Weidan Peng

Claudin-3 gene silencing with siRNA suppresses ovarian tumor growth and metastasis

Claudin-3 (CLDN3) is a tight junction protein that is overexpressed in 90% of ovarian tumors. Previous in vitro studies have indicated that CLDN3 overexpression promotes the migration, invasion, and survival of ovarian cancer cells. Here, we investigated the efficacy of lipidoid-formulated CLDN3 siRNA in 3 different ovarian cancer models. Intratumoral injection of lipidoid/CLDN3 siRNA into OVCAR-3 xenografts resulted in dramatic silencing of CLDN3, significant reduction in cell proliferation, reduction in tumor growth, and a significant increase in the number of apoptotic cells. Intraperitoneal injection of lipidoid-formulated CLDN3 siRNA resulted in a substantial reduction in tumor burden in MISIIR/TAg transgenic mice and mice bearing tumors derived from mouse ovarian surface epithelial cells. Ascites development was reduced in CLDN3 siRNA-treated mice, suggesting the treatment effectively suppressed metastasis. Toxicity was not observed after multiple i.p. injections. Importantly, treatment of mice with nonimmunostimulatory 2′-OMe modified CLDN3 siRNA was as effective in suppressing tumor growth as unmodifed siRNA. These results suggest that lipidoid-formulated CLDN3 siRNA has potential as a therapeutic for ovarian cancer.

Nanoparticle-Delivered Suicide Gene Therapy Effectively Reduces Ovarian Tumor Burden in Mice

There is currently no effective therapy for patients with advanced ovarian cancer. To address the need for a more effective treatment for this deadly disease, we conducted preclinical tests in ovarian tumor-bearing mice to evaluate the therapeutic efficacy of using a cationic biodegradable poly(beta-amino ester) polymer as a vector for nanoparticulate delivery of DNA encoding a diphtheria toxin suicide protein (DT-A). The promoter sequences of two genes that are highly active in ovarian tumor cells, MSLN and HE4, were used to target DT-A expression to tumor cells. Administration of DT-A nanoparticles directly to s.c. xenograft tumors and to the peritoneal cavity of mice bearing primary and metastatic ovarian tumors resulted in a significant reduction in tumor mass and a prolonged life span compared to control mice. Minimal nonspecific tissue and blood chemistry toxicity was observed following extended treatment with nanoparticles. DT-A nanoparticle therapy suppressed tumor growth more effectively than treatment with clinically relevant doses of cisplatin and paclitaxel. Our findings suggest that i.p. administration of polymeric nanoparticles to deliver DT-A encoding DNA, combined with transcriptional regulation to target gene expression to ovarian tumor cells, holds promise as an effective therapy for advanced-stage ovarian cancer.

Rapid Optimization of Gene Delivery by Parallel End-modification of Poly(β-amino ester)s

Poly(β-amino ester)s are cationic degradable polymers that have significant potential as gene delivery vectors. Here we present a generalized method to modify poly(β-amino ester)s at the chain ends to improve their delivery performance. End-chain coupling reactions were developed so that polymers could be synthesized and tested in a high-throughput manner, without the need for purification. In this way, many structural variations at the polymer terminus could be rapidly evaluated. End-modification of the terminal amine structure of a previously optimized poly(β-amino ester), C32, significantly enhanced its in vitro transfection efficiency. In vivo, intraperitoneal (IP) gene delivery using end-modified C32 polymers resulted in expression levels over one order of magnitude higher than unmodified C32 and jet-polyethylenimine (jet-PEI) levels in several abdominal organs. The rapid end-modification strategy presented here has led to the discovery of many effective polymers for gene delivery and may be a useful method to develop and optimize cationic polymers for gene therapy.

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth

Growing evidence shows that cancer cells use mRNA-binding proteins and miRNAs to posttranscriptionally regulate signaling pathways to adapt to harsh tumor microenvironments. In ovarian cancer, cytoplasmic accumulation of mRNA-binding protein HuR (ELAVL1) is associated with poor prognosis. In this study, we observed high HuR expression in ovarian cancer cells compared with ovarian primary cells, providing a rationale for targeting HuR. RNAi-mediated silencing of HuR in ovarian cancer cells significantly decreased cell proliferation and anchorage-independent growth, and impaired migration and invasion. In addition, HuR-depleted human ovarian xenografts were smaller than control tumors. A biodistribution study showed effective tumor-targeting by a novel Cy3-labeled folic acid (FA)-derivatized DNA dendrimer nanocarrier (3DNA). We combined siRNAs against HuR with FA-3DNA and found that systemic administration of the resultant FA-3DNA-siHuR conjugates to ovarian tumor-bearing mice suppressed tumor growth and ascites development, significantly prolonging lifespan. NanoString gene expression analysis identified multiple HuR-regulated genes that function in many essential cellular and molecular pathways, an attractive feature of candidate therapeutic targets. Taken together, these results are the first to demonstrate the versatility of the 3DNA nanocarrier for in vivo-targeted delivery of a cancer therapeutic and support further preclinical investigation of this system adapted to siHuR-targeted therapy for ovarian cancer.

DNA nanotherapy for pre-neoplastic cervical lesions

OBJECTIVE This study aims to test the hypothesis that targeted nanoparticle delivery of DNA encoding HPV16-regulated diphtheria toxin (DT-A) will result in the death of HPV16-infected cells. MATERIALS AND METHODS Plasmid constructs containing a HPV16 Long Control Region (LCR) DNA sequence upstream of DT-A or luciferase reporter (Luc) DNA sequences were used to formulate poly(β-amino ester) nanoparticles. The effect on tumor growth of HPV/DT-A-nanoparticle injection directly into HPV16(+) CaSki human cervical cancer cell-derived xenografts in mice was determined. To evaluate the ability of the HPV16 LCR regulatory sequence to activate gene expression specifically in HPV16-infected cells, mice underwent bioluminescent optical imaging following intraperitoneal injection of HPV/Luc-nanoparticles. The use of Lutrol F127, a thermal-sensitive gel, to target delivery of nanoparticles and subsequent gene expression to cervical epithelial cells was evaluated in ex vivo cultures of mouse cervix and following intravaginal delivery of nanoparticle/gel in mice, as well as in ex vivo cultures of surgical LEEP samples. RESULTS The selected HPV16 LCR regulatory sequence activates gene expression in both HPV16-infected cells and non-infected cells. However, in the cervix, it is specifically active in epithelial cells. Following exposure of cervical cells to HPV/DT-A-nanoparticles mixed with Lutrol F127 gel, DT-A is expressed and cells die. CONCLUSIONS An HPV16 DNA sequence that targets gene expression specifically to HPV16-infected cells remains to be discovered. Topical application of a Lutrol F127 thermal gel/nanoparticle mix is illustrative of how to restrict exposure of cells to therapeutic nanoparticles, thereby allowing for targeted DNA delivery to cervical pre-cancerous lesions.

Insights from HuR biology point to potential improvement for second-line ovarian cancer therapy

This retrospective study aimed to investigate the role that an RNA-binding protein, HuR, plays in the response of high-grade serous ovarian tumors to chemotherapeutics. We immunohistochemically stained sections of 31 surgically-debulked chemo-naïve ovarian tumors for HuR and scored the degree of HuR cytoplasmic staining. We found no correlation between HuR intracellular localization in tumor sections and progression free survival (PFS) of these patients, 29 of whom underwent second-line gemcitabine/platin combination therapy for recurrent disease. Ribonucleoprotein immunoprecipitation (RNP-IP) analysis of ovarian cancer cells in culture showed that cytoplasmic HuR increases deoxycytidine kinase (dCK), a metabolic enzyme that activates gemcitabine. The effects of carboplatin treatment on HuR and WEE1 (a mitotic inhibitor) expression, and on cell cycle kinetics, were also examined. Treatment of ovarian cancer cells with carboplatin results in increased HuR cytoplasmic expression and elevated WEE1 expression, arresting cell cycle G2/M transition. This may explain why HuR cytoplasmic localization in chemo-naïve tumors is not predictive of therapeutic response and PFS following second-line gemcitabine/platin combination therapy. These results suggest treatment of recurrent ovarian tumors with a combination of gemcitabine, carboplatin, and a WEE1 inhibitor may be potentially advantageous as compared to current clinical practices.

Abstract 700: Systemic administration of DNA nanoparticles containing the diphtheria toxin gene reduces pancreatic tumor load in mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite years of research and hundreds of clinical trials, our ability to treat metastatic pancreatic ductal adenocarcinoma (PDA) effectively has been thwarted due to the numerous genetic mutations in multiple core pathways that are characteristically present in pancreatic tumors, as well as the ability of tumors to develop resistance to chemotherapeutic drugs and small molecules. To avoid these complications, we have developed a suicide gene therapy that targets the death of PDA tumor cells regardless of their genetic mutation profile while avoiding unwanted harmful effects to healthy cells. A novel DNA nanoparticle, 3DNA, serving as a delivery vector is derivatized by conjugation to a DNA construct (MSLN promoter-driven diphtheria toxin A, pMSLN/DTA) as well as to a tumor-targeting molecule. Targeted DTA-induced tumor cell death is achieved in two ways: targeted nanoparticle delivery to tumor cells via docking and internalization, and targeted expression to cells that have an active mesothelin (MSLN) promoter (MSLN is highly overexpressed in ∼50% of PDA tumors). In preclinical tests, these formulations are systemically administered to PDA tumor-bearing mice. In an initial experiment, after confirming that MSLN is highly expressed in murine Pan02-luc PDA cells, we generated Pan02-luc orthografts in C57BL/6 mice and treated mice 2X/week with ∼50μg cargo DNA/i.v. injection for 4 weeks with either MSLN/DTA-TFP (n=7) or with MSLN/XX-TFP (n=5) dendrimer formulations (TFP is a peptide with strong affinity for the human transferrin receptor (TR), and weaker affinity for murine TR; XX = no coding sequence). Tumor load was assessed weekly by optical imaging and luciferase bioluminescence quantitation. Fold increase in relative light units (RLU) (baseline to 4 days after the last injection) was used to measure therapeutic efficacy. After 4 weeks of treatment, the mean RLU ratio for the MSLN/DTA-TFP treated mice was 0.51 +/-0.20 while that of the MSLN/XX-TFP treated mice was 7.1 +/-8.22, a significant 15-fold difference in tumor load (p=0.04). This quantitative analysis of tumor load as well as gross observations of tumors upon sacrifice of the mice showed that tumors in mice systemically treated with MSLN/DTA-TFP dendrimer formulation shrunk significantly to the point where tumor load was negligible in contrast to rapid growth of tumors in mice treated with the MSLN/XX-TFP formulation. This experiment demonstrates that systemically-administered DNA nanoparticle-delivered DTA effectively kills tumor cells with little to no apparent systemic toxicity as assessed by body weight and TUNEL assays of multiple normal tissues. In ongoing experiments to further validate this approach for the treatment of metastatic PDA, other targeting molecules such as folate and TFP are being used to target delivery to Pan02-luc orthografts and human BxPC3-luc2 xenografts. Citation Format: Janet A. Sawicki, Weidan Peng, Kelly Rhodes, Robert Getts. Systemic administration of DNA nanoparticles containing the diphtheria toxin gene reduces pancreatic tumor load in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2014-700