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US-12618068-B2 - Replicase cycling reaction (RCR) and the related SamRNA designs thereof

US12618068B2US 12618068 B2US12618068 B2US 12618068B2US-12618068-B2

Abstract

This invention generally relates to a novel composition of RNA/mRNA medicines as well as vaccines produced by using replicase- and/or RNA-dependent RNA polymerase (RdRp)-mediated RNA cycling reaction (RCR). The present invention is useful for developing a variety of self-amplifying RNA/mRNA (samRNA) medicines and vaccines containing at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both, of any desired RNA molecule, including but not limited to antisense RNA (aRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)/miRNA precursor, long non-coding RNA (lnRNA) and mRNA. These RNA molecules can be either in single-stranded or in double-stranded, or mixed, conformation. The samRNA so obtained is useful not only for producing RNA-based vaccines and/or medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under a proper in-vitro or in-cell translation condition. The replicase/RdRp-binding sites used in samRNA are derived or modified from coronaviral (e.g. COVID-19) and/or hepatitis C viral (HCV) RNA-dependent RNA polymerases (RdRp) in either single-stranded or double-stranded compositions.

Inventors

  • Shi-Lung Lin
  • Sam Lin
  • Chun-Hung Lin

Assignees

  • Shi-Lung Lin
  • Sam Lin
  • Chun-Hung Lin

Dates

Publication Date
20260505
Application Date
20230118

Claims (14)

  1. 1 . A novel composition of self-amplifying RNA (samRNA), comprising: at least an isolated RNA sequence flanked with at least a 5′-end RdRp-binding site and at least a 3′-end RdRp-binding site, so as to form at least a samRNA platform construct; wherein said 5′-end RdRp-binding site contains a nucleotide sequence of UCWCYWA (SEQ ID NO: 2) and said 3′-end RdRp-binding site contains a nucleotide sequence of UWRGWR (SEQ ID NO: 4) and wherein said isolated RNA sequence is not a viral genome, wherein W comprises A/U; wherein Y comprises U/C; and wherein R comprises A/G.
  2. 2 . The composition as defined in claim 1 , wherein said 5′-end and 3′-end RdRp-binding sites contain over 57% complementarity to each other.
  3. 3 . The composition as defined in claim 1 , wherein said isolated RNA is single-stranded.
  4. 4 . The composition as defined in claim 1 , wherein said isolated RNA is double stranded.
  5. 5 . The composition as defined in claim 1 , wherein said isolated RNA contains modified nucleotide analogs.
  6. 6 . The composition as defined in claim 1 , wherein said isolated RNA is a pharmaceutical compound or composition.
  7. 7 . The composition as defined in claim 1 , wherein a plurality of said 5′-end and 3′-end RdRp-binding sites are used together in a combination.
  8. 8 . The composition as defined in claim 1 , wherein said isolated RNA is further formulated with at least a delivery agent for facilitating intracellular transfection.
  9. 9 . The composition as defined in claim 8 , wherein said delivery agent is liposomal nanoparticles (LNP).
  10. 10 . The composition as defined in claim 1 , wherein said samRNA is mRNA.
  11. 11 . The composition as defined in claim 1 , wherein said samRNA is precursor microRNA (pre-miRNA).
  12. 12 . The composition as defined in claim 1 , wherein said isolated RNA comprises a 5′-cap molecule.
  13. 13 . The composition as defined in claim 1 , wherein said isolated RNA comprises modified uridine nucleotides.
  14. 14 . The composition as defined in claim 1 , wherein said isolated RNA comprises at least a Kozak sequence.

Description

PRIORITY The present invention claims priority to U.S. Provisional Patent Applications No. 63/302,163 filed on Jan. 24, 2022, and No. 63/338,881 filed on May 5, 2022, both of which are entitled “Novel Replicase Cycling Reaction (RCR)”. The present invention also claims priority to U.S. Provisional Patent Application No. 63/429,150 filed on Dec. 1, 2022, which is entitled “Novel Replicase Cycling Reaction (RCR) and The Related SamRNA Designs Thereof”. Additionally, the present application is a continuation-in-part application of the U.S. patent application Ser. No. 17/489,357 filed on Sep. 29, 2021, which is entitled “Novel mRNA Composition and Production Method for Use in Anti-Viral and Anti-Cancer Vaccines”. The present application is also a continuation-in-part application of the U.S. patent application Ser. No. 17/648,336 filed on Jan. 19, 2022, which is entitled “Novel Replicase Cycling Reaction (RCR)”. Moreover, the present application is a continuation-in-part application of the U.S. patent application Ser. No. 17/648,340 filed on Jan. 19, 2022, which is entitled “Novel RNA Composition and Production Method for Use in iPS Cell Generation”. All aforementioned six prior patent applications are hereby incorporated by reference as if fully set forth herein. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (027706-00057-Sequence Listing.xml; Size: 6,879 bytes; and Date of Creation: Jan. 18, 2023) are herein incorporated by reference in its entirety. FIELD OF INVENTION This invention generally relates to a novel non-replicon-based self-amplifying RNA/mRNA (samRNA) composition capable of being produced by using RNA replicase- and/or RNA-dependent RNA polymerase (RdRp)-mediated cycling reaction (RCR). The present invention is useful for designing and developing a variety of self-amplifying RNA/mRNA (samRNA) medicines as well as vaccines containing at least a replicase/RdRp-binding site in the 5′-end or 3′-end, or both, of any desired RNA molecule, including but not limited to antisense RNA (aRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)/miRNA precursor (pre-miRNA), long noncoding RNA (lnRNA), and messenger RNA (mRNA). The conformation of these desired RNA molecules can be either single strand or double strand, or a mixture (like shRNA or pre-miRNA) thereof. The samRNA so obtained is useful not only for producing RNA/mRNA-based vaccines and medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under a proper in-vitro or in-cell translation condition. The replicase/RdRp-binding sites used in samRNA constructs are derived or modified from coronaviral (e.g. COVID-19) and/or hepatitis C viral (HCV) RdRp-binding sites in either single-stranded or double-stranded DNA/RNA compositions. BACKGROUND Prior polymerase chain reaction (PCR) is a method using thermostable DNA polymerases to amplify double-stranded DNA sequences from DNA templates, no involvement of any RNA material. Unlike PCR, newly invented RNA replicase/RdRp-mediated cycling reaction (RCR) uses RNA-dependent RNA polymerases (RdRp) to amplify single-stranded and/or double-stranded RNA/mRNA sequences from desired samRNA platform templates, no requirement of any DNA template. Clearly, PCR and RCR are very different methods and thus not comparable to each other. Hence, previous PCR studies are not related to RCR. As a result, the RCR-amplified RNA/mRNA (samRNA) products are herein neither related nor comparable to the PCR products as well. Lin et al. first reported RCR in year 2002 (WO2002/092774 to Lin). Lin had found that using a special design of 5′-cap-capture primers can trigger some viral and/or bacteriophage replicase-mediated RNA amplification from single-stranded or double-stranded RNA templates. This prior RCR mechanism may mimic the replication/amplification mechanisms of certain RNA viruses or bacteriophages, but not representing all kinds of RNA viruses. Particularly, the requirement of specific 5′-cap-capture primers limits its use because many RNA species do not carry 5′-cap molecules. Also, the linked 5′-cap-capture molecules may contaminate the resulting RNA products. For serving as RNA/mRNA medicines or vaccines, this contamination is problematic because removal of the 5′-cap-capture molecules from the RNA products is tedious and may cause RNA degradation. Hence, a new RCR method without using any 5′-cap-capture primer or molecule is highly desirable. In year 2012, Ahn et al reported a pair of 5′- and 3′-end RNA-dependent RNA polymerase (RdRp) binding sites isolated from severe acute respiratory syndrome coronavirus (SARS-CoV) (Ahn et al., Arch. Virol. 157:2095-2104, 2012). This pair of SARS-CoV RdRp-binding sites consists of minimal 36-37-nucleotide (nt)-long hairpin-like stem-loop RNA structures, which are however not compatible with PCR due to their lengthy and highly structured sequences. In order to prevent replicon/plasmi