Therapeutic double stranded RNA and methods for producing the same
Inventors
EQUELS, Thomas K. • ATODARIA, Vishwajeetsinh M. • SCOTT, Victoria G. • Strayer, David R. • RODINO, Peter W.
Assignees
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Publication Number
US-12312376-B2
Publication Date
2025-05-27
Expiration Date
Abstract
Disclosed is a method for the synthesis of a therapeutic double-stranded RNA (tdsRNA), comprising: a) synthesizing a first single-stranded RNA (first ssRNA) in a first synthesis reaction with PNPase as the only RNA polymerase; b) synthesizing a second single-stranded RNA (second ssRNA) in a second synthesis reaction with PNPase as the only RNA polymerase; and c) hybridizing the first ssRNA with the second ssRNA to form the tdsRNA; wherein step a) and step b) are performed in any order. Also disclosed is a product produced by the method.
Core Innovation
The invention relates to therapeutic double-stranded RNA (tdsRNA) synthesized by separately producing a first single-stranded RNA (first ssRNA) and a second single-stranded RNA (second ssRNA), and then hybridizing the first ssRNA with the second ssRNA to form the tdsRNA. The first ssRNA is synthesized in a first synthesis reaction using polynucleotide phosphorylase (PNPase) as the only RNA polymerase, and the second ssRNA is synthesized in a second synthesis reaction using PNPase as the only RNA polymerase. The order of performing the first and second synthesis reactions is interchangeable.
The synthesis of the first ssRNA is performed with inosine diphosphate (rIDP) as the only free ribonucleotide, while the synthesis of the second ssRNA is performed with cytidine diphosphate (rCDP) and uridine diphosphate (rUDP) as the only two free ribonucleotides. The method is performed without lyophilization. The tdsRNA formulas include rI_n·r(CxU)_n, rI_n·r(CxG)_n, and rA_n·rU_n, including rugged dsRNA that is partially hybridized and denaturation-resistant.
The invention further documents an approach for preparing the final tdsRNA without undesired nucleotide species and optionally purifying ssRNAs prior to hybridizing, followed by downstream handling such as filtering. Biological support described in the patent includes therapeutic evaluation in relation to SARS-CoV-2, including antiviral evaluation of rintatolimod and in vivo mouse survival and viral titer trends when combined with SARS-CoV-2 spike protein immunization.
Claims Coverage
The independent claim set contains 1 independent claim focused on a synthesis method for therapeutic tdsRNA. The inventive features center on using PNPase as the only RNA polymerase for separately making two ssRNAs, specifying the free ribonucleotide compositions, hybridizing the ssRNAs to form tdsRNA, allowing the synthesis steps to be performed in any order, and performing the method without lyophilization.
Pnpase-only synthesis of first and second ssRNA
Synthesizing a first single-stranded RNA (first ssRNA) in a first synthesis reaction with PNPase as the only RNA polymerase, and synthesizing a second single-stranded RNA (second ssRNA) in a second synthesis reaction with PNPase as the only RNA polymerase, wherein the steps are performed in any order.
Specified free ribonucleotide composition for first synthesis
The first synthesis reaction comprises inosine diphosphate (rIDP) as the only free ribonucleotide.
Specified free ribonucleotide composition for second synthesis
The second synthesis reaction comprises cytidine diphosphate (rCDP) and uridine diphosphate (rUDP) as the only two free ribonucleotides.
Hybridization to form therapeutic tdsRNA without lyophilization
Hybridizing the first ssRNA with the second ssRNA to form the tdsRNA, wherein the method is performed without lyophilization.
Ordered hybridization conditions
Hybridizing at 62°C to 68°C for 5 to 30 minutes, then hybridizing at 50°C for more than 30 minutes.
Optional ssRNA purification before hybridization
Purifying first and/or second ssRNA after synthesis and before hybridization, where the purification removes the ssRNA from PNPase and free ribonucleotides.
Post-hybridization filtering of tdsRNA
Purifying tdsRNA by filtering it through a 0.2 micron filter after the hybridization step.
The claim coverage centers on separately synthesizing first and second ssRNAs using PNPase as the only RNA polymerase, with rIDP alone in the first reaction and rCDP/rUDP as the only two free ribonucleotides in the second reaction, then hybridizing to form therapeutic tdsRNA without lyophilization. Additional features specify hybridization conditions, optional ssRNA purification before hybridization, and filtering after hybridization.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Therapeutic evaluation related to SARS-CoV-2, including antiviral evaluation of rintatolimod and in vivo mouse survival and viral titer trends when combined with SARS-CoV-2 spike protein immunization.
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