Patent Application
20230099592
The contents of the electronic sequence listing (sequencelisting.xml; Size: 8,723 bytes; and Date of Creation: Jul. 25, 2022) is herein incorporated by reference in its entirety.
This invention generally relates to a novel composition and method for RNA/mRNA production as well as amplification using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes and the use of associated RNA/mRNA products thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase/RdRp-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp) in either modified or non-modified mRNA and/or protein compositions, particularly including coronaviral (e.g. COVID-19) and hepatitis C viral (HCV) RdRp enzymes.
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, RNA replicase-mediated cycling reaction (RCR) uses RNA-dependent RNA polymerases (RdRp) to amplify single-stranded RNA sequences from RNA templates, no involvement of any DNA material. Clearly, PCR and RCR are very different and definitely not comparable. Hence, previous PCR studies are not related to RCR.
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-molecule-linked primers can trigger some viral and/or bacteriophage repliase-mediated RNA amplification from single-stranded RNA templates. This RCR mechanism mimics some viral or bacteriophage replication/amplification mechanisms, but not all. Nevertheless, the requirement of specific 5′-cap-capture-molecule-linked primers limits its use because many RNA species do not carry 5′-cap molecules. Also, the linked 5′-cap-capture molecules contaminate the resulting RNA products. For mRNA vaccine production, this 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-molecule-linked primer is highly desirable.
In year 2012, Ahn et al reported a pair of 5′- and 3′-end RNA-dependent RNA polymerase (RdRp) binding sites for initiating RNA synthesis using severe acute respiratory syndrome coronavirus (SARS-CoV) RdRp enzymes (Ahn et al., Arch. Virol. 157:2095-2104, 2012). This pair of SARS-CoV RdRp-binding sites consist of minimal 13˜37-nucleotide (nt)-long hairpin-like stem-loop RNA structures, which are however not compatible with PCR due to the lengthy and high structured sequences thereof. In routine practice, a conventional methodology of polymerase chain reaction-in vitro transcription (PCR-IVT) is commonly used to generate RdRp-amplifiable RNA templates, or called self-amplifying mRNAs/RNAs or samRNAs (
Recently, another RCR-like methodolgy may be anticipated by Bloom et al. (Gene Therapy 28:117-129, 2021), using Alphavirus RdRp and its associated binding/recognition site, an about 19-nt-long 3′-conserved sequence element (3′-CSE). Yet, no practical detail was provided for fulfilling cycling RNA amplification due to lack of any 5′-end binding site. Although Bloom's method may not use any 5′-cap-capture primer, the proposed 3′-CSE is too long and too structural to be incorporated into a desired RNA template using PCR. Particularly, the 19-nt 3′-CSE is too long and too structural to be placed into PCR primers. Also, because the 3′-CSE is a highly structured RNA sequence, it hinders RNA transcription (McDowell et al., Science 266:822-825, 1994) and thus can not be efficiently used in or with traditional IVT methods. Most problematically, the 3′-CSE is specifically recognized only by Alphavirus RdRp, which contains at least four distinct subunits and is however not commercially available, resulting in further hindering the development of its related technology. Given that the properties of different viral replicase/RdRp species are different, it is desirable to search and use another kind of replicase/RdRp enzymes with a more concise and less structural binding stie for overcoming the problems of prior RCR methods.
In view of the drawbacks of previous RCR methods, it is herein highly desirable to develop a novel RCR methodology not only using a more concise and less structural binding/recognition stie of replicase and/or RdRp but also without using any 5′-cap-capture primer for achieving highly efficient RNA/mRNA amplification and production.
The principle of the present invention is relied on the incorporation of at least a coronaviral (e.g. COVID-19) and/or hepatitis C viral (HCV) replicase/RdRp-binding (recognition) site into the 5′- or 3′-ends, or both, of desired RNA templates, leading to the cycling amplification of either the sense (+) strands or antisense (−) strands, or both, of the desired RNA sequences. In RCR, the defined replicase/RdRp-binding sites serve as a promoter and/or enhancer for initiating replicase/RdRp activities. As shown in
To prepare RCR-ready RNA templates, we first use reverse transcription-polymerase chain reaction (RT-PCR) or only PCR to incorporate at least a coronaviral (e.g. COVID-19) and/or HCV replicase/RdRp-binding site into the 5′- or 3′-ends, or both, of the complementary DNAs (cDNA) of desired RNA sequences. In our special design, at least a replicase/RdRp-binding site is synthetically embedded in each of the PCR primers (called RCR-ready PCR primers) and hence the cDNAs of RCR-ready RNA templates are formed after RT-PCR or PCR with the designed replicase/RdRp-binding sites incorporated in the 5′- or 3′-ends thereof, or both. Alternatively, the resulting cDNAs can be cloned into a plasmid or viral vector for further IVT reaction and/or storage preservation. For generating RCR-ready templates of desired RNAs, an IVT reaction is then performed to produce desired RCR-ready RNA templates from the cDNAs.
After that, the resulting RCR-ready RNA templates can be used in RCR to repeatedly amplify and produce the desired RNA sequences. Alternatively, in real practice, since the IVT and RCR can also be performed together simultaneously under exactly the same buffer condition, the replicase/RdRp-binding site-incorporated cDNAs (called RCR-ready cDNA templates) are herein also preferred to be used as a starting material for amplifying the desired RNA sequences in a combined IVT-RCR reaction.
After computer screening through over 17 strains of coronaviral and HCV RNA genomes, the present inventors had identified several conserved homologs of replicase/RdRp-binding sites, including 5′- and 3′-end RdRp-binding sites, respectively. As shown in our prior studies as well as in the claimed priority invention U.S. patent application Ser. No. 17/648,336, the sequences of coronaviral (e.g. COVID-19) and HCV RdRp-binding sites share very high similarity and compatibility, indicating that COVID-19 infection may significantly increase the sympton severity of HCV patients and vice versa. In addition to those previously found RdRp-binding sites described in our priority U.S. patent application Ser. No. 17/648,336 and Ser. No. 17/648,340, the present invention further provides a new design pair of 5′- and 3′-end RdRp-binding sites. The new 5′-end RdRp-binding site contains at least a sequence of 5′-UUCWACGCGU AG-3′ (SEQ ID NO:1) or 5′-UUCWWACGCG UAG-3′ (SEQ ID NO:2), or both, while the new 3′-end RdRp-binding site contains at least a sequence of SEQ ID NO:1 or SEQ ID NO:2, or both. This new pair of 5′- and 3′-end RdRp-binding sites is specially designed by the current inventors, modified from a reported stem-loop RdRp-binding site located in coronaviral mRNA (Hillen et al., Nature 584:154-159, 2020). Also, this new pair of RdRp-binding sites can be combined and/or used with the previously identified 5′- and 3′-end RdRp-binding sites described in our priority U.S. patent application Ser. No. 17/648,336 and Ser. No. 17/648,340. Notably, due to our modification, this new pair of RdRp-binding sites can provide multiple cycles of RCR reactions, whereas the previously reported Hillen's stem-loop binding site only achieves 3′-end RNA extension of one strand, but not cycling amplification of both strands because the reported large stem-loop structure can not be placed in the 5′-end of desired RNA templates (usually defined as the sense (+) strands), of which the position is complementary to the 3′-end of the resulting synthesized RNAs (usually defined as the antisense (−) strands). It is noted that the resulting antisense (−) strand RNAs will serve as new templates for the next cycle amplification of the sense-strand (+) RNAs. Since RdRp starts transcription from the 3′- to 5′-orientation (in the complementary direction) of an RNA template, any large (>7˜9-bp) stem-loop structure in the 3′-end of the resulting synthesized (−) RNAs (equivalent to the 5′-end of the desired (+) RNAs) will hinder the initiation of next cycle RNA synthesis of the desired RNAs.
Also, for facilitating RCR-ready PCR primer designs, the uridine/uracil (U) contents of these RdRp-binding sites can be replaced by thymidine (dT) and/or deoxyuridine (dU) in the primers. Moreover, for enhancing RNA stability, the uridine/uracil (U) contents of these RdRp-binding sites can be further replaced by pseudouridine, 5-methyluridine, 5-methoxyuridine, or any other proper modified nucleotide analog during IVT and/or RCR. Due to our findings and further designs of these novel RdRp-binding sites, the currently available coronaviral (COVID-19) and HCV RdRp enzymes can be used to efficiently transcribe and amplify either the sense (+) or antisense (−) strands, or both, of desired RNA/mRNA sequences in vitro, ex vivo as well as in vivo.
Based on our studies, a “Door-Lock” model theory has been proposed to demonstrate the interaction between RdRp and its 5′-/3′-end RdRp-binding sites. In this theory, from the view of a starting RNA template [defined as the sense (+) strand], the 3′-end RdRp-binding site on the starting RNA template is responsible for binding and locking RdRp into a stable position for new RNA synthesis to initiate and thus make a 5′-end RdRp-binding site in each of 5-′ ends of the newly synthesized RNAs [defined as the antisense (−) strand], while the 5′-end RdRp-binding site, which is complementary to the 3′-end RdRp-binding site, is responsible for releasing RdRp as well as the newly synthesized RNAs from the 3′-end RdRp-binding site and then pushing the RNA synthesis forward continuously on the antisense (complementary) strands from the 3′- to 5′-orientation. After that, the 5′- and 3′-end RdRp-binding sites of the newly synthesized (−) RNAs in turns serve the same functions as those of the RNA template to synthesize and amplify the desired sense-strand (+) RNAs. As a result, all of the synthesized RNAs from every cycle of RCR amplification carry the 5′- and 3′-end RdRp-binding sites in the both ends, ready for further cycling RNA synthesis Notably, the RNA templates and the resulting synthesized RNAs may contain the same or different 5′-end and/or 3′-end RdRp-binding sites, respectively. Also, the RNA templates and the resulting synthesized RNAs may contain single or multiple 5′-end and/or 3′-end RdRp-binding sites. The complementarity between 5′-end and 3′-end RdRp-binding sites can be partial (>57%˜99%) or perfect (100%) match to each other. Noteworthily, the combination of multiple 5′-end and/or 3′-end RdRp-binding sites can further enhance the RNA synthesis efficiency of replicase/RdRp enzymes. Theoretically, the synthesized RNAs and the desired RNA templates may form a double-stranded RNA conformation, which may also enhance RdRp-mediated RNA synthesis as well. Hence, the starting RNA templates may be used in either single- or double-strand conformation, or both.
In view of the proposed “Door-Lock” theory, it clearly shows the differences between the present invention and the previous Ahn's and Bloom's methodologies. Both Ahn and Bloom et al consider that highly structured hairpin-like stem-loop binding sites are required to initiate RdRp activity, while our findings demonstrate that only the right pairs of complementary 5′- and 3′-end RdRp-binding sites are required, but not the loop structures. Our studies demonstrate that the 5′- and 3′-end RdRp-binding sites form a complementary pair to stabilize the binding of RdRp on a desired RNA template and then form a reaction circle to activate the RNA synthesis of the reverse, complementary strand RNAs and vice versa. Hence, any loop structure in the 5′-end RdRp-binding site will hinder the reaction circle to start the next cycle of RNA synthesis. As a result, Ahn's and Bloom's methodologies can only provide 3′-end RNA extension of one strand, while the present invention can achieve multiple cycles of amplification of both sense (+) and antisense (−) RNA strands.
As shown in the present invention as well as our priority invention U.S. patent application Ser. No. 17/648,336, the combinations of various 5′- and 3′-end RdRp-binding sites can provide different RNA amplification rates, depending on the length and structural complexity of the desired RNA sequences. Due to such a different amplification preference, we can design and use different combinations of these RdRp-binding sites to selectively amplify one RNA strand over the other strand or one kind of RNA strands over the other kind of RNA strands. By this means, a relatively pure single-stranded and/or double-stranded RNA products of the desired RNA/mRNA sequence(s) can be generated, obtained and collected in RCR and then further purified by other methodologies.
In one preferred embodiment, the desired RNA sequence (i.e, mRNA and/or microRNA, or any other kind of RNA species) contains at least an RdRp-binding site in both of its 5′- and 3′-end regions. Since both ends of the desired RNA carry at least an RdRp-binding site for RNA amplification with replicase/RdRp activities, the sense-strand RNA sequences can be used to amplify its complementary antisense RNAs (cRNA or aRNA), while the antisense-strand RNA sequences can be used to amplify the sense RNAs as well, so as to form an amplification cycle of both of the sense- and antisense-strand RNAs and thus resulting in a maximal amplification rate of the desired RNAs. The desired RNAs so obtained can be in either single-stranded or double-stranded conformation, depending on the stop point of RCR. Also, the resulting sense- and antisense-strand RNAs may further form double-stranded RNAs, facilitating the generation of siRNAs, shRNAs, miRNAs, and/or piRNAs of the desired RNA sequences.
Alternatively, in another preferred embodiment, the desired RNA sequence contains at least an RdRp-binding site in its either 5′-end or 3′-end region. In this way, we can selectively amplify either the sense- or antisense-strand of the desired RNA, leading to more specific amplification of the desired RNA strand. Particularly, this approach is useful for generating and amplifying either the mRNA or the antisense RNA (aRNA) of a specific functional protein, viral antigen or antibody, facilitating the development of mRNA vaccines and/or RNA/antibody-based medicines. The mRNA vaccines and RNA/antibody-based medicines so obtained may help to treat a variety of human diseases, including but not limited to Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, myocardial infraction, hemophilia, anemia, leukemia, and many kinds of cancers as well as many kinds of viral and bacterial infections.
Conceivably, our new RCR methodology can be used to produce and amplify all varieties of RNA species carrying at least an RdRp binding site, particularly viral antigen mRNAs and/or known functional RNAs/mRNAs, which are useful for developing anti-viral and/or anti-disease vaccines as well as medicines, and likely many more. For example, by co-transfection of RCR-ready RNA templates and an isolated coronaviral RdRp mRNA into human somatic cells, our two US priority patent applications (U.S. Provisional Patent Applications No. 63/270,034 and No. 63/280,226 to Lin) had demonstrated a novel method for iPS cell generation. To this, an ordinary skill person in the art can anticipate the use of three-/four-Yamanaka-factor mRNAs (i.e. Oct4/3, Sox2, Nanog and/or Lin-28) to replace the claimed miR-302 precursor microRNA (pre-miRNA) for iPS cell generation as well. Alternatively, as shown in our another priority patent application (U.S. patent application Ser. No. 17/489,357 to Lin), we had developed a new design of RdRp-mediated self-amplifiable RNAs (saRNA) for generating novel mRNA vaccines as well as medicines for treating viral infections and cancers, respectively. Moreover, the RCR-amplified mRNAs can be further used in an in-vitro translation system for producing the encoded proteins, peptides and/or antibodies of interest. In view of these prior invention-proved achievements, many more developments of potential applications of the present invention are highly expected.
To efficiently produce highly structured RNA templates and RdRp mRNA, our priority patent application (U.S. patent application Ser. No. 17/489,357 to Lin) had developed a novel PCR-IVT methodology for overcoming the low efficiency problem of highly structured RNA generation. Traditionally, it is not reasonable for an ordinary skill person in the art to anticipate the effective generation of highly structured RNAs in vitro because it is known that the presence of hairpin- and/or stem-loop-like RNA structures greatly hinders RNA transcription. In fact, hairpin-like stem-loop structures are signals of intrinsic transcription termination for prokaryotic RNA polymerases (McDowell et al, Science 266:822-825, 1994). To solve this problem, our priority method adopts a new IVT system with a mixture of RNA polymerase and helicase activities. The additional helicase activity used in IVT (and likely in RCR as well) markedly reduces the secondary structures of both DNA/RNA templates and the resulting RNA products for far more efficiently producing highly structured RNAs. Accordingly, an improved buffer system is also used to maintain and enhance the efficiency of mixed RNA polymerase/replicase and helicase activities in IVT (and RCR as well). Interestingly, although several prior studies had reported that helicase may be involved in prokaryotic transcription termination, our studies however demonstrate a totally different functionality of helicase in RNA amplification during IVT (and IVT-RCR).
For facilitating intracellular delivery/transfection in vitro, ex vivo or in vivo, the RCR-ready cDNA/RNA template(s) and RdRp mRNA can be mixed, conjugated, encapsulated and/or formulated with at least a delivery/transfection agent selected from, but not limited to, glycylglycerin-derived chemicals, liposomes, nanoparticles, liposomal nanoparticles (LNP), conjugating molecules, infusion/transfusion chemicals, gene gun materials, electroporation agents, transposons/retrotransposons, and a combination thereof.
The advantages of using RCR-ready cDNA/RNA templates for RNA/mRNA production and amplification include (1) high RNA yield rate, (2) high RNA purity, (3) easy preparation in that all reaction materials can be made into a biochemical enzyme kit for performing RCR and/or combined IVT-RCR reactions, (4) simple reaction procedure compatible with other RT-PCR and IVT reactions, (5) simple equipment requirement which can be easily accomplished using a PCR machine or a temperature-controlled incubator, and (6) a variety of potential applications. As a result, it is conceivable that the RCR-ready cDNA/RNA templates of the present invention are very useful for producing and amplifying a variety of desired RNA/mRNA sequences, which can then be used in all sorts of pharmaceutical and therapeutic applications, including but not limited to the development of mRNA vaccines and RNA/microRNA-associated medicines as well as protein/peptide/antibody generation.
To facilitate understanding of the invention, a number of terms are defined below:
Nucleic Acid: a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), either single or double stranded.
Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of the pentose) and that combination of base and sugar is a nucleoside. A nucleoside containing at least one phosphate group bonded to the 3′ or 5′ position of the pentose is a nucleotide. DNA and RNA are consisted of different types of nucleotide units called deoxyribonucleotide and ribonucleotide, respectively.
Deoxyribonucleoside Triphosphates (dNTPs): the building block molecules for DNA synthesis, including dATP, dGTP, dCTP, and dTTP and sometimes may further containing some modified deoxyribonucleotide analogs.
Ribonucleoside Triphosphates (rNTPs): the building block molecules for RNA synthesis, including ATP, GTP, CTP, and UTP and sometimes may further containing pseudouridine, 5′ methyluridine, methoxyuridine, and/or some other modified ribonucleotide analogs.
Nucleotide Analog: a purine or pyrimidine nucleotide that differs structurally from adenine (A), thymine (T), guanine (G), cytosine (C), or uracil (U), but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule.
Oligonucleotide: a molecule comprised of two or more monomeric units of DNA and/or RNA, preferably more than three, and usually more than ten. An oligonucleotide longer than 13 nucleotide monomers is also called polynucleotiude. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, RNA transcription, reverse transcription, or a combination thereof.
Nucleic Acid Composition: a nucleic acid composition refers to an oligonucleotide or polynucleotide such as a DNA or RNA sequence, or a mixed DNA/RNA sequence, in either a single-stranded or a double-stranded molecular structure.
Gene: a nucleic acid composition whose oligonucleotide or polynucleotide sequence codes for an RNA and/or a polypeptide (protein). A gene can be either RNA or DNA. A gene may encode a non-coding RNA, such as small hairpin RNA (shRNA), microRNA (miRNA), rRNA, tRNA, snoRNA, snRNA, and their RNA precursors as well as derivatives. Alternatively, a gene may encode a protein-coding RNA essential for protein/peptide synthesis, such as messenger RNA (mRNA) and its RNA precursors as well as derivatives. In some cases, a gene may encode a protein-coding RNA that also contains at least a microRNA or shRNA sequence.
Primary RNA Transcript: an RNA sequence that is directly transcribed from a gene without any RNA processing or modification.
Precursor messenger RNA (pre-mRNA): primary RNA transcripts of a protein-coding gene, which are produced by eukaryotic type-II RNA polymerase (Pol-II) machineries in eukaryotes through an intracellular mechanism termed transcription. A pre-mRNA sequence contains a 5′-untranslated region (UTR), a 3′-UTR, exons and introns.
Intron: a part or parts of a gene transcript sequence encoding non-protein-reading frames, such as in-frame intron, 5′-UTR and 3′-UTR.
Exon: a part or parts of a gene transcript sequence encoding protein-reading frames (cDNA), such as cDNA for cellular genes, growth factors, insulin, antibodies and their analogs/homologs as well as derivatives.
Messenger RNA (mRNA): assembly of pre-mRNA exons, which is formed after intron removal by intracellular RNA splicing machineries (e.g. spliceosomes) and served as a protein-coding RNA for peptide/protein synthesis. The peptides/proteins encoded by mRNAs include, but not limited, enzymes, growth factors, insulin, antibodies and their analogs/homologs as well as derivatives.
Complementary DNA (cDNA): a single-stranded or double-stranded DNA that contains a sequence complementary to an mRNA sequence and does not contain any intronic sequence.
Sense: a nucleic acid molecule in the same sequence order and composition as the homologous mRNA. The sense conformation is indicated with a “+”, “s” or “sense” symbol.
Antisense: a nucleic acid molecule complementary to the respective mRNA molecule. The antisense conformation is indicated as a “—” symbol or with an “a” or “antisense” in front of the DNA or RNA, e.g., “aDNA” or “aRNA”.
Base Pair (bp): a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA, uracil (U) is substituted for thymine. Generally the partnership is achieved through hydrogen bonding. For example, a sense nucleotide sequence “5′-A-T-C-G-U-3” can form complete base pairing with its antisense sequence “5′-A-C-G-A-T-3”.
5′-end: a terminus lacking a nucleotide at the 5′ position of successive nucleotides in which the 5′-hydroxyl group of one nucleotide is joined to the 3′-hydroyl group of the next nucleotide by a phosphodiester linkage. Other groups, such as one or more phosphates, may be present on the terminus.
3′-end: a terminus lacking a nucleotide at the 3′ position of successive nucleotides in which the 5′-hydroxyl group of one nucleotide is joined to the 3′-hydroyl group of the next nucleotide by a phosphodiester linkage. Other groups, most often a hydroxyl group, may be present on the terminus.
Template: a nucleic acid molecule being copied by a nucleic acid polymerase. A template can be single-stranded, double-stranded or partially double-stranded, RNA or DNA, depending on the polymerase. The synthesized copy is complementary to the template, or to at least one strand of a double-stranded or partially double-stranded template. Both RNA and DNA are synthesized in the 5′ to 3′ direction. The two strands of a nucleic acid duplex are always aligned so that the 5′ ends of the two strands are at opposite ends of the duplex (and, by necessity, so then are the 3′ ends).
Nucleic Acid Template: a double-stranded DNA molecule, double-stranded RNA molecule, hybrid molecules such as DNA-RNA or RNA-DNA hybrid, or single-stranded DNA or RNA molecule.
Conserved: a nucleotide sequence is conserved with respect to a pre-selected (referenced) sequence if it non-randomly hybridizes to an exact complement of the pre-selected sequence.
Homologous or Homology: a term indicating the similarity between a polynucleotide and a gene or mRNA sequence. A nucleic acid sequence may be partially or completely homologous to a particular gene or mRNA sequence, for example. Homology may be expressed as a percentage determined by the number of similar nucleotides over the total number of nucleotides.
Complementary or Complementarity or Complementation: a term used in reference to matched base pairing between two polynucleotides (i.e. sequences of an mRNA and a cDNA) related by the aforementioned “base pair (bp)” rules. For example, the sequence “5′-A-G-T-3” is complementary to not only the sequence “5′-A-C-T-3” but also to “5′-A-C-U-3”. Complementation can be between two DNA strands, a DNA and an RNA strand, or between two RNA strands. Complementarity may be “partial” or “complete” or “total”. Partial complementarity or complementation occurs when only some of the nucleic acid bases are matched according to the base pairing rules. Complete or total complementarity or complementation occurs when the bases are completely or perfectly matched between the nucleic acid strands. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as in detection methods that depend on binding between nucleic acids. Percent complementarity or complementation refers to the number of mismatch bases over the total bases in one strand of the nucleic acid. Thus, a 50% complementation means that half of the bases were mismatched and half were matched. Two strands of nucleic acid can be complementary even though the two strands differ in the number of bases. In this situation, the complementation occurs between the portion of the longer strand corresponding to the bases on that strand that pair with the bases on the shorter strand.
Complementary Bases: nucleotides that normally pair up when DNA or RNA adopts a double stranded configuration.
Complementary Nucleotide Sequence: a sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to that on another single strand to specifically hybridize between the two strands with consequent hydrogen bonding.
Hybridize and Hybridization: the formation of duplexes between nucleotide sequences which are sufficiently complementary to form complexes via base pairing. Where a primer (or splice template) “hybridizes” with target (template), such complexes (or hybrids) are sufficiently stable to serve the priming function required by a DNA polymerase to initiate DNA synthesis. There is a specific, i.e. non-random, interaction between two complementary polynucleotides that can be competitively inhibited.
Posttranscriptional Gene Silencing: a targeted gene knockout or knockdown effect at the level of mRNA degradation or translational suppression, which is usually triggered by either foreign/viral DNA or RNA transgenes or small inhibitory RNAs.
RNA Interference (RNAi): a posttranscriptional gene silencing mechanism in eukaryotes, which can be triggered by small inhibitory RNA molecules such as microRNA (miRNA), small hairpin RNA (shRNA) and small interfering RNA (siRNA). These small RNA molecules usually function as gene silencers, interfering with expression of intracellular genes containing either completely or partially complementarity to the small RNAs.
MicroRNA (miRNA): single-stranded RNAs capable of binding to targeted gene transcripts that have partial complementarity to the miRNA. MiRNA is usually about 17-27 oligonucleotides in length and is able to either directly degrade its intracellular mRNA target(s) or suppress the protein translation of its targeted mRNA, depending on the complementarity between the miRNA and its target mRNA. Natural miRNAs are found in almost all eukaryotes, functioning as a defense against viral infections and allowing regulation of gene expression during development of plants and animals.
Precursor MicroRNA (Pre-miRNA): hairpin-like single-stranded RNAs containing stem-arm and stem-loop regions for interacting with intracellular RNaseIII endoribonucleases to produce one or multiple microRNAs (miRNAs) capable of silencing a targeted gene or genes complementary to the microRNA sequence(s). The stem-arm of a pre-miRNA can form either a perfectly (100%) or a partially (mis-matched) hybrid duplexes, while the stem-loop connects one end of the stem-arm duplex to form a circle or hairpin-loop conformation. In the present invention, however, precursor of microRNA may also includes pri-miRNA.
Small interfering RNA (siRNA): short double-stranded RNAs sized about 18-27 perfectly base-paired ribonucleotide duplexes and capable of degrading target gene transcripts with almost perfect complementarity.
Small or short hairpin RNA (shRNA): single-stranded RNAs that contain a pair of partially or completely matched stem-arm nucleotide sequences divided by an unmatched loop or bubble oligonucleotide to form a hairpin-like structure. Many natural miRNAs are derived from small hairpin-like RNA precursors, namely precursor microRNA (pre-miRNA).
Vector: a recombinant nucleic acid composition such as recombinant DNA (rDNA) capable of movement and residence in different genetic environments. Generally, another nucleic acid is operatively linked therein. The vector can be capable of autonomous replication in a cell in which case the vector and the attached segment is replicated. One type of preferred vector is an episome, i.e., a nucleic acid molecule capable of extrachromosomal replication. Preferred vectors are those capable of autonomous replication and expression of nucleic acids. Vectors capable of directing the expression of genes encoding for one or more polypeptides and/or non-coding RNAs are referred to herein as “expression vectors” or “expression-competent vectors”. Particularly important vectors allow cloning of cDNA from mRNAs produced using a reverse transcriptase. A vector may contain components consisting of a viral or a type-II RNA polymerase (Pol-II or pol-2) promoter, or both, a Kozak consensus translation initiation site, polyadenylation signals, a plurality of restriction/cloning sites, a pUC origin of replication, a SV40 early promoter for expressing at least an antibiotic resistance gene in replication-competent prokaryotic cells, an optional SV40 origin for replication in mammalian cells, and/or a tetracycline responsive element. The structure of a vector can be a linear or circular form of single- or double-stranded DNA selected form the group consisting of plasmid, viral vector, transposon, retrotransposon, DNA transgene, jumping gene, and a combination thereof.
Promoter: a nucleic acid to which a polymerase molecule recognizes, perhaps binds to, and initiates RNA transcription. For the purposes of the instant invention, a promoter can be a known polymerase binding site, an enhancer and the like, any sequence that can initiate synthesis of RNA transcripts by a desired polymerase.
RNA Processing: a cellular mechanism responsible for RNA maturation, modification and degradation, including RNA splicing, intron excision, exosome digestion, nonsense-mediated decay (NMD), RNA editing, RNA processing, 5′-capping, 3′-poly(A) tailing, and a combination thereof.
Gene Delivery: a genetic engineering method selected from the group consisting of polysomal transfection, liposomal transfection, chemical (nanoparticle) transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion, microinjection, gene-gun penetration, and a combination thereof.
Genetic Engineering: a DNA recombination method selected from the group consisting of DNA restriction and ligation, homologous recombination, transgene incorporation, transposon insertion, jumping gene integration, retroviral infection, and a combination thereof.
Transfected Cell: a single or a plurality of eukaryotic cells after being artificially inserted with at least a nucleic acid sequence or protien/peptide molecule into the cell(s), selected from the group consisting of a somatic cell, a tissue cell, a stem cell, a germ-line cell, a tumor cell, a cancer cell, a virus-infected cell, and a combination thereof.
Antibody: a peptide or protein molecule having a pre-selected conserved domain structure coding for a receptor capable of binding a pre-selected ligand.
Pharmaceutical and/or therapeutic Application: a biomedical utilization and/or apparatus useful for stem cell generation, drug/vaccine development, non-transgenic gene therapy, cancer therapy, disease treatment, wound healing, tissue/organ repair and regeneration, and high-yield production of proteins/peptides/antibodies, drug ingredients, medicines, vaccines and/or food supplies, and a combination thereof.
A novel composition and method for RNA replicase-mediated RNA/mRNA production and amplification, comprising:
(a) providing at least an RNA sequence, wherein said RNA sequence contains at least a 5′-end and at least a 3′-end RdRp binding sites;
(b) providing at least an RNA replicase, wherein said RNA replicase is isolated or modified from the RNA-dependent RNA polymerases (RdRp) of COVID-19 coronavirus and/or hepatitis C virus (HCV); and
(c) mixing the RNA sequence of (a) and the RNA replicase of (b) under a buffer condition, so as to elicit RNA replicase-mediated production and amplification of said RNA sequence, wherein said buffer condition contains ribonucleoside triphosphate molecules (rNTPs) required for RNA synthesis and is in a pH range from 6.0 to 8.0 as well as in a temperature range from 20° C. to 45° C.
For coronaviral COVID-19 and/or HCV-derived RdRp enzymes, the 5′-end RdRp-binding site contains at least a sequence of 5′-UUCWACGCGU AG-3′ (SEQ ID NO:1) or 5′-UUCWWACGCG UAG-3′ (SEQ ID NO:2), or both, while the 3′-end RdRp-binding site contains at least a sequence of SEQ ID NO:1 or SEQ ID NO:2, or both For incorporating these RdRp-binding sites into PCR primers, the uridine/uracil (U) contents of these RdRp-binding sites can be replaced by thymidine (dT) and/or deoxyuridine (dU) in the primers. Also, for enhancing RNA stability, the uridine/uracil (U) contents of these RdRp-binding sites as well as the resulting RNA/mRNA products can be replaced by pseudouridine, 5-methyluridine, 5-methoxyuridine, or other modified nucleotide analogs.
Also, the designed new pair of SEQ ID NO:1 and SEQ ID NO:2 RdRp-binding sites can be combined and/or used with the previously identified 5′- and 3′-end RdRp-binding sites described in our priority U.S. patent application Ser. No. 17/648,336 and Ser. No. 17/648,340. In details, the previously identified 5′-end RdRp-binding site is a nucleotide motif containing at least a sequence of either 5′-AU(G/C)(U/-)G(A/U)-3′ (i.e. 5′-AUSUGW-3′; SEQ ID NO:7) or 5′-U(C/-) (U/A)C(U/C)(U/A)A-3′ (i.e. 5′-UCWCYWA-3′; SEQ ID NO:8), or both. Preferably, the 5′-end RdRp-binding site is selected from a sequence containing homologs of 5′-AUCUGU-3′ (SEQ ID NO:9), 5′-UCUCUAA-3′ (SEQ ID NO:10), 5′-UCUCCUA-3′ (SEQ ID NO:11), and/or 5′-UUCAA-3′ (SEQ ID NO:12), or a combination thereof. On the other hand, the previously identified 3′-end RdRp-binding site is a nucleotide motif containing at least a sequence of either 5′-(U/A)C(A/-)(C/G)AU-3′ (i.e. 5′-WCASAU-3′; SEQ ID NO:13) or 5′-U(A/U)(A/G)G(A/U)(G/-)A-3′ (i.e. 5′-UWRGWR-3′; SEQ ID NO:14), or both. Preferably, the 3′-end RdRp-binding site is selected from a sequence containing homologs of 5′-ACAGAU-3′ (SEQ ID NO:15), 5′-UUAGAGA-3′ (SEQ ID NO:16), 5′-UAGGAGA-3′ (SEQ ID NO:17), and/or 5′-UUGAA-3′ (SEQ ID NO:18), or a combination thereof.
Referring particularly to the drawings for the purpose of illustration only and not limitation, there is illustrated:
Starting tissue cells can be obtained from either enzymatically dissociated skin cells using Aasen's protocol (Nat. Protocols 5, 371-382, 2010) or simply from the buffy coat fraction of heparin-treated peripheral blood cells. The isolated tissue samples must be kept fresh and used immediately by mixing with 4 mg/mL collagenase I and 0.25% TrypLE for 15-45 min, depending on cell density, and rinsed by HBSS containing trypsin inhibitor two times and then transferred to a new sterilized microtube containing 0.3 mL of feeder-free SFM culture medium (IrvineScientific, CA). After that, cells were further dissociated by shaking in a microtube incubator for 1 min at 37° C. and then transferred the whole 0.3 mL cell suspension to a 35-mm Matrigel-coated culture dish containing 1 mL of feeder-free SFM culture medium supplemented with formulated pre-miR-302+RdRp mRNA mixture, LIF, and bFGF/FGF2, or other optional defined factors. The concentrations of pre-miR-302+RdRp mRNA mixture, LIF, bFGF/FGF2, and other optional defined factors are ranged from 0.1 to 500 microgram (μg)/mL, respectively, in the cell culture medium. The cell culture medium and all of the supplements must be refreshed every 2-3 days and the cells are passaged at about 50%-60% confluence by exposing the cells to trypsin/EDTA for 1 min and then rinsing two times in HBSS containing trypsin inhibitor. For ASC expansion, the cells were replated at 1:51:500 dilution in fresh feeder-free MSC Expansion SFM culture medium supplemented with formulated pre-miR-302+RdRp mRNA mixture, LIF, bFGF/FGF2, and/or other optional defined factors. For culturing keratinocytes, cells are isolated from skin tissues and cultivated in EpiLife serum-free cell culture medium supplemented with human keratinocyte growth supplements (HKGS, Invitrogen, Carlsbad, Calif.) in the presence of proper antibiotics at 37° C. under 5% CO2. Culture cells are passaged at 50%-60% confluency by exposing cells to trypsin/EDTA solution for 1 min and rinsing once with phenol red-free DMEM medium (Invitrogen), and the detached cells are replated at 1:10 dilution in fresh EpiLife medium with HKGS supplements. Human cancer and normal cell lines A549, MCF7, PC3, HepG2, Colo-829 and BEAS-2B were obtained either from the American Type Culture Collection (ATCC, Rockville, Md.) or our collaborators and then maintained according to manufacturer's or provider's suggestions. After reprogramming, the resulting iPS cells (iPSCs) were cultivated and maintained following either Lin's feeder-free or Takahashi's feeder-based iPSC culture protocols (Lin et al., RNA 14:2115-2124, 2008; Lin et al., Nucleic Acids Res. 39:1054-1065, 2011; Takahashi K and Yamanaka S, Cell 126:663-676, 2006).
For intracellular delivery/transfection, 0.5˜200 μg of RCR-amplified RNA/mRNA (i.e. pre-miR-302 or coronaviral S protein mRNA) and RdRp mRNA mixture (ratio ranged from about 20:1 to 1:20) is dissolved in 0.5 ml of fresh cell culture medium and mixed with 1-50 μl of In-VivoJetPEI or other similar transfection reagents. After 10˜30 min incubation, the mixture is then added into a cell culture containing 50%-60% confluency of the cultivated cells. The medium is reflashed every 12 to 48 hours, depending on cell types. This transfection procedure may be performed repeatedly to increase transfection efficiency.
3. Preparation of RCR-Ready cDNA/RNA Templates
Reverse transcription (RT) of desired RNA/mRNA is performed by adding about 0.01 ng-10 microgram (μg) of isolated RNA/mRNA into a 20˜50 μL RT reaction (SuperScript III cDNA RT kit, ThermoFisher Scientific, Mass., USA), following the manufacturer's suggestions. Depending on the RNA/mRNA amount, the RT reaction mixture further contains about 0.01˜20 nmole RT primer, a proper amount of deoxyribonucleoside triphosphate molecules (dNTPs) and reverse transcriptase in 1× RT buffer. Then, the RT reaction is incubated at 37˜65° C. for 1-3 hours (hr), depending on the length and structural complexity of the desired RNA/mRNA sequences, so as to make the complementary DNA (cDNA) templates thereof for the next step of PCR. For isolation of viral RdRp mRNA, we have designed and used an RT-reverse primer 5′-GACAACAGGT GCGCTCAGGT CCT-3′ (SEQ ID NO:3) to generate the coronaviral RdRp cDNA sequence, which already possesses internal motif sequences similar to SEQ ID NO:1.
Next, polymerase chain reaction (PCR) is performed by adding about 0.01 pg˜10 μg of the RT-derived cDNAs into a 20˜50 μL PCR preparation mixture (High-Fidelity PCR master kit, ThermoFisher Scientific, Mass., USA), following the manufacturer's suggestions. Then, the PCR mixture is first incubated in five to twenty (5˜20) cycles of denaturation at 94° C. for 1 mim, annealing at 30˜55° C. for 30 sec-1 min, and then extension at 72° C. for 1-3 min, depending on the structure and length of the desired cDNA sequences. After that, another ten to twenty (10˜20) cycles of PCR are performed with a series of sequential cycling steps of denaturation at 94° C. for 1 mim, annealing at 50˜58° C. for 30 sec, and then extension at 72° C. for 1-3 min, depending on the structure and length of the resulting PCR products. Finally, the resulting PCR products are used as cDNA templates for IVT and RCR. For IVT-RCR template preparation, we design and use a specific pair of RCR-ready PCR primers for incorporating the identified RdRp-binding sites into the PCR-derived RdRp cDNA templates, including SEQ ID NO:3 and 5′-GATATCTAAT ACGACTCACT ATAGGGAGAG GTATGGTACT TGGTAGTT-3′ (SEQ ID NO:4). Later, a 5′-cap molecule may be further incorporated in the resulting mRNA products of IVT-RCR. On the other hand, we also design and use another pair of RCR-ready PCR primers for incorporating the identified RdRp-binding sites into the PCR-derived cDNA templates of human pre-miR-302 familial cluster (pre-miR-302), including 5′-GATATCTAAT ACGACTCACT ATAGGGAGAT CTGTGGGAAC TAGTTCAGGA AGGTAA-3′ (SEQ ID NO:5) and 5′-GTTCTCCTAA GCCTGTAGCC AAGAACTGCA CA-3′ (SEQ ID NO:6). In the primer design, various sequences and combinations of RNA promoters and RdRp-binding sites can be used, such as T7, T3, M13 and/or SP6 promoter, and at least an RdRp binding site has been incorporated in the 5′- and/or 3′-end primers.
For generating RCR-ready RNA/mRNA templates, since at least a promoter and at least an RdRp-binding site have been incorporated into the resulting PCR-derived cDNA products (served as RCR-ready cDNA templates), an IVT-RCR reaction can then be performed to amplify desired RNA/mRNA sequences from the cDNA templates. The IVT-RCR reaction mixture contains 0.01 ng˜10 μg of the PCR-derived cDNA product, 0.1˜50 U of isolated coronaviral RdRp/helicase (Abcam, Mass., USA/Creative Enzymes, N.Y.), a proper amount of ribonucleoside triphosphate molecules (rNTPs) and RNA polymerase (i.e. T7, T3, M13 and/or SP6) in 1× transcription buffer. The transcription buffer is commercially available and may be further adjusted according to the manufacturer's suggestions. Preferably, the 1× transcription buffer may further contain 0.001˜10 mM of betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), and/or 3-(N-morpholino)propane sulfonic acid (MOPS), and/or a combination thereof. Then, the IVT-RCR reaction is incubated at 30˜40° C. for 1˜6 hr, depending on the stability and activity of the used RdRp and RNA polymerase enzymes.
The starting RCR mixture contains about 0.01 ng-10 μg of the RCR-ready RNA/mRNA templates, about 0.1˜50 U of isolated coronaviral RdRp/helicase, and a proper amount of rNTPs in 1× transcription buffer. RdRp/helicase is either an RdRp enzyme with an additional RNA unwinding activity or a mixture of RdRp and helicase. The transcription buffer is commercially available in the market and may be further adjusted according to the manufacturer's suggestions. Additionally, the 1× transcription buffer may further conatin 0.001˜10 mM of betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), and/or 3-(N-morpholino)propane sulfonic acid (MOPS), and/or a combination thereof, which facilitates the denaturation of highly structured RNA/DNA sequences, such as hairpins and stem-loop structures. After that, the RCR reaction is incubated at 20˜45° C. for 1˜6 hr, depending on the stability and activity of the used RdRp enzymes.
Desired RNAs (10 μg) are isolated with a mirVana™ RNA isolation kit (Ambion, Austin, Tex.) or similar purification filter column, following the manufacturer's protocol, and then further purified by using either 5%˜10% TBE-urea polyacrylamide or 1%˜3.5% low melting point agarose gel electrophoresis. For Northern blot analysis, the gel-fractionated RNAs are electroblotted onto a nylon membrane. Detection of the RNA and its IVT template (the PCR-derived cDNA product) is performed with a labeled [LNA]-DNA probe complementary to a target sequence of the desired RNA. The probe is further purified by high-performance liquid chromatography (HPLC) and tail-labeled with terminal transferase (20 units) for 20 min in the presence of either a dye-labeled nucleotide analog or [32P]-dATP (>3000 Ci/mM, Amersham International, Arlington Heights, Ill.).
Cells (106) are lysed with a CelLytic-M lysis/extraction reagent (Sigma) supplemented with protease inhibitors, Leupeptin, TLCK, TAME and PMSF, following the manufacturer's suggestion. Lysates are centrifuged at 12,000 rpm for 20 min at 4° C. and the supernatant is recovered. Protein concentrations are measured using an improved SOFTmax protein assay package on an E-max microplate reader (Molecular Devices, CA). Each 30 μg of cell lysate are added to SDS-PAGE sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT) conditions, and boiled for 3 min before loading onto a 6-8% polyacylamide gel. Proteins are resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto a nitrocellulose membrane and incubated in Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 2 hr at room temperature. Then, a primary antibody is applied to the reagent and incubated the mixture at 4° C. After overnight incubation, the membrane is rinsed three times with TBS-T and then exposed to goat anti-mouse IgG conjugated secondary antibody to Alexa Fluor 680 reactive dye (1:2,000; Invitrogen—Molecular Probes), for 1 hr at the room temperature. After three additional TBS-T rinses, fluorescent scanning of the immunoblot and image analysis are conducted using Li-Cor Odyssey Infrared Imager and Odyssey Software v. 10 (Li-Cor).
Cell/Tissue samples are fixed in 100% methanol for 30 min at 4° C. and then 4% paraformaldehyde (in 1×PBS, pH 7.4) for 10 min at 20° C. After that, the samples are incubated in 1×PBS containing 0.1%˜0.25% Triton X-100 for 10 min and then washed in 1× PBS three times for 5 min. For immunostaining, primary antibodies were purchased from Invitrogen (CA, USA) and Sigma-Aldrich (MO, USA), respectively. Dye-labeled goat anti-rabbit or horse anti-mouse antibody are used as the secondary antibody (Invitrogen, Calif., USA). Results are examined and analyzed at 100× or 200× magnification under a fluorescent 80i microscopic quantitation system with a Metamorph imaging program (Nikon).
The mixture of RCR-amplified RNA/mRNA and RdRp mRNA (ratio ranged from about 20:1 to 1:20) is mixed well with a proper amount of delivery agent, such as an In-VivoJetPEI transfection reagent or other similar LNP-based delivery/transfection agents, following the manufacturer's protocol, and then injected into blood veins or muscles of an animal, depending the purpose of applications. The delivery/transfection agent is used for mixing, conjugating, encapsulating or formulating the amplified RNA/mRNA and RdRp mRNA mixture, so as to not only protect the RNA contents from degradation but also facilitate the delivery/transfection of the RNA/mRNA and RdRp mRNA mixture into specific target cells of interest in vitro, ex vivo and/or in vivo.
All data were shown as averages and standard deviations (SD). Mean of each test group was calculated by AVERAGE of Microsoft Excel. SD was performed by STDEV. Statistical analysis of data was performed by One-Way ANOVA. Tukey and Dunnett's t post hoc test were used to identify the significance of data difference in each group. p<0.05 was considered significant (SPSS v12.0, Claritas Inc).
The present application claims priority to U.S. Provisional Patent Application No. 63/270,034 filed on Oct. 20, 2021, and No. 63/280,226 filed on Nov. 17, 2021, both of which are entitled “Novel RNA Composition and Production Method for Use in iPS Cell Generation”. The present application further claims priority to U.S. Provisional Patent Application 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)”. Additionally, the present application is a continuation-in-part of 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 of U.S. patent application Ser. No. 17/648,336 filed on Jan. 19, 2022, which is entitled “Novel Replicase Cycling Reaction (RCR)”. The present application is further a continuation-in-part application of 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”. The disclosure of each of the aforementioned patent applications is hereby incorporated by reference in its entirety as if fully set forth herein.
| Number | Date | Country | |
|---|---|---|---|
| 63270034 | Oct 2021 | US | |
| 63280226 | Nov 2021 | US | |
| 63302163 | Jan 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17489357 | Sep 2021 | US |
| Child | 17930292 | US | |
| Parent | 17648336 | Jan 2022 | US |
| Child | 17489357 | US | |
| Parent | 17648340 | Jan 2022 | US |
| Child | 17648336 | US |
