Invented by Chiang Jia Li, Xiangao Sun, Harry Rogoff, Youzhi Li, 1 GLOBE HEALTH INSTITUTE LLC, 1Globe Health Institute LLC

Asymmetric interfering RNA (aiRNA) is a relatively new class of RNA molecules that has been gaining attention in recent years due to its potential applications in gene therapy and drug development. AiRNA is a type of small interfering RNA (siRNA) that is designed to target specific genes and inhibit their expression. Unlike traditional siRNA, aiRNA is asymmetric, meaning that it has different lengths and structures on each end. This asymmetry allows aiRNA to be more effective at targeting specific genes and reducing off-target effects.

The market for compositions and applications of aiRNA is still in its early stages, but it is expected to grow rapidly in the coming years. According to a report by MarketsandMarkets, the global market for RNA interference (RNAi) technologies, which includes aiRNA, is expected to reach $1.2 billion by 2022, growing at a compound annual growth rate (CAGR) of 7.5% from 2017 to 2022.

One of the main applications of aiRNA is in gene therapy, which involves using RNA molecules to treat genetic diseases by targeting and correcting specific genes. AiRNA has several advantages over traditional siRNA in this regard, including its ability to target specific genes more effectively and reduce off-target effects. Several companies are currently developing aiRNA-based gene therapies for a variety of genetic diseases, including Huntington’s disease, cystic fibrosis, and Duchenne muscular dystrophy.

Another potential application of aiRNA is in drug development, particularly in the development of targeted cancer therapies. AiRNA can be used to target specific genes that are involved in cancer growth and metastasis, allowing for more precise and effective treatments. Several companies are currently developing aiRNA-based cancer therapies, including Alnylam Pharmaceuticals and Arrowhead Pharmaceuticals.

The market for aiRNA compositions and applications is still relatively small, but it is expected to grow rapidly in the coming years as more companies invest in research and development in this area. The potential applications of aiRNA in gene therapy and drug development are vast, and the technology has the potential to revolutionize the way we treat genetic diseases and cancer. As the market for aiRNA continues to grow, it will be interesting to see what new applications and innovations emerge in this exciting field.

The 1 GLOBE HEALTH INSTITUTE LLC, 1Globe Health Institute LLC invention works as follows

The present invention provides duplex asymmetrical RNA molecules capable of silencing specific genes based on sequence. The RNA molecule consists of a first and second strand. The first strand of RNA is longer than the other strand. The RNA molecule includes a double-stranded area formed by the first and second strands, as well as two ends selected independently from the group consisting 5?-overhangs, 3??-overhangs, and blunt ends. The RNA molecules can be used for research and/or as therapeutics.

Background for Compositions and applications of asymmetric interferingRNA

Gene Silencing Through RNAi (RNA Interference) By Use of Small or Short Interfering RNA” (siRNA), has emerged as a powerful molecular tool and could be used to treat diseases (de Fougerolles et. al., 2007, Kim and Rossi 2007).

Theoretically, RNAi could be used to silence or knockdown any disease gene. RNAi can be used to treat a wide range of diseases including genetic, epigenetic and infectious, as long as a gene causing the disease is identified.

However, aside from the prominent delivery problem, the development RNAi-based drug faces challenges due to the limited efficacy siRNA and non-specific effects siRNA like interferon-like reactions and sense-strand mediated gene silencing off-target, as well as the prohibitive or expensive cost associated with siRNA synthesis. For most genes in mammalian cell, siRNA’s gene-silencing effectiveness is about 50% or lower. These molecules are expensive to produce (much more than antisense deoxynucleotides), require chemical modification, and are inefficient. The observation that extracellular administrations of synthetic siRNAs trigger interferon like responses has created a significant barrier to RNAi research and RNAi based therapeutic development.

RNAi is selectively triggered either by synthetic short interfering (siRNA) or by genetic elements that encode short-hairpin (shRNA) RNAs, which are then cleaved to siRNAs by Dicer (a ribonuclease III like enzyme). Although the biochemical mechanism for gene silencing is not fully understood, it appears to involve a multiprotein RNA-induced silencing complex (RISC). RISC binds to, unwinds and incorporates anti-sense strands of siRNA, which recognize and target perfectly complementary mRNAs. This reduces gene expression. The catalytic properties in the RISC complex are responsible for the potent gene silencing (from 1-10 days). The precision of RNAi is what gives it its power. Off-target RNAi is known to occur. The activation of interferon-like responses by siRNA is another major side effect. This is mediated through dsRNA dependent protein kinase receptors (PKR) as well as Toll-like (TLR). It is the length of siRNA that determines its ability to trigger an interferon response. (ibid.)

The current state of the art states that siRNA, for gene silencing within mammalian cell, is a double-stranded RNA of symmetrical structure with a length between 19 and 21 nucleotides. It also has a 3? “For gene silencing in mammalian cell, the current art teaches that the structure of siRNA is a symmetric double stranded RNA with a length of 19-21 nucleotides and 3? responses. (ibid.) This ‘optimal’ structure is well-established in the field. This ‘optimal?

The authors state that “there is a necessity to develop novel approaches for effective RNAi therapy in mammalian cell through a novel siRNA design with better efficacy, potency, rapid onset, durability and a shorter duplex RNA to avoid a non-specific response like interferon and to reduce cost of synthesis.

The references cited in this document are not considered prior art for the claimed invention.

The present invention concerns a discovery of a new class of small duplexed RNAs that are capable of silencing genes in mammalian cell, and which we call asymmetrical interference RNAs (aiRNA). This novel class of RNAi inducers is characterized by the asymmetry in length of the two RNA strands. This novel structural design not only has a high level of effectiveness in silencing genes, but also offers several advantages compared to the current siRNAs. The aiRNA structure can be much shorter than current siRNAs, reducing the cost of synthesis, and abrogating/reducing the length-dependent activation of nonspecific interferon responses. Asymmetry in the aiRNA structures also reduces or eliminates off-target effects mediated by the sense-strand. AiRNA induces gene silencing more effectively, quickly, and durably than siRNA. AiRNA is applicable in all the areas where siRNA and shRNA are currently being used or considered to be used. This includes biology research, R&D in biotechnology, pharmaceutical industry and RNAi-based therapy.

The present invention is a duplexed RNA molecule. The duplexed RNA molecules comprises a first and second strands with lengths ranging from 18 to 23 nucleotides. The second strand forms a double-stranded area with the first, with the first having a 3? overhang between 1-9 nucleotides. In one embodiment, the sequence of the first strand is substantially complementary to that of a target mRNA. In another embodiment, the sequence of the first strand is at least 70% complementary to that of a target sequence. In another embodiment, eukaryotic cells are mammalian or avian cells.

In one embodiment, a nucleotide from the sequence 5? The overhang of the sequence A, U, or dT is chosen from this group.

In one embodiment, the GC is between 20% and 70% in the double-stranded region.

In an embodiment, the length of the first strand is between 19 and 22 nucleotides.

In one embodiment, the length of the first strand is 21 nucleotides. In another embodiment, the length of the second strand is 14-16 nucleotides.

In one embodiment, the length of the first strand is 21 nucleotides and that of the second strand is 15 nucleotides. In another embodiment, the first-strand has a 3-?-overhang between 2-4 nucleotides. In a further embodiment, the first nucleotide has a 3-nucleotide overhang.

In one embodiment, the duplexed RNA molecule has at least one nucleotide modified or an analogue. In another embodiment, at least one nucleotide is modified by sugar, base, or backbone. In a further embodiment, a backbone-modified nucleotide is modified in a linkage phosphodiester with another ribonucleotide. In one embodiment, at least one heteroatom of nitrogen or sulfur is added to the phosphodiester. In another embodiment, a nucleotide analog is a modified ribonucleotide having a phosphothioate backbone.

In one embodiment, at least one nucleotide modified or its analog is an unusual or modified base. In another embodiment, at least one nucleotide modified or its analog is inosine or a tritylated base.

In another embodiment, the nucleotide analog is a sugar modified ribonucleotide. The 2?-OH is replaced by one of the following groups: H, OR R, Halo, SH, NHR, NHR2, NR2, CN. Each R is independently C1 to C6 alkyl alkenyl alkynyl. And halo can be F, Cl Br, or I.

In one embodiment, the first deoxynucleotide is present in at least one strand. In another embodiment, at least one of the deoxynucleotides is located in one or more regions chosen from the group consisting 3?-overhangs, 5? overhangs, and double-stranded regions. In another embodiment, at least one deoxynucleotide is present in the second strand.

The present invention provides a method for modulating gene expression within a cell, or in an organism. This involves contacting the cell or organism under conditions that allow selective gene silencing to occur and directing the selective gene silencer effected by this duplexed RNA towards a gene or nucleic acids with a sequence part substantially matching the double-stranded DNA. In another embodiment, the contacting step includes the introduction of said duplex-RNA molecule to a cell or organism that can be used for selective gene silencing. In a further embodiment, the introducing process is chosen from the group consisting transfection, lipofections, electroporation infection, injections, oral administration, topical administration and regional administration. In another embodiment, an excipient or carrier that is pharmaceutically acceptable can be used to introduce the drug. These include a positive-charge carrier (a liposome), a protein carrier (a polymer), a nanoparticle (a nanoemulsion), a lipid and a Lipoid.

In one embodiment, modulating methods are used to determine the function or utility a gene has in a cellular or organism.

In one embodiment, modulating methods are used to treat or prevent a disease or undesirable condition.

Click here to view the patent on Google Patents.