANP_SS(Cas9/sgGDF15) led to an increased presence of M1-like TAMs and GZMB⁺CD8⁺ T cells in the TME (Fig. 6F-I). These findings suggest that inhibiting GDF15 genetically along with α-PD-1 therapy significantly hinders tumor growth, suggesting a promising therapeutic approach for immunologically ‘cold’ GBM.

Synergistic efficacy of ANP_SS(Cas9/sgGDF15) combined with an α-PD-1 antibody. (A) Fluorescence images of orthotopic GL261-bearing C57BL/6 mice following treatment with saline, ANP_SS(Cas9/sgGDF15), an α-PD-1 antibody, or ANP_SS(Cas9/sgGDF15) combined with the α-PD-1 antibody (n = 8). Tumor volumes of the different groups of mice at 20 days after implantation of GL261-Luc. (B) Individual GL261-Luc tumor growth curves of the mice after different treatments. (C) Kaplan–Meier survival curves of the mice that received different treatments. (D) The results of DNA sequencing revealed GDF15 gene editing in orthotopic GL261 tumors excised from mice treated with ANP_SS(Cas9/sgGDF15) in combination with α-PD-1 treatment. (E) Immunohistochemistry analysis was conducted to assess the expression of GDF15, Caspase-3, and Ki67 in tumor tissues. Scale bar,50 μm. F-H. Flow cytometric quantification of CD206⁺ TAMs, CD86⁺ TAMs, and CD3⁺CD8⁺GZMB⁺ T cells in tumors. I. Multiplex immunofluorescence analysis of PanCK, CD8, GZMB, CD86, and CD206 expression. Scale bar, 50 μm

ANPSS(Cas9/sgGDF15) exhibits a favorable safety profile

Owing to possible safety issues related to off-target effects, toxicity, and immunogenicity, a thorough evaluation is necessary for genome editing employing CRISPR/Cas9 technology. Initially, we examined the impact of ANP_SS(Cas9/sgGDF15) on the proliferation of diverse healthy cells, which encompass hepatic stellate cells LX-2, hepatocytes AML12, cardiomyocytes AC16, cardiac muscle cells HL-1, lung epithelial cells MLE12, and renal tubular cells TCMK-1. Notably, no significant alterations in the growth patterns of any of the cellular populations were detected (Supplementary Fig. 16). Afterward, a comprehensive examination of off-target effects was conducted by pinpointing the locations in the tumor tissue with the greatest potential for off-target changes in the genomic sequence, with a specific emphasis on GDF15. Following the administration of ANP_SS(Cas9/sgGDF15) to mice with GL261 tumors, NGS analysis indicated minimal gene disruption at the suspected locations within the tumor tissue. The mutation frequency was found to be less than 0.5% across all 5 hypothesized target sites in these models. Given the tendency of nanoparticles to accumulate in the brain, heart, liver, and kidneys, we further examined these organs to assess potential off-target effects. Interestingly, the mutation frequencies at the 5 potential off-target sites were also below 0.5% in the brain, heart, liver, and kidneys of mice bearing GL261 tumors (Supplementary Fig. 17A). Next, ANP_SS(Cas9/sgGDF15) was administered intravenously to healthy C57BL/6 mice on alternate days, a total of 4 times, in order to evaluate both the immune response and toxicity. Throughout the treatment period, biochemical profiles of the mice treated with ANP_SS(Cas9/sgGDF15) were virtually identical to those observed in the saline-treated group, suggesting that ANP_SS(Cas9/sgGDF15) exerted minimal to no negative effects on kidney and liver functions (Supplementary Fig. 17B-C). Moreover, cachexia caused by cancer is a common concern that associated with reduced quality of life and shortened lifespan. Elevated levels of GDF15 in the bloodstream is also related to cachexia and decreased survival rates in cancer patients [23]. We found that treatment with ANP_SS(Cas9/sgGDF15) prevents tumor-driven weight loss (Supplementary Fig. 18). These results suggest that the systemic delivery of ANP_SS(Cas9/sgGDF15) at therapeutic doses is safe and does not trigger an immune response. Nonetheless, a thorough evaluation of possible toxic effects is required for advancing to preclinical stages.