Doherty, Caroline

Abstract Background and Objectives Cholangiocarcinoma (CCA) is an aggressive, highly lethal cancer of the biliary tract epithelium for which tumor resection is the only curative treatment. However, approximately half of patients present with nonresectable disease and median survival of these patients is just 8-12 months. A significant need exists for tumor-targeted therapeutics for patients with advanced disease who currently have limited treatment options. We aimed to address this need by developing an aptamer-targeted therapy for delivery of siRNAs inhibiting undruggable components of the Hippo pathway, a key driver of cholangiocarcinoma. Method We applied cell-SELEX against human CCA model cell line HuCCTl with negative selection against normal hepatocytes and cholangiocytes to identify an aptamer that selectively bound tumor cells. This aptamer, termed MC7122, was chemically modified to include fluorescent labels that enabled whole-animal biodistribution studies to assess in vivo selectivity. MC7122 was then deployed onto the surface of siRNA-loaded nanovesicles to drive tumor-targeted nucleic acid delivery. Tumor-bearing mice were treated with siRNAs three times per week for two weeks alone or in combination with standard of care chemotherapy. Results Combined treatment with siRNAs and standard of care chemotherapy reduced tumor burden in mice versus vehicle controls and slowed the spread of metastasis. Importantly, we found tumor-specific target gene knockdown without corresponding off-target knockdown in healthy surrounding liver tissue. Further, we demonstrated that sites in the lung, the primary location of metastasis in this model, were reduced not because of global target knockdown in lung but due to tumor-specific knockdown at the site of liver implantation. Conclusions Aptamer-guided vesicles represent a promising platform for targeted therapy delivery for previously undruggable targets. With this platform, tumor-selective therapies can be designed to exhibit exceptionally potent cytotoxicity, as healthy tissues remain unaffected by off-target effects. Future work will focus on refinement of therapeutic siRNA target selection and modification of nanovesicle components for enhanced levels of tumor specificity.

Abstract Glioblastoma (GBM) is an aggressive, high-grade glioma with a near-universally fatal prognosis. Therapeutic failure is often attributed to the highly selective blood brain barrier (BBB), the diffuse infiltrative nature of the tumor, and the marked intratumoral heterogeneity of GBM. Although antibody drug conjugates ADCs have shown promise for high grade gliomas such as GBM, efficacy is limited by ADC size. Aptamers—short, synthetic, single-stranded DNA or RNA molecules—can be ∼6-fold lower in molecular weight than IgG antibodies and have the potential to cross the intact BBB. Unlike other nucleic acid-based therapies, aptamer function arises from three-dimensional shape rather than genetic coding. Here we aim to replace the targeting component of the ADC paradigm with a DNA aptamer, thus creating an aptamer-drug conjugate (ApDC). We employed in vivo SELEX using an orthotopic patient-derived xenograft (PDX) GBM mouse model and a vast (∼100 trillion 80-mer sequences) ApDC library. We report the results from this first in vivo ApDC selection of its kind. We characterize target tissue binding ex vivo , cell association, biodistribution, and pharmacokinetics from this selection. This study exemplifies an unbiased approach to a problem that rational design has yet to overcome, offering a new direction for GBM therapeutic development.

ABSTRACT Cellular senescence is an irreversible form of cell‐cycle arrest caused by excessive stress or damage. While various biomarkers of cellular senescence have been proposed, there are currently no universal, stand‐alone indicators of this condition. The field largely relies on the combined detection of multiple biomarkers to differentiate senescent cells from non‐senescent cells. Here we introduce a new approach: unbiased cell culture selections to identify senescent cell‐specific folded DNA aptamers from vast libraries of trillions of random 80‐mer DNAs. Senescent mouse adult fibroblasts and their non‐senescent counterparts were employed for selection. We demonstrate aptamer specificity for senescent mouse cells in culture, identify a form of fibronectin as the molecular target of two selected aptamers, show increased aptamer staining in naturally aged mouse tissues, and demonstrate decreased aptamer staining when p16 expressing cells are removed in a transgenic INK‐ATTAC mouse model. This work demonstrates the value of unbiased cell‐based selections to identify new senescence‐specific DNA reagents.

Abstract Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor of adults. Current therapeutic options yield dismal prognoses that have remained essentially unchanged over nearly two decades. Diffuse growth patterns, high intratumoral heterogeneity, and variable blood–brain barrier integrity limit treatment efficacy, creating challenges that rational small molecule design has not overcome. Antibody–drug conjugates have shown some promise, leading us to hypothesize that smaller folded DNA aptamers, developed in vivo via principles of natural selection, might eventually have advantages for drug delivery. Here, we document the first in vivo DNA aptamer selection involving an orthotopic patient-derived xenograft GBM mouse model to identify tumor-homing DNA aptamers. We demonstrate the preferential accumulation of these aptamers in the tumor relative to other tissues 4 h after intraperitoneal injection. The aptamers can be detected by quantitative polymerase chain reaction, fluorescent tumor staining, and stain GBM tumor section from untreated mice and the GBM tumor cells in culture. Two of three candidates are selective for the target cell line in vitro and do not bind other human tumor cells. In vivo selection of tumor-specific DNA aptamers demonstrates a novel approach for diagnostics or toxin delivery that might allow for the development of individualized therapies.

Abstract Despite recent advances in the understanding of brain tumor pathophysiology, challenges associated with tumor location and characteristics have prevented significant improvement in neuro-oncology therapies. Aptamers are short, single-stranded DNA or RNA oligonucleotides that fold into sequence-specific, 3-dimensional shapes that, like protein antibodies, interact with targeted ligands with high affinity and specificity. Aptamer technology has recently been applied to neuro-oncology as a potential approach to innovative therapy. Preclinical research has demonstrated the ability of aptamers to overcome some obstacles that have traditionally rendered neuro-oncology therapies ineffective. Potential aptamer advantages include their small size, ability in some cases to penetrate the blood-brain barrier, inherent lack of immunogenicity, and applicability for discovering novel biomarkers. Herein, we review recent reports of aptamer applications in neuro-oncology including aptamers found by cell- and in vivo- Systematic Evolution of Ligands by Exponential Enrichment approaches, aptamer-targeted therapeutic delivery modalities, and aptamers in diagnostics and imaging. We further identify crucial future directions for the field that will be important to advance aptamer-based drugs or tools to clinical application in neuro-oncology.

Lung cancer is among the most common and lethal cancers and warrants novel therapeutic approaches to improving patient outcomes. Although immune checkpoint inhibitors (ICIs) have demonstrated substantial clinical benefits, most patients remain unresponsive to currently approved ICIs or develop resistance after initial response. Many ongoing clinical studies are investigating combination therapies to address the limited efficacy of ICIs. Here, we have assessed whether p53 gene therapy via a tumor-targeting nanomedicine (termed SGT-53) can augment anti-programmed cell death-1 (PD-1) immunotherapy to expand its use in non-responding patients. Using syngeneic mouse models of lung cancers that are resistant to anti-PD-1, we demonstrate that restoration of normal p53 function potentiates anti-PD-1 to inhibit tumor growth and prolong survival of tumor-bearing animals. Our data indicate that SGT-53 can restore effective immune responses against lung cancer cells by reducing immuno-suppressive cells (M2 macrophages and regulatory T cells) and by downregulating immunosuppressive molecules (e.g., galectin-1, a negative regulator of T cell activation and survival) while increasing activity of cytotoxic T cells. These results suggest that combining SGT-53 with anti-PD-1 immunotherapy could increase the fraction of lung cancer patients that responds to anti-PD-1 therapy and support evaluation of this combination particularly in patients with ICI-resistant lung cancers.

Because lung cancer remains the most common and lethal of cancers, novel therapeutic approaches are urgently needed. RB94 is a truncated form of retinoblastoma tumor suppressor protein with elevated anti-tumor efficacy. Our investigational nanomedicine (termed scL-RB94) is a tumor-targeted liposomal formulation of a plasmid containing the gene encoding RB94. In this research, we studied anti-tumor and immune modulation activities of scL-RB94 nanocomplex in preclinical models of human non-small cell lung cancer (NSCLC). Systemic treatment with scL-RB94 of mice bearing human NSCLC tumors significantly inhibited tumor growth by lowering proliferation and increasing apoptosis of tumor cells in vivo. scL-RB94 treatment also boosted anti-tumor immune responses by upregulating immune recognition molecules and recruiting innate immune cells such as natural killer (NK) cells. Antibody-mediated depletion of NK cells blunted the anti-tumor activity of scL-RB94, suggesting that NK cells were crucial for the observed anti-tumor activity in these xenograft models. Treatment with scL-RB94 also altered the polarization of tumor-associated macrophages by reducing immune-suppressive M2 macrophages to lower immune suppression in the tumor microenvironment. Collectively, our data suggest that the efficacy of scL-RB94 against NSCLC is due to an induction of tumor cell death as well as enhancement of innate anti-tumor immunity.