Paloma H. Giangrande

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type–specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.

The E2F1–3 transcription factors are essential for cellular proliferation

The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.

E2Fs link the control of G1/S and G2/M transcription

Previous work has provided evidence for E2F‐dependent transcription control of both G1/S‐ and G2/M‐regulated genes. Analysis of the G2‐regulated cdc2 and cyclin B1 genes reveals the presence of both positive‐ and negative‐acting E2F promoter elements. Additional elements provide both positive (CCAAT and Myb) and negative (CHR) control. Chromatin immunoprecipitation assays identify multiple interactions of E2F proteins that include those previously shown to activate and repress transcription. We find that E2F1, E2F2, and E2F3 bind to the positive‐acting E2F site in the cdc2 promoter, whereas E2F4 binds to the negative‐acting site. We also find that binding of an activator E2F is dependent on an adjacent CCAAT site that is bound by the NF‐Y transcription factor and binding of a repressor E2F is dependent on an adjacent CHR element, suggesting a role for cooperative interactions in determining both activation and repression. Finally, the kinetics of B‐Myb interaction with the G2‐regulated promoters coincides with the activation of the genes, and RNAi‐mediated reduction of B‐Myb inhibits expression of cyclin B1 and cdc2. The ability of B‐Myb to interact with the cdc2 promoter is dependent on an intact E2F binding site. These results thus point to a role for E2Fs, together with B‐Myb, which is an E2F‐regulated gene expressed at G1/S, in linking the regulation of genes at G1/S and G2/M.

Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and inhibit tumor growth in mice

4-1BB is a major costimulatory receptor that promotes the survival and expansion of activated T cells. Administration of agonistic anti-4-1BB Abs has been previously shown to enhance tumor immunity in mice. Abs are cell-based products posing significant cost, manufacturing, and regulatory challenges. Aptamers are oligonucleotide-based ligands that exhibit specificity and avidity comparable to, or exceeding, that of Abs. To date, various aptamers have been shown to inhibit the function of their cognate target. Here, we have described the development of an aptamer that binds 4-1BB expressed on the surface of activated mouse T cells and shown that multivalent configurations of the aptamer costimulated T cell activation in vitro and mediated tumor rejection in mice. Because aptamers can be chemically synthesized, manufacturing and the regulatory approval process should be substantially simpler and less costly than for Abs. Agonistic aptamers could therefore represent a superior alternative to Abs for the therapeutic manipulation of the immune system.

Myc requires distinct E2F activities to induce S phase and apoptosis.

Previous work has shown that the Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, we now show that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, we propose that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions.

Mapping and Characterization of the Functional Domains Responsible for the Differential Activity of the A and B Isoforms of the Human Progesterone Receptor

In humans, the biological response to progesterone is mediated by two distinct forms of the progesterone receptor (human (h) PR-A, 94 kDa and hPR-B, 114 kDa). These two isoforms are transcribed from distinct estrogen-inducible promoters within a single copy PR gene; the only difference between them is that the first 164 amino acids of hPR-B (B-upstream sequence) are absent in hPR-A. In most cell lines such as MCF-7 (human breast cancer cells), CV-1 (monkey kidney fibroblasts), and HeLa (human cervical carcinoma cells), hPR-A functions as a transcriptional repressor, whereas hPR-B functions as a transcriptional activator of progesterone-responsive genes. Interestingly, in these cell contexts, hPR-A also acts as a trans-dominant repressor of the transcriptional activity of other steroid hormone receptors. In contrast to hPR-A, which functions predominantly as a ligand-dependent transcriptional repressor, we show in this study that the A isoform of the chicken PR (cPR-A) lacks this trans-dominant repressor function and is a transcriptional activator in all contexts examined. By constructing chimeras between the N-terminal domains of the chicken and human PR, we mapped the trans-dominant repressor function of hPR-A to the first 140 amino acids of the protein. Notably, when this 140-amino acid “repressor” domain is placed onto chicken PR-A, the activity of the latter changes from a transcriptional activator to a repressor. Interestingly, however, this “repressor domain” is necessary, but not sufficient, for trans-repression as it is inactive when it is tethered to a heterologous protein. This suggests that the trans-repression function is comprised not only of the repressor domain of hPR-A but also requires the context of the receptor to function. The identification of a discrete inhibitory region within hPR-A which is transferable to another receptor implies that this region interacts with a set of transcription factors or adaptors that are distinct from those recognized by hPR-B, the identification of which will be required to define the mechanism by which hPR-A modulates steroid hormone receptor transcriptional activity. Thus, although chickens and humans both produce two very similar forms of the progesterone receptor, it is clear from these studies that the mechanism of action of progesterone in these two systems is quite different.

Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay

The main reason why tumours are not controlled by the immune system is that, unlike pathogens, they do not express potent tumour rejection antigens (TRAs). Tumour vaccination aims at stimulating a systemic immune response targeted to, mostly weak, antigens expressed in the disseminated tumour lesions. Main challenges in developing effective vaccination protocols are the identification of potent and broadly expressed TRAs and effective adjuvants to stimulate a robust and durable immune response. Here we describe an alternative approach in which the expression of new, and thereby potent, antigens are induced in tumour cells by inhibiting nonsense-mediated messenger RNA decay (NMD). Small interfering RNA (siRNA)-mediated inhibition of NMD in tumour cells led to the expression of new antigenic determinants and their immune-mediated rejection. In subcutaneous and metastatic tumour models, tumour-targeted delivery of NMD factor-specific siRNAs conjugated to oligonucleotide aptamer ligands led to significant inhibition of tumour growth that was superior to that of vaccination with granulocyte–macrophage colony-stimulating factor (GM-CSF)-expressing irradiated tumour cells, and could be further enhanced by co-stimulation. Tumour-targeted NMD inhibition forms the basis of a simple, broadly useful, and clinically feasible approach to enhance the antigenicity of disseminated tumours leading to their immune recognition and rejection. The cell-free chemically synthesized oligonucleotide backbone of aptamer–siRNAs reduces the risk of immunogenicity and enhances the feasibility of generating reagents suitable for clinical use.

Therapeutic Applications of DNA and RNA Aptamers

Structured single-stranded nucleic acids, or aptamers, bind target molecules with high affinity and specificity, which translates into unique therapeutic possibilities. Currently, aptamers can be identified to most proteins, including blood-clotting factors, cell-surface receptors, and transcription factors. Chemical modifications to the oligonucleotides enhance their pharmacokinetics and pharmacodynamics, thus extending their therapeutic potential. Several aptamers have entered the clinical pipeline for applications and diseases such as macular degeneration, coronary artery bypass graft surgery, and various types of cancer. Furthermore, the functional repertoire of aptamers has expanded with the descriptions of multivalent agonistic aptamers and aptamers-siRNA chimeras. This review highlights those aptamers and aptamer-based approaches with particular likelihood of achieving therapeutic application.

Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers

Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2+-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating ‘cell-type specific’, ‘cell-internalizing RNA ligands (aptamers)’ capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2+-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2+-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

Identification of E-Box Factor TFE3 as a Functional Partner for the E2F3 Transcription Factor

ABSTRACT Various studies have demonstrated a role for E2F proteins in the control of transcription of genes involved in DNA replication, cell cycle progression, and cell fate determination. Although it is clear that the functions of the E2F proteins overlap, there is also evidence for specific roles for individual E2F proteins in the control of apoptosis and cell proliferation. Investigating protein interactions that might provide a mechanistic basis for the specificity of E2F function, we identified the E-box binding factor TFE3 as an E2F3-specific partner. We also show that this interaction is dependent on the marked box domain of E2F3. We provide evidence for a role for TFE3 in the synergistic activation of the p68 subunit gene of DNA polymerase α together with E2F3, again dependent on the E2F3 marked box domain. Chromatin immunoprecipitation assays showed that TFE3 and E2F3 were bound to the p68 promoter in vivo and that the interaction of either E2F3 or TFE3 with the promoter was facilitated by the presence of both proteins. In contrast, neither E2F1 nor E2F2 interacted with the p68 promoter under these conditions. We propose that the physical interaction of TFE3 and E2F3 facilitates transcriptional activation of the p68 gene and provides strong evidence for the specificity of E2F function.