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Most cases of pancreatic cancer are not diagnosed until they are no longer curable with surgery. Therefore, it is critical to develop a sensitive, preferably noninvasive, method for detecting the disease at an earlier stage. In order to identify biomarkers for pancreatic cancer, we devised an in vitro positive/negative selection strategy to identify RNA ligands (aptamers) that could detect structural differences between the secretomes of pancreatic cancer and non-cancerous cells. Using this molecular recognition approach, we identified an aptamer (M9-5) that differentially bound conditioned media from cancerous and non-cancerous human pancreatic cell lines. This aptamer further discriminated between the sera of pancreatic cancer patients and healthy volunteers with high sensitivity and specificity. We utilized biochemical purification methods and mass-spectrometric analysis to identify the M9-5 target as cyclophilin B (CypB). This molecular recognition-based strategy simultaneously identified CypB as a serum biomarker and generated a new reagent to recognize it in body fluids. Moreover, this approach should be generalizable to other diseases and complementary to traditional approaches that focus on differences in expression level between samples. Finally, we suggest that the aptamer we identified has the potential to serve as a tool for the early detection of pancreatic cancer., Introduction At the time of diagnosis, the vast majority of patients with pancreatic cancer have locally advanced or distant metastatic disease that is not resectable (1). Earlier detection of the [...]

With an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most [...]

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., Introduction In addition to antigen-dependent TCR signaling, multiple costimulatory pathways regulate the potency, duration, and type of the T cell response. Experimental manipulation of costimulatory pathways offers an attractive way [...]

Intimal hyperplasia (IH) and restenosis limit the long-term utility of bypass surgery and angioplasty due to pathological proliferation and migration of vascular smooth muscle cells (VSMCs) into the intima of treated vessels. Consequently, much attention has been focused on developing inhibitory agents that reduce this pathogenic process. The E2F transcription factors are key cell cycle regulators that play important roles in modulating cell proliferation and cell fate. Nonselective E2F inhibitors have thus been extensively evaluated for this purpose. Surprisingly, these E2F inhibitors have failed to reduce IH. These findings prompted us to evaluate the roles of different E2Fs during IH to determine how selective targeting of E2F isoforms impacts VSMC proliferation. Importantly, we show that E2F3 promotes proliferation of VSMCs leading to increased IH, whereas E2F4 inhibits this pathological response. Furthermore, we use RNA probes to show that selective inhibition of E2F3, not global inhibition of E2F activity, significantly reduces VSMC proliferation and limits IH in murine bypass grafts. cell cycle | RNA therapeutics

Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent.

Author(s): Christopher P. Rusconi (corresponding author) [1]; Elizabeth Scardino [1]; Juliana Layzer [1]; George A. Pitoc [1]; Thomas L. Ortel [2]; Dougald Monroe [3]; Bruce A. Sullenger (corresponding author) [1] [...]

Author(s): Bruce A. Sullenger (corresponding author); Eli Gilboa The concept of using RNA molecules as therapeutic agents is relatively new, but has received increasing attention during the past decade. Much [...]

Research was conducted to examine the problem of low sequence specificity when using Tetrahymena group I ribozyme as an RNA repair agent. A systematic approach to solving the problem of low specificity was carried out using a ribozyme derivative with two mutations, each affecting catalysis differently. Results reveal that a detailed understanding of the biochemistry of a catalytic RNA can facilitate the design of customized ribozymes with improved properties for therapeutic applications.

A simple in vitro trans-splicing model system where both the cleavage and ligation steps of group I intron splicing take place is described. The exchange of ligands in the guanosine-binding site that must take place between the two steps also occur. It is shown by the model system that exogenous guanosine competes with omegaG for access to the guanosine-binding site during cleavage, while it is unable to compete with omegaG during ligation. The ribozyme orchestrates the relative affinities of the guanosine-binding site for free guanosine and omegaG, thus allowing the progression of the splicing reaction.

Sickle cell anemia is the most common heritable hematological disease, yet no curative treatment exists for this disorder. Moreover, the intricacies of globin gene expression have made the development of treatments for hemoglobinopathies based on gene therapy difficult. An alternative genetic approach to sickle cell therapy is based on RNA repair. A trans-splicing group I ribozyme was used to alter mutant Β-globin transcripts in erythrocyte precursors derived from peripheral blood from individuals with sickle cell disease. Sickle Β-globin transcripts were converted into messenger RNAs encoding the anti-sickling protein [Gamma]-globin. These results suggest that RNA repair may become a useful approach in the treatment of genetic disorders., The genetic basis of sickle cell disease is an A to T transversion in the sixth codon of the Β-globin gene (1). Individuals who are homozygous for this mutation accumulate [...]

A newly identified RNA sequence that binds a mouse monoclonal antibody for an autoantigenic epitope of human insulin receptor structurally resembles the antigenic epitope on the insulin receptor protein. The RNA ligand binds with high affinity only to the anti-insulin receptor antibody. The RNA also prevents the antibody from binding the insulin receptor and cross-reacts with autoantibodies upon high insulin resistance.

Targeted trans-splicing of ribozymes enables them to function as substitutes for defective RNA sections in a functional sequence, thus helping in the development of new treatments for genetic diseases. The self-splicing group I intron from Tetrahymena thermophila helps in the trans-splicing by reproducing the coding capacity of truncated lacZ transcripts. The in vitro trans-splicing yields translatable lacZ messages and constitutes a new method for the sequence modification of transcripts.

Cellular compartmentalization of RNAs is thought to influence their susceptibility to ribozyme cleavage. As a test of this idea, two retroviral vectors--one encoding a hammerhead ribozyme designed to cleave lacZ transcripts and another encoding the lacZ messenger RNA--were coexpressed inside retroviral packaging cells. Because of the retroviral packaging signal, the ribozyme would be expected to colocalize with the lacZ-containing viral genomic RNA but not with the lacZ messenger RNA. The ribozyme was found to reduce the titer of infectious virus containing lacZ by 90 percent, but had no effect on translation of lacZ messenger RNA. These results indicate that sorting gene inhibitors to appropriate intracellular sites may increase their effectiveness., The ability to target ribozymes to cleave viral RNAs in vitro has led to speculation about their potential therapeutic value as antiviral agents in vivo (1). To develop ribozymes for [...]

Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a [is greater than] 14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy., Group II introns are catalytic RNAs that function as mobile genetic elements by inserting directly into target sites in double-stranded DNA (1, 2). This mobility is mediated by a multifunctional [...]

Several groups have attempted to develop gene therapy strategies to treat cancer via introduction of the wild-type (wt) p53 cDNA into cancer cells. Unfortunately, these approaches do not result in regulated expression of the p53 gene and do not reduce expression of the mutant p53 that is overexpressed in cancerous cells. These shortcomings may greatly limit the utility of this gene replacement approach. We describe an alternative strategy with trans-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wt p53 activity in various human cancers. The ribozyme accomplished such conversion by repairing defective p53 mRNAs with high fidelity and specificity. The corrected transcripts were translated to produce functional p53 that can transactivate p53-responsive promoters and down-modulate expression of the multidrug resistance (MDR1) gene promoter. The level of wt p53 activity generated was significant, resulting in a 23-fold induction of a p53-responsive promoter and a 3-fold reduction in MDR1 promoter expression in transfected cancer cells. Once efficient delivery systems are developed, this strategy should prove useful for making human cancers more responsive to p53 activity and more sensitive to chemotherapeutic agents.

Destroy-the-messenger approach as opposed to a save-the-messenger is used to show that by turning the riboswitch from the destruction mode to the salvage mode, gene expression can be changed from very low levels to relatively high levels. To turn the riboswitch to the salvage mode, a regulator is added that binds the self-destruction motif, allowing the messenger RNA (mRNA) to survive and be translated into protein.