Last week, the Nobel Committees announced the much-anticipated Nobel Prize winners of 2023, honouring the contributions that, as per Alfred Nobel’s will of 1895, “have conferred the greatest benefit to humankind”. In this series of articles, we present selected patents of these winners, that, at least to some extent, result from or lead to their celebrated works. For those with an interest in the most commemorated scientific and technological achievements in 2023 and intellectual property, this series makes for interesting reading.
On 2 October 2023, the Nobel Assembly at Karolinska Institute announced that the 2023 Nobel Prize in Physiology or Medicine was rewarded to Katalin Karikó and Drew Weissman “for their discoveries concerning nucleoside base modifications that enabled the development of effective mRNA vaccines against COVID-19”.
The Committee commended that:
The discoveries by the two Nobel Laureates were critical for developing effective mRNA vaccines against COVID-19 during the pandemic that began in early 2020. Through their groundbreaking findings, which have fundamentally changed our understanding of how mRNA interacts with our immune system, the laureates contributed to the unprecedented rate of vaccine development during one of the greatest threats to human health in modern times.
Specifically, the groundbreaking discoveries of Karikó and Weissman that mRNAs with base modifications lead to reduced inflammatory responses and increased protein production compared with unmodified ones, have paved the way for clinical applications of mRNA.
We present herein two patent families with Karikó and Weissman listed as the inventors. These two families relate to RNAs containing modified nucleosides and their applications.
Family 1 – RNA Containing Modified Nucleosides and Methods of Use Thereof
Family 1 patents claim priority from US Provisional Application No. 60/710,164 and have an earliest priority date of 23 August 2005. Granted US Patent No. 8,691,966, for example, has one independent claim that states:
1. A composition comprising an in vitro-synthesized modified RNA comprising an open reading frame that encodes a protein of interest for translation in a mammalian cell, wherein said in vitro-synthesized modified RNA comprises a modified nucleoside selected from the group consisting of (i) 1-methylpseudouridine (m1Ψ) and (ii) pseudouridine (Ψ).
Notably, 1-methylpseudouridine (m1Ψ) is the most commonly modified base used in mRNA vaccine production these days, including in the COVID-19 vaccines.
The role of nucleoside modifications on the immuno-stimulatory potential and on the translation efficiency of RNA was unknown at the time of filing of this patent. Family 1 discloses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleosides. It also discloses the use of these molecules in gene therapy vectors, gene therapy methods and gene transcription silencing methods. It further discloses methods of reducing immunogenicity of RNA, oligoribonucleotides and polyribonucleotides, and methods of synthesizing the modified oligoribonucleotide and polyribonucleotide molecules. It was demonstrated in these patents that:
- the immunogenicity of RNA is affected by the extent of nucleoside modification, with a greater degree of modification tending to decrease immunogenicity, for example, nucleoside modifications such as m6A s2U, m5C, m5U, Ψ, reduce the immunogenicity of RNA as mediated by TLR3 signalling; and
- pseudouridine modification increases RNA translation efficiency in vitro and in vivo—in multiple animal models and by multiple routes of administration;
- modified RNA of the invention can be used to treat or prevent diseases or conditions including restenosis, cystic fibrosis, X-linked agammaglobulinemia, organ rejection, Niemann-Pick Disease, mucopolysaccharidosis and other metabolic disorders, and vasospasm.
Other patents in Family 1 are directed to different aspects of the invention. For example, granted US Patent No. 8,748,089 has one independent claim which reads as follows:
1. A method for inducing a mammalian cell to produce a protein of interest comprising repeatedly administering to said mammalian cell an in vitro-synthesized modified RNA comprising an open reading frame that encodes a protein of interest, wherein said in vitro-synthesized modified RNA comprises at least one modified nucleoside selected from the group consisting of 1-methylpseudouridine (m1Ψ) and pseudouridine (Ψ).
Granted US Patent No. 9,750,824 has the following independent claim:
1. A method for reducing the immunogenicity of in vitro-synthesized RNA comprising an open reading frame that encodes a functional protein, the method comprising:
replacing a nucleotide of the in vitro-synthesized RNA with at least one modified nucleotide comprising a modified nucleoside selected from the group consisting of:
pseudouridine (Ψ), 1-methylpseudouridine (m1Ψ), 5-methyluridine (m5U), 2-thiouridine (s2U), and 5-methylcytidine (m5C),
wherein when pseudouridine (Ψ) is selected, it replaces uridine, and
wherein when 1-methylpseudouridine (m1Ψ) is selected, it replaces uridine, and
wherein when 5-methyluridine (m5U) is selected, it replaces uridine, and
wherein when 2-thiouridine (s2U) is selected, it replaces uridine, and
wherein when 5-methylcytidine (m5C) is selected, it replaces cytidine,
thereby generating in vitro-synthesized RNA comprising a modified nucleotide and reducing the immunogenicity of said in vitro-synthesized RNA.
Family 1 was also filed in Europe. The granted European patent was validated in jurisdictions including Belgium, Bulgaria, Switzerland, Germany, Estonia, France, United Kingdom, Greece, Croatia, Ireland, Luxembourg, Monaco, Republic of North Macedonia, Serbia, and Sweden.
Family 2 – Purification and purity assessment of RNA molecules synthesized with modified nucleosides
Family 2 patents claim priority from US Provisional Application No. 61/783,645 and have an earliest priority date of 14 March 2013. Granted US Patent No. 11,060,107, for example, has two independent claims:
1. A purified preparation of RNA, the RNA comprising at least one modified nucleoside selected from the group consisting of a 1-methyl-pseudouridine, m5C, m5U, m6A, s2U, Ψ, and 2′-O-methyl-U, wherein about 95% to about 99.9% of RNA in the purified preparation is messenger RNA, and wherein the purified preparation is prepared by subjecting a preparation of messenger RNA to enzymatic digestion with 0.001 units of at least one enzyme selected from the group consisting of RNase III, RNase V1, Dicer, and Chipper.
3. A purified preparation of RNA, the RNA comprising at least one modified nucleoside selected from the group consisting of a 1-methyl-pseudouridine, m5C, m5U, m6A, s2U, Ψ, and 2′-O-methyl-U, wherein about 95% to about 99.9% of RNA in the purified preparation is messenger RNA, and wherein the purified preparation is prepared by subjecting a preparation of messenger RNA to liquid chromatography using a linear gradient of 38% Buffer B (0.1 M triethylammonium acetate, 25% acetonitrile, pH 7.0) to 55% Buffer B in Buffer A (0.1 M triethylammonium acetate, pH 7.0) over 22 minutes.
Family 2 indicates that modified nucleoside-containing RNA transcribed by phage RNA polymerases still retains a low level of activation of innate immune receptors. It is speculated that this is possibly due to modified nucleosides that do not completely suppress the ability of the RNA molecule to activate sensors, or due to dsRNA contaminants which activate the receptors. The specification further notes that RNA transcribed in vitro by phage polymerase contains multiple aberrant RNAs, including short RNAs as a result of abortive transcription initiation events and, dsRNAs generated by RNA dependent RNA polymerase activity, RNA-primed transcription from RNA templates and self-complementary 3′ extension.
The aim in the Family 2 patents seems to be to provide purified preparations of an RNA, oligoribonucleotide, or polyribonucleotide comprising pseudouridine or a modified nucleoside. These have been prepared by subjecting a purified preparation of a modified RNA to enzymatic digestion with 0.001 units of at least one enzyme selected from the group consisting of RNase III, RNase V1, Dicer, and Chipper. Alternatively, the result may be achieved by purifying the modified RNA by liquid chromatography using a linear gradient of 38% Buffer B (0.1 M triethylammonium acetate, 25% acetonitrile, pH 7.0) to 55% Buffer B in Buffer A (0.1 M triethylammonium acetate, pH 7.0) over 22 minutes. Although they are claimed as separate and distinct processes, the liquid chromatographic separation and enzyme digestion may be used alone or in any combination. It has been demonstrated that purification results in mRNA that ranges between 98% and 99.9% purity starting with in vitro transcribed RNA that typically has 70-93% purity and this potentially improves the efficacy of vaccine.
Family 2 was filed as a PCT application but appears to have only entered national phase in the US.
Patents and works of Karikó and Weissman are well worth a more detailed review for interested parties.