RNA-Mediated Reprogramming of Primary Adult Human Dermal Fibroblasts into c-kit(+) Cardiac Progenitor Cells.

Cardiovascular disease is the leading cause of death in the United States. Heart failure is a common, costly, and potentially fatal condition that is inadequately managed by pharmaceuticals. Cardiac repair therapies are promising alternative options. A potential cardiac repair therapy involves reprogramming human fibroblasts toward an induced cardiac progenitor-like state. We developed a clinically useful and safer reprogramming method by nonintegrative delivery of a cocktail of cardiac transcription factor-encoding mRNAs into autologous human dermal fibroblasts obtained from skin biopsies. Using this method, adult and neonatal dermal fibroblasts were reprogrammed into cardiac progenitor cells (CPCs) that expressed c-kit, Isl-1, and Nkx2.5. Furthermore, these reprogrammed CPCs differentiated into cardiomyocytes (CMs) in vitro as judged by increased expression of cardiac troponin T, α-sarcomeric actinin, RyR2, and SERCA2 and displayed enhanced caffeine-sensitive calcium release. The ability to reprogram patient-derived dermal fibroblasts into c-kit(+) CPCs and differentiate them into functional CMs provides clinicians with a potential new source of CPCs for cardiac repair from a renewable source and an alternative therapy in the treatment of heart failure.

Targeted disruption of ß-arrestin 2-mediated signaling pathways by aptamer chimeras leads to inhibition of leukemic cell growth.

UNLABELLED: ß-arrestins, ubiquitous cellular scaffolding proteins that act as signaling mediators of numerous critical cellular pathways, are attractive therapeutic targets because they promote tumorigenesis in several tumor models. However, targeting scaffolding proteins with traditional small molecule drugs has been challenging. Inhibition of ß-arrestin 2 with a novel aptamer impedes multiple oncogenic signaling pathways simultaneously. Additionally, delivery of the ß-arrestin 2-targeting aptamer into leukemia cells through coupling to a recently described cancer cell-specific delivery aptamer, inhibits multiple ß-arrestin-mediated signaling pathways known to be required for chronic myelogenous leukemia (CML) disease progression, and impairs tumorigenic growth in CML patient samples. The ability to target scaffolding proteins such as ß-arrestin 2 with RNA aptamers may prove beneficial as a therapeutic strategy. HIGHLIGHTS: An RNA aptamer inhibits ß-arrestin 2 activity.Inhibiting ß-arrestin 2 impedes multiple tumorigenic pathways simultaneously.The therapeutic aptamer is delivered to cancer cells using a cell-specific DNA aptamer.Targeting ß-arrestin 2 inhibits tumor progression in CML models and patient samples.

Identification and characterization of an agonistic aptamer against the T cell costimulatory receptor, OX40.

Induction of an effective immune response that can target and eliminate malignant cells or virus-infected cells requires the stimulation of antigen-specific effector T cells. A productive and long-lasting memory response requires 2 signals: a specific signal provided by antigen recognition through engagement of the T cell receptor and a secondary signal via engagement of costimulatory molecules (such as OX40) on these newly activated T cells. The OX40-OX40-ligand interaction is critical for the generation of productive effector and memory T cell functions. Thus agonistic antibodies that stimulate OX40 on activated T cells have been used as adjuvants to augment immune responses. We previously demonstrated that an aptamer modified to stimulate murine OX40 enhanced vaccine-mediated immune responses in a murine melanoma model. In this study, we describe the development of an agonistic aptamer that targets human OX40 (hOX40). This hOX40 aptamer was isolated using systematic evolution of ligands by exponential enrichment and binds the target purified protein with high affinity [dissociation constants (K(d))<10 nM]. Moreover, the hOX40 aptamer-streptavidin complex has an apparent binding affinity of ~50 nM for hOX40 on activated T cells as determined by flow cytometry and specifically binds activated human T cells. A multivalent version of the aptamer, but not a mutant version of the aptamer, was able to stimulate OX40 on T cells and enhance cell proliferation and interferon-gamma production. Future studies will assess the therapeutic potential of hOX40 aptamers for ex vivo stimulation of antigen specific T cells in conjunction with dendritic cell-based vaccines for adoptive cellular therapy.

Aptamer-mediated delivery of splice-switching oligonucleotides to the nuclei of cancer cells.

To reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer-mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing.

Ty1 reverse transcriptase does not read through the proposed 2',5'-branched retrotransposition intermediate in vitro.

2',5'-branched RNA was recently proposed as a key Ty1 retrotransposition intermediate, for which cleavage by lariat debranching enzyme (Dbr1p) enables reverse transcription to continue synthesizing the complete Ty1 cDNA. Because dbr1 cells can produce substantial Ty1 cDNA despite lacking Dbr1p, the obligatory intermediacy of branched RNA would require that Ty1 reverse transcriptase (RT) can read through the proposed branch site with considerable efficiency. Here we have used deoxyribozyme-synthesized 2',5'-branched RNA corresponding exactly to the proposed Ty1 branch site for a direct test of this read-through ability. Using an in vitro assay that incorporates all components known to be required for Ty1 cDNA synthesis (including the TyA chaperone protein), Ty1 RT can elongate up to the branch site. Strand transfer from the 2'-arm to the 3'-arm of the branch is observed when the Ty1 RT is RNase H+ (i.e., wild-type) but not when the Ty1 RT is RNase H-. When elongating from either the 2'-arm or the 3'-arm, Ty1 RT reads through the branch site with

A deoxyribozyme that synthesizes 2',5'-branched RNA with any branch-site nucleotide.

RNA molecules with internal 2',5'-branches are intermediates in RNA splicing, and branched RNAs have recently been proposed as retrotransposition intermediates. A broadly applicable in vitro synthetic route to branched RNA that does not require self-splicing introns or spliceosomes would substantially improve our ability to study biochemical processes that involve branched RNA. We recently described 7S11, a deoxyribozyme that was identified by in vitro selection and has general RNA branch-forming ability. However, an important restriction for 7S11 is that the branch-site RNA nucleotide must be a purine (A or G), because a pyrimidine (U or C) is not tolerated. Here, we describe the compact 6CE8 deoxyribozyme (selected using a 20 nt random region) that synthesizes 2',5'-branched RNA with any nucleotide at the branch site. The Mn2+-dependent branch-forming ligation reaction is between an internal branch-site 2'-hydroxyl nucleophile on one RNA substrate with a 5'-triphosphate on another RNA substrate. The preference for the branch-site nucleotide is U > C congruent with A > G, although all four nucleotides are tolerated with useful ligation rates. Nearly all other nucleotides elsewhere in both RNA substrates allow ligation activity, except that the sequence requirement for the RNA strand with the 5'-triphosphate is 5'-pppGA, with 5'-pppGAR (R = purine) preferred. These characteristics permit 6CE8 to prepare branched RNAs of immediate practical interest, such as the proposed branched intermediate of Ty1 retrotransposition. Because this branched RNA has two strands with identical sequence that emerge from the branch site, we developed strategies to control which of the two strands bind with the deoxyribozyme during the branch-forming reaction. The ability to synthesize the proposed branched RNA of Ty1 retrotransposition will allow us to explore this important biochemical pathway in greater detail.