Multivalent RNA Composition Analysis Using IDR Sequences
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Solution Overview
Problem
Current methods for producing multivalent mRNA constructs are time and resource intensive, especially at Good Manufacturing Practice (GMP) scale, due to the need for individual transcription and purification of each mRNA product before mixing.
Innovation Solution
Incorporating unique identification and/or ratio determination (IDR) sequences into distinct RNA species allows for the analysis and characterization of multivalent RNA compositions by digesting RNA with RNase H guide oligonucleotides to release identifiable fragments, enabling the quantification of RNA species and determining their ratios, even when they share similar lengths or coding sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If individual transcription and purification of each mRNA product is performed before mixing, then the multivalent RNA composition can be produced with high purity and accuracy, but the production time and cost increase significantly
Solution Approach 1:
The patent divides the RNA species into distinct groups, each containing unique identification sequences (IDRs) at specific positions. This segmentation allows for simultaneous transcription of multiple RNA species from a single DNA template while maintaining the ability to individually identify and quantify each RNA type through its unique IDR signature, thereby eliminating the need for separate purification steps.
Solution Approach 2:
The patent introduces unique identification sequences (IDRs) as intermediary elements within each RNA species. These IDRs serve as mediators that enable differentiation and quantification of individual RNA species within the multivalent composition without requiring separate purification. The IDRs can be detected and measured to determine the presence and ratio of each RNA type in the mixture.
2Ease of manufacture
If multiple RNA species with similar lengths and coding sequences are mixed, then the multivalent composition can be simplified, but the difficulty of detecting and measuring individual RNA species increases
Solution Approach 1:
The patent applies local quality by placing unique identification sequences (IDRs) at specific local positions within each RNA species, such as in the 5' UTR, 3' UTR, or other non-coding regions. This localized differentiation allows each RNA species to maintain its functional coding sequence while possessing a unique identifiable marker at a specific location, enabling easy detection and measurement without affecting the overall simplicity of the multivalent composition.
Solution Approach 2:
The patent uses unique identification sequences (IDRs) as distinct markers for each RNA species, analogous to color changes. Each IDR sequence acts as a unique 'color' or signature that can be detected and differentiated, allowing for straightforward identification and quantification of individual RNA species within the multivalent composition even when they have similar lengths and coding sequences.
3Reliability
If coding sequences are modified to alter therapeutic protein structure, then the therapeutic efficacy can be improved, but the complexity of analyzing and characterizing the RNA composition increases
Solution Approach 1:
The patent incorporates unique identification sequences (IDRs) into each RNA species during the initial design and construction phase, before any coding sequence modifications are made. This preliminary action ensures that each RNA species has a built-in identifier that remains constant regardless of subsequent coding sequence modifications, allowing for consistent and simplified analysis and characterization even as therapeutic efficacy is improved through coding sequence optimization.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach simplifies the analysis and production of multivalent RNA compositions by allowing for the characterization and quantification of RNA species based on unique IDR sequences, reducing the complexity and cost associated with distinguishing and measuring multiple RNA species, and enabling the modification of coding sequences independently.
Implementation Method 1
contacting a multivalent RNA composition, comprising a first RNA species and a second RNA species, with two or more RNase H guide oligonucleotides
Implementation Method 2
digesting the first RNA species and the second RNA species with an RNase H enzyme to release a plurality of first RNA fragments and second RNA fragments
Data Source
AI summary
Aspects of the disclosure relate to methods for analyzing compositions comprising RNA species with unique nucleotide sequences for identification and/or ratio determination of RNAs. The disclosure is based, in part, on methods of cleaving identifying sequences from RNAs in a composition, and detecting the abundance of each identifying sequence to quantify the abundance of corresponding RNA species. Other aspects relate to methods for producing compositions comprising more than two RNA species, such as multivalent RNA compositions. The disclosure is based, in part, on methods of determining the proper amount of input DNA for in vitro transcription (IVT) reactions that will result in RNA being transcribed in a predetermined ratio. In some aspects, the disclosure relates to pharmaceutical compositions comprising multivalent RNA compositions produced by methods described, by the disclosure.