NIH/NCI 356: Tools and Technologies for Monitoring RNA

Fast-Track proposals will not be accepted.

Direct-to-Phase II will not be accepted.

Number of anticipated awards: 3-5

Budget (total costs, per award):

Phase I: $250,000 for 9 months;

Phase II: $1,500,000 for 2 years

PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED. 
 

Summary

Chemical modifications play a crucial role in the regulation of biological processes. Protein function is often modulated by tagging with phosphates, sugars, or lipids, while epigenomic marks on DNA or histones can regulate gene expression up or down. One area that lags behind is the mechanistic understanding of the role of RNA chemical modifications, sometimes referred to as the ‘epitranscriptome’.

The RNA Modification Database lists more than 60 RNA modifications that occur in eukaryotic cells. Transfer and ribosomal RNA have been shown to be heavily modified, and some of these same modifications also occur in messenger RNA and non-coding RNAs. However, the vast majority of these modifications have not been well-studied in messenger and non-coding RNAs. Even though much about RNA modifications remains to be elucidated, there is emerging evidence that RNA modifications are functionally significant and play important roles in biological processes and diseases in vertebrates.

Several RNA chemical modifications or the enzymes that catalyze the addition of modifications (writers), the removal of modifications (erasers), or translate the effects of modifications (readers) have been associated with a variety of cancers. For example, certain mutations in the N6-methyladenosine (m6A) demethylase (or ‘eraser’) FTO are associated with melanoma and breast cancer risk. Additionally, mutations in the pseudouridine ‘writer’ DKC1 cause dyskeratosis congenita, a disease associated with premature aging and increased tumor susceptibility. Furthermore, specific DKC1 mutations have been identified in human pituitary adenomas.

These early findings linking the disruption of RNA modifications to cancer initiation and progression highlight the potential importance of the field of epitranscriptomics to understanding cancer biology. However, a lack of experimental tools for monitoring RNA modifications has slowed the potential progress. The purpose of this topic is to incentivize small businesses to generate tools and technologies for monitoring covalently modified eukaryotic RNA.

Project Goals

The major obstacles hampering efforts to better understand RNA modifications are fundamentally technical in nature. Presently, we lack appropriate tools and technologies for investigating the epitranscriptome broadly and at single nucleotide resolution. Additionally, there is evidence that the availability of tools will drive research in this field. For example, an antibody-based assay for monitoring the m6A modification was developed in 2012, and by 2014 there had been a four-fold increase in the number of m6A publications.

Despite the growing interest in and importance of RNA modifications, the available tools that scientists have to monitor modified RNAs are limited. The purpose of this contract topic is to incentivize small businesses to generate tools, technologies, and products for monitoring covalently modified eukaryotic RNA, including messenger RNA and regulatory RNA. In the long term, these tools and products will allow the investigation of how altered RNA modifications contribute to the initiation and progression of cancer and potentially identify a new class of cancer biomarkers.

Potential tools, technologies, or products would include, but are not limited to:

• Well-validated antibodies, affinity reagents, or affinity-based assay kits for detection, quantitation, or immunoprecipitation of modified RNAs, or enzymes that write, erase, or bind to these modifications.
• Systems or kits that enable high-throughput mapping of specific RNA modifications to residues in individual RNA species using genome-wide sequencing approaches (i.e., approaches analogous to the bisulfite sequencing assays used for detecting methylcytosine or hydroxymethylcytosine in DNA).
• Approaches that enable researchers to sequence RNA without a cDNA intermediate or that otherwise preserve or amplify the RNA modification information.  This could include the development or adaptation of nanoscale sequencing devices or other equipment for direct identification and quantitation of sequence-specific RNA modifications.
• Approaches that exploit the ability of certain RNA modifications to disrupt reverse transcription.
• Assay systems or reagents that facilitate the discovery, detection or quantitation of modified RNAs.
• Products or systems that enable simultaneous detection of many types of RNA modifications at high sensitivity.
• Assay systems or reagents that enable researchers to monitor the effect of an RNA modification on the structure or function of an individual RNA.
• Products that would enable the in vitro or in vivo imaging of modified RNA molecules.
• The development of analytical software tools to facilitate the identification of modified, circular, or edited RNA from high-throughput sequencing datasets.  This could include algorithms that improve our ability to identify which base on a given RNA is modified.

Phase I Activities and Deliverables

The goal of Phase I is to develop proof-of-concept or prototype tools, technologies, or products for monitoring specific RNA modification(s). Activities and deliverables include:

• Identify and justify development of a tool or technology for monitoring a specific RNA modification or set of RNA modifications.
• Describe the current state of the art technologies, if any, for monitoring the specific RNA Modification(s) and outline the advantages that their approach will provide.
• Develop and characterize the tool or technology for monitoring the specific RNA Modification(s).
• Specify and justify quantitative milestones that can be used to evaluate the success of the tool or technology being developed.
• Develop an assay or system for testing and benchmarking the specificity and sensitivity of the tool or technology and comparing the tool or technology to existing approaches if applicable.
• Provide a proof-of-concept SOP for the tool or technology.

Phase II Activities and Deliverables

The goal of Phase II is an optimized commercial resource, product, reagent, kit, or device for monitoring specific RNA modification(s).

Decisions for continued product development into Phase II will be based on:

• Demonstration of the reliability and robustness of the tool, technology, or product. Offerors shall provide a technical evaluation and quality assurance plan with specific detail on shelf life, best practices for use, equipment required for use.
• Demonstration that the tool, technology, or product can be scaled up at a price point that is compatible with market success and widespread adoption by the basic research community.
• Demonstration of preliminary proof-of-concept data demonstrating the monitoring of the specific RNA Modification(s) in cell or animal cancer models with the potential to benchmark data across a variety of cancer models.

Deliverables and activities include:

• Scale up the synthesis and/or manufacture of necessary agents, chemicals, devices, or products.
• Design and implement quality assurance controls and assays to validate production.
• Validate scaled up tool, technology, or product. Specifically, demonstrate the utility, reliability, sensitivity, and specificity of the tool, technology, or product across relevant in vitro and/or in vivo cancer models (e.g., 2D and 3D tissue culture systems, in vivo animal models of cancer).
• Refine SOPs to allow for user friendly implementation of the tool, technology, or product by the target market.