Immunoliposome-based targeted delivery of the CRISPR/Cas9gRNA-IL30 complex inhibits prostate cancer and prolongs survival

Fieni, Cristiano; Sorrentino, Carlo; Ciummo, Stefania Livia; Fontana, Antonella; Lotti, Lavinia Vittoria; Scialis, Sofia; Calvo Garcia, Darien; Caulo, Massimo; Di Carlo, Emma

doi:10.1038/s12276-024-01310-2

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Published: 04 September 2024

Immunoliposome-based targeted delivery of the CRISPR/Cas9gRNA-IL30 complex inhibits prostate cancer and prolongs survival

Cristiano Fieni^1,2,
Carlo Sorrentino ORCID: orcid.org/0000-0002-6553-7429^1,2,
Stefania Livia Ciummo^1,2,
Antonella Fontana^3,4,
Lavinia Vittoria Lotti⁵,
Sofia Scialis²,
Darien Calvo Garcia^6,7,
Massimo Caulo^6,7 &
…
Emma Di Carlo ORCID: orcid.org/0000-0001-7778-1042^1,2

Experimental & Molecular Medicine volume 56, pages 2033–2051 (2024)Cite this article

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Abstract

The development of selective and nontoxic immunotherapy targeting prostate cancer (PC) is challenging. Interleukin (IL)30 plays immunoinhibitory and oncogenic roles in PC, and its tumor-specific suppression may have significant clinical implications. CRISPR/Cas9-mediated IL30 gene deletion in PC xenografts using anti-PSCA antibody-driven lipid nanocomplexes (Cas9gRNA-hIL30-PSCA NxPs) revealed significant genome editing efficiency and circulation stability without off-target effects or organ toxicity. Biweekly intravenous administration of Cas9gRNA-hIL30-PSCA NxPs to PC-bearing mice inhibited tumor growth and metastasis and improved survival. Mechanistically, Cas9gRNA-hIL30-PSCA NxPs suppressed ANGPTL 1/2/4, IL1β, CCL2, CXCL1/6, SERPINE1-F1, EFNB2, PLG, PF4, VEGFA, VEGFD, ANG, TGFβ1, EGF and HGF expression in human PC cells while upregulated CDH1, DKK3 and PTEN expression, leading to low proliferation and extensive ischemic necrosis. In the syngeneic PC model, IL30-targeting immunoliposomes downregulated NFKB1 expression and prevented intratumoral influx of CD11b⁺Gr-1⁺MDCs, Foxp3⁺Tregs, and NKp46⁺RORγt⁺ILC3, and prolonged host survival by inhibiting tumor progression. This study serves as a proof of principle that immunoliposome-based targeted delivery of Cas9gRNA-IL30 represent a potentially safe and effective strategy for PC treatment.

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Introduction

Prostate cancer (PC) is a leading cause of cancer-related death in men worldwide. Although its incidence has remained stable over the past decade, a 4 to 6% annual increase in the incidence of advanced disease has been observed, and the proportion of patients diagnosed with PC in advanced stages has subsequently increased from 3.9 to 8.2%¹. Furthermore, given that PC mainly affects patients older than 50 years of age, an increase in new cases is expected in the near future due to population aging. However, great strides in therapy have not been made.

The development of personalized treatments that are well-tolerated by elderly patients with comorbidities is a major challenge and would have a high impact on the health care system.

Recent advances in nanobiotechnology could fulfill these needs by providing safe and flexible nanostructures suitable for precision medicine².

Nanoliposomes, or submicron bilayer lipid vesicles, are a state-of-the-art tool suitable for the targeted delivery of bioactive agents that can overcome the toxicity of traditional drugs³. Due to their biodegradability, high entrapment efficiency and possibility for surface functionalization, nanoliposomes are very promising smart drug delivery vehicles⁴ and are currently being investigated for the treatment of aggressive and highly metastatic tumors⁵.

We recently discovered the tumor-promoting potential of an immunoregulatory molecule, the p28 subunit of interleukin (IL)27, also known as IL30, which is expressed by activated macrophages and myeloid-derived suppressor cells^6,7,8,9, but is also found as a membrane-anchored cytokine in the cytoplasmic membrane of cancer cells^10,11,12.

IL30 expression has been detected in approximately 77% of metastatic PC patients, localized in cancer and/or infiltrating myeloid cells, and is correlated with disease grade and stage⁷. Targeted deletion of the IL30 gene in PC cells using CRISPR/Cas9 genome editing inhibits a cascade of autocrine and paracrine signals, which foster cancer proliferation, migration, and immune evasion, resulting in tumor suppression and prolonged host survival¹². CRISPR/Cas9 is the most developed and widely used tool for current genome editing strategies¹³, because it is highly specific, efficient, and versatile¹⁴, and has entered clinical trials for the treatment of different human diseases, such as hematological disorders, cancer, inherited eye disease, diabetes, infectious and inflammatory diseases, and protein-folding pathologies (https://innovativegenomics.org/news/crispr-clinical-trials-2022/). However, many challenges remain to be addressed to increase its efficacy, among which the delivery system plays a key role.

In this study, using a microfluidic device, we designed and synthesized a nonimmunogenic biocompatible cationic lipid nanocomplex coated with PEG (NxP) and conjugated with anti-PSCA antibodies (Abs) for the selective delivery of the Cas9 guide (g) RNA-IL30 complex to human (h)-derived PSCA⁺IL30⁺ PC xenografts and in a syngeneic, fully immunocompetent, PSCA⁺IL30⁺ PC model. We demonstrated the efficient uptake of Ab-conjugated Cas9gRNA-IL30 NxPs in PC cells both in vitro and in vivo, after systemic administration. The NxPs led to high-efficiency editing, tumor-selective suppression of IL30 signaling pathways and remodeling of the intratumoral immune cell context, resulting in substantial tumor growth inhibition and improved survival without evident toxicity. Our investigation provides a proof of concept that immunoliposome delivery of the Cas9gRNA-hIL30 complex for IL30 genome editing at the tumor site is a clinically valuable tool for the safe and effective immunotherapy of PC.

Materials and methods

Cell lines

Human PC cell lines derived from the metastases of high-grade PCs, DU145^12,15 and PC3¹⁶ cells, and the murine PC cell line TRAMP-C1 cells isolated from a 32-week-old C57BL/6J male TRAMP mouse¹⁷ were purchased from the ATCC (Manassas, VA, USA) and were authenticated using STR profile analysis. The generation of the Il30 expression, or empty, lentiviral vector and its transfection into TRAMP-C1 cells to obtain IL30-TRAMP-C1 and EV-TRAMP-C1 cells were performed as described previously¹².

The cell lines were cultured in RPMI-1640 with 10% FCS (Seromed, Biochrom KG, Berlin, Germany), confirmed to be mycoplasma-free by PCR analysis, and passaged for fewer than 6 months after initial thawing.

CRISPR/Cas9-mediated IL30 gene knockout in PC cells

CRISPR/Cas9 technology was used to generate IL30 gene knockout (IL30KO) in both DU145 and PC3 cells, and abrogation of IL30 expression was validated by western blotting, as previously described¹².

Flow cytometry and antibody conjugation efficiency

PSCA expression on DU145, PC3, wild-type TRAMP-C1 and IL30-TRAMP-C1 cells and the conjugation efficiency between the anti-PSCA Abs and the aldehyde-modified DSPE-PEG2000 lipid present on the external layer of the NxP were assessed using flow cytometry, as described in the Supplementary Methods.

Synthesis of nanoliposomes and Cas9IL30 NxPs and functionalization with anti-PSCA Abs

Cationic lipid nanocomplexes coated with PEG (NxP) were synthesized using a microfluidic device from Dolomite Microfluidics (Royston, UK) and lipids, as described in detail in the Supplementary Methods. All components were approved, for clinical use, by the European Medicines Agency (EMA) and Food and Drug Administration (FDA).

Physical characterization of nanoliposomes

The size and zeta potential of the nanoliposomes were analyzed using dynamic laser light scattering (DLS). Their size and shape were also investigated using transmission electron microscopy (TEM), as described in the Supplementary Methods. Details on the assessment of their serum and pH stability are reported in the Supplementary Methods.

Encapsulation efficiency and release rate of the Cas9IL30 NxPs

The encapsulation efficiency of Cas9gRNA-IL30 into nanoliposomes and the release rate of the Cas9IL30 NxPs were determined as reported in the Supplementary Methods.

Editing quantification and analysis of off-target cleavage in vitro and in vivo

The true guide synthetic (s)gRNAs used for IL30 gene editing were designed and synthesized by Thermo Fisher Scientific (Waltham, MA, USA) using a validated proprietary algorithm that predicts high-efficiency editing (Supplementary Figs. 1, 2 and Supplementary Table 1). Editing quantification and evaluation of off-target cleavage events for the selected sgRNAs (identified using COSMID software, https://crispr.bme.gatech.edu, and the CRISPR-Cas9 guide RNA design checker (https://eu.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE) (Supplementary Table 2) were performed as described in the Supplementary Methods. Whole-genome next-generation sequencing (NGS) was performed by Lexogen GmbH (Wien, Austria) using the Illumina NovaSeq 6000 platform system and Dragen software.

Efficiency of genome editing in Cas9IL30-PSCA NxP-treated human and murine PC cells by tracking indels using decomposition analysis or inference of CRISPR edits

The efficiency of genome editing was quantified using the Tracking of Indels by Decomposition (TIDE) method in DU15 and PC3 cells or the Inference of CRISPR Edits (ICE) method in TRAMP-C1 cells, as previously reported^18,19 and briefly described in the Supplementary Methods.

Ultrastructural analyses of PC cells treated with Cas9IL30-PSCA NxPs

The intracellular uptake of NxPs by PC cells in vitro was analyzed using TEM, as reported in the Supplementary Methods.

Cytotoxicity and proliferation assay

PC cell viability and proliferation after treatment with empty NxPs or Cas9IL30-loaded NxPs were assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (#G3582; Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Biosafety and distribution of nanoliposomes in vivo

Assessment of markers of organ and hematological toxicity, analyses of the immunogenicity of NxPs and analyses of weight changes due to the treatment were performed as described in the Supplementary Methods. To assess the immunogenicity of NxPs and any weight changes due to treatment, four groups of five fully immunocompetent BALB/c male mice were intravenously (i.v.) injected with PBS, the naked Cas9hIL30 complex, empty-PSCA NxPs or Cas9hIL30-PSCA NxPs. Blood samples were collected at 6 and 24 hrs post injection to measure IL6 and TNFα levels, as described in the Supplementary Methods. Mice weight was constantly monitored for 63 days, consistent with the longest treatment schedule duration (12 administrations, 6 weeks), to assess the systemic toxicity of NxPs.
Histopathological and ultrastructural analyses of the intracellular uptake and toxicity of NxPs in vivo were performed as described in the Supplementary Methods.
Laser scanning confocal (LSC) microscopy analyses of the lung transit and tumor uptake of the NxPs were performed as described in the Supplementary Methods.

Pharmacokinetic studies

For pharmacokinetic studies, 3 groups of 5 PC3 and 3 groups of 5 DU145 tumor-bearing NSG mice and 3 groups of 5 TRAMP-C1 tumor-bearing C57BL/6J mice were i.v. treated when the tumors reached a mean volume of ~700 mm³, with Cas9hIL30 NxPs or Cas9mIL30 NxPs conjugated or not conjugated with anti-PSCA Abs or free Cas9 (in PBS) at a dose of 1.5 mg of Cas9 equivalent/kg. Subsequently, blood was collected at predetermined time intervals (5 min, 15 min, 30 min, 1 hr, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs, and 12 hrs) for ELISA (Cas9 ELISA Kit, # PRB-5079, Cell Biolabs, San Diego, CA, USA).

Magnetic resonance imaging (MRI)

The biodistribution of NxPs and their efficiency in targeting the tumor site were determined by using a 3T scanner (Philips Medical Systems, Nederland), as described in the Supplementary Methods.

Western blot quantification of the tissue distribution of Cas9 protein

Analysis of Cas9 protein in tumors and organs from NSG mice treated with anti-hPSCA Ab-conjugated or unconjugated NxPs was performed as reported in the Supplementary Methods.

In vivo testing of Cas9gRNA-IL30 NxP treatment in PC models

Eight-week-old NSG male mice were purchased from Charles River (Wilmington, MA, USA) and housed under high barrier conditions according to the Jackson Laboratory’s guidelines (https://www.jax.org/jax-mice-and-services/find-and-order-jax-mice/nsg-portfolio/housing-and breeding-considerations-for-nsg-mice) in the animal facility of the Center for Advanced Studies and Technology at the “G. d’Annunzio” University of Chieti-Pescara, Italy.

To study the effects of IL30 target treatment on tumor growth and progression, three groups of twenty-seven 8-week-old, NSG male mice were subcutaneously (s.c.) injected with 5 × 10⁵ wild-type (CTRL) DU145 cells or with 1 × 10⁶ PC3 cells. To study the effects of IL30-targeted treatment on the tumor immune microenvironment and tumor behavior, three groups of twenty-seven 8-week-old C57BL/6J male mice were s.c. injected with 5 × 10⁵ mIL30 lentiviral-DNA-transfected TRAMP-C1 cells (IL30-TRAMP-C1).

Mice were treated with Cas9IL30-PSCA NxPs, empty-PSCA NxPs, or PBS as soon as the tumors became palpable and twice a week thereafter, as shown in Supplementary Fig. 3. Two additional groups of twenty-seven 8-week-old NSG male mice were s.c. injected with nontargeting guide RNA-transfected (NTgRNA) or IL30 knockout (IL30KO) DU145 or PC3 cells, whereas an additional group of twenty-seven 8-week-old C57BL/6J male mice was s.c. injected with wild-type TRAMP-C1 cells. These control groups were monitored for tumor growth but were left untreated. Tumors were measured with calipers as soon as they were palpable (2 mm in diameter). Based on the tumor growth and progression rates, 12 mice from each group were euthanized at (4) key time points (3 mice per point) for histopathological and ultrastructural analyses. The remaining 15 mice per group were kept alive until tumors reached 2 cm³ or evidence of suffering was observed. Autopsy and histopathological examinations of the different organs (heart, liver, lungs, kidney, spleen and prostate) were performed.

An overall sample size of 15 mice per group allowed the detection of a statistically significant difference in tumor growth among the five groups (ANOVA) with 80% power at a 0.05 significance level (G*Power, RRID:SCR_013726). The animal procedures were performed in accordance with the European Community and ARRIVE guidelines and were approved by the Institutional Animal Care Committee of “G. d’Annunzio” University and by the Italian Ministry of Health (Authorization n. 892/2018-PR).

Histology, immunohistochemistry, and TUNEL assays

Histology, immunohistochemistry (performed with the Abs listed in Supplementary Table 3), assessment of apoptotic (TUNEL assay) and proliferation (Ki-67 immunostaining) indices, and evaluation of the microvessel density (MVD) in tumors were performed as described in the Supplementary Methods.

Statistics

For in vitro and in vivo studies, between-group differences were assessed using the Student’s t test or ANOVA, followed by Tukey’s HSD test. Survival curves were constructed using the Kaplan‒Meier method, and survival differences were analyzed using the log-rank test. All the statistical tests were evaluated at an α level of 0.05 using Stata, version 13 (StataCorp, College Station, TX, USA; RRID:SCR_012763). For MRI data assessment, the mean signal intensities of the tumor, prostate, lungs, liver, kidneys, spleen, heart, bones, and brain on both the T1- and T2-weighted images were calculated at scheduled time points for each group. The results are expressed as the mean ± standard deviation, and Prism 6 (GraphPad Software Inc., La Jolla, CA) was used to perform all the statistical analyses.

Study approval

All animal procedures were performed in accordance with the European Community and ARRIVE guidelines and were approved by the Institutional Animal Care Committee of “G. d’Annunzio” University and by the Italian Ministry of Health (Authorization n. 892/2018-PR).

The human prostate tissue samples for immunostaining with anti-PSCA Abs were obtained from the institutional Biobank of the Local Health Authority n. 2 Lanciano - Vasto - Chieti (Italy). The personal data processing complied with the data protection laws.

Results

Cationic lipid nanocomplexes were loaded with Cas9gRNA-hIL30 to delete the IL30 gene and functionalized by conjugation with anti-hPSCA Abs to target PC cells

The well-established antitumor efficacy of IL30 gene deletion in PC cells¹² has led to the development of a therapeutic approach to selectively inhibit its expression at the tumor site, minimizing the risk of side effects.

Prostate stem cell antigen (PSCA) is a prostate-specific glycosylphosphatidylinositol-anchored protein that is minimally detected in normal prostatic tissue (Fig. 1a, a) and is upregulated in most PCs. Its expression is positively related to disease grade and stage and to androgen independence^20,21. Both AR⁺DU145 and AR^-PC3 cells, as well as tumors originating from their subcutaneous (s.c.) implantation in NSG mice (Fig. 1a, b and c), express PSCA (Fig. 1b, Supplementary Fig. 4a, b); therefore, to increase NxP accumulation at the tumor site, anti-human (h) PSCA Abs were conjugated to the PEG derivatives present on the external bilayer of preformed NxPs using an aldehyde-maleimide reaction. The specificity of the binding of the anti-hPSCA Ab-conjugated/rhodamine B-labeled NxP (RhB-hPSCA-NxP) to the PSCA on the surface of PC cells was determined using flow cytometry (Fig. 1c).

**Fig. 1: PSCA expression in PC3 and DU145 cells and specific binding and uptake of nanoliposomes conjugated with anti-hPSCA Ab by tumor cells.**

NxPs were loaded with the Cas9gRNA-hIL30 complex, which demonstrated a fair entrapment efficiency (58%) when considering the substantial size of the Cas9 protein (160 kDa), and then conjugated with anti-hPSCA Abs to generate Cas9gRNA-hIL30-hPSCA NxPs, hereinafter referred to as Cas9hIL30-hPSCA NxPs. Alternatively, NxPs were left empty and conjugated with anti-PSCA Abs to generate empty-hPSCA NxPs, which were used as controls.

DLS measurements of the average particle size and zeta electromotive force determined that the empty-hPSCA NxPs and Cas9hIL30-hPSCA NxPs were submicron vesicles (Fig. 1d–f; Supplementary Table 4). Both immunoliposomes had a spherical structure and uniform size, and no particle precipitation was observed. These features were confirmed by TEM analyses of PC cells treated with Cas9hIL30-hPSCA NxPs, which were identified as round nanostructures that were homogeneous in size (0.1 μm) and shape and in close contact with PC cells (Fig. 1g). More rapid cellular uptake was noted for Cas9hIL30-hPSCA NxPs than for unconjugated Cas9hIL30 NxPs given that after 2 hrs of coculture with PC cells, most of them had been endocytosed, whereas the unconjugated Cas9hIL30 NxPs appeared largely crowded on the cell surface, suggesting that conjugation with Abs improves nanoparticle uptake by cancer cells (Fig. 1h–k). By the third hour of coculture, both the Ab-conjugated and unconjugated NxPs had been endocytosed by the tumor cells.

Cas9hIL30-hPSCA NxPs demonstrate genome editing efficiency in human PC cells, no off-target activity and high serum and pH stability

One of the potential issues associated with CRISPR/Cas9 therapies is the genotoxicity caused by nonspecific genome cleavage. We evaluated cleavage specificity by performing deep sequencing of the off-target regions (Supplementary Table 2) of the selected single-guide RNAs (sgRNAs) in PC3 and DU145 cell cultures, and in NSG mice treated with Cas9hIL30-hPSCA NxPs (10 mg/ml, 48 hrs) or PBS. Our results showed that the frequency of variants of the off-target regions in cell cultures (Fig. 2a, Supplementary Fig. 5a) and in mice (Fig. 2b, c; Supplementary Fig. 5b, c) treated with Cas9hIL30-hPSCA NxPs was comparable to that observed in cells or mice treated with PBS (controls) (<0.1%), confirming the specificity of genome editing.

**Fig. 2: Physical characterization and biodistribution of Cas9hIL30-hPSCA NxPs.**

TIDE analyses revealed efficient genome editing in both DU145 cells and, particularly, in PC3 cells. In PC3 cells, the total editing efficiency of the targeted IL30 gene was 63.8% for the forward strand and 62.6% for the reverse strand (Supplementary Fig. 6), whereas in DU145 cells, the total editing efficiency of the targeted IL30 gene was 41.1% for the forward strand and 45.5% for the reverse strand (Supplementary Fig. 7). Deep sequencing of the IL30 gene region in tumor tissues confirmed in vivo these data and allowed us to analyze the mutation patterns and the allelic mutation frequency of Cas9hIL30-hPSCA NxPs (Supplementary Figs. 8 and 9). The total editing efficiency of the targeted IL30 gene was 59% in PC3 tumors and 40% in DU145 tumors (Fig. 2b, c).

Given that the physical stability of lipid nanocomplexes in the blood seriously affects their use as CRISPR/Cas9 drug delivery systems, we next evaluated the serum stability of our immunoliposomes by measuring overtime changes in their size in the presence of medium containing 10% FBS. Cas9hIL30-hPSCA NxPs exhibited great serum stability, and their size was relatively stable over a wide range of pH values, as measured over 24 hrs (Fig. 2d, e). This finding suggests that microenvironmental pH does not influence the physical stability of NxPs. As shown in Fig. 2f and Supplementary Fig. 10, at 30 min, the release rates of Cas9 by Cas9hIL30 NxPs and Cas9hIL30-hPSCA NxPs were 28% and 32%, respectively. These findings comply with the liposome formulation guidelines in the international pharmacopoeia (https://digicollections.net/phint/2020/index.html#d/b.1), which recommends a release rate of less than 40% at 0.5 hr.

Toxicity screening of the immunoliposomes assessed in both DU145 and PC3 cells exposed to increasing concentrations (0.2, 0.4, or 1 mg/ml) of empty-hPSCA NxPs for 48 hrs, revealed that the viability of PC cells treated with NxPs and PBS (or untreated) was comparable (ANOVA: p > 0.05), indicating that the immunoliposome formulation had no obvious toxicity (Fig. 2g).

Importantly, pharmacokinetic studies revealed that Cas9hIL30-hPSCA NxPs exhibited prolonged blood circulation, with a half-life (t1/2) of 1 hr, which was significantly longer than that of free Cas9, which was eliminated quickly from the bloodstream (t1/2 = 5 min) (ANOVA, p < 0.001) (Fig. 2h). Unconjugated NxPs showed a half-life comparable to that of Ab-conjugated NxPs (1 hr), suggesting that the prolonged circulation time was due to the lipidic shell, which efficiently protects the CRISPR/Cas9gRNA complex from enzymatic degradation. The pharmacokinetic results obtained from the DU145 tumor-bearing NSG mice were comparable to those from the PC3 tumor-bearing mice (Fig. 2h).

Intravenously administered Cas9hIL30-hPSCA NxPs specifically targets and is efficiently taken up by PC xenografts

To assess the biodistribution of NxPs and their efficiency in selectively targeting the tumor site, magnetic resonance imaging (MRI) of tumor-bearing mice was performed before and 5 min, 15 min, 30 min, 1 hr 30 min, 2 hrs 30 min, 4 hrs, and 5 hrs 30 min after intravenous (i.v.) inoculation of core-encapsulated (CE) gadolinium (Gd) nanoliposomes conjugated or not with anti-PSCA Abs, herein referred to as CE-Gd-hPSCA NxP and CE-Gd NxP, respectively, or Gd-DOTA, which served as a control contrast agent. T1-weighted MR revealed that both CE-Gd-hPSCA NxP and CE-Gd NxP, as well as Gd-DOTA, were detectable in the tumor as early as 5 min after administration (Fig. 2i). A greater contrast effect was observed in the tumor xenografts of mice inoculated with CE-Gd-hPSCA NxPs than in the tumors of mice inoculated with CE-Gd NxPs starting 30 min after inoculation, and a progressive increase in the difference in signal intensity, which peaked at 2 hrs 30 min, was evident (Fig. 2i, j). The distribution of the Cas9 protein, as assessed by western blotting, in tumors from mice treated with Cas9hIL30-hPSCA NxPs versus tumors from mice treated with unconjugated NxPs, at different time points after nanoparticle injection, confirmed these data (Supplementary Fig. 11).

In animals treated with CE-Gd NxPs, the signal intensity, which is reported as the dose in tissue sample/injected dose × 100 (percentage of the injected dose (%ID), was significantly greater in the tumor than in the other organs 5 min and 15 min after i.v. injection and subsequently decreased (Fig. 2k). After NxP injection, the signal intensity progressively increased in the liver and kidney, the organs primarily involved in liposome metabolism and clearance²², in which a greater contrasting effect, compared to the other organs, was detected starting 1 hr 30 min after inoculation of CE-Gd NxPs or CE-Gd-hPSCA NxPs (Fig. 2k, l). Unlike tumors from mice that received unconjugated NxPs, tumors from mice inoculated with Abs-conjugated NxPs generated a consistently greater signal intensity than all other organs at all time points (Fig. 2l). Western blot (WB) quantification of the Cas9 protein in tumors versus organs from mice treated with unconjugated or anti-hPSCA Ab-conjugated NxPs, at different time points post injection, was consistent with the MRI results (Supplementary Figs. 12 and 13).

NxP uptake by PC xenografts was also investigated using laser scanning confocal (LSC) microscopy and TEM. LSC images of lungs from GFP-labeled DU145 or PC3 tumor-bearing NSG mice inoculated i.v. with rhodamine B (RhB)-labeled NxP conjugated or not with anti-PSCA Abs, namely, RhB-hPSCA-NxP and RhB-NxP, respectively, showed a distinct RhB fluorescence signal 5 min after inoculation, its strong decline 10 min later, and its nearly lost after 30 min (Fig. 2m, n). No signs of intravascular aggregation of NxPs were observed. A faint RhB signal was detected in the tumors as early as 5 min post-NxP inoculation, but its intensity progressively increased and differed substantially between the RhB-NxP- and RhB-hPSCA-NxP-treated tumors, which generated a stronger signal starting 30 min after injection and peaking 2 hrs 30 min after injection (Fig. 3a–e), confirming the MRI data. At this time point (i.e., 2 hrs 30 min post NxP injection), TEM images of the tumors (Fig. 3f, g) revealed that the uptake and internalization of NxPs in neoplastic cells were very efficient when they were conjugated with anti-hPSCA Abs (Fig. 3f). In contrast, the uptake and internalization of NxPs was poor when they were not conjugated (Fig. 3g) because unconjugated NxPs remained largely located among the neoplastic cells.

**Fig. 3: Tumor uptake of intravenously injected rhodamine B (RhB)-labeled NxPs (RhB-NxP) and RhB-labeled NxPs conjugated with anti-hPSCA Abs (RhB-hPSCA-NxP) in tumor-bearing NSG mice.**

Cas9hIL30-hPSCA NxP treatment inhibits tumor growth and improves survival in NSG mice bearing human PC xenografts

Given that MRI and LSC microscopy revealed an increased ability of Ab-conjugated versus unconjugated nanoliposomes to selectively reach tumor masses expressing PSCA (Fig. 1a, b and c), the effects of anti-hPSCA Ab-conjugated Cas9hIL30 NxPs were compared to those of anti-hPSCA Ab-conjugated empty NxPs to assess the antitumor potential of targeted delivery of the Cas9gRNA-hIL30 complex.

The treatment schedule consisted of two i.v. injections of immunoliposomes per week (250 µl dose with a 10 mg/ml lipid concentration) starting from the onset of a palpable tumor (~2 mm Ø) and continuing throughout the lifespan of the mice (Fig. 4a).

**Fig. 4: Tumor growth and survival in PC xenograft-bearing mice treated with Cas9hIL30-hPSCA NxPs.**

To compare the effects on tumor growth and progression of the immunoliposome-based delivery of CRISPR/Cas9gRNA-hIL30 complex versus a direct IL30 gene editing in PC cells, which generated IL30KO-DU145 and IL30KO-PC3 cells, in addition to three groups of NSG (NOD scid gamma) mice s.c. implanted with wild-type DU145 or PC3 cells, and then treated with Cas9hIL30-hPSCA NxPs, or empty-hPSCA NxPs, or PBS; two groups of mice implanted with IL30KO-DU145 or IL30KO-PC3 cells, and then treated with PBS, were included in the study.

In both PC xenograft models, treatment with Cas9hIL30-hPSCA NxPs substantially inhibited tumor growth compared to treatment with empty-hPSCA NxPs or PBS (ANOVA: p = 0.0001; Fig. 4b, c). The growth curve of PC3 tumors in mice treated with Cas9hIL30-hPSCA NxPs matched that of IL30KO-PC3 tumors. In both groups, the mean tumor volume was lower than that in PBS- or empty-hPSCA-treated mice (ANOVA: p = 0.0001; Fig. 4b). Kaplan–Meier analysis revealed that both groups of mice survived longer than mice treated with PBS or empty-hPSCA NxPs (63 versus 47 days, log-rank test: p < 0.000001; Fig. 4d). DU145 tumor-bearing mice, treated with Cas9hIL30-hPSCA NxPs, also survived longer than mice treated with PBS or empty-hPSCA NxPs (74 versus 58 days, log-rank test: p < 0.000001) but less than mice bearing slow-growing IL30KO-DU145 tumors (94 days) (Fig. 4c, e).

In the PC3 model, Cas9hIL30-hPSCA NxP treatment had no effect on lung metastases (lesions >500 µm in size). Although the IL30KO tumor-bearing mice had only micrometastases (≤500 µm in size, ref. ²³), all the mice eventually developed metastases (Fig. 4f, g, Supplementary Fig. 14). In the DU145 model, 60% (9/15) of the mice treated with Cas9hIL30-hPSCA NxPs and 53% (8/15) of the mice bearing IL30KO tumors were metastasis free, whereas all the mice treated with empty-hPSCA NxPs or PBS developed lung metastases (Fisher’s exact test: p = 0.0003; Fig. 4h, i). In mice that developed metastatic disease, no difference was observed in the mean number of metastases per mouse between the groups (Supplementary Table 5).

Cas9hIL30-hPSCA NxPs dramatically suppresses IL30 expression and inhibits proliferation, vascularization, and cancer driver genes in PC xenografts without evidence of toxicity or immunogenicity

Immunopathological analysis of DU145 and PC3 tumors revealed a lack of IL30 expression following Cas9hIL30-hPSCA NxP treatment, as observed in IL30KO tumors, and extensive areas of ischemic hemorrhagic necrosis in association with poor vascularization and reduced tumor cell proliferation (Fig. 5a, b and Supplementary Table 6), compared with tumors from PBS- or empty-hPSCA NxP-treated mice. In addition, few apoptotic events occurred, as found in tumors from PBS- or empty-hPSCA NxP-treated mice (Supplementary Table 6). The histopathological features of wild-type tumors from Cas9hIL30-hPSCA NxP-treated mice and IL30KO tumors from PBS-treated mice were comparable (Fig. 5a, b). Macrophage infiltration, mostly localized to the tumor edge, was comparable between DU145 or PC3 tumors from mice treated with empty or Cas9hIL30-loaded immunoliposomes or with PBS, suggesting the non-immunogenicity of the NxP formulations (Supplementary Fig. 15).

**Fig. 5: Immunopathological and molecular features of PC xenografts from mice treated with Cas9hIL30-hPSCA NxPs or Empty-hPSCA NxPs.**

The molecular events driving Cas9hIL30-hPSCA NxP-dependent inhibition of tumor proliferation and angiogenesis were investigated by assessing PC cell viability and angiogenic profile in response to nanoliposomes. DU145 and PC3 cell proliferation was substantially inhibited after 72 hrs of treatment with Cas9hIL30-hPSCA NxPs. These results were similar to that obtained with CRISPR/Cas9gRNA-mediated editing of the IL30 gene in PC cells (Fig. 5c). Albeit to a lesser extent when compared to CRISPR/Cas9gRNA-mediated IL30 gene editing, nanoliposome-based delivery of CRISPR/Cas9gRNA-hIL30 complex dramatically inhibited the angiogenic potential of both PC cell lines, because it reshaped their transcriptional profiles and downregulated a wide range of proangiogenic genes, including ANGPTL 1/2/4, IL1β, CCL2, CXCL1, CXCL6, SERPINE1, SERPINF1, EFNB2, PLG, PF4, VEGFA, VEGFD, ANG, TGFβ1, EGF, and HGF, and a few negative regulators of vascular network formation, such as ADGRB1, COL18A1, CXCL10, and TIMP2 (Fig. 5d, e). The substantial downregulation of IGF1, CCN2, CXCL5, CXCL8, EDN1, ITGAV, TNF, MMP14, FGF1, and FGF2 in both DU145 and PC3 cells was a shared effect between direct and nanoliposome-mediated IL30 gene editing (Fig. 5f). Immunopathological detection of the downregulation, or lack of expression, of angiogenic drivers in IL30KO tumors and nanoparticle-treated tumors substantiated the molecular in vitro findings.

In addition to IL30 expression, treatment with Cas9hIL30-hPSCA NxPs effectively suppressed the expression of previously identified IL30-regulated oncogenes¹², such as IGF1 (Fig. 6a, b; Supplementary Tables 7 and 8), which also functions as an angiogenic factor, and PTGS2 and NFKB1, which promote cancer cell survival and inflammation-associated cancer²⁴. In contrast, the expression of tumor suppressors, such as CDH1, DKK3, and PTEN (which were assessed only in DU145 tumors because PC3 cells have a homozygous deletion of the PTEN gene and are therefore negative for PTEN expression, ref. ²⁵), was markedly upregulated, as we described previously in IL30KO-DU145 tumors¹² and confirmed here in IL30KO-PC3 tumors (Fig. 6c, d; Supplementary Tables 7 and 8).

**Fig. 6: Immunopathological aspects of PC xenografts from mice treated with Cas9hIL30-hPSCA NxPs or Empty-hPSCA NxPs.**

Ultrastructural analyses, using TEM, revealed that both tumors treated with empty-hPSCA NxPs and those treated with Cas9hIL30-hPSCA NxPs showed intracytoplasmic spherical membranes. These membranes were visualized both in the peripheral and deep areas of the tumor masses, but no signs of damage to the cytoplasmic organelles or cytotoxicity were detected in non necrotic neoplastic areas (Fig. 7a, b).

**Fig. 7: Morphological features of PC xenografts and organs of mice treated with Cas9hIL30-hPSCA NxPs or empty-hPSCA NxPs.**

Histopathological analyses, performed both at the end of the experiment and at different time points of the treatment, showed that the lungs, heart, liver, kidney, spleen, and prostate from Cas9hIL30-hPSCA NxP- or empty-hPSCA NxP-treated animals were histologically normal and comparable to the organs obtained from PBS-treated animals (Fig. 7c, d). TEM investigations of the liver (to monitor the main organ that expresses key enzymes for lipid metabolism) and kidney tissues (to assess the risk of lipotoxic renal damage) confirmed the absence of cellular damage at the ultrastructural level (Fig. 7a, b).

The safety of Cas9hIL30-hPSCA NxPs was further assessed using serological, hemolytic, and immunogenicity tests. To assess the toxicity of the treatment in specific organs, the levels of the following markers were measured 21 days after starting treatment (after 6 treatments): aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are markers of liver function; blood urea nitrogen (BUN) and creatinine (Cr), which are markers of renal function; and cardiac troponin-1 (cTnI), creatine kinase (CK) and lactate dehydrogenase (LDH), which are markers of cardiac function. The serum parameters of the animals treated with empty-hPSCA NxPs or Cas9hIL30-hPSCA NxPs were comparable to those of the PBS-treated mice, thus indicating the lack of specific organ damage (Supplementary Table 9). Furthermore, as shown in Supplementary Fig. 16, Cas9hIL30-hPSCA NxPs had no obvious hemolytic effect.

Assessment of NxP immunogenicity in fully immunocompetent BALB/c mice harboring the genetic background of the NSG mice used for treatment revealed that i.v. inoculation of naked Cas9gRNA-hIL30 complex, empty-hPSCA NxPs, or Cas9hIL30-hPSCA NxPs stimulated only minimal TNFα and IL6 responses, which returned to baseline PBS levels 24 hrs post injection. These findings suggest that the nanoliposomes, or their Cas9gRNA-hIL30 content, had no lasting adverse immune or inflammatory activity. In addition, the average weight of mice treated with naked Cas9gRNA-hIL30 complex, empty-hPSCA NxPs, unconjugated Cas9hIL30 NxPs or Cas9hIL30-hPSCA NxPs was comparable (ANOVA: p > 0.05) to that of mice treated with PBS (Supplementary Table 10), strongly suggesting the absence of systemic toxicity²⁶. Overall, the immunoliposome-based targeted delivery of the Cas9gRNA-hIL30 complex, which selectively inhibits tumor IL30 expression, has antiangiogenic and antiproliferative effects in both PC models and has antimetastatic efficacy in DU145 tumor model, without evidence of hematological or organ toxicity.

Immunoliposomes loaded with CRISPR/Cas9gRNA to delete murine Il30 exhibit genome editing efficiency in murine PC cells without off-target or cytotoxic effects

To assess whether IL30-targeting immunoliposomes could reverse the immunosuppressive effects of IL30^9,11,27 and restore antitumor immunity, we tested this therapeutic approach in a fully immunocompetent syngeneic murine model involving the s.c. implantation of TRAMP-C1 cells engineered to overexpress IL30 (IL30-TRAMP-C1, the clone releasing 458,00 ± 12,06 ng/mL of mIL30, ref. ¹⁴). These cells produced rapidly progressing and highly metastatic tumors, resulting in decreased survival compared to that of mice bearing control tumors (wild-type TRAMP-C1 and EV-TRAMP-C1).

Given that IL30-TRAMP-C1 and control tumors express PSCA (Fig. 8a), nanoparticles synthesized to treat murine IL30-producing tumors were functionalized, after loading with the Cas9gRNA-mIL30 complex (hereinafter referred to as the Cas9mIL30 complex), with anti-mouse (m) PSCA Abs (hereinafter referred to as Cas9mIL30-mPSCA NxP), as above reported for treating human IL30-producing PC xenografts. These NxPs specifically bound to mPSCA expressed by tumor cells (Fig. 8b). The liposomal loading capacity of the Cas9mIL30 complex was 63%. Both empty-mPSCA NxPs and Cas9mIL30-mPSCA NxPs were submicron (Supplementary Table 11, Fig. 8c) round nanostructures that were uptaken by tumor cells in vitro more efficiently than unconjugated NxPs (Fig. 8c).

**Fig. 8: Physical characterization and biodistribution of Cas9mIL30-mPSCA NxPs and their effect on tumor growth and survival in a fully immunocompetent host.**

Assessment of cleavage specificity for the selected sgRNA in IL30-TRAMP-C1 cell cultures and in C57BL/6J mice treated with Cas9mIL30-mPSCA NxPs (10 mg/ml, 48 h) or with PBS demonstrated the specificity of genome editing given that the frequency of variants of the off-target regions in cell cultures (Fig. 8d) and in mice (Fig. 8e) treated with Cas9mIL30-mPSCA NxPs was comparable to that observed in cells or mice treated with PBS (<0.1%).

Genome editing was assessed by ICE (Inference of CRISPR Edits), which can significantly expedite CRISPR editing workflows¹⁹. The findings revealed that the total editing efficiency of the targeted mIl30 gene was 82% (Fig. 8f). The results of deep sequencing of the Il30 gene region in IL30-TRAMP-C1 tumor tissues are consistent with these data because the total editing efficiency of the targeted mIl30 gene reached 76% (Fig. 8e). The mutation pattern introduced by CRISPR/Cas9 editing in mIl30 gene is described in the Supplementary Fig. 8. The serum and pH stabilities of the Cas9mIL30-mPSCA NxPs were comparable to those of the Cas9hIL30-hPSCA NxPs, and the release profile of Cas9 by Cas9mIL30 NxPs was comparable to that of Cas9hIL30 NxPs.

Importantly, the immunoliposomes used to target TRAMP-derived cancer cells were demonstrated to be nontoxic in vitro given that the viabilities of IL30-TRAMP-C1 cells treated with Empty-mPSCA NxPs or PBS (or untreated) were comparable (ANOVA: p > 0.05) (Supplementary Fig. 17). The pharmacokinetics of Cas9mIL30-mPSCA NxPs in IL30-TRAMP-C1 tumor-bearing C57BL/6J mice were comparable to those of Cas9hIL30-hPSCA NxPs in DU145 tumor- and PC3 tumor-bearing mice (Fig. 8g).

Cas9gRNA-mIL30 targeting of IL30-overexpressing PCs in immunocompetent hosts decreases the recruitment of myeloid-derived cells, Tregs and ILC3s, inhibits NFKB1 expression, and hinders tumor progression

The effects of immunoliposomes targeting tumor-produced murine IL30, in a fully immunocompetent host, were tested by applying the conditions and schedule used for the treatment of PC xenografts (Supplementary Fig. 3). TEM images demonstrated that NxPs functionalized with anti-mPSCA Abs reached the tumor site as early as 30 min after i.v. administration and were efficiently uptaken and internalized by neoplastic cells (Fig. 8h).

NxP uptake by PC xenograft was also investigated by LSC microscopy. As in NSG mice bearing DU145 and PC3 tumors, a faint RhB signal was detected in tumors as early as 5 min post NxP inoculation, and its intensity progressively increased and showed a substantial difference between RhB-NxP and RhB-mPSCA-NxP treated tumors, which generated a stronger signal, starting 30 min after injection and peaking 2 hrs 30 min later (Supplementary Fig. 18).

The effect of Cas9mIL30-mPSCA NxPs on tumor production of IL30, as well as on the tumor microenvironment and biological behavior, was assessed. This experiment included one group of mice implanted with wild-type TRAMP-C1 cells and then treated with PBS in addition to three groups of C57BL/6J mice implanted with IL30-TRAMP-C1 cells and then treated with Cas9mIL30-mPSCA NxPs, empty-mPSCA NxPs, or PBS.

In mice bearing IL30-overproducing PCs, treatment with IL30-targeting immunoliposomes consistently slowed tumor growth compared to that in mice bearing IL-30-TRAMP-C1 tumors, treated with empty-mPSCA NxPs or PBS, and mice bearing wild-type TRAMP-C1 tumors treated with PBS (ANOVA: p < 0.0001) (Fig. 8i). At the end of the experiment, all the mice (15/15) bearing IL30-TRAMP-C1 tumors and treated with Empty-mPSCA NxPs or PBS had metastases, in contrast, 47% (7/15) of the mice bearing wild-type TRAMP-C1 tumors and treated with PBS developed lung metastases, and only 40% (6/15) of the mice bearing IL30-TRAMP-C1 tumors and treated with Cas9mIL30-mPSCA NxPs developed metastases (Fig. 8j). Kaplan–Meier analyses revealed that mice bearing IL30-TRAMP-C1 tumors and treated with Cas9mIL30-mPSCA NxPs survived longer (92 days) than controls (PBS- and empty-mPSCA-treated) (64 days) and PBS-treated TRAMP-C1 tumor-bearing mice (78 days) (log-rank test: p < 0.000001) (Fig. 8k). The organs from the Cas9mIL30-mPSCA NxP- or empty-mPSCA NxP-treated animals were histologically normal (Supplementary Fig. 19) and free of ultrastructural cell damage (Fig. 8l). Hematological analysis confirmed the absence of specific organ toxicity (Supplementary Table 12). Assessment of NxP immunogenicity in fully immunocompetent C57BL/6J mice demonstrated that i.v. inoculation of naked Cas9gRNA-mIL30 complex, empty-mPSCA NxPs, or Cas9mIL30-mPSCA NxPs induced a minimal increase in the blood levels of TNFα and IL6, which returned to baseline within 24 hrs.

Treatment with Cas9mIL30-mPSCA NxPs suppressed IL30 expression and resulted in multiple foci of necrobiosis and ischemia associated with impaired microvessel density (MVD) and reduced cancer cell proliferation (ANOVA: p < 0.0001) compared with those in tumors treated with empty-mPSCA NxPs or PBS (Fig. 8m and Supplementary Table 13). Immunopathological studies demonstrated that treatment with Cas9mIL30-mPSCA NxPs prevented the intratumoral recruitment of Foxp3⁺ regulatory T lymphocytes (Tregs) (Fig. 8n) and nonspecific immune cells, such as F4/80⁺ macrophages, CD11b⁺Gr-1⁺ myeloid-derived cells (MDCs) and Ly-6G⁺ granulocytes, which characterize the IL30-TRAMP-C1 tumor microenvironment (TME). In contrast, CD3⁺CD8⁺ and CD3⁺CD4⁺ T lymphocyte levels were comparable to those observed in empty-mPSCA NxP-treated tumors or PBS-treated IL30-overexpressing and wild-type tumors (automated immune cell quantitation in the TME is reported in Supplementary Fig. 20). Furthermore, NKp46⁺RORγt⁺ cells, which are known as innate lymphoid cells 3 (ILC3), heavily infiltrated untreated and empty-mPSCA NxP- or PBS-treated IL30-TRAMP-C1 tumors, as observed in IL30-overexpressing syngeneic breast cancers¹¹, whereas their number was consistently reduced in Cas9mIL30-mPSCA NxP-treated IL30-TRAMP-C1 tumors (Fig. 8n, Supplementary Fig. 20). The reduced intratumoral immune cell infiltration due to IL30-targeting treatment was accompanied by substantial inhibition in cancer cells expression of NFKB1 (Fig. 8n), which is a key player in cancer-immune cell crosstalk and in modulating antitumor immunity²⁸.

Discussion

Immunotherapy has been a breakthrough in the treatment of advanced tumors²⁹, but limited results have been obtained in PC. The identification of new targetable immunosuppressive oncogenes would be useful.

IL30 has demonstrated “prostate cancer driving” properties. Indeed, the IL30/IL6R/gp130 axis promotes inflammatory, immunosuppressive and cancer progression programs and is critical for PC onset and metastasis^7,9,12,30. Given that PC mainly affects the fragile and progressively expanding elderly population, a tumor-selective and well-tolerated therapeutic approach is especially needed. Nanobiotechnology can provide innovative tools for the development of precision cancer immunotherapy, and IL30 represents a potential therapeutic target in PC.

In this study, we used a microfluidic platform and EMA- and FDA-approved biocompatible components suitable for clinical use to produce sterically stable submicron particles consisting of self-assembled lipids and an anionic ribonucleoprotein complex formed by Cas9 and sgRNA³¹ to knock out the IL30 gene.

The encapsulation efficiency, which was not particularly high due to the large size of the loaded protein³², nevertheless ensures a good level of genome editing and efficient deletion of the IL30 gene, without off-target effects both in vitro and in vivo. PEGylation prevents nanoliposome opsonization and clearance and allows the nanoparticles to retain their size and integrity under a wide range of pH conditions^33,34; therefore, PEGylation provides high serum stability and a sufficiently long circulation time after intravenous injection, suggesting resistance to macrophage uptake and enzymatic degradation³⁵. Moreover, the lipid formulation we used, including DOTAP, DOPE, and cholesterol, was selected to promote both bilayer stability, which ensures the structural integrity of liposomes, and endolysosomal escape³⁶. Nanoparticle functionalization with Abs³⁷ that specifically recognize and bind to PSCA, which is overexpressed by most PCs²⁰, ensures efficient site-specific accumulation and favors internalization into PC cells, as revealed by ultrastructural studies.

Our nanocarriers are ineffective on cancer cell viability. When loaded with the Cas9gRNA-IL30 complex and conjugated with antitumor-specific Abs, that promote their uptake by PC cells, nanoparticles exert antiproliferative effects on both cell cultures and palpable tumors. Histopathological analysis of the liver, kidneys, spleen, heart, lungs, and prostate of immunoliposome-treated mice, together with the ultrastructural study of the liver and kidneys, the organs primarily involved in lipid metabolism²², excluded the possibility of organ damage or obvious inflammatory responses. These data were substantiated by the lack of significant changes in weight and serum levels of inflammatory cytokines and markers of cardiac, kidney, and liver function, confirming the absence of side effects.

Chronic biweekly treatment with immunoliposomes, starting at tumor onset, substantially inhibited tumor growth and progression. After traveling through the pulmonary circulation, intravenously administered immunoliposomes enter the systemic circulation but exclusively accumulate in PSCA⁺ tumors, yielding inhibitory effects on tumor progression programs.

In the PC3 model, the antitumor effect of the treatment was comparable to that obtained from knocking out the IL30 gene in cancer cells using classical CRISPR/Cas9-mediated gene editing. In the DU145 tumor model, the efficacy of the nanotherapy in slowing tumor growth and improving survival was lower than what was observed when the IL30 gene was deleted in cancer cells before xenograft implantation, however, the antimetastatic effect was comparable to that of DU145 IL30KO tumors of the same size and resulted in more than half of the animals being free from lung metastases. In contrast, all control mice developed lung metastasis. These results corroborate data on the antimetastatic potential of targeting IL30^9,12,30.

Notably, in both the AR⁺ (DU145) and AR^- (PC3) IL30-expressing tumor models, treatment with CRISPR/Cas9gRNA-hIL30-loaded immunoliposomes substantially improved survival. Large areas of ischemic necrosis associated with severely compromised vascularization, due to the overwhelming downmodulation of proangiogenic genes compared to antiangiogenic genes, which reprogram PC-endothelium crosstalk³⁸, and reduced tumor cell proliferation are the hallmarks of nanotherapy-induced tumor growth inhibition. The extensive remodeling of the gene expression signature of PC cells include the suppression of a broad range of angiogenic and angiocrine factors, such as IGF1³⁹, CCN2⁴⁰, CXCL5, CXCL8⁴¹, EDN1⁴², ITGAV⁴³, TNF⁴⁴, MMP14⁴⁵, FGF1, and FGF2⁴⁶, associated with the downregulation of oncogenes, such as PTGS2⁴⁷, and a key driver of inflammation and PC cell survival, namely, NFKB1²⁴, and the upregulation of tumor suppressors, such as CDH1⁴⁸, DKK3^49,50, and PTEN⁵¹. These pathways are among the molecular mechanisms triggered by both classical and nanotherapy-mediated IL30 genome editing, which underlies its antitumor efficacy. These mechanisms are also largely involved in the response to treatment with the IL30-targeting immunoliposome in the syngeneic model of IL30-overexpressing PC in fully immunocompetent hosts, of which the inhibition of angiogenesis and proliferation and the downmodulation of NFKB1²⁸ are hallmarks. In addition, in the syngeneic PC model, treatment with Cas9gRNA-mIL30-loaded nanoparticles prevents intratumoral infiltration of macrophages, MDCs and granulocytes and inhibits Treg⁵² and ILC3^53,54,55 influx, thus abrogating the immune cell landscape orchestrated by IL30 overproducing tumor¹².

Remodeling the TME in immunocompetent hosts is associated with substantial inhibition of tumor growth and metastasis, leading to improved survival of IL30-targeting immunoliposome-treated mice and strengthening the results obtained in xenograft tumor models.

Notably, human IL30 is membrane-anchored, whereas its murine counterpart,¹² with which it shares 73% homology, is a secreted cytokine. However, the consequences of IL30 gene editing on xenograft and syngeneic tumor behavior largely overlap. As observed for many antiangiogenic drugs, particularly those targeting the VEGF/VEGFR pathway^56,57, the occurrence of adaptive resistance and tumor regrowth due to the selective pressure exerted by an IL30-targeting therapy cannot be excluded. Although the molecular mechanisms that may cause treatment failure remain to be investigated, the emergence of treatment resistance and resumption of tumor progression could be limited by combining IL30 suppression with immunotherapy or other personalized treatments, as observed in the combined treatment protocols that have been successfully used for the management of different types of advanced stage cancer^58,59.

To date, different liposome-based formulations are approved for clinical use in the treatment of various advanced stage tumors^60,61. However, immunoliposomes that selectively target tumors and deliver a CRISPR/Cas9 complex to edit tumor-promoting and immunoregulatory genes are absent from the current therapeutic landscape.

By rescuing the TME from immunosuppression, IL30-targeting nanotherapy exhibits the potential to overcome resistance to T-cell-based immunotherapies⁶¹, which have thus far been unsuccessful in the treatment of PC^62,63. Immunocompetent models of PC may provide insight into whether targeting IL30 in the context of combination therapy may reverse the disappointing results (presented at the ESMO Congress 2023) achieved in the latest clinical trials with Sipuleucel-T (Provenge®), the autologous dendritic cell vaccine, or with Pembrolizumab, the humanized anti-PD-1 monoclonal Ab, both of which are FDA-approved for the treatment of metastatic castration-resistant PC (https://dailyreporter.esmo.org/esmo-congress-2023/genitourinary-cancers).

Immunopathological and clinical data show that among patients who underwent prostatectomy due to high-grade and locally advanced PC, those with IL30^negative cancer had a better tumor immune response and a longer disease-free survival than patients with IL30^positive cancer⁴³, emphasizing the clinical value of an IL30-targeting therapeutic strategy.

The dramatic disruption of proangiogenic and tumor progression programs and the overcoming the immunosuppressive TME, attributed to the selective nanoliposome-mediated deletion of the IL30 gene in cancer cells, paves the way for the development of a modern and safe immuno-nanotherapy for advanced PC that meets the needs of healthy aging.

Data availability

The data generated in this study are available upon request from the corresponding author.

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Acknowledgements

The research leading to these results has received funding from the following: AIRC, under IG 2019 - ID. 23264 project to P.I. Emma Di Carlo; MUR (Ministero dell’Università e della Ricerca) from the National Operational Program for Research and Innovation/Programma Operativo Nazionale Ricerca e Innovazione 2014–2020 (PON R&I) to Carlo Sorrentino; the European Union - Next Generation EU under the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2-M4C2 – Investment, 1.5-Call for tender No. 3277, December 30, 2021, Italian Ministry of University, Award Number: ECS00000041, Project title: “Innovation, digitalisation and sustainability for the diffused economy in central Italy” to Antonella Fontana. The authors thank Dr. Samanta Moffa for helping in the preparation of vesicles and their characterization.

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Authors and Affiliations

Department of Medicine and Sciences of Aging, “G. d’Annunzio” University of Chieti-Pescara, 66100, Chieti, Italy
Cristiano Fieni, Carlo Sorrentino, Stefania Livia Ciummo & Emma Di Carlo
Anatomic Pathology and Immuno-Oncology Unit, Center for Advanced Studies and Technology (CAST), “G. d’Annunzio” University of Chieti-Pescara, 66100, Chieti, Italy
Cristiano Fieni, Carlo Sorrentino, Stefania Livia Ciummo, Sofia Scialis & Emma Di Carlo
Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, 66100, Chieti, Italy
Antonella Fontana
UDA-TECHLAB Research Center, “G. d’Annunzio” University of Chieti-Pescara, 66100, Chieti, Italy
Antonella Fontana
Department of Experimental Medicine, La Sapienza University of Rome, 00161, Rome, Italy
Lavinia Vittoria Lotti
Department of Neuroscience, Imaging and Clinical Sciences, “G. d’Annunzio” University of Chieti-Pescara, 66100, Chieti, Italy
Darien Calvo Garcia & Massimo Caulo
Institute for Advanced Biomedical Technologies (ITAB), “G. d’Annunzio” University of Chieti-Pescara, 66100, Chieti, Italy
Darien Calvo Garcia & Massimo Caulo

Authors

Cristiano Fieni
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Carlo Sorrentino
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Stefania Livia Ciummo
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Antonella Fontana
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Lavinia Vittoria Lotti
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Sofia Scialis
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Darien Calvo Garcia
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Massimo Caulo
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Emma Di Carlo
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Contributions

E.D.C. conceived the study. C.F., C.S., S.L.C., A.F., and S.S. performed the experiments. L.V.L. performed the electron microscopy. D.C.G. and M.C. performed the magnetic resonance imaging. E.D.C. and C.F. performed the data analyses. E.D.C. interpreted the data, wrote the manuscript and was responsible for the overall content. All authors reviewed the manuscript.

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Correspondence to Emma Di Carlo.

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Fieni, C., Sorrentino, C., Ciummo, S.L. et al. Immunoliposome-based targeted delivery of the CRISPR/Cas9gRNA-IL30 complex inhibits prostate cancer and prolongs survival. Exp Mol Med 56, 2033–2051 (2024). https://doi.org/10.1038/s12276-024-01310-2

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Received: 05 February 2024
Revised: 20 June 2024
Accepted: 24 June 2024
Published: 04 September 2024
Issue Date: September 2024
DOI: https://doi.org/10.1038/s12276-024-01310-2