Lee Adam Wheeler

Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras.

The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4⁺ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.

Durable Knockdown and Protection From HIV Transmission in Humanized Mice Treated With Gel-formulated CD4 Aptamer-siRNA Chimeras

The continued spread of HIV underscores the need to interrupt transmission. One attractive strategy, in the absence of an effective vaccine, is a topical microbicide, but the need for application around the time of sexual intercourse leads to poor patient compliance. Intravaginal (IVAG) application of CD4 aptamer-siRNA chimeras (CD4-AsiCs) targeting the HIV coreceptor CCR5, gag, and vif protected humanized mice from sexual transmission. In non-dividing cells and tissue, RNAi-mediated gene knockdown lasts for several weeks, providing an opportunity for infrequent dosing not temporally linked to sexual intercourse, when compliance is challenging. Here, we investigate the durability of gene knockdown and viral inhibition, protection afforded by CCR5 or HIV gene knockdown on their own, and effectiveness of CD4-AsiCs formulated in a gel in polarized human cervicovaginal explants and in humanized mice. CD4-AsiC-mediated gene knockdown persisted for several weeks. Cell-specific gene knockdown and protection were comparable in a hydroxyethylcellulose gel formulation. CD4-AsiCs against CCR5 or gag/vif performed as well as a cocktail in humanized mice. Transmission was completely blocked by CCR5 CD4-AsiCs applied 2 days before challenge. Significant, but incomplete, protection also occurred when exposure was delayed for 4 or 6 days. CD4-AsiCs targeting gag/vif provided some protection when administered only after exposure. These data suggest that CD4-AsiCs are a promising approach for developing an HIV microbicide.

HIV DNA is heavily uracilated, which protects it from autointegration

Human immune cells infected by HIV naturally contain high uracil content, and HIV reverse transcriptase (RT) does not distinguish between dUTP and dTTP. Many DNA viruses and retroviruses encode a dUTPase or uracil-DNA glycosylase (UNG) to counteract uracil incorporation. However, although HIV virions are thought to contain cellular UNG2, replication of HIV produced in cells lacking UNG activity does not appear to be impaired. Here we show that HIV reverse transcripts generated in primary human immune cells are heavily uracilated (>500 uracils per 10 kb HIV genome). We find that HIV DNA uracilation, rather than being dangerous, may promote the early phase of the viral life cycle. Shortly after reverse transcription, the ends of the HIV DNA are activated by the viral integrase (IN) in preparation for chromosomal insertion. However, the activated ends can attack the viral DNA itself in a suicidal side pathway, called autointegration. We find here that uracilation of target DNA inhibits the strand transfer of HIV DNA ends by IN, thereby inhibiting autointegration and facilitating chromosomal integration and viral replication. When uracilation is increased by incubating uracil-poor cells in the presence of increasing concentrations of dUTP or by infecting with virus that contains the cytosine deaminase APOBEC3G (A3G), the proportion of reverse transcripts that undergo suicidal autointegration decreases. Thus, HIV tolerates, or even benefits from, nonmutagenic uracil incorporation during reverse transcription in human immune cells.

Gene Knockdown by EpCAM Aptamer–siRNA Chimeras Suppresses Epithelial Breast Cancers and Their Tumor-Initiating Cells

Effective therapeutic strategies for in vivo siRNA delivery to knockdown genes in cells outside the liver are needed to harness RNA interference for treating cancer. EpCAM is a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells (TIC, also known as cancer stem cells). Here, we show that aptamer–siRNA chimeras (AsiC, an EpCAM aptamer linked to an siRNA sense strand and annealed to the siRNA antisense strand) are selectively taken up and knock down gene expression in EpCAM+ cancer cells in vitro and in human cancer biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony and mammosphere formation (in vitro TIC assays) and tumor initiation by EpCAM+ luminal and basal-A triple-negative breast cancer (TNBC) cell lines, but not EpCAM− mesenchymal basal-B TNBCs, in nude mice. Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM+ Her2+ and TNBC tumors and suppress their growth. Thus, EpCAM-AsiCs provide an attractive approach for treating epithelial cancer. Mol Cancer Ther; 14(10); 2279–91. ©2015 AACR.

Phase II multicenter study of gene-mediated cytotoxic immunotherapy as adjuvant to surgical resection for newly diagnosed malignant glioma

BACKGROUND Despite aggressive standard of care (SOC) treatment, survival of malignant gliomas remains very poor. This Phase II, prospective, matched controlled, multicenter trial was conducted to assess the safety and efficacy of aglatimagene besadenovec (AdV-tk) plus valacyclovir (gene-mediated cytotoxic immunotherapy [GMCI]) in combination with SOC for newly diagnosed malignant glioma patients. METHODS Treatment cohort patients received SOC + GMCI and were enrolled at 4 institutions from 2006 to 2010. The preplanned, matched-control cohort included all concurrent patients meeting protocol criteria and SOC at a fifth institution. AdV-tk was administered at surgery followed by SOC radiation and temozolomide. Subset analyses were preplanned, based on prognostic factors: pathological diagnosis (glioblastoma vs others) and extent of resection. RESULTS Forty-eight patients completed SOC + GMCI, and 134 met control cohort criteria. Median overall survival (OS) was 17.1 months for GMCI + SOC versus 13.5 months for SOC alone (P = .0417). Survival at 1, 2, and 3 years was 67%, 35%, and 19% versus 57%, 22%, and 8%, respectively. The greatest benefit was observed in gross total resection patients: median OS of 25 versus 16.9 months (P = .0492); 1, 2, and 3-year survival of 90%, 53%, and 32% versus 64%, 28% and 6%, respectively. There were no dose-limiting toxicities; fever, fatigue, and headache were the most common GMCI-related symptoms. CONCLUSIONS GMCI can be safely combined with SOC in newly diagnosed malignant gliomas. Survival outcomes were most notably improved in patients with minimal residual disease after gross total resection. These data should help guide future immunotherapy studies and strongly support further evaluation of GMCI for malignant gliomas. CLINICAL TRIAL REGISTRY ClinicalTrials.gov NCT00589875.

P19-22. CD4-targeted delivery of HIV and CCR5 siRNAs by aptamer-siRNA chimeras suppresses HIV infection in primary cells and in human cervical explants

Background The therapeutic use of small interfering RNAs (siRNA) to prevent or treat HIV infection requires an effective means for in vivo delivery into susceptible target cells. Transfection of lymphocytes is especially difficult, even in vitro. Aptamers, which are small structured nucleic acid sequences that bind with high specificity to individual proteins, provide an attractive approach for cell-specific targeting.

RNAi as a potential new therapy for HIV infection

Controlling HIV infection continues to be a major clinical and scientific challenge. Despite the therapeutic benefits associated with HAART, the need for novel treatment approaches to combat HIV-1 remains. Effective inhibition of HIV-1 infection has been achieved by harnessing the endogenous RNAi pathway in a variety of cell types, including primary T cells and macrophages. Here we discuss the opportunities and challenges associated with translating these findings into clinically relevant therapeutic approaches.

Abstract LB-299: Gene knockdown by EpCAM aptamer-siRNA chimeras suppresses epithelial breast cancers and their tumor-initiating cells

Effective therapeutic strategies for in vivo siRNA delivery to knockdown genes in cells outside the liver are needed to harness RNA interference for treating cancer. EpCAM is a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells (T-IC, also known as cancer stem cells). Here we show that aptamer-siRNA chimeras (AsiC, an EpCAM aptamer linked to an siRNA sense strand and annealed to the siRNA antisense strand) are selectively taken up and knockdown gene expression in EpCAM+ cancer cells in vitro and in human cancer biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony and mammosphere formation (in vitro T-IC assays) and tumor initiation by EpCAM+ luminal and basal-A triple negative breast cancer (TNBC) cell lines, but not EpCAM- mesenchymal basal-B TNBCs, in nude mice. Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM+ Her2+ and TNBC tumors and suppress their growth. Thus EpCAM-AsiCs provide an attractive approach for treating epithelial cancer. Note: This abstract was not presented at the meeting. Citation Format: Adi Gilboa-Geffen, Peter Hamar, Lee Adam Wheeler, Radiana Trifonova, Fabio Petrocca, Anders Wittrup, Judy Lieberman. Gene knockdown by EpCAM aptamer-siRNA chimeras suppresses epithelial breast cancers and their tumor-initiating cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-299. doi:10.1158/1538-7445.AM2015-LB-299

Abstract CN01-02: Targeting basal-like TNBCs and epithelial tumor-initiating cells with aptamer-siRNA chimeras.

RNA interference (RNAi) offers the exciting therapeutic possibility of selectively knocking down disease-causing genes. Recent Phase I and II clinical trials of small interfering RNAs (siRNAs) have shown promising gene knockdown and clinical benefit in a handful of diverse diseases caused by aberrant liver gene expression. In these studies siRNAs were targeted to the liver by encapsulation in lipoplexes or by conjugation with a sugar selectively recognized by hepatocytes. The major obstacle to harnessing RNAi for treating cancer is the difficulty delivering RNAs into disseminated cancer cells. Giangrande, McNamara and Gilboa developed a flexible and effective multifunctional RNA delivery platform using aptamer-siRNA chimeras (AsiC), to accomplish this goal. RNA aptamers, structured RNAs that bind with high affinity to a protein, covalently linked to siRNAs, cause knockdown selectively in cells bearing the receptor recognized by the aptamer. Although treatment for breast cancer has improved with the development of targeted therapeutics, there is still a need for new approaches for poor prognosis breast cancers. In particular, there is no targeted therapy for triple negative breast cancers (TNBC), which often relapse after current therapy; Her2+ breast cancers frequently develop resistance to targeted therapy with trastuzumab or lapatinib. We chose EpCAM as the receptor for aptamer-targeted treatment of basal-like TNBC because EpCAM is highly expressed on the most malignant epithelial cancers and their tumor-initiating cells (T-ICs). In normal epithelia, EpCAM is only expressed on basolateral gap junctions, where it may not be accessible to drugs. In epithelial cancers, it is not only much more abundant (by several orders of magnitude), but is also distributed along the whole cell membrane. All basal-A TNBC and luminal breast cancer cell lines examined were strongly EpCAM+, while a normal breast cancer epithelial line and mesenchymal TNBCs had close to background EpCAM levels. RNA uptake, gene knockdown and the cytotoxic effect of EpCAM-AsiCs that knockdown PLK1 were robust in basal-like TNBC and luminal breast cancer lines, but not in normal epithelial cells and mesenchymal cells. In fact gene knockdown and antitumor effect correlated with EpCAM expression. EpCAM-AsiCs targeting PLK1, but not control siRNAs, eliminated T-ICs from epithelial TNBC and luminal breast cancer cell lines as assessed by in vitro assays for T-IC function-mammosphere and colony formation and survival of Aldefluor+ cells. These results suggest that EpCAM-AsiCs could be used to treat epithelial breast tumors and the T-ICs within them, sparing normal cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN01-02. Citation Format: Adi Gilboa-Geffen, Lee Adam Wheeler, Anders Wittrup, Fabio Petrocca, Judy Lieberman. Targeting basal-like TNBCs and epithelial tumor-initiating cells with aptamer-siRNA chimeras. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN01-02.