Proteomic features of soft tissue tumours in adolescents and young adults.

BACKGROUND: Adolescents and young adult (AYA) patients with soft tissue tumours including sarcomas are an underserved group with disparities in treatment outcomes. METHODS: To define the molecular features between AYA and older adult (OA) patients, we analysed the proteomic profiles of a large cohort of soft tissue tumours across 10 histological subtypes (AYA n = 66, OA n = 243), and also analysed publicly available functional genomic data from soft tissue tumour cell lines (AYA n = 5, OA n = 8). RESULTS: Biological hallmarks analysis demonstrates that OA tumours are significantly enriched in MYC targets compared to AYA tumours. By comparing the patient-level proteomic data with functional genomic profiles from sarcoma cell lines, we show that the mRNA splicing pathway is an intrinsic vulnerability in cell lines from OA patients and that components of the spliceosome complex are independent prognostic factors for metastasis free survival in AYA patients. CONCLUSIONS: Our study highlights the importance of performing age-specific molecular profiling studies to identify risk stratification tools and targeted agents tailored for the clinical management of AYA patients.

Soft tissue tumours are cancers that develop in the connective and supporting tissues of the body, such as muscle or fat. These tumours arise in patients across the entire age range. However, improvements in survival outcomes in adolescent and young adult (AYA) patients have lagged behind outcomes in older adults (OA) and children. To better understand the biology of AYA patients with soft tissue tumours, we analysed protein profiles across 10 different types. We identified biological differences between AYA and OA patients and report an age-specific signature that can potentially be used to help predict which AYA patients are more likely to have aggressive cancers that will spread to other parts of the body. Our study highlights the importance of performing age-specific studies to identify new tools to predict patient outcomes and potentially find more suitable treatments.

Clinical implications and molecular features of extracellular matrix networks in soft tissue sarcomas.

PURPOSE: The landscape of extracellular matrix (ECM) alterations in soft tissue sarcomas (STS) remains poorly characterised. We aimed to investigate the tumour ECM and adhesion signalling networks present in STS and their clinical implications. EXPERIMENTAL DESIGN: Proteomic and clinical data from 321 patients across 11 histological subtypes were analysed to define ECM and integrin adhesion networks. Subgroup analysis was performed in leiomyosarcomas (LMS), dedifferentiated liposarcomas (DDLPS) and undifferentiated pleiomorphic sarcomas (UPS). RESULTS: This analysis defined subtype-specific ECM profiles including enrichment of basement membrane proteins in LMS and ECM proteases in UPS. Across the cohort, we identified three distinct co-regulated ECM networks which are associated with tumour malignancy grade and histological subtype. Comparative analysis of LMS cell line and patient proteomic data identified the LCP1 cytoskeletal protein as a prognostic factor in LMS. Characterisation of ECM network events in DDLPS revealed three subtypes with distinct oncogenic signalling pathways and survival outcomes. Evaluation of the DDLPS subtype with the poorest prognosis nominates ECM remodelling proteins as candidate anti-stromal therapeutic targets. Finally, we define a proteoglycan signature which is an independent prognostic factor for overall survival in DDLPS and UPS. CONCLUSIONS: STS comprise heterogeneous ECM signalling networks and matrix-specific features have utility for risk stratification and therapy selection which could in future guide precision medicine in these rare cancers.

Translational Aspects of Epithelioid Sarcoma: Current Consensus.

Epithelioid sarcoma (EpS) is an ultra-rare malignant soft-tissue cancer mostly affecting adolescents and young adults. EpS often exhibits an unfavorable clinical course with fatal outcome in ∼50% of cases despite aggressive multimodal therapies combining surgery, chemotherapy, and irradiation. EpS is traditionally classified in a more common, less aggressive distal (classic) type and a rarer aggressive proximal type. Both subtypes are characterized by a loss of nuclear INI1 expression, most often following homozygous deletion of its encoding gene, SMARCB1-a core subunit of the SWI/SNF chromatin remodeling complex. In 2020, the EZH2 inhibitor tazemetostat was the first targeted therapy approved for EpS, raising new hopes. Still, the vast majority of patients did not benefit from this drug or relapsed rapidly. Further, other recent therapeutic modalities, including immunotherapy, are only effective in a fraction of patients. Thus, novel strategies, specifically targeted to EpS, are urgently needed. To accelerate translational research on EpS and eventually boost the discovery and development of new diagnostic tools and therapeutic options, a vibrant translational research community has formed in past years and held two international EpS digital expert meetings in 2021 and 2023. This review summarizes our current understanding of EpS from the translational research perspective and points to innovative research directions to address the most pressing questions in the field, as defined by expert consensus and patient advocacy groups.

The proteomic landscape of soft tissue sarcomas.

Soft tissue sarcomas (STS) are rare and diverse mesenchymal cancers with limited treatment options. Here we undertake comprehensive proteomic profiling of tumour specimens from 321 STS patients representing 11 histological subtypes. Within leiomyosarcomas, we identify three proteomic subtypes with distinct myogenesis and immune features, anatomical site distribution and survival outcomes. Characterisation of undifferentiated pleomorphic sarcomas and dedifferentiated liposarcomas with low infiltrating CD3 + T-lymphocyte levels nominates the complement cascade as a candidate immunotherapeutic target. Comparative analysis of proteomic and transcriptomic profiles highlights the proteomic-specific features for optimal risk stratification in angiosarcomas. Finally, we define functional signatures termed Sarcoma Proteomic Modules which transcend histological subtype classification and show that a vesicle transport protein signature is an independent prognostic factor for distant metastasis. Our study highlights the utility of proteomics for identifying molecular subgroups with implications for risk stratification and therapy selection and provides a rich resource for future sarcoma research.

Molecular profiling in desmoplastic small round cell tumours.

Desmoplastic small round cell tumour (DSRCT) is an ultra-rare soft tissue sarcoma that is characterised by aggressive disease and dismal patient outcomes. Despite multi-modal therapy, prognosis remains poor and there are currently no effective targeted therapies available for patients with this disease. Advances in comprehensive molecular profiling approaches including next generation sequencing and proteomics hold the promise of identifying new therapeutic targets and biomarkers. In this review, we provide an overview of the current status of molecular profiling studies in DSRCT patient specimens and cell lines, highlighting the key genomic, epigenetic and proteomic findings that have contributed to our biological knowledge base of this recalcitrant disease. In-depth analysis of these molecular profiles has led to the identification of promising novel and repurposed candidate therapies that are suitable for translation into clinical trials. We further provide a perspective on how future integrated studies including proteogenomics could further enrich our understanding of this ultra-rare entity and deliver progress that will ultimately impact the outcomes of patients with DSRCT.

Lipid-Nanoparticle-Mediated Delivery of Docetaxel Prodrug for Exploiting Full Potential of Gold Nanoparticles in the Treatment of Pancreatic Cancer.

Current chemoradiation therapy suffers from normal tissue toxicity. Thus, we are proposing incorporating gold nanoparticles (GNPs) and docetaxel (DTX), as they have shown very promising synergetic radiosensitization effects. Here, we explored the effect of a DTX prodrug encapsulated in lipid nanoparticles (LNPDTX-P) on GNP uptake in pancreatic cancer models in vitro and in vivo. For the in vitro experiment, a pancreatic cancer cell line, MIA PaCa-2, was cultured and dosed with 1 nM GNPs and 45 nM free DTX or an equivalent dose of LNPDTX-P. For the in vivo experiment, MIA PaCa-2 cells were implanted subcutaneously in NRG mice, and the mice were dosed with 2 mg/kg of GNPs and 6 mg/kg of DTX or an equivalent dose of LNPDTX-P. The results show that LNPDTX-P-treated tumour samples had double the amount GNPs compared to control samples, both in vitro and in vivo. The results are very promising, as LNPDTX-P have superior targeting of tumour tissues compared to free DTX due to their nanosize and their ability to be functionalized. Because of their minimal toxicity to normal tissues, both GNPs and LNPDTX-P could be ideal radiosensitization candidates in radiotherapy and would produce very promising synergistic therapeutic outcomes.

Development of lipid nanoparticles and liposomes reference materials (II): cytotoxic profiles.

Lipid based nanocarriers are one of the most effective drug delivery systems that is evident from the recent COVID-19 mRNA vaccines. The main objective of this study was to evaluate toxicity of six lipid based formulations with three surface charges-anionic, neutral or cationic, to establish certified reference materials (CRMs) for liposomes and siRNA loaded lipid nanoparticles (LNP-siRNA). Cytotoxicity was assessed by a proliferation assay in adherent and non-adherent cell lines. High concentration of three LNP-siRNAs did not affect viability of suspension cells and LNP-siRNAs were non-toxic to adherent cells at conventionally used concentration. Systematic evaluation using multiple vials and repeated test runs of three liposomes and three LNP-siRNA formulations showed no toxicity in HL60 and A549 cells up to 128 and 16 µg/mL, respectively. Extended treatment and low concentration of LNPs did not affect the viability of suspension cells and adherent cells at 96 h. Interestingly, 80% of A549 and HL60 cells in 3D conditions were viable when treated with cationic LNP-siRNA for 48 h. Taken together, anionic, cationic and neutral lipid formulations were non-toxic to cells and may be explored further in order to develop them as drug carriers.

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy.

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active. Here we explore the use of RNA interference (RNAi) to target a universally conserved region of all AR splice variants for cleavage and degradation, thereby eliminating protein level resistance mechanisms. To this end, we tested five siRNA sequences designed against exon 1 of the AR mRNA and identified several that induced potent knockdown of full-length and truncated variant ARs in the 22Rv1 human prostate cancer cell line. We then demonstrated that 2'O methyl modification of the top candidate siRNA (siARvm) enhanced AR and AR-V7 mRNA silencing potency in both 22Rv1 and LNCaP cells, which represent two different prostate cancer models. For downstream in vivo delivery, we formulated siARvm-LNPs and functionally validated these in vitro by demonstrating knockdown of AR and AR-V7 mRNA in prostate cancer cells and loss of AR-mediated transcriptional activation of the PSA gene in both cell lines following treatment. We also observed that siARvm-LNP induced cell viability inhibition was more potent compared to LNP containing siRNA targeting full-length AR mRNA (siARfl-LNP) in 22Rv1 cells as their proliferation is more dependent on AR splice variants than LNCaP and PC3 cells. The in vivo biodistribution of siARvm-LNPs was determined in 22Rv1 tumor-bearing mice by incorporating 14C-radiolabelled DSPC in LNP formulation, and we observed a 4.4% ID/g tumor accumulation following intravenous administration. Finally, treatment of 22Rv1 tumor bearing mice with siARvm-LNP resulted in significant tumor growth inhibition and survival benefit compared to siARfl-LNP or the siLUC-LNP control. To best of our knowledge, this is the first report demonstrating therapeutic effects of LNP-siRNA targeting AR splice variants in prostate cancer.

Synthesis and Characterization of Hybrid Lipid Nanoparticles Containing Gold Nanoparticles and a Weak Base Drug.

Hybrid lipid nanoparticles containing gold nanoparticles (LNP-GNPs) and drugs have potential for imaging applications as well as triggered release of LNP contents in response to pulsed laser or X-ray radiation mediated by the GNPs. However, methods to synthesize LNP-GNP systems that efficiently entrap GNPs (the potential triggered release and imaging agent) and then load and retain the drug cargo in a manner that may have clinical applications have proven elusive. Here, we develop a straightforward "bottom-up" approach to manufacture drug-loaded LNP-GNP systems. We show that negatively charged GNPs of 5 nm diameter can be stably loaded into LNPs containing 10 mol % ionizable cationic lipid using an ethanol dilution, rapid mixing approach and that these systems also exhibit aqueous compartments. Further, we show that such systems can also entrap ammonium sulfate, enabling pH-dependent loading of the weak base anti-cancer drug doxorubicin into the aqueous compartments. Cryo-transmission electron microscopy (Cryo-TEM) imaging clearly demonstrates the presence of GNPs in the interior of the resulting hybrid nanostructures as well as the formation of electron-dense drug precipitates in the aqueous core of the LNP-GNPs. The approach described here is a robust and straightforward method to generate hybrid LNP-GNP-drug and other LNP-metal nanoparticle-drug systems with potential applications for a variety of triggered release protocols.

Bcar1/p130Cas is essential for ventricular development and neural crest cell remodelling of the cardiac outflow tract.

AIMS: The adapter protein p130Cas, encoded by the Bcar1 gene, is a key regulator of cell movement, adhesion, and cell cycle control in diverse cell types. Bcar1 constitutive knockout mice are embryonic lethal by embryonic days (E) 11.5-12.5, but the role of Bcar1 in embryonic development remains unclear. Here, we investigated the role of Bcar1 specifically in cardiovascular development and defined the cellular and molecular mechanisms disrupted following targeted Bcar1 deletions. METHODS AND RESULTS: We crossed Bcar1 floxed mice with Cre transgenic lines allowing for cell-specific knockout either in smooth muscle and early cardiac tissues (SM22-Cre), mature smooth muscle cells (smMHC-Cre), endothelial cells (Tie2-Cre), second heart field cells (Mef2c-Cre), or neural crest cells (NCC) (Pax3-Cre) and characterized these conditional knock outs using a combination of histological and molecular biology techniques. Conditional knockout of Bcar1 in SM22-expressing smooth muscle cells and cardiac tissues (Bcar1SM22KO) was embryonically lethal from E14.5-15.5 due to severe cardiovascular defects, including abnormal ventricular development and failure of outflow tract (OFT) septation leading to a single outflow vessel reminiscent of persistent truncus arteriosus. SM22-restricted loss of Bcar1 was associated with failure of OFT cushion cells to undergo differentiation to septal mesenchymal cells positive for SMC-specific α-actin, and disrupted expression of proteins and transcription factors involved in epithelial-to-mesenchymal transformation (EMT). Furthermore, knockout of Bcar1 specifically in NCC (Bcar1PAX3KO) recapitulated part of the OFT septation and aortic sac defects seen in the Bcar1SM22KO mutants, indicating a cell-specific requirement for Bcar1 in NCC essential for OFT septation. In contrast, conditional knockouts of Bcar1 in differentiated smooth muscle, endothelial cells, and second heart field cells survived to term and were phenotypically normal at birth and postnatally. CONCLUSION: Our work reveals a cell-specific requirement for Bcar1 in NCC, early myogenic and cardiac cells, essential for OFT septation, myocardialization and EMT/cell cycle regulation and differentiation to myogenic lineages.

The perplexing role of immuno-oncology drugs in osteosarcoma.

Osteosarcoma is a rare, primary tumour of bone. Curative treatment consists of multi-agent chemotherapy and complete surgical resection. Despite the use of multi-agent chemotherapy, the risk of recurrence is high. Survival outcomes for patients with osteosarcoma have not changed since the 1980's. Based on biologic rationale, there has been interest in adding immunotherapies to upfront curative intent chemotherapy, including mifamurtide (a macrophage activator) and interferon. However, results to date have been disappointing. In the metastatic setting, checkpoint inhibitors alone have not proven effective. Ongoing translational work is needed to further understand which patients may benefit from immune-oncology approaches with standard cytotoxic chemotherapy.

Modular Lipid Nanoparticle Platform Technology for siRNA and Lipophilic Prodrug Delivery.

Successfully employing small interfering RNA (siRNA) therapeutics requires the use of nanotechnology for efficient intracellular delivery. Lipid nanoparticles (LNPs) have enabled the approval of various nucleic acid therapeutics. A major advantage of LNPs is the interchangeability of its building blocks and RNA payload, which allow it to be a highly modular system. In addition, drug derivatization approaches can be used to synthesize lipophilic small molecule prodrugs that stably incorporate in LNPs. This provides ample opportunities to develop combination therapies by co-encapsulating multiple therapeutic agents in a single formulation. Here, it is described how the modular LNP platform is applied for combined gene silencing and chemotherapy to induce additive anticancer effects. It is shown that various lipophilic taxane prodrug derivatives and siRNA against the androgen receptor, a prostate cancer driver, can be efficiently and stably co-encapsulated in LNPs without compromising physicochemical properties or gene-silencing ability. Moreover, it is demonstrated that the combination therapy induces additive therapeutic effects in vitro. Using a double-radiolabeling approach, the pharmacokinetic properties and biodistribution of LNPs and prodrugs following systemic administration in tumor-bearing mice are quantitatively determined. These results indicate that co-encapsulating siRNA and lipophilic prodrugs into LNPs is an attractive and straightforward plug-and-play approach for combination therapy development.

Synthesis and Anti-HCV Activities of 4'-Fluoro-2'-Substituted Uridine Triphosphates and Nucleotide Prodrugs: Discovery of 4'-Fluoro-2'- C-methyluridine 5'-Phosphoramidate Prodrug (AL-335) for the Treatment of Hepatitis C Infection.

We report the synthesis and biological evaluation of a series of 4'-fluoro-2'- C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC50 values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.

Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA.

Lipid nanoparticles (LNPs) containing distearoylphosphatidlycholine (DSPC), and ionizable amino-lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) are potent siRNA delivery vehicles in vivo. Here we explore the utility of similar LNP systems as transfection reagents for plasmid DNA (pDNA). It is shown that replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro. Further, these formulations exhibited excellent transfection properties in a variety of mammalian cell lines and transfection efficiencies approaching 90% in primary cell cultures. These transfection levels were equal or greater than achieved by Lipofectamine, with much reduced toxicity. Finally, microinjection of LNP-eGFP into the limb bud of a chick embryo resulted in robust reporter-gene expression. It is concluded that LNP systems containing ionizable amino lipids can be highly effective, non-toxic pDNA delivery systems for gene expression both in vitro and in vivo.

The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA.

The therapeutic applications of lipid nanoparticle (LNP) formulations of small interfering RNA (siRNA), are hampered by inefficient delivery of encapsulated siRNA to the cytoplasm following endocytosis. Recent work has shown that up to 70% of endocytosed LNP-siRNA particles are recycled to the extracellular medium and thus cannot contribute to gene silencing. Niemann-Pick type C1 (NPC1) is a late endosomal/lysosomal membrane protein required for efficient extracellular recycling of endosomal contents. Here we assess the influence of NP3.47, a putative small molecule inhibitor of NPC1, on the gene silencing potency of LNP-siRNA systems in vitro. Intracellular uptake and colocalization studies revealed that the presence of NP3.47 caused threefold or higher increases in accumulation of LNP-siRNA in late endosomes/lysosomes as compared with controls in a variety of cell lines. The gene silencing potency of LNP siRNA was enhanced up to fourfold in the presence of NP3.47. Mechanisms of action studies are consistent with the proposal that NP3.47 acts to inhibit NPC1. Our findings suggest that the pharmacological inhibition of NPC1 is an attractive strategy to enhance the therapeutic efficacy of LNP-siRNA by trapping LNP-siRNA in late endosomes, thereby increasing opportunities for endosomal escape.

A Glu-urea-Lys Ligand-conjugated Lipid Nanoparticle/siRNA System Inhibits Androgen Receptor Expression In Vivo.

The androgen receptor plays a critical role in the progression of prostate cancer. Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor. Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration. Through these features, and by using a structurally refined cationic lipid and an optimized small interfering RNA payload, a lipid nanoparticle system with improved potency and significant therapeutic potential against prostate cancer and potentially other solid tumors was developed. Decreases in serum prostate-specific antigen, tumor cellular proliferation, and androgen receptor levels were observed in a mouse xenograft model following intravenous injection. These results support the potential clinical utility of a prostate-specific membrane antigen-targeted lipid nanoparticle system to silence the androgen receptor in advanced prostate cancer.

IGFBP2 is neither sufficient nor necessary for the physiological actions of leptin on glucose homeostasis in male ob/ob mice.

The ability of leptin to improve metabolic abnormalities in models of leptin deficiency, lipodystrophy, and even type 1 diabetes is of significant interest. However, the mechanism by which leptin mediates these effects remains ill-defined. Leptin was recently reported to regulate insulin-like growth factor-binding protein-2 (IGFBP2), and adenoviral overexpression of pharmacological levels of IGFBP2 ameliorates diabetic symptoms in many models of diabetes. We sought to determine the role of physiological levels of IGFBP2 in the glucoregulatory action of leptin. To investigate whether physiological levels of IGFBP2 are sufficient to mimic the action of leptin, we treated male ob/ob mice with low-dose IGFBP2 adenovirus (Ad-IGFBP2) or low-dose leptin. Despite similar levels of circulating IGFBP2, leptin but not Ad-IGFBP2 lowered body weight and plasma insulin and improved glucose and insulin tolerance. To elucidate the role of IGFBP2 in normal glucose homeostasis, we knocked down IGFBP2 in male C57BL/6 mice using small interfering RNA to determine whether this would recapitulate any aspect of the ob/ob phenotype. Despite successful IGFBP2 knockdown, body weight, blood glucose, and plasma insulin were unchanged. Finally, to determine whether IGFBP2 is required for the glucoregulatory actions of leptin, we prevented leptin-mediated increases in IGFBP2 in male ob/ob mice using RNA interference. Even though increases in IGFBP2 were blocked, the ability of leptin to decrease body weight, blood glucose, and plasma insulin levels were unaltered. In conclusion, physiological levels of IGFBP2 are neither sufficient to mimic nor required for the physiological action of leptin.

Influence of cationic lipid composition on uptake and intracellular processing of lipid nanoparticle formulations of siRNA.

The in vivo gene silencing potencies of lipid nanoparticle (LNP)-siRNA systems containing the ionizable cationic lipids DLinDAP, DLinDMA, DLinKDMA, or DLinKC2-DMA can differ by three orders of magnitude. In this study, we examine the uptake and intracellular processing of LNP-siRNA systems containing these cationic lipids in a macrophage cell-line in an attempt to understand the reasons for different potencies. Although uptake of LNP is not dramatically influenced by cationic lipid composition, subsequent processing events can be strongly dependent on cationic lipid species. In particular, the low potency of LNP containing DLinDAP can be attributed to hydrolysis by endogenous lipases following uptake. LNP containing DLinKC2-DMA, DLinKDMA, or DLinDMA, which lack ester linkages, are not vulnerable to lipase digestion and facilitate much more potent gene silencing. The superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to higher uptake and improved ability to stimulate siRNA release from endosomes subsequent to uptake. FROM THE CLINICAL EDITOR: This study reports on the in vivo gene silencing potency of lipid nanoparticle-siRNA systems containing ionizable cationic lipids. It is concluded that the superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to their higher uptake thus improved ability to stimulate siRNA release from endosome.

Small molecule ligands for enhanced intracellular delivery of lipid nanoparticle formulations of siRNA.

Gene silencing activity of lipid nanoparticle (LNP) formulations of siRNA requires LNP surface factors promoting cellular uptake. This study aimed to identify small molecules that enhance cellular uptake of LNP siRNA systems, then use them as LNP-associated ligands to improve gene silencing potency. Screening the Canadian Chemical Biology Network molecules for effects on LNP uptake into HeLa cells found that cardiac glycosides like ouabain and strophanthidin caused the highest uptake. Cardiac glycosides stimulate endocytosis on binding to plasma membrane Na(+)/K(+) ATPase found in all mammalian cells, offering the potential to stimulate LNP uptake into various cell types. A PEG-lipid containing strophanthidin at the end of PEG (STR-PEG-lipid) was synthesized and incorporated into LNP. Compared to non-liganded systems, STR-PEG-lipid enhanced LNP uptake in various cell types. Furthermore, this enhanced uptake improved marker gene silencing in vitro. Addition of STR-PEG-lipid to LNP siRNA may have general utility for enhancing gene silencing potency. FROM THE CLINICAL EDITOR: In this study, the authors identified small molecules that enhance cellular uptake of lipid nanoparticle siRNA systems, then used them as LNP-associated ligands to improve gene silencing potency.

Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo.

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using short-hairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP AR-siRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo.