CN117337330A - TMEM173 saRNA compositions and methods of use - Google Patents

Abstract

The present disclosure relates to saRNA useful for upregulating expression of a target gene and therapeutic compositions comprising the same, wherein the target gene is TMEM173. Methods of using the saRNA and the therapeutic compositions are also provided.

Description

TMEM173 saRNA compositions and methods of use

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/166,390 entitled TMEM173 SARNA composition and method of use filed on month 26 of 2021 and U.S. provisional application No. 63/318,927 entitled TMEM173 SARNA composition and method of use filed on month 11 of 2022, each of which is incorporated herein by reference in its entirety.

Reference to sequence Listing

The present application is filed with a sequence listing in electronic format. The submitted sequence Listing file is named 2058_10343TE_SL.txt, created at 3/23/2022, and is 34,380 bytes in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to oligonucleotides (particularly saRNA) for modulating gene expression, compositions, and methods of using the compositions in diagnostic and therapeutic applications.

Background

Recently it was discovered that small duplex RNAs increase gene expression by targeting ncRNAs overlapping with gene promoters (Janowski et al, nature Chemical Biology, vol.3:166-173 (2007), the contents of which are incorporated herein by reference in their entirety). Any short RNA that causes up-regulation of target gene expression by any mechanism is referred to as a short activating RNA or small activating RNA (saRNA).

Many solid cancers contain a dysfunctional immune microenvironment. Modulators that initiate immune responses to foreign pathogens may be promising therapeutic agents that induce an effective response against tumors. There remains a need for compositions and methods for targeted modulation of immunostimulatory genes with saRNA.

Brief description of the drawings

The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the disclosure, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the disclosure.

FIG. 1 is a schematic diagram illustrating the relationship between nucleic acid portions involved in the function of the saRNA of the present disclosure.

FIG. 2 shows TMEM173mRNA expression in HepG2 cells treated with TMEM 173-saRNA.

FIG. 3 shows TMEM173 mRNA expression in HepG2 cells treated with TMEM173-Pr-70, TMEM173-Pr-70-invAb-Se-m2, TMEM173-Pr-70-invAb-Se-m1, TMEM173-Pr-70-invAb-Se-0, TMEM173-Pr-70-invAb-Se-p1 and TMEM173-Pr-70-invAb-Se-p 2.

FIG. 4 shows TMEM173 mRNA expression in A549 cells treated with various TMEM173-saRNA and controls.

FIGS. 5A-5C show changes in TMEM173 mRNA and TMEM173 protein levels in A549 cells after treatment with TMEM 173-saRNA.

FIG. 6 shows TMEM173 mRNA expression in A549 cells treated with TMEM173-Pr-70-invAb-Se-m1 and TMEM173-Pr-70-m1-emod 51.

FIGS. 7A-7C show TMEM173 mRNA changes in A549 cells treated with various doses of TMEM173-saRNA at different times.

Disclosure of Invention

The present disclosure provides synthetic isolated small activating RNAs (sarnas) that up-regulate expression of a target gene, wherein the target gene is TMEM173 (STING). In some embodiments, the saRNA comprises an antisense strand that is at least 80% complementary to a region on the targeting sequence of the target gene, wherein the targeting sequence is selected from the group consisting of SEQ ID NOs 2-15, and wherein the antisense strand has 14-30 nucleotides. Pharmaceutical compositions, kits, and devices comprising such sarnas are also provided.

The disclosure also provides methods of up-regulating expression of a target gene in a subject, wherein the target gene is TMEMI73 (STING). Methods of modulating immune signaling pathways and methods of treating diseases associated with TMEM173, such as, but not limited to, cancer, are also provided. The method comprises administering a saRNA of the present disclosure to a subject.

Details of various embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

Detailed Description

The present disclosure provides compositions, methods, and kits for modulating target gene expression and/or function for therapeutic purposes. The compositions, methods, and kits comprise at least one saRNA that upregulates target gene expression.

I.design and Synthesis of sarna

One aspect of the present disclosure provides a method of designing and synthesizing a saRNA.

In the context of the present disclosure, the term "small activating RNA", "short activating RNA" or "saRNA" refers to single-or double-stranded RNA that up-regulates or has a positive effect on the expression of a specific gene. The saRNA may be single stranded with 14 to 30 nucleotides (e.g., 19, 20, 21, 22, or 23 nucleotides). The saRNA may also be double stranded, each strand comprising 14 to 30 nucleotides, e.g. 19, 20, 21, 22 or 23 nucleotides. The gene is called the target gene of saRNA. As used herein, a target gene is a double stranded DNA comprising a coding strand and a template strand. For example, a saRNA that upregulates expression of TMEM173 gene is referred to as "TMEM173-saRNA", and TMEM173 gene is a target gene of TMEM 173-saRNA. The target gene may be any target gene. In some embodiments, the target gene has a promoter region on the template strand.

"up-regulation" or "activation" of a gene refers to an increase in the level of gene expression, or an increase in the level of a polypeptide encoded by the gene or its activity, or an increase in the level of an RNA transcript transcribed from the template strand of the gene, as compared to that observed when the saRNA of the present disclosure is not present. The saRNA of the present disclosure may have a direct up-regulating effect on the expression of the target gene.

The saRNA of the present disclosure may have an indirect upregulation of RNA transcripts transcribed from the template strand of the target gene and/or polypeptides encoded by the target gene or mRNA. RNA transcripts transcribed from a target gene are hereinafter referred to as target transcripts. The target transcript may be an mRNA of the target gene. The target transcript may be present in mitochondria. The saRNA of the present disclosure may have a downstream effect on a biological process or activity. In such embodiments, a saRNA targeting a first transcript may have a (up-or down-regulating) effect on a second transcript (i.e., a non-target transcript).

Targeting sequences

In some embodiments, the saRNA comprises an antisense strand that is at least 80% complementary to a region on the template strand or coding strand of the target gene. This region on the template strand or coding strand that hybridizes or binds to this strand of saRNA is referred to as the "targeting sequence (targeted sequence)" or "target site". In some embodiments, the target region is located on the coding strand. In some embodiments, the target region is located on a template strand. FIG. 1 illustrates the relationship between the antisense strand and the targeting region on the template strand.

The term "complementary to … …" in this context means capable of hybridizing under stringent conditions. It will be appreciated that thymidine of DNA is replaced in RNA by uridine, and that this difference does not alter the understanding of the term "complementarity".

The antisense strand of the saRNA (whether single-stranded or double-stranded) can have at least 80%, 90%, 95%, 98%, 99% or 100% identity to the reverse complement of the targeting sequence. Thus, the reverse complement of the antisense strand of saRNA has a high degree of sequence identity to the targeting sequence. The targeting sequence may have the same length, i.e., the same number of nucleotides, as the saRNA and/or the reverse complement of the saRNA.

In some embodiments, the targeting sequence comprises at least 14 and less than 30 nucleotides.

In some embodiments, the targeting sequence has 19, 20, 21, 22, or 23 nucleotides.

In some embodiments, the location of the targeting sequence is within the promoter region of the template strand.

In some embodiments, the targeting sequence is located within the TSS (transcription initiation site) core of the template strand. As used herein, "TSS core" or "TSS core sequence" refers to the region between 2000 nucleotides upstream and 2000 nucleotides downstream of the TSS (transcription initiation site). Thus, the TSS core comprises 4001 nucleotides and the TSS is located 2001 from the 5' end of the TSS core sequence. The term "transcription initiation site" (TSS) as used herein means a nucleotide on a template strand of a gene that corresponds to or marks the transcription initiation site. The TSS may be located in a promoter region on a gene module strand.

In some embodiments, the targeting sequence is located between 1000 nucleotides upstream and 1000 nucleotides downstream of the TSS.

In some embodiments, the targeting sequence is located between 500 nucleotides upstream and 500 nucleotides downstream of the TSS.

In some embodiments, the targeting sequence is located between 250 nucleotides upstream and 250 nucleotides downstream of the TSS.

In some embodiments, the targeting sequence is located between 100 nucleotides upstream and 100 nucleotides downstream of the TSS.

In some embodiments, the targeting sequence is located upstream of the TSS in the TSS core. The targeting sequence may be less than 2000, less than 1000, less than 500, less than 250, or less than 100 nucleotides upstream of the TSS.

In some embodiments, the targeting sequence is located downstream of the TSS in the TSS core. The targeting sequence may be less than 2000, less than 1000, less than 500, less than 250, or less than 100 nucleotides downstream of the TSS.

In some embodiments, the targeting sequence is located +/-50 nucleotides around the TSS of the TSS core. In some embodiments, the targeting sequence substantially overlaps with the TSS of the TSS core. In some embodiments, the targeting sequence starts or ends at the TSS of the TSS core. In some embodiments, the targeting sequence overlaps the TSS of the TSS core by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in the upstream or downstream direction.

The position of the targeting sequence on the template strand is defined by the position of the 5' end of the targeting sequence. The 5' end of the targeting sequence can be at any position of the TSS core and the targeting sequence can begin at any position selected from position 1 to position 4001 of the TSS core. For reference herein, a targeting sequence is considered upstream of a TSS when the 5 'end of the targeting sequence is located between position 1 and position 2000 of the TSS core, and downstream of the TSS when the 5' end of the targeting sequence is located from position 2002 to position 4001. When the 5' end of the targeting sequence is located at nucleotide 2001, the targeting sequence is considered to be the TSS center sequence and is neither upstream nor downstream of the TSS.

For further reference, for example, when the 5' end of the targeting sequence is located at position 1600 of the TSS core, i.e., it is the 1600 th nucleotide of the TSS core, the targeting sequence starts at position 1600 of the TSS core and is considered upstream of the TSS.

saRNA design

In one embodiment, the saRNA of the present disclosure is a single stranded saRNA. The single stranded saRNA may be at least 14 or at least 18, e.g., 19, 20, 21, 22 or 23 nucleotides in length, as oligonucleotide duplexes exceeding this length may have an increased risk of inducing an interferon response. Preferably, the single stranded saRNA is less than 30 nucleotides in length. In some embodiments, the single stranded saRNA is 19 to 25 nucleotides in length. In one embodiment, the single stranded saRNA may be exactly 19 nucleotides in length. In another embodiment, the single stranded saRNA may be exactly 20 nucleotides in length. In another embodiment, the single stranded saRNA may be exactly 21 nucleotides in length. In another embodiment, the single stranded saRNA may be exactly 22 nucleotides in length. In another embodiment, the single stranded saRNA may be exactly 23 nucleotides in length. In some embodiments, a single stranded saRNA of the present disclosure comprises a sequence of at least 14 nucleotides and less than 30 nucleotides that has at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to a targeting sequence. In one embodiment, the sequence having at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to the targeting sequence is at least 15, 16, 17, 18 or 19 nucleotides in length, or 18 to 22, or 19 to 21, or just 19 nucleotides in length.

In another embodiment, the saRNA of the present disclosure has two strands forming a duplex, one strand being the antisense strand or the guide strand. The saRNA duplex is also referred to as double stranded saRNA. As used herein, a double-stranded saRNA or saRNA duplex is a saRNA comprising more than one strand, and preferably two strands, wherein strand hybridization may form a region of duplex structure. The two strands of double-stranded saRNA are referred to as the antisense or guide strand, and the sense or passenger strand.

Each strand of the duplex may be at least 14, or at least 18, e.g., 19, 20, 21, or 22 nucleotides in length. The duplex may hybridize over a length of at least 12, or at least 15, or at least 17, or at least 19 nucleotides. Each strand may be exactly 19, 20, 21, 22 or 23 nucleotides in length. Preferably, each strand of the saRNA is less than 30 nucleotides in length, as oligonucleotide duplex beyond this length may have an increased risk of inducing an interferon response. In one embodiment, each strand of the saRNA is 19 to 25 nucleotides in length. The strands forming the saRNA duplex may be of equal or unequal length.

In one embodiment, the antisense strand of a saRNA of the present disclosure comprises a sequence of at least 14 nucleotides and less than 30 nucleotides in length (e.g., exactly 19, 20, 21, 22, or 23 nucleotides) that has at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to the targeting sequence. In one embodiment, the sequence having at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to the targeting sequence is at least 15, 16, 17, 18 or 19 nucleotides, or 18 to 22 or 19 to 21, or exactly 19 nucleotides in length.

The antisense strand can have no more than 5, or no more than 4 or 3, or no more than 2, or no more than 1 mismatches with the targeting sequence on the template strand. Thus, the antisense strand has a high degree of complementarity to the targeting sequence on the template strand. The sense strand of the saRNA duplex has a high degree of sequence identity to the targeting sequence on the template strand.

The relationship between the saRNA duplex, target gene, coding strand of target gene, template strand of target gene, target transcript, targeting sequence/target site and TSS is shown in figure 1.

In the context of the present disclosure, "strand" means a continuous sequence of nucleotides, including non-naturally occurring nucleotides or modified nucleotides. The two or more chains may be separate molecules or each form part of separate molecules, or they may be covalently linked, for example, by a linker such as a polyethylene glycol linker. At least one strand of the saRNA may comprise a region complementary to a region on the guide strand of the target gene (the targeting sequence) and having sequence identity to a region on the coding strand of the target gene. Such strands are referred to as antisense or guide strands of the saRNA duplex. The second strand of the saRNA, which comprises a region complementary to the antisense strand of the saRNA, is referred to as the sense strand or passenger strand.

The saRNA duplex may also be formed from single molecules that are at least partially self-complementary to form hairpin structures (including duplex regions). In this case, the term "strand" refers to one of the regions of the saRNA that is complementary to another internal region of the saRNA. The guide strand of the saRNA will have no more than 5, or no more than 4 or 3, or no more than 2, or no more than 1 mismatches with sequences within the region on the template strand of the target gene (the targeting sequence).

In some embodiments, the passenger strand of the saRNA may comprise at least one nucleotide that is not complementary to a corresponding nucleotide on the guide strand, referred to as a mismatch to the guide strand. Mismatches to the guide strand may facilitate preferential loading of the guide strand (Wu et al, PLoS ONE, vol.6 (12): e28580 (2011), the contents of which are incorporated herein by reference in their entirety). In one embodiment, at least one mismatch to the guide strand may be located at the 3' end of the passenger strand. In one embodiment, the 3' end of the passenger strand may comprise 1-5 mismatches with the guide strand. In one embodiment, the 3' end of the passenger strand may comprise 2-3 mismatches with the guide strand. In one embodiment, the 3' end of the passenger strand may comprise 6-10 mismatches with the guide strand.

The saRNA duplex may have siRNA-like (siRNA-like) complementarity to a targeting sequence on the template strand; that is, there is 100% complementarity between nucleotides 2-6 of the 5' end of the guide strand in the saRNA duplex and the region on the targeting sequence. In addition, other nucleotides of the saRNA may have at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to a region of the targeting sequence. For example, nucleotide 7 (counted from the 5 'end) up to the 3' end of the saRNA may have at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to a region of the targeting sequence.

The term "small interfering RNA" or "siRNA" in this context means double stranded RNA, typically 20-25 nucleotides long, that is involved in the RNA interference (RNAi) pathway and that interferes with or inhibits the expression of a particular gene. The gene is a target gene of siRNA. siRNA is typically about 21 nucleotides long, with a 3' overhang (e.g., 2 nucleotides) at each end of both strands.

In some embodiments, the saRNA may comprise a number of unpaired nucleotides at the 3 'end of each strand to form a 3' overhang or tail. The number of unpaired nucleotides forming the 3' overhang of each strand may be in the range of 1 to 5 nucleotides or 1 to 3 nucleotides, or 2 nucleotides.

Thus, in some embodiments, the saRNA of the present disclosure may be single stranded and consist of: (i) A sequence having at least 80% complementarity to a targeting sequence on a template strand of a target gene; and (ii) a 3 'tail of 1-5 nucleotides (overhang), which may comprise uracil residues, such as UU, UUU or mUmU (m stands for 2' -OMe modification). In some embodiments, a saRNA of the present disclosure can be double-stranded and consist of a first strand comprising (i) a first sequence having at least 80% complementarity to a targeting sequence on a template strand of a target gene; and (ii) 3' overhang of 1-5 nucleotides; the second strand comprises (i) a second sequence that forms a duplex with the first sequence and (ii) a 3' overhang of 1-5 nucleotides. Such 3' tails (overhangs) should not be considered mismatches in terms of determining complementarity between the antisense strand of the saRNA and the targeting sequence. The saRNA of the present disclosure may have complementarity to the targeting sequence over its entire length, except for the 3' tail (overhang), if present.

The saRNA of the present disclosure may contain flanking sequences. Flanking sequences may be inserted into the 3 'or 5' end of the saRNA of the present disclosure. In one embodiment, the flanking sequences are the sequences of mirnas, giving the saRNA a miRNA configuration and can be processed with Drosha and Dicer. In one non-limiting example, the saRNA of the present disclosure has two strands and is cloned into a microRNA (microRNA) precursor, such as a miR-30 backbone flanking sequence.

The saRNA of the present disclosure may comprise a restriction enzyme substrate or recognition sequence. The restriction enzyme recognition sequence may be located at the 3 'end or the 5' end of the saRNA of the present disclosure. Non-limiting examples of restriction enzymes include NotI and AscI.

Target gene and saRNA

As described above, the antisense strand of the saRNA has a high degree of sequence identity to the reverse complement of the targeting sequence. In addition to being "complementary to a targeting sequence," the antisense strand of the saRNA of the present disclosure can also be defined as having "identity" to a region on the coding strand of a target gene. Thus, the genomic sequence of the target gene can be used to design the saRNA.

In some embodiments, the target gene of the saRNA of the present disclosure is TMEM173 (STING). The sequences of the target gene, the protein and mRNA encoded by the target gene, and the TSS core of the target gene are provided in table 1.

TABLE 1 target genes and the sequences of proteins and mRNAs encoded by the target genes

Table 2 describes the targeting sequence of the saRNA, the genomic position of the targeting sequence, and the relative position of the saRNA without 3' overhang. In Table 2, the targeting sequence is defined as the region on the template strand of the target gene. The relative position is the distance from the 5' end of the targeting sequence to the TSS. The negative number indicates a location upstream of the TSS and the positive number indicates a location downstream of the TSS.

TABLE 2 targeting sequences of saRNA

The saRNA may be single stranded and comprise 14-30 nucleotides. The sequence of the single stranded saRNA may have at least 60%, 70%, 80% or 90% identity to a sequence selected from the antisense strand sequences in table 3. In one embodiment, the single stranded saRNA comprises a sequence selected from the antisense strand sequences in table 3. In one embodiment, the single stranded saRNA may have a 3' tail (overhang). The sequence of the single stranded saRNA having a 3' tail (overhang) may have at least 60%, 70%, 80% or 90% identity to a sequence selected from the group consisting of the antisense strand sequences in table 4. In one embodiment, the single stranded saRNA comprises a sequence selected from the antisense strand sequences in table 4.

The saRNA may be double stranded. The two strands form a duplex and each strand comprises 14-30 nucleotides. The first strand of the double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity to a sequence selected from the antisense strand sequences in table 3. In one embodiment, the first strand of the double-stranded saRNA comprises a sequence selected from the antisense strand sequences in table 3. The second strand of the double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity to a sequence selected from the sense strand sequences in table 3. In one embodiment, the second strand of the double-stranded saRNA comprises a sequence selected from the sense strand sequences in table 3. In one embodiment, the double stranded saRNA may have a 3' overhang on each strand. The first strand of the double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity to a sequence selected from the antisense strand sequences in table 4. In one embodiment, the first strand of the double-stranded saRNA comprises a sequence selected from the antisense strand sequences in table 4. The second strand of the double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity to a sequence selected from the sense strand sequences in table 4. In one embodiment, the second strand of the double-stranded saRNA comprises a sequence selected from the sense strand sequences in table 4.

The saRNA may be modified or unmodified.

TABLE 3 sequence of saRNA (without chemical modification or overhang)

TABLE 4 sequence of saRNA (with chemical modifications and/or overhangs)

-u means 2 'o-methyl-uracil (2' -OMe). The 3 'overhang UU in the sequence may be replaced with any other 3' overhang such as UU (unmodified uracil) or UUU. A5 'overhang, such as dT, ddT or invAb, may also be added to the 5' position.

-mN (n= A, C, G or U) means 2' -OMe modified N.

The method disclosed in US2013/0164846 filed on 6/23 2011 (saRNA algorithm) can also be used to design saRNA, the content of which is incorporated herein by reference in its entirety. The design of saRNA is also disclosed in U.S. patent No. 8,324,181 and U.S. patent No. 7,709,566 to Corey et al; U.S. patent publication No. 2010/0210707 to Li et al; and voutilla et al, mol Ther Nucleic Acids, vol.1, e35 (2012), the contents of each of which are incorporated herein by reference in their entirety.

The saRNA of the present disclosure may be produced by any suitable method, for example, by synthesis or expression in cells using standard molecular biology techniques well known to those of ordinary skill in the art. For example, the saRNA of the present disclosure may be chemically synthesized or recombinantly produced using methods known in the art.

Bifunctional oligonucleotides

Bifunctional or dual-function oligonucleotides, such as saRNA, may be designed to up-regulate expression of a first gene and down-regulate expression of at least one second gene. One strand of the dual function oligonucleotide activates expression of a first gene and the other strand inhibits expression of a second gene. Each strand may further comprise a Dicer substrate sequence.

Chemical modification of saRNA

Herein, in saRNA, the term "modified" or "modified" where appropriate refers to structural and/or chemical modifications to A, G, U or C ribonucleotides. The nucleotides in the saRNA of the present disclosure may comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. The saRNA of the present disclosure may include any useful modification, such as modification of a sugar, nucleobase, or internucleoside linkage (e.g., linkage phosphate/phosphodiester linkage/phosphodiester backbone). One or more atoms of the pyrimidine nucleobase may be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol, an optionally substituted alkyl group (e.g., methyl or ethyl), or a halogen (e.g., chloro or fluoro). In certain embodiments, a modification (e.g., one or more modifications) is present in each of the sugar and internucleoside linkages. The modification of the present disclosure may be that ribonucleic acid (RNA) is modified to deoxyribonucleic acid (DNA), threose Nucleic Acid (TNA), glycol Nucleic Acid (GNA), peptide Nucleic Acid (PNA), locked Nucleic Acid (LNA) or hybrids thereof. In one non-limiting example, the 2'-OH of U is replaced with 2' -QMe.

In one embodiment, the saRNA of the present disclosure can comprise at least one modification described herein.

In another embodiment, the saRNA is a saRNA duplex and the sense strand and the antisense sequence may independently comprise at least one modification. As one non-limiting example, the sense sequence may comprise a modification, while the antisense strand may be unmodified. As another non-limiting example, the antisense sequence can comprise a modification, while the sense strand can be unmodified. As yet another non-limiting example, the sense sequence can comprise more than one modification, and the antisense strand can comprise one modification. As one non-limiting example, the antisense sequence can comprise more than one modification, and the sense strand can comprise one modification.

The saRNA of the present disclosure may include a combination of modifications to sugar, nucleobase, and/or internucleoside linkages. These combinations may include any one or more of the modifications described herein or in international application publication WO2013/052523 filed 10/2012, the contents of which are specifically incorporated herein by reference in their entirety, particularly formulas (Ia) - (Ia-5), (Ib) - (If), (IIa) - (IIp), (IIb-1), (IIb-2), (IIc-1) - (IIc-2), (IIn-1), (IIn-2), (IVa) - (IVl), and (IXa) - (IXr).

The saRNA of the present disclosure may or may not be uniformly modified along the entire length of the molecule (uniformly modified). For example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or any or more or all of A, G, U, C) may or may not be uniformly modified in the saRNA of the present disclosure. In some embodiments, all nucleotides X in a saRNA of the present disclosure are modified, wherein X can be any one of nucleotides A, G, U, C, or any one of the combinations a+ G, A + U, A + C, G + U, G + C, U + C, A +g+ U, A +g+ C, G +u+c or a+g+c.

Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) can be present at different positions in the saRNA. One of ordinary skill in the art will appreciate that nucleotide analogs or other modifications can be located at any position of the saRNA such that the function of the saRNA is not substantially reduced. The saRNA of the present disclosure may contain about 1% to about 100% modified nucleotides (either relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U or C) or any intermediate percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 95%, from 20% to 100%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 10% to 70%, from 10% to 80%, from 80% to 80%, from 50% to 95%, from 50% to 80%, from 80% to 95%, from 80% to 80%, from 50% to 95%, from 80% to 80% from 80% to 95% from 50% to 95% from).

In some embodiments, the saRNA of the present disclosure can be modified to a circular nucleic acid. The ends of the saRNA of the present disclosure can be linked by chemical or enzymatic reagents, thereby producing a circular saRNA without a free end. The circular saRNA is expected to be more stable than its linear counterpart and to be resistant to digestion by RNase R exonuclease. The circular saRNA may also comprise other structural and/or chemical modifications with respect to A, G, U or C ribonucleotides.

The saRNA of the present disclosure may be modified with any modification of the oligonucleotides or polynucleotides disclosed in PCT publication WO2013/151666, pages 136 to 247, published 10, 2013, 10, the disclosure of which is incorporated herein by reference in its entirety.

The saRNA of the present disclosure may comprise a combination of modifications. The saRNA may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 modifications for each strand.

In some embodiments, the saRNA is at least 50% modified, e.g., at least 50% of the nucleotides are modified. In some embodiments, the saRNA is at least 75% modified, e.g., at least 75% of the nucleotides are modified. In some embodiments, both strands of the saRNA may be modified over the entire length (100% modified). It will be appreciated that since the nucleotides (sugar, base and phosphate moieties, e.g. linkers) may each be modified, any modification to any portion of the nucleotide or nucleoside will constitute a modification.

In some embodiments, the saRNA is at least 10% modified in only one component of the nucleotide selected from the group consisting of nucleobases, sugars, or linkages between nucleosides. For example, the modification of the saRNA can be performed on at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of nucleobases, sugars or linkages of the saRNA.

In some embodiments, the saRNA comprises at least one sugar modification. Non-limiting examples of sugar modifications can include the following:

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in some embodiments, at least one 2 'position of the sugar of the nucleotide of the saRNA (OH in RNA or H in DNA) is substituted with-OMe, referred to as 2' -OMe.

In some embodiments, at least one 2 'position of the sugar of the nucleotide of the saRNA (OH in RNA or H in DNA) is substituted with-F, referred to as 2' -F.

In some embodiments, the saRNA comprises at least one phosphorothioate linkage or methylphosphonate linkage between nucleotides.

In some embodiments, the saRNA comprises a 3 'and/or 5' cap or overhang. In some embodiments, a saRNA of the present disclosure may comprise at least one inverted deoxyribonucleoside or dideoxyribonucleoside overhang (e.g., dT or ddT). The inverted overhang, e.g., dT, may be located at the 5 'end or 3' end of the passenger (sense) strand. In some embodiments, the saRNA of the present disclosure may comprise an inverted abasic (invAb) modification on the passenger strand. The at least one inverted abasic modification may be located at the 5 'end, or the 3' end, or both, of the passenger strand. Reverse abasic modification can facilitate preferential loading of the guide (antisense) strand.

In some embodiments, the saRNA comprises at least one 5'- (E) -vinylphosphonate (5' -E-VP) modification.

In some embodiments, the saRNA comprises at least one Glycol Nucleic Acid (GNA), an acyclic nucleic acid analog, as a modification.

saRNA conjugates and combinations

Conjugation can result in increased stability and/or half-life, and is particularly useful for targeting the saRNA of the present disclosure to a particular site in a cell, tissue, or organism. The saRNA of the present disclosure may be designed to be conjugated to other polynucleotides, dyes, intercalators (e.g., acridine), cross-linking agents (e.g., psoralen (psoralene), mitomycin C), porphyrins (TPPC 4, texaphyrin, sapphirin)), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [ MPEG ]] ₂ A polyamino group, an alkyl group, a substituted alkyl group, a radiolabel, an enzyme, a hapten (e.g., biotin), a transport/absorption enhancer (e.g., aspirin, vitamin E, folic acid), a synthetic ribonuclease, a protein such as a glycoprotein, or a peptide such as a molecule having specific affinity for a co-ligand (co-ligand), or an antibody such as an antibody that binds to a specified cell type (e.g., cancer cells, endothelial cells, or bone cells), a hormone and hormone receptor, a non-peptide species such as a lipid, lectin, carbohydrate, vitamin, cofactor, or drug. Suitable conjugates of nucleic acid molecules are disclosed in international publication WO 2013/090648 submitted at 12/14 2012, the contents of which are incorporated herein by reference in their entirety.

According to the present disclosure, the saRNA of the present disclosure may be administered with or further include one or more of the following to achieve different functions: RNAi agents, small interfering RNAs (siRNA), small hairpin RNAs (shRNA), long non-coding RNAs (lncRNA), enhancer RNAs, enhancer-derived RNAs or enhancer-driven RNAs (enona), micrornas (miRNA), miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like. The one or more RNAi agents, small interfering RNAs (sirnas), small hairpin RNAs (shrnas), long non-coding RNAs (lncrnas), micrornas (mirnas), miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, trnas, RNAs that induce triple helix formation, aptamers, or vectors may comprise at least one modification or substitution.

In some embodiments, the modification is selected from the group consisting of a chemical substitution of a nucleic acid at a sugar position, a chemical substitution at a phosphate position, and a chemical substitution at a base position. In other embodiments, the chemical modification is selected from the incorporation of modified nucleotides; 3' capping; conjugation to a high molecular weight non-immunogenic compound; conjugation to a lipophilic compound; and incorporating phosphorothioates into the phosphate backbone. In one embodiment, the high molecular weight non-immunogenic compound is a polyalkylene glycol or polyethylene glycol (PEG).

In one embodiment, the saRNA of the present disclosure can be attached to a transgene, so that it can be co-expressed from the RNA polymerase II promoter. In one non-limiting example, the saRNA of the present disclosure is attached to a green fluorescent protein Gene (GFP).

In one embodiment, the saRNA of the present disclosure can be attached to DNA or RNA aptamers, thereby producing saRNA-aptamer conjugates. An aptamer is an oligonucleotide or peptide with high selectivity, affinity and stability. They take on a specific and stable three-dimensional shape, providing a highly specific tight binding to the target molecule. An aptamer may be a nucleic acid substance that has been engineered to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms by repeated rounds of in vitro selection or equivalent SELEX (exponential enriched ligand system evolution). Nucleic acid aptamers have specific binding affinities for molecules through interactions other than classical Watson-Crick base pairing. Nucleic acid aptamers, such as peptides or monoclonal antibodies (mabs) produced by phage display, are capable of specifically binding to a selected target and blocking its ability to function by binding. In some cases, the aptamer may also be a peptide aptamer. For any particular molecular target, the nucleic acid aptamer may be identified from a combinatorial library of nucleic acids, for example by SELEX. The yeast two-hybrid system can be used to identify peptide aptamers. Thus, the skilled artisan is able to design suitable aptamers for delivering the saRNA or cells of the present disclosure to target cells, such as hepatocytes. DNA, RNA and peptide aptamers are contemplated. The saRNA of the present disclosure is preferably administered to the liver using liver-specific aptamers.

As used herein, a typical aptamer is about 10-15kDa (20-45 nucleotides) in size, binds its target with at least nanomolar affinity, and distinguishes closely related targets. The aptamer may be ribonucleic acid, deoxyribonucleic acid, or a mixture of ribonucleic acid and deoxyribonucleic acid. The aptamer may be single-stranded ribonucleic acid, deoxyribonucleic acid, or a mixture of ribonucleic acid and deoxyribonucleic acid. The aptamer may comprise at least one chemical modification.

Suitable nucleotide lengths for the aptamer range from about 15 to about 100 nucleotides (nt), and in various other embodiments, any of the lengths 15-30nt, 20-25nt, 30-100nt, 30-60nt, 25-70nt, 25-60nt, 40-60nt, 25-40nt, 30-40nt,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40nt, or 40-70nt. However, the sequence may be designed to be sufficiently flexible that it can accommodate the interaction of the aptamer with two targets at the distances described herein. The aptamer may be further modified to provide protection against nucleases and other enzymatic activities. The aptamer sequence may be modified by any suitable method known in the art.

The saRNA-aptamer conjugate may be formed using any known method for linking two moieties, such as direct chemical bond formation, linking by a linker such as streptavidin, and the like.

In one embodiment, the saRNA of the present disclosure may be attached to an antibody. Methods for generating antibodies to target cell surface receptors are well known. The saRNA of the present disclosure may be attached to such antibodies using known methods, for example using RNA carrier proteins. The resulting complex can then be administered to a subject and taken up by the target cell via receptor-mediated endocytosis.

In one embodiment, the saRNA of the present disclosure may be conjugated to: lipid moieties such as cholesterol moieties (Letsinger et al, proc. Natl. Acid. Sci. USA,1989, 86:6553-6556); bile acids (Manoharan et al, biorg. Med. Chem. Let.,1994, 4:1053-1060); thioethers, for example beryl-5-tritylthiol (Manoharan et al, ann.N. Y. Acad. Sci.,1992,660:306-309; manoharan et al, biorg. Med. Chem. Let.,1993, 3:2765-2770); mercaptocholesterol (Oberhauser et al, nucleic acids Res.,1992, 20:533-538); aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al, EMBO J,1991,10:1111-1118; kabanov et al, FEBS Lett.,1990,259:327-330; svinarchuk et al, biochimie,1993, 75:49-54); phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1, 2-di-O-hexadecyl-rac-glyceryl-3-H phosphonate (Manoharan et al, tetrahedron Lett.,1995,36:3651-3654; shea et al, nucleic acids Res.,1990, 18:3777-3783); polyamine or polyethylene glycol chains (Manoharan et al, nucleosides & Nucleosides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al, tetrahedron Lett.,1995, 36:3651-3654); palm-based moieties (Mishra et al, biochim. Biophys. Acta,1995, 1264:229-237); or a stearylamine or hexylamino-carbonyloxy cholesterol moiety (Crooke et al, J.Pharmacol. Exp. Ther.,1996, 277:923-937), the respective contents of which are incorporated herein by reference in their entirety.

In one embodiment, the saRNA of the present disclosure is conjugated to a ligand. In one non-limiting example, the ligand may be any of the ligands disclosed in US20130184328 to Manoharan et al, the contents of which are incorporated herein by reference in their entirety. The conjugate has the formula: ligand- [ linker] _Optional - [ tether (tether)] _Optional -an oligonucleotide agent. The oligonucleotide agent may comprise a subunit of formula (I) as disclosed in US20130184328 to Manoharan et al, the contents of which are incorporated by referenceFor the purpose of being incorporated herein in its entirety. In another non-limiting example, the ligand may be any of the ligands disclosed in US20130317081 to Akinc et al (the contents of which are incorporated herein by reference in their entirety), such as lipid, protein, hormone or carbohydrate ligands of formulas II-XXVI. The ligand may be coupled to the saRNA through a divalent or trivalent branched linker of formulas XXXI-XXXV as disclosed in Akinc.

Representative U.S. patents teaching the preparation of such nucleic acid/lipid conjugates include, but are not limited to: U.S. patent No. 4,828,979;4,948,882;5,218,105;5,525,465;5,541,313;5,545,730;5,552,538;5,578,717,5,580,731;5,591,584;5,109,124;5,118,802;5,138,045;5,414,077;5,486,603;5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136;5,082,830;5,112,963;5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241,5,391,723;5,416,203,5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5,585,481;5,587,371;5,595,726;5,597,696;5,599,923;5,599,928 and 5,688,941, the contents of each of which are incorporated herein by reference in their entirety.

The saRNA of the present disclosure may be provided in combination with other active ingredients known to have utility in the particular method contemplated. The additional active ingredient may be administered simultaneously, separately or sequentially with the saRNA of the present disclosure. In one embodiment, the saRNA of the present disclosure is administered with a saRNA that modulates a different target gene.

In one embodiment, the saRNA is conjugated to a carbohydrate ligand, such as any of the carbohydrate ligands disclosed in U.S. patent nos. 8106022 and 8828956 to Manoharan et al (Alnylam Pharmaceuticals), the contents of which are incorporated herein by reference in their entirety. For example, the carbohydrate ligand may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide. These carbohydrate-conjugated RNA agents can target parenchymal cells of the liver. In one embodiment, the saRNA is conjugated to more than one carbohydrate ligand, preferably two or three carbohydrate ligands. In one embodiment, the saRNA is conjugated to one or more galactose moieties. In another embodiment, the saRNA is conjugated to at least one (e.g., two or three or more) lactose molecules (lactose is glucose coupled to galactose). In another embodiment, the saRNA is conjugated to at least one (e.g., two or three or more) N-acetyl-galactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate). In one embodiment, the saRNA is conjugated to at least one mannose ligand, and the conjugated saRNA targets macrophages.

In one embodiment, the saRNA of the present disclosure is administered with a small interfering RNA or siRNA that inhibits gene expression.

In one embodiment, the saRNA of the present disclosure is administered with one or more drugs for therapeutic purposes.

Compositions of the present disclosure

One aspect of the present disclosure provides a pharmaceutical composition comprising a small activating RNA (saRNA) that upregulates a target gene and at least one pharmaceutically acceptable carrier.

Formulation, delivery, administration and dosing

The pharmaceutical formulation may additionally comprise pharmaceutically acceptable excipients, which as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonizing agents, thickening or emulsifying agents, preservatives, and the like, suitable for the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing such compositions are known in the art (see Remington: the Science and Practice of Pharmacy, 21 st edition, a.r. gennaro, lippincott, williams & Wilkins, baltimore, MD,2006; which is incorporated herein by reference in its entirety). It is contemplated within the scope of the present disclosure to use conventional excipient mediums, except for the following: any conventional excipient medium may be incompatible with the substance or derivative thereof, for example by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any of the other components of the pharmaceutical composition.

In some embodiments, the composition is administered to a human, human patient, or subject. For the purposes of this disclosure, the phrase "active ingredient" generally refers to the saRNA to be delivered as described herein.

Although the description of pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, such as a non-human animal, e.g., a non-human mammal. Modification of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to a variety of animals is well known and the ordinarily skilled veterinary pharmacologist can design and/or make such modifications by mere routine experimentation, if any. Subjects contemplated for administration of the pharmaceutical composition thereto include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds, such as poultry, chickens, ducks, geese, and/or turkeys.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the pharmacological arts or later developed. Generally, such preparation methods include the step of combining the active ingredient with excipients and/or one or more other auxiliary ingredients, and then, if necessary and/or desired, dividing, shaping and/or packaging the product into the required single or multiple dose units.

Pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or marketed in bulk as single unit doses and/or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient is typically equal to the dose of active ingredient to be administered to the subject and/or a suitable fraction of the dose, e.g. one half or one third of the dose.

The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical compositions of the present disclosure will vary depending upon the nature, size, and/or condition of the subject being treated and further depending upon the route of administration of the composition. For example, the composition may comprise from 0.1% to 100%, e.g., from.5% to 50%, 1-30%, 5-80%, at least 80% (w/w) active ingredient.

In some embodiments, a formulation described herein can contain at least one saRNA. As one non-limiting example, a formulation may contain 1, 2, 3, 4, or 5 sarnas having different sequences. In one embodiment, the formulation contains at least three sarnas having different sequences. In one embodiment, the formulation contains at least five sarnas having different sequences.

The saRNA of the present disclosure may be formulated using one or more excipients to: (1) increased stability; (2) increasing cell transfection; (3) Allowing sustained or delayed release (e.g., from a depot formulation of saRNA); (4) Altering the biodistribution (e.g., targeting saRNA to a specific tissue or cell type); (5) increasing in vivo translation of the encoded protein; and/or (6) altering the in vivo release profile of the encoded protein.

In addition to conventional excipients such as any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present disclosure may include, but are not limited to: lipids (lipidoid), liposomes, lipid nanoparticles, polymers, lipid complexes (lipoplex), core-shell nanoparticles, peptides, proteins, cells transfected with saRNA (e.g., for implantation into a subject), hyaluronidases, nanoparticle mimics, and combinations thereof. Thus, the formulations of the present disclosure may include one or more excipients, each in an amount that, together, increases stability of the saRNA and/or increases cell transfection of the saRNA. Furthermore, the saRNA of the present disclosure may be formulated using self-assembled nucleic acid nanoparticles. Pharmaceutically acceptable carriers, excipients and delivery agents that can be used for nucleic acids formulated with the saRNA of the present disclosure are disclosed in international publication WO 2013/090648, filed 12/14 2012, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the saRNA of the present disclosure comprises two single RNA strands, each 21 nucleotides in length, that are annealed to form a double stranded saRNA as the active ingredient. The composition also contained a salt buffer containing 50mM Tris-HCl (pH 8.0), 100mM NaCl and 5mM EDTA.

In another embodiment, the saRNA of the present disclosure can be delivered with a dendrimer (dendrimer). Dendrimers are highly branched macromolecules. In one embodiment, the saRNA of the present disclosure is complexed with a structurally flexible poly (amidoamine) (PAMAM) dendrimer for targeted in vivo delivery. This complex is called a saRNA-dendrimer. Dendrimers have a high degree of molecular homogeneity, narrow molecular weight distribution, specific size and shape characteristics, and highly functionalized terminal surfaces. The manufacturing process is a series of repeated steps starting from a central initiator core. Each subsequent growth step represents a new generation of polymers with larger molecular diameters and molecular weights and more reactive surface sites than the previous generation.

PAMAM dendrimers are efficient nucleotide delivery systems with primary amine groups on the surface and tertiary amine groups within the structure. Primary amine groups participate in nucleotide binding and promote cellular uptake thereof, while buried tertiary amino groups act as proton sponges in endosomes and enhance release of nucleic acids into the cytoplasm. These dendrimers can protect the saRNA they carry from ribonuclease degradation and achieve large release of saRNA over a long period of time via endocytosis for efficient gene targeting. The in vivo efficacy of these nanoparticles has been previously evaluated, with biodistribution studies indicating that dendrimers preferentially accumulate in peripheral blood mononuclear cells and survive, with no appreciable toxicity (see Zhou et al, molecular ter.201101vol.19, 2228-2238, the contents of which are incorporated herein by reference in their entirety). The PAMAM dendrimer may comprise a Triethanolamine (TEA) core, a Diaminobutane (DAB) core, a cystamine core, a diaminohexane (HEX) core, a diaminododecane (DODE) core, or an Ethylenediamine (EDA) core. In one embodiment, the PAMAM dendrimer comprises a TEA core or a DAB core.

Lipid-like material

Lipid synthesis has been widely described and formulations containing these compounds are particularly suitable for delivery of oligonucleotides or nucleic acids (see Mahon et al, bioconjug chem.2010:1448-1454; schroeder et al, J International Med.2010:267-9-21; akinec et al, nat Biotechnol.2008:561-569; love et al, proc Natl Acad Sci U S A.2010 107:1864-1869; siegwart et al, proc Natl Acad Sci U S A.2011:12996-3001; all of which are incorporated herein in their entirety).

While these lipids have been used to efficiently deliver double-stranded small interfering RNA molecules in rodents and non-human primates (see Akine et al, nat Biotechnol.2008:561-569; frank-Kamenotsky et al, proc Natl Acad Sci U S A.2008:11915-11920; akinec et al, mol Ther.2009:17:872-879; love et al, proc Natl Acad Sci U S A.2010 107:1864-1869; leuschner et al, nat Biotechnol.2011:1005-1010; all of which are incorporated herein in their entirety), the present disclosure contemplates their formulation and use for delivering saRNA. Complexes, micelles, liposomes or particles containing these lipids can be prepared, and thus effective delivery of saRNA can be achieved after injection of lipid formulations via local and/or systemic administration routes. Lipid complexes of saRNA can be administered by a variety of means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.

In vivo delivery of nucleic acids may be affected by a number of parameters including, but not limited to, formulation composition, nature of particle pegylation, degree of loading, ratio of oligonucleotide to lipid, and biophysical parameters such as, but not limited to, particle size (Akinec et al, mol Ther.2009:872-879; the contents of which are incorporated herein by reference in their entirety). For example, small changes in the anchor chain length of polyethylene glycol (PEG) lipids may have a significant impact on in vivo efficacy. Formulations having different lipids including, but not limited to, penta [3- (1-laurylaminopropionyl) ] -triethylenetetramine hydrochloride (TETA-5 LAP; also known as 98N12-5, see Murugaiah et al, analytical Biochemistry,401:61 (2010), the contents of which are incorporated herein by reference in their entirety), C12-200 (including derivatives and variants), and MD1 can be tested for in vivo activity.

A lipid referred to herein as "98N12-5" is disclosed by Akinec et al, mol Ther.20097:872-879, the contents of which are incorporated by reference in their entirety.

Lipids referred to herein as "C12-200" are disclosed by Love et al, proc Natl Acad Sci U S A.2010 107:1864-1869, and Liu and Huang, molecular therapy.2010 669-670; the contents of both of these documents are incorporated herein by reference in their entirety. The lipid formulation may comprise particles comprising 3 or 4 or more components other than saRNA. As one example, a formulation with certain lipids includes, but is not limited to 98N12-5 and may contain 42% lipid, 48% cholesterol, and 10% peg (C14 alkyl chain length). As another example, a formulation with certain lipids includes, but is not limited to, C12-200, and may contain 50% lipid, 10% distearoyl phosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG.

In one embodiment, a saRNA formulated with lipids for systemic intravenous administration can target the liver. For example, a final optimized intravenous formulation using saRNA can result in the formulation being distributed to more than 90% of the liver, the formulation comprising a lipid molar composition of 42%98n12-5, 48% cholesterol, and 10% PEG-lipid, having a final weight ratio of total lipid to saRNA of about 7.5 to 1, PEG lipid being C14 alkyl chain length, and having an average particle size of about 50-60nm (see, akinc et al, mol ter.2009:872-879; the contents of which are incorporated herein by reference in their entirety). In another example, an intravenous formulation using a C12-200 (see published International application WO2010129709, the contents of which are incorporated herein by reference in their entirety) lipid may be effective for delivering a sarNA, the formulation may have a molar ratio of C12-200/distearoyl phosphatidylcholine/cholesterol/PEG-DMG of 50/10/38.5/1.5, a total lipid to nucleic acid weight ratio of 7:1, and an average particle size of 80nm (see Love et al, proc Natl Acad Sci U S A.2010 107:1864-1869, the contents of which are incorporated herein by reference in their entirety).

In another embodiment, the formulation comprising MD1 lipid can be used to effectively deliver saRNA to hepatocytes in vivo. The characteristics of optimized lipid formulations for intramuscular or subcutaneous routes can vary significantly depending on the target cell type and the ability of the formulation to diffuse through the extracellular matrix into the blood stream. Although particle sizes less than 150nm may be required for effective hepatocyte delivery due to the size of the endothelial fenestration (endothelial fenestrae) (see, akine et al, mol Ther.2009.17:872-879, the contents of which are incorporated herein by reference in their entirety), the use of lipid formulated saRNA to deliver the formulation to other cell types, including but not limited to endothelial cells, myeloid cells, and muscle cells, may not be limited by similar sizes.

The use of lipid formulations to deliver siRNA in vivo to other non-liver cells such as myeloid cells and endothelium has been reported (see Akine et al, nat Biotechnol.2008:26-561-569; leuschner et al, nat Biotechnol.2011:29-1005-1010; cho et al, adv. Function. Mater. 20099:3112-3118; international Judah Folkman conference, cambridge, MA 10 month 8-9 days, 2010; the contents of each of which are incorporated herein by reference in their entirety). The lipid formulations may have similar molar ratios of components for effective delivery to myeloid cells such as monocytes. Different ratios of lipids and other components (including but not limited to distearoyl phosphatidylcholine, cholesterol, and PEG-DMG) can be used to optimize formulations of saRNA for delivery to different cell types, including but not limited to hepatocytes, myeloid cells, myocytes, and the like. For example, the component molar ratios may include, but are not limited to, 50% C12-200, 10% distearoyl phosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG (see Leuschner et al, nat Biotechnol 2011 29:1005-1010; the contents of which are incorporated herein by reference in their entirety). The use of lipid formulations to deliver nucleic acids locally to cells (e.g., without limitation, adipocytes and myocytes) via subcutaneous or intramuscular delivery may not require all of the formulation components required for systemic delivery, and thus may comprise only lipid and saRNA.

Liposomes, lipid complexes, and lipid nanoparticles

The saRNA of the present disclosure may be formulated using one or more liposomes, lipid complexes, or lipid nanoparticles. In one embodiment, the pharmaceutical composition of saRNA comprises a liposome. Liposomes are artificially prepared vesicles that consist primarily of lipid bilayers and can be used as delivery vehicles for administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes, such as, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments; small single cell vesicles (SUVs), which may be less than 50nm in diameter; and Large Unilamellar Vesicles (LUVs), which may be between 50 and 500nm in diameter. Liposome designs may include, but are not limited to, opsonin (opsonin) or ligands to improve the attachment or activation events of the liposome to unhealthy tissues, such as, but not limited to, endocytosis. Liposomes can contain low or high pH values to improve delivery of the pharmaceutical formulation.

The formation of liposomes can depend on physicochemical characteristics such as, but not limited to, the entrapped pharmaceutical formulation and the liposomal composition; the nature of the medium in which the lipid vesicles are dispersed; effective concentration of the entrapped material and its potential toxicity; any additional processes involved during vesicle application and/or delivery; optimal size, polydispersity and shelf life of vesicles for the intended application; and batch-to-batch reproducibility and possibility of mass production of safe and efficient liposome products.

In one embodiment, the pharmaceutical compositions described herein may include, but are not limited to, liposomes, such as those composed of 1, 2-dioleyloxy-N, N-dimethylaminopropane (DODMA), diLa2 liposomes from Marina Biotech (Bothenl, WA), 1, 2-dioleyloxy-3-dimethylaminopropane (DLin-DMA), 2-dioleyloxy-4- (2-dimethylaminoethyl) - [1,3]Dioxolane (DLin-KC 2-DMA) and MC3 (US 20100324120; the contents of which are incorporated herein by reference in their entirety), and liposomes that can deliver small molecule drugs, such as, but not limited to, those from Janssen Biotech, inc. (Horsham, pa.)

In one embodiment, the pharmaceutical compositions described herein may include, but are not limited to, liposomes, e.g., as synthetically formed from stable plasmid-lipid particles (SPLPs) or Stable Nucleic Acid Lipid Particles (SNALPs) that have been previously described and shown to be suitable for in vitro and in vivo oligonucleotide delivery (see Wheeler et al Gene therapy.1999 6:271-281; zhang et al Gene therapy.1999 6:1438-1447; jeffs et al Pharm Res.2005 22:362-372; morrissey et al, nat Biotechnol.2005 2:1002-1007; zimmermann et al, nature.441:111-114; heyes et al J Rel.107:276-287; semple et al, nature Biotech.2010:172-176; judge et al J Clin investment.20019: 673;deFougerolles Hum Gene Ther.2008 19:125-132; and the contents of each of which are incorporated herein in their entirety). The initial manufacturing method of Wheeler et al was a detergent dialysis method, later modified by Jeffs et al, and was called the self-foaming method. In addition to saRNA, the liposomal formulation may also include 3 to 4 lipid components. As one example, liposomes can contain, but are not limited to, 55% cholesterol, 20% distearoyl phosphatidylcholine (DSPC), 10% peg-S-DSG, and 15%1, 2-dioleyloxy-N, N-dimethylaminopropane (DQDMA), as described by Jeffs et al. In another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, wherein the cationic lipid may be 1, 2-distearoyloxy-N, N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1, 2-dioleyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al. In another example, the nucleic acid-lipid particles may comprise cationic lipids, which comprise about 50mol% to about 85mol% of the total lipids present in the particles; a non-cationic lipid comprising from about 13mol% to about 49.5mol% of the total lipid present in the particle; and conjugated lipids that inhibit aggregation of the particles, which constitute from about 0.5mol% to about 2mol% of the total lipids present in the particles, as described in WO2009127060 to Maclachlan et al, the contents of which are incorporated herein by reference in their entirety. In another example, the nucleic acid-lipid particle may be any of the nucleic acid-lipid particles disclosed in US2006008910 to Maclachlan et al, the contents of which are incorporated herein by reference in their entirety. As one non-limiting example, the nucleic acid-lipid particles may comprise a cationic lipid of formula I, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of the particles.

In one embodiment, the saRNA of the present disclosure may be formulated in lipid vesicles that may have cross-links between functionalized lipid bilayers.

In one embodiment, the liposome may contain a sugar modified lipid as disclosed in US5595756 to Bally et al, the contents of which are incorporated herein by reference in their entirety. The lipid may be ganglioside (ganglioside) and cerebroside (cerebroside) in an amount of about 10 mole%.

In one embodiment, the saRNA of the present disclosure can be formulated in a liposome comprising a cationic lipid. The molar ratio of nitrogen atoms in the cationic lipid to phosphate in the saRNA (N: P ratio) in the liposome may be from 1:1 to 20:1, as described in international publication No. WO2013006825, the contents of which are incorporated herein by reference in their entirety. In another embodiment, the liposome may have an N to P ratio of greater than 20:1 or less than 1:1.

In one embodiment, the saRNA of the present disclosure can be formulated in a lipid-polycation complex. The formation of lipid-polycation complexes may be accomplished by methods known in the art and/or as described in U.S. publication No. 20120178702, the contents of which are incorporated herein by reference in their entirety. As one non-limiting example, the polycation may include a cationic peptide or polypeptide, such as, but not limited to, polylysine, polyornithine and/or polyarginine, and the cationic peptides described in international publication No. WO 2012013326; incorporated herein by reference in its entirety. In another embodiment, the saRNA may be formulated in a lipid-polycation complex, which may also include neutral lipids such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

Liposome formulations may be affected by, but are not limited to, the following: the choice of cationic lipid component, the degree of saturation of the cationic lipid, the nature of the pegylation, the proportions of all components and biophysical parameters such as size. In one example of Semple et al (Semple et al, nature Biotech.2010:172-176; the contents of which are incorporated herein by reference in their entirety), the liposome formulation consisted of 57.1% cationic lipid, 7.1% dipalmitoyl phosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.

In some embodiments, the proportion of PEG in the Lipid Nanoparticle (LNP) formulation may be increased or decreased, and/or the carbon chain length of the PEG lipid may be modified between C14 and C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulation. As one non-limiting example, the LNP formulation may contain PEG-c-DOMG in a lipid molar ratio of 1-5% compared to cationic lipid, DSPC and cholesterol. In another embodiment, PEG-c-DOMG may be replaced by a PEG lipid such as, but not limited to, PEG-DSG (1, 2-distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG (1, 2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200, and DLin-KC2-DMA.

In one embodiment, the saRNA of the present disclosure may be formulated in a lipid nanoparticle, such as the lipid nanoparticle described in international publication No. WO2012170930, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the cationic lipids useful in the formulations of the present disclosure may be selected from, but are not limited to, the cationic lipids described in the following documents: international publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865 and WO2008103276; U.S. patent nos. 7,893,302, 7,404,969 and 8,283,333; US patent publication nos. US20100036115 and US20120202871; the respective content of which is incorporated herein by reference in its entirety. In another embodiment, the cationic lipid may be selected from, but is not limited to, formula a described in international publication nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365 and WO 2012044638; the contents of each are incorporated by reference herein in their entirety. In yet another embodiment, the cationic lipid may be selected from, but is not limited to, the formula CLI-CLXXIX of international publication No. WO2008103276, the formula CLI-CLXXIX of US patent No. 7,893,302, the formula CLI-clxxxii of US patent No. 7,404,969, and the formulas I-VI of US patent publication No. US 20100036115; the contents of each are incorporated by reference herein in their entirety. In yet another embodiment, the cationic lipid may be a multivalent cationic lipid, such as disclosed in U.S. patent No. 7223887 to Gaucheron et al, the contents of which are incorporated herein by reference in their entirety. Cationic lipids can have a positively charged head group comprising two quaternary ammonium groups and a hydrophobic moiety comprising four hydrocarbon chains, as described in U.S. patent No. 7223887 to Gaucheron et al, the contents of which are incorporated herein by reference in their entirety. In yet another embodiment, the cationic lipid may be biodegradable, such as the biodegradable lipid disclosed in US20130195920 to Maier et al, the contents of which are incorporated herein by reference in their entirety. The cationic lipid may have one or more biodegradable groups located in the lipid portion of the cationic lipid, as described in formulas I-IV in US20130195920 to Maier et al, the contents of which are incorporated herein by reference in their entirety.

As a non-limiting example, the cationic lipid may be selected from the group consisting of (20Z, 23Z) -N, N-dimethyltwenty-1, 15-dien-4-amine, (17Z, 20Z) -N, N-dimethyltwenty-17, 20-dien-9-amine, (1Z, 19Z) -N, 5N-dimethyltwenty-1, 19-dien-8-amine, (13Z, 16Z) -N, N-dimethyltwenty-13, 16-dien-5-amine, (12Z, 15Z) -N, N-dimethyltwenty-1, 15-dien-4-amine, (14Z, 17Z) -N, N-dimethyltwenty-14, 17-dien-6-amine, (15Z, 18Z) -N, N-dimethyltwenty-15, 18-dien-7-amine, (Z, 21Z) -N, N-dimethyltwenty-18, 21-dien-10-amine, (15Z, 18Z) -N, N-dimethyltwenty-12, 15-14-dien-4-amine, (14Z, 17-dimethyltwenty-6-amine, (18Z, 18Z) -N, 17-dimethyltwenty-8-dien-amine, (18Z ) -N, N-dimethyltwenty-18-14-dien-amine, (18Z, 17-N, 17-dimethyltwenty-14-amine, n-dimethylhexacosane-17, 20-dien-7-amine, (16Z, 19Z) -N, N-dimethylhexapentadec-16, 19-dien-6-amine, (22Z, 25Z) -N, N-dimethylheptadecade-22, 25-dien-10-amine, (21Z, 24Z) -N, N-dimethylheptadecade-21, 24-dien-9-amine, (18Z) -N, N-dimethylheptadecade-18-en-10-amine, (17Z) -N, N-dimethylhexahexadeca-17-en-9-amine, (19Z, 22Z) -N, N-dimethylheptadecade-10-amine, (20Z, 23Z) -N-ethyl-N-methylheptadecade-20, 23-dien-10-amine, 1- [ (11Z, 14Z) -1-nonacosyl-11, 14-dien-1-yl ] pyrrole, (17Z) -N, N-dimethylheptadecade-9-amine, (19, 22Z) -N, N-dimethylheptadecade-10-amine, (20Z) -N-dimethylheptadecade-10-amine, n-dimethylicosacarbon-17-en-10-amine, (24Z) -N, N-dimethyltricridec-24-en-10-amine, (20Z) -N, N-dimethyltricridec-20-en-10-amine, (22Z) -N, N-dimethyltricyclohexadec-22-en-10-amine, (16Z) -N, N-dimethyltricyclopentadeca-16-en-8-amine, (12Z, 15Z) -N, N-dimethyl-2-nonyldi-undec-12, 15-dien-1-amine, (13Z, 16Z) -N, N-dimethyl-3-nonyldocodecyl-13, 16-dien-1-amine, N-dimethyl-1- [ (1S, 2R) -2-octylcyclopropyl ] heptadec-8-amine, 1- [ (1S, 2R) -2-hexylcyclopropyl ] -N, N-dimethylnona-10-amine, N-dimethyl-1- [ (2R) -2-octylcyclopropyl ] deca-13, 16-dien-1-amine, N-dimethyl-1- [ (1S, 16Z) -N-dimethyl-1-N-octylcyclopropyl ] deca-1-amine, n-dimethyl-1- [ (1S, 2S) -2- { [ (1R, 2R) -2-pentylcyclopropyl ] methyl } cyclopropyl ] nonadecan-10-amine, N-dimethyl-1- [ (1S, 2R) -2-octylcyclopropyl ] hexadeca-8-amine, N-dimethyl- [ (1R, 2S) -2-undecylcyclopropyl ] tetradeca-5-amine, N-dimethyl-3- {7- [ (1S, 2R) -2-octylcyclopropyl ] heptyl } dodeca-1-amine, 1- [ (1R, 2S) -2-heptylcyclopropyl ] -N, N-dimethyloctadeca-9-amine, 1- [ (1S, 2R) -2-decylcyclopropyl ] -N, N-dimethylpentadecan-6-amine, N-dimethyl-1- [ (1S, 2R) -2-octylcyclopropyl ] pentadecan-8-amine, R-N, N-dimethyl-1- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] -3- (octyloxy) propan-2-amine, S-N, N-dimethyl-1- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] -3- (octyloxy) propan-2-amine, 1- {2- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] -1- [ (octyloxy) methyl ] ethyl } pyrrolidine, (2S) -N, N-dimethyl-1- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] -3- [ (5Z) -oct-5-en-1-yloxy ] propan-2-amine, 1- {2- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] -1- [ (octyloxy) methyl ] ethyl } azetidine, (2S) -1- (hexyloxy) -N, N-dimethyl-3- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] propan-2-amine, (2S) -1- (heptyloxy) -N, N-dimethyl-3- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] propan-2-amine, N-dimethyl-1- (nonyloxy) -3- [ (9Z, 12-dien-1-yloxy ] propan-2-amine, N-dimethyl-3- [ (nonyloxy) -3, Z) -9, 12-dien-2-yloxy ] propan-amine, n-dimethyl-1- [ (9Z) -octadec-9-en-1-yloxy ] -3- (octyloxy) propan-2-amine; (2S) -N, N-dimethyl-1- [ (6Z, 9Z, 12Z) -octadec-6, 9, 12-trien-1-yloxy ] -3- (octyloxy) propan-2-amine, (2S) -1- [ (11Z, 14Z) -eicosan-11, 14-dien-1-yloxy ] -N, N-dimethyl-3- (pentyloxy) propan-2-amine, (2S) -1- (hexyloxy) -3- [ (11Z, 14Z) -eicosan-11, 14-dien-1-yloxy ] -N, N-dimethylpropan-2-amine, 1- [ (11Z, 14Z) -eicosan-11, 14-dien-1-yloxy ] -N, N-dimethyl-3- (octyloxy) propan-2-amine, 1- [ (Z, 16Z) -docosan-3- (octyloxy) propan-2-amine, (2S) -1- [ (13Z) -1-dien-1-yloxy ] -N, N-dimethyl-3- (octyloxy) propan-2-amine, N-dimethyl-1- [ (13Z) -didodecyl-1-yloxy ] -N, 16Z-dien-1-yloxy ] -N, 16-dien-1-yloxy ] -2-amine, (2S) -1- [ (13Z) -docosan-13-en-1-yloxy ] -3- (hexyloxy) -N, N-dimethylpropan-2-amine, 1- [ (13Z) -docosan-13-en-1-yloxy ] -N, N-dimethyl-3- (octyloxy) propan-2-amine, 1- [ (9Z) -hexadeca-9-en-1-yloxy ] -N, N-dimethyl-3- (octyloxy) propan-2-amine, (2R) -N, N-dimethyl-H (1-methyloctyl) oxy ] -3- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] propan-2-amine, (2R) -1- [ (3, 7-dimethyloctyl) oxy ] -N, N-dimethyl-3- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] propan-2-amine, N-dimethyl-1- (oxy) -3- ({ 8- [ (1S, 2R) -2- (2R) cyclopropyl ] cyclopropyl } amine, n-dimethyl-1- { [8- (2-octylcyclopropyl) octyl ] oxy } -3- (octyloxy) propan-2-amine and (11E, 20Z, 23Z) -N, N-dimethyl-icosadeca-11,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the lipid may be a cleavable lipid, such as those described in international publication No. WO2012170889, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the nanoparticle described herein can comprise at least one cationic polymer described herein and/or known in the art.

In one embodiment, the cationic lipid may be synthesized by methods known in the art and/or as described in the following documents: international publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 and WO201021865; the respective content of which is incorporated herein by reference in its entirety.

In one embodiment, the LNP formulation of sarNA may contain a 3% lipid molar ratio of PEG-c-DOMG. In another embodiment, the LNP formulation of sarNA can contain a 1.5% lipid molar ratio of PEG-c-DOMG.

In one embodiment, the pharmaceutical composition of saRNA may include at least one pegylated lipid described in international publication No. 2012099755, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the LNP formulation may contain PEG-DMG 2000 (1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000). In one embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, and at least one other component. In another embodiment, the LNP formulation may contain PEG-DMG 2000, cationic lipids known in the art, DSPC, and cholesterol. As one non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol. As another non-limiting example, an LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol in a molar ratio of 2:40:10:48 (see, e.g., gel et al Nonviral delivery of self-amplifying RNA vaccines, PNAS2012; PMID:22908294; incorporated herein by reference in its entirety). As another non-limiting example, the saRNA described herein can be formulated in nanoparticles for delivery by parenteral route, as described in U.S. publication No. 20120207845; the contents of which are incorporated herein by reference in their entirety. The cationic lipids may also be those disclosed in US20130156845 to Manoharan et al, and US20130129785 to Manoharan et al, WO 2012047656 to Wasan et al, WO 2010144740 to Chen et al, WO 2013086322 to Ansell et al, or WO 2012016184 to Manoharan et al, the respective contents of which are incorporated herein by reference in their entirety.

In one embodiment, the saRNA of the present disclosure may be formulated with a variety of cationic lipids, for example, first and second cationic lipids as described in US20130017223 to Hope et al, the contents of which are incorporated herein by reference in their entirety. The first cationic lipid may be selected based on a first property and the second cationic lipid may be selected based on a second property, wherein the properties may be determined as outlined in US20130017223, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the first and second properties are complementary.

In another embodiment, the saRNA may be formulated with a lipid particle comprising one or more cationic lipids and one or more secondary lipids, and one or more nucleic acids, wherein the lipid particle comprises a solid core, as described in U.S. patent publication No. US20120276209 to Cullis et al, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the saRNA of the present disclosure may be complexed with a cationic amphiphile in an oil-in-water (o/w) emulsion, such as described in EP2298358 to Satishchandran et al, the contents of which are incorporated herein by reference in their entirety. The cationic amphiphile may be a cationic lipid, modified or unmodified spermine (spimine), bupivacaine (bupivacaine) or benzalkonium chloride, and the oil may be a vegetable oil or an animal oil. As one non-limiting example, at least 10% of the nucleic acid-cationic amphiphile complex is in the oil phase of an oil-in-water emulsion (see, e.g., the complex described in european publication No. EP2298358 to Satishchandran et al, the contents of which are incorporated herein by reference in their entirety).

In one embodiment, the saRNA of the present disclosure can be formulated with a composition comprising a mixture of cationic compound and neutral lipid. As one non-limiting example, the cationic compound may be of formula (I) disclosed in WO 1999010390 to Ansell et al, the contents of which are incorporated herein by reference in their entirety, and the neutral lipid may be selected from the group consisting of diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, and sphingomyelin. In another non-limiting example, the lipid formulation may comprise a cationic lipid of formula a, a neutral lipid, a sterol, and a PEG or PEG-modified lipid as disclosed in US20120101148 to Akinc et al, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the LNP formulation may be formulated by the methods described in international publication No. WQ2011127255 or WO2008103276, the respective contents of which are incorporated herein by reference in their entirety. As one non-limiting example, the saRNA of the present disclosure may be encapsulated in any Lipid Nanoparticle (LNP) formulation described in WO2011127255 and/or WO20G 8103276; the respective content of which is incorporated herein by reference in its entirety.

In one embodiment, the LNP formulations described herein can comprise a polycationic composition. As one non-limiting example, the polycationic composition may be selected from formulas 1-60 of U.S. patent publication No. US 20050222064; the contents of which are incorporated herein by reference in their entirety. In another embodiment, LNP formulations comprising polycationic compositions can be used to deliver the saRNA described herein in vivo and/or in vitro.

In one embodiment, the LNP formulations described herein may additionally comprise a permeability-enhancing molecule. Non-limiting permeability facilitating molecules are described in U.S. patent publication No. US 20050222064; the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the pharmaceutical composition may be formulated in a liposome, such as, but not limited to, diLa2 liposome (Marina Biotech, bothenll, WA),NOV340 (Marina Biotech, bopall, WA), neutral DOPC (1, 2-dioleoyl-sn-glycero-3-phosphorylcholine) based liposomes (e.g., siRNA delivery for ovarian Cancer (Landen et al Cancer)&Therapeutic 2006 5 (12) 1708-1713); the contents of which are incorporated by reference in their entiretyHerein) and hyaluronic acid coated liposomes (Quiet Therapeutics, israel).

In some embodiments, the pharmaceutical composition may be formulated with any of the amphiphilic liposomes disclosed in WO 2008/043575 to Panzner and US 8580297 to Essler et al (Marina Biotech), the contents of which are incorporated herein by reference in their entirety. The amphipathic liposome may comprise a lipid mixture comprising a cationic amphiphile, an anionic amphiphile, and optionally one or more neutral amphiphiles. The amphiphilic liposomes may comprise amphiphilic compounds based on amphiphilic molecules, the head groups of which are substituted with one or more amphiphilic groups. In some embodiments, the pharmaceutical compositions may be formulated with an amphipathic lipid comprising one or more amphipathic groups having isoelectric points between 4 and 9, as disclosed in US20140227345 to Essler et al (Marina Biotech), the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the pharmaceutical composition may be formulated with liposomes comprising sterol derivatives as disclosed in US 7312206 to Panzner et al (Novosom), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the pharmaceutical composition may be formulated with an amphipathic liposome comprising at least one amphipathic cationic lipid, at least one amphipathic anionic lipid, and at least one neutral lipid, or a liposome comprising at least one amphipathic lipid having both positive and negative charges and at least one neutral lipid, wherein the liposome is stable at pH 4.2 and pH 7.5, as disclosed in U.S. patent No. 7780983 to Panzner et al (Novosom), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the pharmaceutical composition may be formulated with liposomes comprising a serum-stable mixture of lipids capable of encapsulating the saRNA of the present disclosure, as taught in US20110076322 to Panzner et al, the contents of which are incorporated herein by reference in their entirety. The lipid mixture comprises phosphatidylcholine and phosphatidylethanolamine in a ratio ranging from about 0.5 to about 8. The lipid mixture may also include pH sensitive anionic and cationic amphiphiles such that the mixture is amphoteric, negatively charged or neutral at pH 7.4, and positively charged at pH 4. The drug/lipid ratio can be tailored to target the liposome to a specific organ or other part of the body. In some embodiments, liposomes loaded with the saRNA of the present disclosure as an cargo are prepared by the method disclosed in US20120021042 to Panzner et al, the contents of which are incorporated herein by reference in their entirety. The method comprises the steps of mixing an aqueous solution of a polyanionic active agent with an alcoholic solution of one or more amphiphiles and buffering the mixture to an acidic pH, wherein the one or more amphiphiles are susceptible to forming amphiphilic liposomes at the acidic pH, thereby forming amphiphilic liposomes in suspension form encapsulating the active agent.

The nanoparticle formulation can be a carbohydrate nanoparticle comprising a carbohydrate carrier and a nucleic acid molecule (e.g., saRNA). As one non-limiting example, the carbohydrate carrier may include, but is not limited to, anhydride modified phytoglycogen or glycogen type substances, octenyl phytoglycogen succinate, phytoglycogen beta-dextrin, anhydride modified phytoglycogen beta-dextrin. (see, e.g., international publication No. WO 2012109121; the contents of which are incorporated herein by reference in their entirety).

Lipid nanoparticle formulations can be improved by replacing cationic lipids with biodegradable cationic lipids known as rapid elimination lipid nanoparticles (reLNP). Ionizable cationic lipids such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA have been shown to accumulate in plasma and tissues over time and can be potential sources of toxicity. The rapid metabolism of rapidly eliminated lipids can improve the tolerability and therapeutic index of lipid nanoparticles by 1 order of magnitude from a 1mg/kg dose to a 10mg/kg dose in rats. The inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolic characteristics of the cationic component while still maintaining the activity of the reLNP formulation. The ester linkage may be internal to the lipid chain, or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.

In one embodiment, the saRNA is formulated as a lipid complex, such as, but not limited to, atupex ^TM System, DACC system, DBTC system and other siRNA-liposome complex technology from Silence Therapeutics (london, uk), fromSTEMFECT of (Cambridge, mass.) ^TM And Polyethyleneimine (PEI) or protamine (protamine) based targeted and non-targeted nucleic acid delivery (Aleku et al Cancer Res.2008 68:9788-9798; strumarg et al Int J Clin Pharmacol Ther:2012 50-76-78; santel et al, gene Ther 200613:1222-1234; santel et al, gene Ther 2006:1360-1370; gutbier et al, pulm Phacol. Ther.201023:334-344; kaufmann et al micro-vasc Res 080:286-293; weide et al J Immunother.2009:498-507; weide et al JImmunother.2008:180-188;Pascolo Expert Opin.Biol.Ther.4:1285-1294; fotin-Mleck et al, 2011.34:1-15; sozeng et al, nature Pehnol.2005:709-709, A.95:6283; and the like, each of which is incorporated herein by reference in its entirety).

In one embodiment, such formulations or altered compositions can also be constructed so that they are passively or actively directed to different cell types in vivo, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akine et al Mol Ther.201018:1357-1364; song et al, nat Biotechnol.2005:709-717; judge et al, JClin invest.2009:661-673; kaufmann et al, microvasc Res 201080:286-293; santel et al, gene Ther 2006:1222-1234; santel et al, gene Ther 13:1360-1370; guter et al, pulm Phacol. Ther.023:334-344; basha et al, mol. Ther.20119:2186-Pensand Cuper patent, and Peper patent No. 2011:2186-Peper fig. plug, and Peper patent No. 2011:1138:1138; peper patent application, and by use of the teachings of this disclosure to the present invention, and to the whole of this disclosure, and to the invention, by use of the teachings of the disclosure). One example of a formulation that passively targets hepatocytes includes lipid nanoparticle formulations based on DLin-DMA, DLin-KC2-DMA, and DLin-MC3-DMA, which have been shown to bind to apolipoprotein E and promote binding of these formulations and their uptake into hepatocytes in vivo (Akinc et al Mol ter.2010:1357-1364; the contents of which are incorporated herein by reference in their entirety). Formulations can also be selectively targeted by expressing different ligands on their surfaces, such as but not limited to folic acid, transferrin, N-acetylgalactosamine (GalNAc) and antibody targeting protocols (Kolhatkar et al, curr Drug discovery technology 20118:197-206; musacchi and Torchilin, front biosci.20116:1388-1412; yu et al, mol membrane biol.2010 27:286-298; patil et al, crit Rev Ther Drug Carrier Syst.2008:25-1-61; benoit et al, biomacromolecules.2011:12:2708-2714; zhao et al, exert Opin Drug Deliv.2008 5:309-319; akinec et al, mol Ther.2010:1357-1364; srinivasan et al, methods Mol biol.2012:105-116; beam-Arie et al, methods Mol biol.2012:497-507;Peer 2010J Control Release.20:63-68; peer et al, proc Natl Acad Sci U S A.2007104:4095-0; kim et al, methods Mol biol.2011-2029:319; pekine et al, mol Ther.2012:1357-1364; sriniva et al, methods Mol. 2012:2012:2012-2012, and Gene-insulator.2012:2012-electric motor-insulator.2012:2012; the contents of each of the above are incorporated herein by reference in their entirety).

In one embodiment, the saRNA is formulated as a solid lipid nanoparticle. The Solid Lipid Nanoparticles (SLNs) may be spherical with an average diameter between 10 and 1000 nm. SLNs possess a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and/or emulsifiers. In yet another embodiment, the lipid nanoparticle may be a self-assembled lipid-polymer nanoparticle (see Zhang et al, ACS Nano,2008,2 (8), pp 1696-1702; the disclosure of which is incorporated herein by reference in its entirety).

In one embodiment, the saRNA of the present disclosure can be formulated for controlled release and/or targeted delivery. As used herein, "controlled release (controlled release)" refers to a pharmaceutical composition or compound release profile that follows a particular release pattern to affect the outcome of a treatment. In one embodiment, the saRNA may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term "encapsulate" means to enclose, encase, or encase. When referring to formulations of the compounds of the present disclosure, encapsulation may be substantial, complete, or partial. The term "substantially encapsulated" means that at least greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater than 99.999% of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent. By "partially encapsulated" is meant that less than 10, 20, 30, 40, 50 or less of the pharmaceutical compositions or compounds of the present disclosure can be enclosed, surrounded or encased within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or activity of the pharmaceutical composition or compound of the present disclosure using fluorescence and/or electron micrographs. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or greater than 99.99% of a pharmaceutical composition or compound of the present disclosure is encapsulated in a delivery agent.

In another embodiment, the SaRNA may be encapsulated into a lipid nanoparticle or a rapid elimination lipid nanoparticle, and the lipid nanoparticle or rapid elimination lipid nanoparticle may then be encapsulated into a polymer, hydrogel, and/or surgical sealant described herein and/or known in the art. As one non-limiting example, the polymer, hydrogel, or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, or a combination thereof,(Nanotherapeutics,Inc.Alachua,FL)、/>(Halozyme Therapeutics, san Diego CA), surgical sealants such as fibrinogen polymer (Ethicon Inc.Cornelia, GA), and->(Baxter International, inc., deifield, IL), PEG-based sealants +.>(Baxter International,Inc.,Deerfield,IL)。

In another embodiment, the lipid nanoparticle may be encapsulated into any polymer known in the art that can form a gel when injected into a subject. As another non-limiting example, the lipid nanoparticle may be encapsulated into a polymer matrix, which may be biodegradable.

In one embodiment, the saRNA formulation for controlled release and/or targeted delivery may further comprise at least one controlled release coating. Controlled release coatings include, but are not limited to Polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT->EUDRAGITAnd cellulose derivatives, such as ethylcellulose aqueous dispersions (++>And->)。

In one embodiment, the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester, which may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly (serine esters), poly (L-lactide-co-L-lysine), poly (4-hydroxy-L-proline esters), and combinations thereof. In another embodiment, the degradable polyester may include PEG conjugation to form a pegylated polymer.

In one embodiment, the saRNA of the present disclosure may be formulated with a targeting lipid having a targeting moiety, such as the targeting moiety disclosed in US20130202652 to Manoharan et al, the contents of which are incorporated herein by reference in their entirety. As one non-limiting example, the targeting moiety of formula I in US20130202652 to Manoharan et al may be selected to facilitate the localization of the lipid to a target organ, tissue, cell type or subpopulation or organelle. Non-limiting targeting moieties contemplated in the present disclosure include transferrin, anisoamide (anilamide), RGD peptides, prostate Specific Membrane Antigen (PSMA), fucose, antibodies, or aptamers.

In one embodiment, the saRNA of the present disclosure may be encapsulated in a therapeutic nanoparticle. Therapeutic nanoparticles may be formulated by methods described herein and known in the art, such as, but not limited to, international publication nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923; U.S. publication nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286 and US20120288541; and U.S. patent nos. 8,206,747, 8,293,276, 8,318,208 and 8,318,211; the contents of each of which are incorporated herein by reference in their entirety. In another embodiment, therapeutic polymer nanoparticles can be identified by the method described in U.S. publication No. US20120140790, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the therapeutic nanoparticle may be formulated for sustained release. As used herein, "sustained release (sustained release)" refers to a pharmaceutical composition or compound that follows a release rate over a particular period of time. The time period may include, but is not limited to, hours, days, weeks, months, and years. As one non-limiting example, the sustained release nanoparticle may comprise a polymer and a therapeutic agent, such as, but not limited to, a saRNA of the present disclosure (see international publication No. 2010075072 and U.S. publication nos. US20100216804, US20110217377, and US20120201859, the contents of each of which are incorporated herein by reference in their entirety).

In one embodiment, the therapeutic nanoparticle may be formulated to be target specific. As one non-limiting example, the therapeutic nanoparticle may include a corticosteroid (see international publication No. WO2011084518; the contents of which are incorporated herein by reference in their entirety). In one embodiment, the therapeutic nanoparticle may be formulated to be cancer specific. As one non-limiting example, therapeutic nanoparticles may be formulated in nanoparticles described in the following patent documents: international publication nos. WO2008121949, WO2010005726, WO2010005725, WO2011084521, and U.S. publication nos. US20100069426, US20120004293 and US20100104655, the contents of each of which are incorporated herein by reference in their entirety.

In one embodiment, the nanoparticles of the present disclosure may comprise a polymer matrix. As one non-limiting example, the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxyacid, polypropylene fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly (orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly (ethyleneimine), poly (serine ester), poly (L-lactide-co-L-lysine), poly (4-hydroxy-L-proline ester), or a combination thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblock copolymer. In one embodiment, the diblock copolymer may comprise PEG in combination with a polymer such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxyacid, polypropylene fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly (orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly (ethyleneimine), poly (serine ester), poly (L-lactide-co-L-lysine), poly (4-hydroxy-L-proline ester), or a combination thereof.

As one non-limiting example, the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see U.S. publication No. US20120004293 and U.S. patent No. 8,236,330, each of which is incorporated herein by reference in its entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth (stealth) nanoparticle comprising PEG and PLA or a diblock copolymer of PEG and PLGA (see U.S. patent No. 8,246,968 and international publication No. WO2012166923, the contents of each of which are incorporated herein by reference in their entirety).

In one embodiment, the therapeutic nanoparticle may comprise a multi-block copolymer, such as, but not limited to, the multi-block copolymers described in U.S. patent nos. 8,263,665 and 8,287,910; the contents of each of which are incorporated herein by reference in their entirety.

In one embodiment, the block copolymers described herein may be included in polyionic complexes containing non-polymeric micelles and block copolymers. (see, e.g., U.S. publication No. 20120076836; the contents of which are incorporated herein by reference in their entirety).

In one embodiment, the therapeutic nanoparticle may comprise at least one acrylic polymer. Acrylic polymers include, but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly (acrylic acid), poly (methacrylic acid), polycyanoacrylates, and combinations thereof.

In one embodiment, the therapeutic nanoparticle may comprise at least one amine-containing polymer, such as, but not limited to, polylysine, polyethylenimine, poly (amidoamine) dendrimers, poly (β -amino esters) (see, e.g., U.S. patent No. 8,287,849; the contents of which are incorporated herein by reference in their entirety), and combinations thereof.

In one embodiment, the therapeutic nanoparticle may comprise at least one degradable polyester that may comprise polycationic side chains. Degradable polyesters include, but are not limited to, poly (serine esters), poly (L-lactide-co-L-lysine), poly (4-hydroxy-L-proline esters), and combinations thereof. In another embodiment, the degradable polyester may include PEG conjugates to form a pegylated polymer.

In another embodiment, the therapeutic nanoparticle may include conjugation of at least one targeting ligand. The targeting ligand may be any ligand known in the art, such as, but not limited to, a monoclonal antibody. (Kirpotin et al, cancer Res.2006:6732-6740; the contents of which are incorporated herein by reference in their entirety).

In one embodiment, the therapeutic nanoparticles may be formulated in aqueous solutions useful for targeting cancer (see international publication No. WO2011084513 and U.S. publication No. US20110294717, the contents of each of which are incorporated herein by reference in their entirety).

In one embodiment, the saRNA may be encapsulated in, attached to, and/or bound to the synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in the following patent documents: international publication Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149411, WO2012149454 and WO2013019669; and U.S. publication nos. US20110262491, US20100104645, US20100087337, and US20120244222, the contents of each of which are incorporated herein by reference in their entirety. Synthetic nanocarriers can be formulated using methods known in the art and/or described herein. As one non-limiting example, the synthetic nanocarriers may be formulated by the methods described in the following patent documents: international publication nos. WO2010005740, WO2010030763 and WO201213501, and U.S. publication nos. US20110262491, US20100104645, US20100087337 and US2012024422, the contents of each of which are incorporated herein by reference in their entirety. In another embodiment, the synthetic nanocarrier formulation may be lyophilized by the methods described in international publication No. WO2011072218 and U.S. patent No. 8,211,473; the contents of each of which are incorporated herein by reference in their entirety.

In one embodiment, the synthetic nanocarriers may contain reactive groups to release the saRNA described herein (see international publication No. WO20120952552 and U.S. publication No. US20120171229, the contents of each of which are incorporated herein by reference in their entirety).

In one embodiment, the synthetic nanocarriers may be formulated for targeted release. In one embodiment, the synthetic nanocarriers may be formulated to release saRNA at a specified pH and/or after a desired time interval. As one non-limiting example, the synthetic nanoparticles may be formulated to release saRNA after 24 hours and/or at pH 4.5 (see international publication nos. WO2010138193 and WO2010138194 and US publication nos. US20110020388 and US20110027217, the contents of each of which are incorporated herein by reference in their entirety).

In one embodiment, the synthetic nanocarriers may be formulated for controlled and/or sustained release of saRNA as described herein. As one non-limiting example, synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein, and/or as described in international publication No. WO2010138192 and U.S. publication No. 20100303850, the contents of each of which are incorporated herein by reference in their entirety.

In one embodiment, the nanoparticle may be optimized for oral administration. The nanoparticle may comprise at least one cationic biopolymer, such as, but not limited to, chitosan or derivatives thereof. As one non-limiting example, nanoparticles may be formulated by the method described in U.S. publication No. 20120282343; the content of this document is incorporated herein by reference in its entirety.

In one embodiment, the saRNA of the present disclosure may be formulated in a modular composition (modular composition), as described in U.S. Pat. No. 8575123 to Manoharan et al, the contents of which are incorporated herein by reference in their entirety. As one non-limiting example, the modular composition can comprise a nucleic acid such as a saRNA of the present disclosure, at least one endosomolytic component, and at least one targeting ligand. The modular composition may have a formulation such as any of the formulations described in US 8575123 to Manoharan et al, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the saRNA of the present disclosure may be encapsulated in a lipid formulation to form a stable nucleic acid-lipid particle (SNALP), such as described in US8546554 to de Fougerolles et al, the contents of which are incorporated herein by reference in their entirety. The lipid may be cationic or non-cationic. In one non-limiting example, the ratio of lipid to nucleic acid (mass/mass ratio) (e.g., lipid to saRNA ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1, or 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1. In another example, SNALP comprises 40%2, 2-diiodo-4-dimethylaminoethyl- [1,3 ] ]Dioxolane (lipid A), 10% dioleoyl phosphatidylcholine (DSPC), 40% cholesterol, 10% polyethylene glycol (PEG) -C-DOMG (mole percent), particle size 63.0.+ -. 20nm, nucleic acid/lipid ratio 0.027. In another embodiment, the saRNA of the present disclosure may be formulated with nucleic acid-lipid particles comprising endosomal membrane destabilizers, as disclosed in US 7189705 to Lam et al, the disclosure of which is incorporated herein by reference in its entirety. As a non-limiting example, the endosomal membrane destabilizer may be Ca ²⁺ Ions.

In one embodiment, the saRNA of the present disclosure may be formulated with formulated lipid particles (flips) as disclosed in US 8148344 to Akinc et al, the contents of which are incorporated herein by reference in their entirety. Akine et al teach that FLiP may comprise at least one of a single-stranded or double-stranded oligonucleotide in which the oligonucleotide has been conjugated to a lipophilic body (lipophile), and at least one of an emulsion or a liposome in which the conjugated oligonucleotide has been aggregated, mixed or otherwise associated. These particles have surprisingly been shown to be effective in delivering oligonucleotides to the heart, lungs and muscles as disclosed in US 8148344 to Akinc et al, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the saRNA of the present disclosure may be delivered to cells using a composition comprising an expression vector in a lipid formulation as described in US 6086913 to Tam et al, the contents of which are incorporated herein by reference in their entirety. The compositions disclosed in Tam are serum stable and comprise an expression vector comprising first and second inverted repeats from an adeno-associated virus (AAV), a rep gene from the AAV, and a nucleic acid fragment. The expression vector in Tam is complexed with lipids.

In one embodiment, the saRNA of the present disclosure may be formulated with the lipid formulation disclosed in US20120270921 to de Fougerolles, the contents of which are incorporated herein by reference in their entirety. In one non-limiting example, the lipid formulation may include a cationic lipid having formula a described in US20120270921, the contents of which are incorporated herein by reference in their entirety. In another non-limiting example, the composition of exemplary nucleic acid-lipid particles disclosed in table a of US20120270921 (the contents of which are incorporated herein by reference in their entirety) can be used with the saRNA of the present disclosure.

In one embodiment, the saRNA of the present disclosure may be fully encapsulated in the lipid particles disclosed in US20120276207 to Maurer et al, the contents of which are incorporated herein by reference in their entirety. The particles may comprise a lipid composition comprising preformed lipid vesicles, a charged therapeutic agent, and a destabilizing agent to form a mixture of preformed vesicles and therapeutic agent in a destabilizing solvent, wherein the destabilizing solvent is effective to destabilize the membrane of the preformed lipid vesicles without destroying the vesicles.

In one embodiment, the saRNA of the present disclosure can be formulated with conjugated lipids. In one non-limiting example, the conjugated lipid can have a formulation such as described in US20120264810 to Lin et al, the contents of which are incorporated herein by reference in their entirety. The conjugate lipid may form a lipid particle that further comprises a cationic lipid, a neutral lipid, and a lipid capable of reducing aggregation.

In one embodiment, the saRNA of the present disclosure may be formulated in a neutral liposome formulation, such as disclosed in US20120244207 to Fitzgerald et al, the disclosure of which is incorporated herein by reference in its entirety. The phrase "neutral liposome formulation" refers to a liposome formulation having a near neutral or neutral surface charge at physiological pH. The physiological pH may be, for example, about 7.0 to about 7.5, or, for example, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, or, for example, 7.3, or, for example, 7.4. An example of a neutral liposome formulation is an Ionizable Lipid Nanoparticle (iLNP). Neutral liposome formulations can include ionizable cationic lipids, such as DLin-KC2-DMA.

In one embodiment, the saRNA of the present disclosure can be formulated with charged lipids or amino lipids. As used herein, the term "charged lipids" is meant to include those lipids having one or two fatty acyl or fatty alkyl chains and a quaternary amino head group. Quaternary amines have a permanent positive charge. The head group may optionally include an ionizable group, such as a primary, secondary, or tertiary amine that may be protonated at physiological pH. The presence of a quaternary amine may alter the pKa of the ionizable group relative to the pKa of the ionizable group in a structurally similar compound lacking the quaternary amine (e.g., the quaternary amine is replaced with a tertiary amine). In some embodiments, the charged lipid is referred to as an "amino lipid". In one non-limiting example, the amino lipid may be any of the amino lipids described in US20110256175 to Hope et al, the contents of which are incorporated herein by reference in their entirety. For example, the amino lipids can have the structures disclosed in tables 3-7 of Hope, such as structure (II), DLin-K-C2-DMA, DLin-K6-DMA, and the like. The resulting pharmaceutical preparation may be lyophilized according to Hope. In another non-limiting example, the amino lipid can be any of the amino lipids described in US20110117125 to Hope et al, the contents of which are incorporated herein by reference in their entirety, such as the lipids of structure (I), DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLin-S-DMA, and the like. In another non-limiting example, the amino lipid may have the structure (I), (II), (III) or (IV) described in WO2009132131 to Manoharan et al, or 4- (R) -DUn-K-DMA (VI), 4- (S) -DUn-K-DMA (V), the contents of which are incorporated herein by reference in their entirety. In another non-limiting example, the charged lipid used in any of the formulations described herein may be any charged lipid described in EP2509636 to Manoharan et al, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the saRNA of the present disclosure can be formulated with association complexes (association complex) containing lipids, liposomes, or lipid complexes. In one non-limiting example, the association complex comprises one or more compounds each having a structure defined by formula (I), PEG-lipids having a structure defined by formula (XV), steroids, and nucleic acids disclosed in US8034376 to Manoharan et al, the contents of which are incorporated herein by reference in their entirety. The saRNA may be formulated with any of the association complexes described in US8034376, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the saRNA of the present disclosure may be formulated with reverse headgroup lipids (reverse head group lipid). As one non-limiting example, the saRNA may be formulated with a zwitterionic lipid (zwitterionic lipid) comprising a headgroup, wherein the positive charge is located near the acyl chain region and the negative charge is located distal to the headgroup, such as the lipid having structure (a) or structure (I) described in WO2011056682 to Leung et al, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the saRNA of the present disclosure can be formulated in a lipid bilayer carrier. As one non-limiting example, the saRNA may be combined with a lipid-detergent mixture comprising an aggregation preventing agent in an amount of about 5mol% to about 20mol%, a cationic lipid in an amount of about 0.5mol% to about 50mol%, and a lipid mixture of fusogenic lipids (fusogenic lipids) and detergents to provide a nucleic acid-lipid-detergent mixture; the nucleic acid-lipid-detergent mixture is then dialyzed against a buffer salt solution having an ionic strength sufficient to encapsulate about 40% to about 80% of the nucleic acid to remove the detergent and encapsulate the nucleic acid in a lipid bilayer carrier and provide a lipid bilayer-nucleic acid composition, as described in WO1999018933 to Cullis et al, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the saRNA of the present disclosure can be formulated in nucleic acid-lipid particles capable of selectively targeting the saRNA to the heart, liver, or tumor tissue site. For example, a nucleic acid-lipid particle may comprise (a) a nucleic acid; (b) 1.0 to 45 mole% of a cationic lipid; (c) 0.0 to 90 mole% of another lipid; (d) 1.0 to 10 mole% of a bilayer stabilizing component; (e) 0.0 to 60 mole% cholesterol; and (f) from 0.0 to 10 mole% of a cationic polymer lipid, as described in EP1328254 to Cullis et al, the contents of which are incorporated herein by reference in their entirety. Cullis teaches that varying the amount of each of the cationic lipid, bilayer stabilizing component, another lipid, cholesterol, and cationic polymer lipid can impart tissue selectivity to heart, liver, or tumor tissue sites, thereby identifying nucleic acid lipid particles that are capable of selectively targeting nucleic acids to heart, liver, or tumor tissue sites.

Polymer, biodegradable nanoparticle and core-shell nanoparticle

The saRNA of the present disclosure may be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers that may be used for delivery include, but are not limited to, DYNAMIC (Arrowhead Research Corp., pasadena, calif.), from +.>Preparation of Bio (Madison, wis.) and Roche Madison (Madison, wis.), PHASERX ^TM Polymer formulations, such as, but not limited to SMARTT POLYMER TECHNOLOGY ^TM (/>Seattle, WA), DMRI/DOPE, poloxamer, +.>Adjuvants, chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimersMolecules and poly (lactic-co-glycolic acid) (PLGA) polymers, RONDEL ^TM (RNAi/oligonucleotide nanoparticle delivery) polymers (Arrowhead Research Corporation, pasadena, calif.) and pH-responsive co-block polymers, such as but not limited to(Seattle,WA)。

One non-limiting example of a chitosan formulation includes a positively charged chitosan core and an outer portion of a negatively charged matrix (U.S. publication No. 20120258176; incorporated herein by reference in its entirety). Chitosan includes, but is not limited to, N-trimethylchitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof.

In one embodiment, the polymers used in the present disclosure have been subjected to a treatment to reduce and/or inhibit the attachment of unwanted substances (e.g., without limitation, bacteria) to the polymer surface. The polymer may be treated by methods known and/or described in the art and/or described in international publication No. WO2012150467 (incorporated herein by reference in its entirety).

One non-limiting example of a PLGA formulation includes but is not limited to PLGA injectable depots (e.g.,it is formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) with the remainder being aqueous solvent and leuprorelin (leuprolide). Upon injection, PLGA and leuprorelin peptide precipitated into the subcutaneous space).

Many of these polymeric methods have demonstrated efficacy in delivering oligonucleotides into the cytoplasm in vivo (reviewed in de Fougerolles Hum Gene Ther.2008 19:125-132; incorporated herein by reference in its entirety). In the case of small interfering RNAs (sirnas), two polymeric methods that have resulted in robust in vivo delivery of nucleic acids are dynamic multi-conjugates (dynamic polyconjugate) and cyclodextrin-based nanoparticles. The first of these delivery methods uses dynamic multi-conjugates and has been shown to efficiently deliver siRNA in vivo in mice and silence endogenous target mRNA in hepatocytes (Rozema et al Proc Natl Acad Sci U S a.2007104:12982-12887; incorporated herein by reference in its entirety). This particular approach is a multicomponent polymer system whose key features include a membrane active polymer, wherein a nucleic acid (in this case an siRNA) is covalently coupled thereto via disulfide bonds, and wherein both PEG (for charge masking) and N-acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (Rozema et al, proc Natl Acad Sci U S a.2007104:12982-12887; incorporated herein by reference in its entirety). Upon binding to hepatocytes and entering the endosome, the polymer complex breaks down in a low pH environment, the polymer exposes its positive charge, causing the endosome to escape and the siRNA to be released from the polymer into the cytoplasm. By replacing the N-acetylgalactosamine group with a mannitol group, it has been shown that targeting can be changed from hepatocytes expressing asialoglycoprotein receptors to hepatic sinus endothelial cells and Kupffer cells. Another polymeric approach involves the use of transferrin-targeted cyclodextrin-containing polycationic nanoparticles. These nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene product in EWS-FLI1 tumor cells expressing transferrin receptor (Hu-Lieskovan et al, cancer res.2005 65:8984-8982; incorporated herein by reference in its entirety), and sirnas formulated in these nanoparticles are well tolerated in non-human primates (Heidel et al Proc Natl Acad Sci USA 2007 104:5715-21; incorporated herein by reference in its entirety). Both of these delivery strategies incorporate rational approaches to use both targeted delivery and endosomal escape mechanisms.

The polymer formulation may allow for sustained or delayed release of the saRNA (e.g., after intramuscular or subcutaneous injection). A change in the release profile of saRNA can result in translation of the encoded protein over an extended period of time, for example. Biodegradable polymers have previously been used to protect Nucleic acids from degradation and have been shown to result in sustained release of a load (payload) in vivo (Rozema et al Proc Natl Acad Sci US A.2007 104:12982-12887; sullivan et al, expert Opin Drug Deliv.20107:1433-1446; convertene et al, biomacromolecules.2010Oct 1; chu et al, acc Chem Res.2012Jan 13; manganiello et al, biomaterials.201233:2301-2309; benoit et al, biomacromolecules.20112:2708-2714; singha et al, nucleic Acid Ther.2011 2:133-147;de Fougerolles Hum Gene Ther.2008 19:125-132; affert and Waer, gene Theer.16:1131-1138; chavedi et al, oppert et al, 201233:2301-2309; benoit et al, biomacromolecules.2011:2708-2714; singha et al, nucleic Acid Ther.2011:133-147;de Fougerolles Hum Gene Ther.2008 19:125-132; gene, 2008:2008, incorporated herein by reference in their entirety).

In one embodiment, the pharmaceutical composition may be a sustained release formulation. In further embodiments, sustained release formulations may be used for subcutaneous delivery. Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamers, and the like, (Nanotherapeutics,Inc.Alachua,FL)、/>(Halozyme Therapeutics, san Diego CA), surgical sealants such as fibrinogen polymer (Ethicon Inc.Cornelia, GA), and->(Baxter International, inc Deerfield, IL), PEG-based sealant and +.>(Baxter International,Inc Deerfield,IL)。

As one non-limiting example, the saRNA can be formulated in PLGA microspheres by preparing PLGA microspheres with an adjustable release rate (e.g., days and weeks) and encapsulating the saRNA in the PLGA microspheres while maintaining the integrity of the saRNA during the encapsulation process. EVAc is a non-biodegradable biocompatible polymer that is widely used in preclinical sustained release implant applications (e.g., time release product Ocusert (a pilocarpine ophthalmic insert for glaucoma) or progessert @A sustained release progesterone intrauterine device; transdermal delivery system Testoderm, duragesic and Selegiline; a catheter). Poloxamer F-407NF is a hydrophilic nonionic surfactant triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosity at temperatures below 5 ℃ and forming a solid gel at temperatures above 15 ℃. The PEG-based surgical sealant comprises two synthetic PEG components mixed in a delivery device, which can be prepared within one minute, sealed within 3 minutes, and reabsorbed within 30 days. And the natural polymer is capable of gelling in situ at the site of application. They have been shown to interact with protein and peptide therapeutic candidates through ionic interactions to provide stabilization.

The polymer formulation may also be selectively targeted by expression of different ligands such as, but not limited to, folic acid, transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al, biomacromolecules.2011 12:2708-2714; rozema et al, proc Natl Acad Sci U S A.2007104:12982-12887;Davis,Mol Pharm.2009 6:659-668;Davis,Nature 2010464:1067-1070, the contents of each of which are incorporated herein by reference in their entirety).

The saRNA of the present disclosure may be formulated with or in polymeric compounds. The polymer may comprise at least one polymer, such as, but not limited to, polyethylene glycol (PEG), poly (L-lysine) (PLL), PEG grafted to PLL, cationic lipid polymers, biodegradable cationic lipid polymers, polyethylenimine (PEI), cross-linked branched poly (alkylene imine), polyamine derivatives, modified poloxamers, biodegradable polymers, elastomeric biodegradable polymers, biodegradable block copolymers, biodegradable random copolymers, biodegradable polyester block random copolymers, multiblock copolymers, linear biodegradable copolymers, poly [ alpha- (4-aminobutyl) -L-glycolic acid) (PAGA), biodegradable cross-linked cationic multiblock copolymers, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylene fumarate, polycaprolactone, polyamides, polyacetals, polyethers, polyesters, poly (orthoesters), polycyanoacrylates, polyvinyl alcohol, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrene, polyamines, polylysines, poly (ethyleneimine), poly (serine esters), poly (L-co-L-glycolic acid) (PAGA), biodegradable polymers, poly (L-proline-4-hydroxy-containing polymers, or derivatives thereof.

As one non-limiting example, the saRNA of the present disclosure can be formulated with a polymeric compound of PEG grafted with PLL as described in U.S. patent No. 6,177,274 (incorporated herein by reference in its entirety). The formulation may be used for in vitro transfection of cells or for in vivo delivery of saRNA. In another example, the saRNA may be suspended in a solution or medium with the cationic polymer, in a dry pharmaceutical composition, or in a solution that can be dried as described in U.S. publication nos. 20090042829 and 20090042825; the contents of each of which are incorporated herein by reference in their entirety.

As another non-limiting example, the saRNA of the present disclosure can be formulated with PLGA-PEG block copolymers (see U.S. publication No. US20120004293 and U.S. patent No. 8,236,330, which are incorporated herein by reference in their entirety) or PLGA-PEG-PLGA block copolymers (see U.S. patent No. 6,004,573, which are incorporated herein by reference in its entirety). As one non-limiting example, the saRNA of the present disclosure may be formulated with PEG and PLA or diblock copolymers of PEG and PLGA (see us patent No. 8,246,968, which is incorporated herein by reference in its entirety).

The polyamine derivatives may be used to deliver nucleic acids or to treat and/or prevent diseases or be included in an implantable or injectable device (U.S. publication No. 20100260817; which is incorporated herein by reference in its entirety). As one non-limiting example, the pharmaceutical composition may comprise a polyamine derivative described in saRNA and U.S. publication No. 20100260817 (which is incorporated herein by reference in its entirety). As another non-limiting example, a polyamide polymer such as, but not limited to, a polymer comprising a 1, 3-even addition polymer prepared by combining a carbohydrate diazide monomer with a dilkyne unit comprising an oligo amine (oligoamine) (U.S. patent No. 8,236,280; incorporated herein by reference in its entirety) may be used to deliver the saRNA of the present disclosure.

In one embodiment, the saRNA of the present disclosure may be formulated with at least one polymer and/or derivative thereof as described in international publication nos. WO2011115862, WO2012082574 and WO2012068187, and U.S. publication No. 20120283427, the contents of each of which are incorporated herein by reference in their entirety. In another embodiment, the saRNA of the present disclosure may be formulated with a polymer of formula Z as described in WO2011115862 (incorporated herein by reference in its entirety). In yet another embodiment, the saRNA may be formulated with a polymer of formula Z, Z' or Z "as described in international publication No. WO2012082574 or WO2012068187 and U.S. publication No. 2012028342; the contents of each of which are incorporated herein by reference in their entirety. The polymers formulated with the saRNA of the present disclosure may be synthesized by the methods described in international publication nos. WO2012082574 or WO2012068187, the contents of each of which are incorporated herein by reference in their entirety.

The saRNA of the present disclosure may be formulated with at least one acrylic polymer. Acrylic polymers include, but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly (acrylic acid), poly (methacrylic acid), polycyanoacrylates, and combinations thereof.

Formulations of the saRNA of the present disclosure may include at least one amine-containing polymer, such as, but not limited to, polylysine, polyethylenimine, poly (amidoamine) dendrimers, or combinations thereof.

For example, the saRNA of the present disclosure may be formulated in pharmaceutical compounds including poly (alkylene imines), biodegradable cationic lipid polymers, biodegradable block copolymers, biodegradable polymers, or biodegradable random copolymers, biodegradable polyester block copolymers, biodegradable polyester polymers, biodegradable polyester random copolymers, linear biodegradable copolymers, PAGAs, biodegradable cross-linked cationic multiblock copolymers, or combinations thereof. Biodegradable cationic lipopolymers can be prepared by methods known in the art and/or described in U.S. patent No. 6,696,038, U.S. application nos. 20030073619 and 20040142474; the contents of each of which are incorporated herein by reference in their entirety. The poly (alkylene imine) may be prepared using methods known in the art and/or as described in U.S. publication No. 20100004315, which is incorporated herein by reference in its entirety. Biodegradable polymers, biodegradable block copolymers, biodegradable random copolymers, biodegradable polyester block copolymers, biodegradable polyester polymers, or biodegradable polyester random copolymers can be prepared using methods known in the art and/or as described in U.S. patent nos. 6,517,869 and 6,267,987, the contents of which are incorporated herein by reference in their entirety. The linear biodegradable copolymers may be prepared using methods known in the art and/or as described in U.S. patent No. 6,652,886. The PAGA polymers may be prepared using methods known in the art and/or as described in U.S. patent No. 6,217,912 (incorporated herein by reference in its entirety). The PAGA polymers may be copolymerized with polymers such as, but not limited to, poly-L-lysine, polyarginine, polyornithine, histones, avidin, protamine, polylactide, and poly (lactide-co-glycolide) to form copolymers or block copolymers. Biodegradable crosslinked cationic multiblock copolymers can be prepared by methods known in the art and/or as described in U.S. patent No. 8,057,821 or U.S. publication No. 2012009145, each of which is incorporated herein by reference in its entirety. For example, a multi-block copolymer may be synthesized using Linear Polyethylenimine (LPEI) blocks having a different pattern than branched polyethylenimine. Further, the composition or pharmaceutical composition may be prepared by methods known in the art, described herein, or as described in U.S. publication No. 20100004315 or U.S. patent nos. 6,267,987 and 6,217,912, each of which is incorporated herein by reference in its entirety.

The saRNA of the present disclosure may be formulated with at least one degradable polyester, which may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly (serine esters), poly (L-lactide-co-L-lysine), poly (4-hydroxy-L-proline esters), and combinations thereof. In another embodiment, the degradable polyester may include PEG conjugation to form a pegylated polymer.

The saRNA of the present disclosure may be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include those known in the art and described in U.S. publication No. 20120269761 (incorporated herein by reference in its entirety).

In one embodiment, the polymers described herein may be conjugated to lipid-terminating (PEG). As one non-limiting example, PLGA may be conjugated with lipid-capped PEG to form PLGA-DSPE-PEG. As another non-limiting example, PEG conjugates for use with the present disclosure are described in international publication No. WO2008103276 (incorporated herein by reference in its entirety). The polymer may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. patent No. 8,273,363 (incorporated herein by reference in its entirety).

In one embodiment, the saRNA described herein can be conjugated to another compound. Non-limiting examples of conjugates are described in U.S. patent nos. 7,964,578 and 7,833,992; each of which is incorporated herein by reference in its entirety. In another embodiment, the saRNA of the present disclosure can be conjugated to a conjugate of formulas 1-122 as described in U.S. patent nos. 7,964,578 and 7,833,992; each of these documents is incorporated herein by reference in its entirety. The saRNA described herein may be conjugated to a metal such as, but not limited to, gold (see, e.g., giljohann et al journal. Amer. Chem. Soc.2009 (6): 2072-2073; incorporated herein by reference in its entirety). In another embodiment, the saRNA described herein may be conjugated and/or encapsulated in gold nanoparticles (international publication No. WO201216269 and U.S. publication No. 20120302940; each of which is incorporated herein by reference in its entirety).

As described in U.S. publication No. 20100004313 (incorporated herein by reference in its entirety), a gene delivery composition can include a nucleotide sequence and a poloxamer. For example, the saRNA of the present disclosure may be used in gene delivery compositions with poloxamers described in us publication No. 20100004313.

In one embodiment, the polymer formulation of the present disclosure may be stabilized by contacting the polymer formulation, which may comprise a cationic carrier, with a cationic lipopolymer, which may be covalently linked to cholesterol and polyethylene glycol groups. The polymer formulation may be contacted with the cationic lipopolymer using the methods described in US publication No. 20090042829 (incorporated herein by reference in its entirety).

Cationic carriers may include, but are not limited to, polyethylenimine, poly (trimethylene imine), poly (tetramethylene imine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-B-cyclodextrin, spermine, spermidine, poly (2-dimethylamino) ethyl methacrylate, poly (lysine), poly (histidine), poly (arginine), cationized gelatin, dendrimer, chitosan, 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), N- [1- (2, 3-dioleoyloxy) propyl ] -N, N-trimethylammonium chloride (DOTMA), 1- [2- (oleoyloxy) ethyl ] -2-oleyl-3- (2-hydroxyethyl) imidazolinium chloride (dotm), 2, 3-dioleoyloxy-N- [2 (spermimidoyl) ethyl ] -N, N-dimethyl-1-propyltrimethylammonium acetate (DOSPA), 3B- [ N- (N ', N' -dimethylamino) -carbamoyl ] cholesterol (DC-N, N-dimethyl-cholestyramid), di- (2-hydroxyethyl) imidazolinium chloride (dotm), 2, 3-dioleoyl-N-2-dioleoyl-3- (2-hydroxyethyl) imidazolinium chloride (dostin (dostim), N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N-dioleyl-N, N-dimethyl ammonium chloride (DODAC), and combinations thereof.

The saRNA of the present disclosure may be formulated in polymeric complexes (polyplex) of one or more polymers (U.S. publication nos. 20120237565 and 20120270927; each of which is incorporated herein by reference in its entirety). In one embodiment, the polymeric composite comprises two or more cationic polymers. The cationic polymer may comprise poly (ethyleneimine) (PEI), such as linear PEI.

The saRNA of the present disclosure may also be formulated as nanoparticles using a combination of polymers, lipids, and/or other biodegradable agents (such as, but not limited to, calcium phosphate). The components may be combined in a core-shell, hybrid, and/or layered structure (layer-by-layer architecture) to allow for fine tuning of the nanoparticle so that delivery of the saRNA may be enhanced (Wang et al, nat mater.2006:791-796; fuller et al, biomaterials.2008:1526-1532; deko et al, adv Drug Deliv rev.20163:748-761; endres et al, biomaterials.2011:7721-7731; su et al, mol pharm.2011jun 6;8 (3): 774-87; incorporated herein by reference in its entirety). As one non-limiting example, the nanoparticle may comprise a variety of polymers, such as, but not limited to, hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG), and/or hydrophilic polymers (international publication No. WO20120225129; incorporated herein by reference in its entirety).

The saRNA can be delivered in vivo using biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers. In one embodiment, the lipid-coated calcium phosphate nanoparticle may also contain a targeting ligand, such as anisoamide, which may be used to deliver the saRNA of the present disclosure. For example, for effective delivery of siRNA in a mouse metastatic lung model, lipid-coated calcium phosphate nanoparticles were used (Li et al, J Contr Rel.2010 142:416-421; li et al, J Contr Rel.2012158:108-114; yang et al, mol Ther.2012:609-615; incorporated herein by reference in its entirety). This delivery system incorporates targeted nanoparticles and an endosomal escape enhancing component calcium phosphate to improve siRNA delivery.

In one embodiment, calcium phosphate with PEG-polyanionic block copolymers may be used to deliver the saRNA of the present disclosure (Kazikawa et al, J Contr Rel.2004:345-356; kazikawa et al, J Contr Rel.2006 111:368-370; incorporated herein by reference in its entirety).

In one embodiment, nanoparticles may be formed using PEG-charge converting polymers (Pitella et al, biomaterials.2011 32:3106-3114) to deliver the sarnas of the present disclosure. The PEG-charge transfer polymer can be modified over the PEG-polyanionic block copolymer by cleavage into polycations at acidic pH to enhance endosomal escape.

The use of core-shell nanoparticles has additionally focused on high-throughput methods to synthesize cationic crosslinked nanogel cores and various shells (Siegwart et al Proc Natl Acad Sci U S a.201108:12996-13001). The complexing, delivery and internalization of polymeric nanoparticles can be precisely controlled by varying the chemical composition in the core and shell components of the nanoparticles. For example, core-shell nanoparticles can be effective in delivering saRNA to mouse hepatocytes after they covalently bind cholesterol to the nanoparticle.

In one embodiment, a hollow lipid core comprising an intermediate PLGA layer and a neutral outer lipid layer comprising PEG may be used to deliver the saRNA of the present disclosure. As one non-limiting example, in mice bearing tumors that express luciferase, it was determined that lipid-polymer-lipid hybrid nanoparticles significantly inhibited luciferase expression compared to conventional lipid complexes (Shi et al, angelw Chem Int ed.2011 50:7027-7031; incorporated herein by reference in its entirety).

In one embodiment, the lipid nanoparticle may include a core and a polymeric shell of the saRNA disclosed herein. The polymeric shell may be any of the polymers described herein and is known in the art. In another embodiment, a polymeric shell may be used to protect the modified nucleic acid in the core.

Core-shell nanoparticles for use with the saRNA of the present disclosure may be formed by the method described in U.S. patent No. 8,313,777 (incorporated herein by reference in its entirety).

In one embodiment, the core-shell nanoparticle may comprise a core and a polymeric shell of the saRNA disclosed herein. The polymeric shell may be any of the polymers described herein and is known in the art. In another embodiment, a polymeric shell may be used to protect the saRNA in the core. As one non-limiting example, core-shell nanoparticles may be used to treat an ocular disease or disorder (see, e.g., U.S. publication No. 20120321719; incorporated herein by reference in its entirety).

In one embodiment, the polymer used with the formulations described herein may be a modified polymer (e.g., without limitation, a modified polyacetal) as described in international publication No. WO2011120053, which is incorporated herein by reference in its entirety.

Delivery of

The present disclosure encompasses the delivery of saRNA by any suitable route for therapeutic, prophylactic, pharmaceutical, diagnostic or imaging use, taking into account possible advances in drug delivery science. Delivery may be bare or formulated.

The saRNA of the present disclosure can be delivered naked to a cell. As used herein, "naked" refers to delivering the saRNA without a transfection-facilitating substance. For example, the saRNA delivered to the cell may not contain modification. Naked saRNA can be delivered to cells using routes of administration known in the art and described herein.

The saRNA of the present disclosure can be formulated using the methods described herein. The formulation may contain saRNA that may be modified and/or unmodified. Formulations may also include, but are not limited to, cell penetrating agents, pharmaceutically acceptable carriers, delivery agents, bioerodible or biocompatible polymers, solvents, and sustained release delivery reservoirs. Formulated saRNA can be delivered to cells using routes of administration known in the art and described herein.

The composition may also be formulated for delivery directly to an organ or tissue in any of several ways in the art, including but not limited to direct infusion or bathing, via a catheter, through a gel, powder, ointment, cream, gel, lotion and/or drop, through the use of a substrate such as a fabric or biodegradable material coated or impregnated with the composition, and the like. The saRNA of the present disclosure may also be cloned into a Retroviral Replicative Vector (RRV) and transduced into cells.

Application of

The saRNA of the present disclosure may be administered by any route that produces a therapeutically effective result. These routes include, but are not limited to, enteral, gastrointestinal, epidural, oral, transdermal, epidural (periepidural), intracerebral (into the brain), intracerebroventricular (into the ventricle), transdermal (applied to the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal (through the nose), intravenous (into the vein), intra-arterial (into the artery), intramuscular (into the muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavernosal injection (into the basal of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (spread across the intact skin for systemic distribution), transmucosal (spread across the mucosa), insufflation (nasal inhalation), sublingual, subchrough, enema, eye drops (onto the conjunctiva), or in ear drops. In particular embodiments, the compositions may be administered in a manner that allows them to cross the blood brain barrier, vascular barrier, or other epithelial barrier. The route of administration disclosed in international publication WO 2013/090648 (the contents of which are incorporated herein by reference in their entirety) filed 12/14 in 2012 can be used to administer the saRNA of the present disclosure.

In some embodiments, the saRNA of the present disclosure is delivered intratumorally.

Dosage form

The pharmaceutical compositions described herein may be formulated into dosage forms described herein, e.g., topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous). The liquid, injectable, pulmonary and solid dosage forms described in international publication WO 2013/090648 (the contents of which are incorporated herein by reference in their entirety) filed 12/14 in 2012 can be used as dosage forms for the saRNA of the present disclosure.

III methods of use

An aspect of the present disclosure provides methods of using the saRNA of the present disclosure and pharmaceutical compositions comprising the saRNA and at least one pharmaceutically acceptable carrier. The saRNA of the present disclosure modulates the expression of its target gene. In one embodiment, a method of modulating expression of a target gene in vitro and/or in vivo is provided, comprising administering a saRNA of the present disclosure. In one embodiment, the expression of a target gene is increased by at least 5%, 10%, 20%, 30%, 40%, or at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% in the presence of a saRNA of the present disclosure as compared to the expression of the target gene in the absence of the saRNA of the present disclosure. In further embodiments, the expression of the target gene is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold, or at least 15, 20, 25, 30, 35, 40, 45, 50-fold, or at least 60, 70, 80, 90, 100-fold in the presence of the saRNA of the present disclosure as compared to the expression of the target gene in the absence of the saRNA of the present disclosure.

STING (TMEM 173) gene

One aspect of the present application provides a method of modulating STING (interferon response CGAMP interacting factor stimulus (Stimulator Of Interferon Response CGAMP Interactor); STING1; TMEM 173) gene expression comprising administering TMEM173-saRNA of the present disclosure. In one embodiment, expression of the TMEM173 gene is increased by at least 20%, 30%, 40%, or at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% in the presence of TMEM173-saRNA of the present disclosure as compared to expression of the TMEM173 gene in the absence of TMEM173-saRNA of the present disclosure. In further embodiments, expression of the TMEM173 gene is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10 fold, or at least 15, 20, 25, 30, 35, 40, 45, 50 fold, or at least 60, 70, 80, 90, 100 fold in the presence of TMEM173-saRNA of the present disclosure as compared to expression of STING gene in the absence of TMEM173-saRNA of the present disclosure. Modulation of TMEM173 gene expression may be reflected or determined by changes in TMEM173 mRNA levels.

The TMEM173 gene encodes an endoplasmic reticulum aptamer protein that is critical for innate immune signaling. It is activated by cyclic GMP-AMP (cGAMP) to trigger downstream innate immune signaling. When cGAS detects intracellular foreign DNA, cGAMP is synthesized and activation of the cGAMP-STING pathway is critical for tumor immunotherapy. STING has been noted to be down-regulated in various types of tumors by promoter hypermethylation. Restoration of STING expression by DNA methylation inhibitors improves control of tumor growth (Kitajima et al, cancer Discovery, vol.9 (1): 34 (2019)). TMEM173-saRNA of the present disclosure may be useful for preventing or treating STING-related diseases or conditions. In some embodiments, the TMEM173-saRNA of the present disclosure is used to prevent or treat a disease, such as cancer, TMEM 173-related vasculopathy, infancy-onset (infartile-onset), and familial lupus chilblain.

In some embodiments, the saRNA of the present invention may be used to treat any disease associated with TMEM173 gene. In various embodiments, methods for treating a subject are provided, wherein the methods comprise administering to a subject having, suspected of having, or having a predisposition to cancer a therapeutically effective amount of a saRNA of the present disclosure. According to the present disclosure, cancer includes any disease or disorder characterized by uncontrolled cellular proliferation, such as hyperproliferation. Cancers may be characterized by tumors such as solid tumors or any neoplasm. In some embodiments, the cancer is a solid tumor.

Furthermore, in some embodiments, the saRNA of the present disclosure is effective for inhibiting tumor growth of multiple types of tumors, whether measured in net size (weight, surface area, or volume) or at a rate that varies over time.

In some embodiments, the tumor size is reduced by about 60% or more after treatment with the saRNA of the present disclosure. In some embodiments, the size of the tumor is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100% as measured by weight and/or area and/or volume.

In the context of a variety of embodiments of the present invention, cancers include, but are not limited to, acoustic neuroma, acute leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia (monocytes, myeloblasts, adenocarcinomas, angiosarcomas, astrocytomas, myelomonocytic and promyelocytic), acute T-cell leukemia, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, brain carcinoma, breast carcinoma, bronchogenic carcinoma, cervical carcinoma, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, colon carcinoma, colorectal carcinoma, craniopharyngeal tumor, cystic adenocarcinoma, diffuse large B-cell lymphoma, burkitt lymphoma dysproliferative changes (dysplasia) and metaplasia (metaplasia)), embryonic cancers, endometrial cancers, endothelial sarcomas, ependymomas, epithelial cancers, erythroleukemia, esophageal cancers, estrogen receptor positive breast cancers, primary thrombocytopoietic hyperplasia, ewing's tumors, fibrosarcomas, follicular lymphomas, germ cell testicular cancers, gliomas, heavy chain diseases (heavy chain disease), angioblastomas, liver cancers, hepatocellular carcinoma, hormone-insensitive prostate cancers, leiomyosarcoma, liposarcoma, lung cancers, lymphangiointimal sarcomas (lymphokines) lymphomas, lymphoblastic leukemias, lymphomas (hodgkins and non-hodgkins); malignant tumors and hyperproliferative disorders of the bladder, breast, colon, lung, ovary, pancreas, prostate, skin and uterus; lymphoid malignancies of T-cell or B-cell origin, leukemias, lymphomas, medullary carcinomas, medulloblastomas, melanomas, meningiomas, mesotheliomas, multiple myeloma, myelogenous leukemia, myeloma, myxosarcoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinomas, pineal tumor, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastomas, rhabdomyosarcomas, sarcomas, sebaceous adenocarcinomas, seminomas, skin cancers, small cell lung cancer, solid tumors (carcinomas and sarcomas), small cell lung cancer, gastric cancer, squamous cell carcinoma, synovial carcinoma, sweat gland carcinoma, thyroid cancer, fahrenheit, macroglobulinemia, testicular tumors, uterine cancer, and Wilms' tumor. Other cancers include primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gall bladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer, kidney cancer, urothelial cancer, female genital tract cancer, uterine cancer, gestational trophoblastic cancer disease, male genital tract cancer, seminal vesicle cancer, testicular cancer, germ cell tumor, endocrine gland tumor, thyroid cancer, adrenal cancer, pituitary cancer, hemangioma, bone and soft tissue sarcoma, kaposi's sarcoma, nerve cancer, eye cancer, meningioma, glioblastoma, neuroma, neuroblastoma (Schwannoma), solid tumors caused by hematopoietic malignancies (such as leukemia), metastatic melanoma, recurrent or persistent ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer, gastrointestinal stromal tumor, colorectal cancer, gastric cancer, melanoma glioblastoma multiforme, non-squamous non-small cell lung cancer, glioblastoma, epithelial ovarian cancer, primary peritoneal serous carcinoma, metastatic liver cancer, neuroendocrine carcinoma, refractory malignancy, triple negative breast cancer, HER2 amplified breast cancer, nasopharyngeal carcinoma, oral cancer, biliary tract cancer, hepatocellular carcinoma, head and neck Squamous Cell Carcinoma (SCCHN), non-medullary thyroid cancer, recurrent multiple glioblastoma, type 1 neurofibromatosis, CNS cancer, liposarcoma, leiomyosarcoma, salivary gland carcinoma, mucosal melanoma, acrophase/freckle-like melanoma, paraganglioma, pheochromocytoma, metastatic carcinoma in advanced stages, solid tumors, triple negative breast cancer, colorectal cancer, sarcoma, melanoma, renal carcinoma, endometrial carcinoma, thyroid cancer, rhabdomyosarcoma, multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal stromal tumors, mantle cell lymphomas, and refractory malignant tumors.

In some embodiments, the cancer is a solid tumor.

In some embodiments, the cancer is liver cancer, such as hepatocellular carcinoma, pancreatic cancer, or ovarian cancer.

Cancers treatable by the methods of the present disclosure typically occur in mammals. Mammals include, for example, humans, non-human primates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs, horses, pigs, sheep, goats, and cattle.

IV kit and device

Kit for detecting a substance in a sample

The present disclosure provides various kits for conveniently and/or efficiently performing the methods of the present disclosure. Typically, the kit will contain sufficient amounts and/or amounts of components to allow a user to perform multiple treatments and/or multiple experiments on a subject.

In one embodiment, the present disclosure provides a kit for modulating gene expression in vitro or in vivo comprising a saRNA of the present disclosure or a combination of sarnas of the present disclosure, saRNA, siRNA, miRNA or other oligonucleotide molecules that modulate other genes.

The kit may further comprise packaging and instructions and/or a delivery agent forming a formulation composition. The delivery agent may comprise saline, a buffer solution, a lipid, a dendrimer, or any of the delivery agents disclosed herein.

In one non-limiting example, the buffer solution may include sodium chloride, calcium chloride, phosphate, and/or EDTA. In another non-limiting example, the buffer solution may include, but is not limited to, saline containing 2mM calcium, 5% sucrose containing 2mM calcium, 5% mannitol containing 2mM calcium, ringer's lactate, sodium chloride with 2mM calcium, and mannose (see U.S. publication No. 20120258046; incorporated herein by reference in its entirety). In yet another non-limiting example, the buffer solution may be precipitated or it may be lyophilized. The amount of each component can be varied to achieve consistent, reproducible high concentration saline or simple buffer formulations. The composition can also be altered to increase the stability of the saRNA in the buffer solution over time and/or under various conditions.

Device and method for controlling the same

The present disclosure provides devices that can incorporate the saRNA of the present disclosure. These devices contain stable formulations that are useful for immediate delivery to a subject in need thereof, such as a human patient.

Non-limiting examples of devices include pumps, catheters, needles, transdermal patches, pressurized olfactory delivery devices, iontophoresis devices (iontophoresis), multilayer microfluidic devices. The device may be used to deliver the saRNA of the present disclosure according to a single, multiple, or split dosing regimen. The device may be used to deliver the saRNA of the present disclosure across biological tissue, transdermally, subcutaneously, or intramuscularly. Further examples of devices suitable for delivering oligonucleotides are disclosed in international publication WO 2013/090648 filed 12/14 2012, the contents of which are incorporated herein by reference in their entirety.

Definition of the definition

For convenience, the meaning of certain terms and phrases used in the specification, examples and appended claims are provided below. If there is a significant difference between the use of terms in other parts of the specification and the definitions provided in this section, the definitions in this section control.

About (about): as used herein, the term "about" refers to +/-10% of the value.

Combination application: as used herein, the term "administration in combination (administered in combination)" or "administration in combination (combined administration)" refers to administration of two or more agents to a subject simultaneously or within an interval such that there may be overlap in the effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minutes of each other. In some embodiments, the administration of the agents are sufficiently closely spaced to each other to achieve a combined (e.g., synergistic) effect.

Amino acid (amino acid): as used herein, the term "amino acid" refers to all naturally occurring L-a-amino acids. Amino acids are identified by one or three letter designations as follows: aspartic acid (Asp: D), isoleucine (Ile: I), threonine (Thr: T), leucine (Leu: L), serine (Ser: S), tyrosine (Tyr: Y), glutamic acid (Glu: E), phenylalanine (Phe: F), proline (Pro: P), histidine (His: H), glycine (Gly: G), lysine (Lys: K), alanine (Ala: A), arginine (Arg: R), cysteine (Cys: C), tryptophan (Trp: W), valine (Val: V), glutamine (Gln: Q), methionine (Met: M), asparagine (Asn: N), wherein amino acids are listed first, followed by three letter and one letter codes, respectively, in brackets.

Animal (animal): as used herein, the term "animal" refers to any member of the kingdom animalia. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, a genetically engineered animal, or a clone.

About (approbamate): as used herein, the term "about" or "approximately" when applied to one or more target values refers to values similar to the reference values. In certain embodiments, the term "about" or "approximately" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (greater than or less than) unless otherwise indicated or apparent from the context (unless the number exceeds 100% of the possible values).

Bind to … … (associated with): as used herein, the terms "bind," "conjugate," "connect," "attach," and "tether," when used with respect to two or more moieties, mean that the moieties are physically bound or connected to each other directly or through one or more additional moieties that act as linkers to form a sufficiently stable structure such that the moieties remain physically bound under conditions in which the structure is used, e.g., physiological conditions. The "binding" need not be strictly achieved by direct covalent chemical bonds. It may also refer to ionic bonding or hydrogen bonding or hybridization-based ligation that is stable enough that the "bound" entity remains physically bound.

Bifunctional (bifunction) or bifunctional (bifunctional): as used herein, the terms "bifunctional" and "bifunctional" refer to any substance, molecule, or moiety capable of having or maintaining at least two functions. These functions may affect the same or different results. The structure of the resulting function may be the same or different. For example, bifunctional sarnas of the present disclosure may comprise cytotoxic peptides (first function), while those nucleosides comprising sarnas are themselves cytotoxic (second function).

Biocompatible: as used herein, the term "biocompatible" refers to a condition that is compatible with living cells, tissues, organs, or systems with little to no risk of causing injury, toxicity, or rejection by the immune system.

Biodegradable (bioodegradable): as used herein, the term "biodegradable" refers to a material that can be broken down into harmless products by the action of living organisms.

Bioactive (biologically active): as used herein, the phrase "bioactive" refers to the characteristic of any substance that is active in a biological system and/or organism. For example, a substance that has a biological effect on an organism when administered to the organism is considered to be biologically active. In particular embodiments, a saRNA of the present disclosure may be considered to be biologically active if even a portion of the saRNA is biologically active or mimics an activity that is considered biologically relevant.

Cancer (cancer): as used herein, the term "cancer" refers to the presence in an individual of cells that are characteristic of oncogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Typically, the cancer cells will be in the form of a tumor, but such cells may be present in the individual alone or may circulate in the blood stream as independent cells (e.g., leukemia cells).

Cell growth (cell growth): as used herein, the term "cell growth" relates primarily to an increase in the number of cells, which occurs by way of cell replication at a rate greater than the rate of cell death (e.g., by apoptosis or necrosis), resulting in an increase in the size of the cell population, although in some cases a small portion of such growth may be due to an increase in the cell size or cell volume of an individual cell. Thus, an agent that inhibits cell growth may alter the balance between these two opposing processes by inhibiting proliferation or stimulating cell death or both to inhibit cell growth.

Cell type (cell type): as used herein, the term "cell type" refers to a cell from a given source (e.g., tissue, organ) or a cell in a given differentiated state, or a cell associated with a given lesion or genetic composition.

Chromosome (chromoname): as used herein, the term "chromosome" refers to the organized structure of DNA and proteins present in a cell.

Complementary (complement): as used herein, the term "complementary" when referring to nucleic acids means that hybridization or base pairing between nucleotides or nucleic acids, e.g., between two strands of a double stranded DNA molecule, or between an oligonucleotide probe and a target, is complementary.

Condition (condition): as used herein, the term "condition" refers to the state of any cell, organ system or organism. The condition may reflect a disease state of the entity or a simple physiological manifestation or condition. The condition may be characterized as a phenotypic condition, such as the macroscopic manifestation of a disease, or a genotypic condition, such as a potential gene or protein expression profile associated with the condition. The condition may be benign or malignant.

Controlled release (controlled release): as used herein, the term "controlled release" refers to the release profile of a pharmaceutical composition or compound that conforms to a particular release pattern to produce a therapeutic result.

Cytostatic (cytostatic): as used herein, "cytostatic" refers to inhibiting, reducing, arresting the growth, division, or proliferation of cells (e.g., mammalian cells (e.g., human cells)), bacteria, viruses, fungi, protozoa, parasites, prions, or combinations thereof.

Cytotoxic (cytoxic): as used herein, "cytotoxic" refers to a killer cell (e.g., a mammalian cell (e.g., a human cell)), a bacterium, a virus, a fungus, a protozoan, a parasite, a prion, or a combination thereof, or a deleterious, toxic, or lethal effect thereof.

Delivery (delivery): as used herein, "delivery" refers to the act or manner of delivering a compound, substance, entity, moiety, load (cargo), or payload.

Delivery agent (delivery agent): as used herein, "delivery agent" refers to any substance that facilitates (at least in part) in vivo delivery of the saRNA of the present disclosure to a targeted cell.

Destabilization (destabilized): as used herein, the term "destabilized", "destabilizing" or "destabilizing region" refers to a region or molecule that is less stable than the original, wild-type or native form of the same region or molecule.

Detectable label (detectable label): as used herein, "detectable label" refers to one or more labels, signals, or moieties that are linked, incorporated, or bound to another entity that is readily detectable by methods known in the art, including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance, and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin, and haptens, quantum dots, and the like. The detectable label may be located at any position in the oligonucleotides disclosed herein. They may be located within a nucleotide or at the 5 'or 3' end.

Encapsulation (encapsulation): as used herein, the term "encasement" refers to enclosing, surrounding or encasing.

Engineered (engineered): as used herein, an embodiment of the present disclosure is "engineered" when it is designed to have a structural or chemical characteristic or property that is different from the starting point, wild-type, or native molecule.

Equivalent subject (equivalent subject): as used herein, an "equivalent subject" may be, for example, a subject having similar age, sex, and health (e.g., liver health or cancer stage), or the same subject prior to treatment of the present disclosure. The equivalent subject is "untreated" in that he does not receive treatment with the saRNA of the present disclosure. However, he may receive conventional anti-cancer therapy provided that the subject treated with the saRNA of the present disclosure also receives the same or equivalent conventional anti-cancer therapy.

Exosomes (exosomes): as used herein, an "exosome" is a vesicle secreted by a mammalian cell.

Expression (expression): as used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) Generating an RNA template from the DNA sequence (e.g., by transcription); (2) Processing the RNA transcript (e.g., by splicing, editing, 5 'cap formation, and/or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

Feature (feature): as used herein, "feature" refers to a property, characteristic, or different element.

Formulation (formulation): as used herein, "formulation" includes at least one saRNA and a delivery agent of the present disclosure.

Fragment (fragment): as used herein, "fragment" refers to a portion. For example, a fragment of a protein may comprise a polypeptide obtained by digestion of a full-length protein isolated from cultured cells. Fragments of an oligonucleotide may comprise nucleotides or regions of nucleotides.

Functional (functional): as used herein, a "functional" biomolecule is a biomolecule in a form that it exhibits characteristics and/or activity that are characteristic thereof.

Gene (gene): as used herein, the term "gene" refers to a nucleic acid sequence that comprises the control sequences, and most typically coding sequences, required for the production of a polypeptide or precursor. However, the gene may not be translated, but rather encodes a regulatory or structural RNA molecule.

The gene may be derived in whole or in part from any source known in the art, including plant, fungal, animal, bacterial genomes or episomes, eukaryotic DNA, nuclear DNA or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA. The gene may comprise one or more modifications in the coding or untranslated regions that may affect the biological activity or chemical structure, the expression rate, or the expression control pattern of the expression product. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. The gene may constitute an uninterrupted coding sequence, or it may comprise one or more introns defined by suitable splicing junctions.

Gene expression (gene expression): as used herein, the term "gene expression" refers to the process by which a nucleic acid sequence undergoes successful transcription and in most cases translation to produce a protein or peptide. For clarity, when reference is made to measuring "gene expression", it is understood to mean measuring transcribed nucleic acid products, such as RNA or mRNA, or translated amino acid products, such as polypeptides or peptides. Methods for measuring the amount or level of RNA, mRNA, polypeptides, and peptides are well known in the art.

Genome (genome): the term "genome" is intended to include the complete DNA complement of an organism, including nuclear DNA components, chromosomal or extra-chromosomal DNA, and cytoplasmic domains (e.g., mitochondrial DNA).

Homology (homology): as used herein, the term "homology" refers to the overall relatedness between polymer molecules, such as between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). According to the present disclosure, two polynucleotide sequences are considered homologous if they encode polypeptides that are at least about 50%, 60%, 70%, 80%, 90%, 95% or even 99% for at least a fragment of at least about 20 amino acids. In some embodiments, the homologous polynucleotide sequences are characterized by the ability to encode fragments of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode fragments of at least 4-5 uniquely specified amino acids. According to the present disclosure, two protein sequences are considered homologous if the protein is at least about 50%, 60%, 70%, 80% or 90% identical for at least one fragment of at least about 20 amino acids.

The term "hyperproliferative cells (hyperproliferative cell)" may refer to any cells that proliferate at an abnormally high rate compared to the proliferation rate of equivalent healthy cells (which may be referred to as "controls"). An "equivalent healthy" cell is the normal healthy counterpart of the cell. Thus, it is a cell of the same type, e.g. from the same organ, performing the same function as the control cell. For example, the proliferation of hyperproliferative hepatocytes should be assessed with reference to healthy hepatocytes, while the proliferation of hyperproliferative prostate cells should be assessed with reference to healthy prostate cells.

By "abnormally high" proliferation rate is meant an increase in proliferation rate of a hyperproliferative cell of at least 20%, 30%, 40%, or at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% as compared to the proliferation rate of an equivalent healthy (non-hyperproliferative) cell. An "abnormally high" proliferation rate may also mean a rate increase of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or at least 15, 20, 25, 30, 35, 40, 45, 50, or at least 60, 70, 80, 90, 100 times compared to the proliferation rate of an equivalent healthy cell.

Hyperproliferative disorder (hyperproliferative disorder): as used herein, a "hyperproliferative disorder" may be any disorder involving hyperproliferative cells as defined above. Examples of hyperproliferative disorders include neoplastic disorders such as cancer, psoriatic arthritis, rheumatoid arthritis, gastric hyperproliferative disorders such as inflammatory bowel disease, skin diseases including psoriasis, reiter's syndrome (Reiter' ssyndrome), pityriasis rubra pilaris (pityriasis rubra pilaris) and hyperproliferative variants of keratosis.

The skilled person is well aware of how to identify hyperproliferative cells. The presence of hyperproliferative cells in an animal can be identified using a scan such as an X-ray, MRI or CT scan. Hyperproliferative cells can also be identified by culturing the sample in vitro, using cell proliferation assays such as MTT, XTT, MTS or WST-1 assays, or assaying proliferation of cells. Flow cytometry can also be used to determine in vitro cell proliferation.

Identity (identity): as used herein, the term "identity" refers to the overall relatedness between polymer molecules, such as between oligonucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. For example, the percent identity of two polynucleotide sequences can be calculated by: the two sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first nucleic acid sequence and the second nucleic acid sequence for optimal alignment, and non-identical sequences may be omitted for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. Then, the nucleotides at the corresponding nucleotide positions are compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences and takes into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Sequence comparison and determination of percent identity between two sequences can be accomplished using mathematical algorithms. For example, the percent identity between two nucleotide sequences can be determined using methods such as those disclosed in: computational Molecular Biology, lesk, a.m., ed., oxford University Press, new York,1988; biocomputing: informatics and Genome Projects, smith, d.w., ed., academic Press, new York,1993; sequence Analysis in Molecular Biology von Heinje, g., academic Press,1987; computer Analysis of Sequence Data, part I, griffin, a.m., and Griffin, h.g., eds., humana Press, new Jersey,1994; and Sequence Analysis Primer, gribskov, m. and Devereux, j., eds., M stock Press, new York,1991; each of which is incorporated herein by reference. For example, the percentage identity between two nucleotide sequences can be determined using the algorithm of Meyers and Millers (CABIOS, 1989, 4:11-17) that has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight remainder table, gap length penalty 12, and gap penalty 4. Alternatively, the percentage identity between two nucleotide sequences may be determined using the GAP program in the GCG software package, using the nwsgapdna. Methods commonly used to determine the percent identity between sequences include, but are not limited to, those disclosed in carllo, h., and Lipman, d., SIAM J Applied mate, 48:1073 (1988), incorporated herein by reference. Techniques for determining identity have been programmed into publicly available computer programs. Exemplary computer software for determining homology between two sequences includes, but is not limited to, GCG package, devereux, j., et al, nucleic Acids Research,12 (1), 387 (1984); BLASTP, BLASTN and FASTA Altschul, s.f. et al, j.molecular.

Inhibition of gene expression (inhibit expression of a gene): as used herein, the phrase "inhibiting gene expression" refers to causing a decrease in the amount of a gene expression product. The expression product may be RNA transcribed from a gene (e.g., mRNA) or a polypeptide translated from mRNA transcribed from a gene. In general, a decrease in mRNA levels results in a decrease in the level of polypeptide translated therefrom. Standard techniques for measuring mRNA or protein can be used to determine expression levels.

In vitro (in vitro): as used herein, the term "in vitro" refers to an event that occurs in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (e.g., an animal, plant, or microorganism).

In vivo (in vivo): as used herein, the term "in vivo" refers to an event that occurs within an organism (e.g., an animal, plant, or microorganism or a cell or tissue thereof).

Isolated (isolated): as used herein, the term "isolated" refers to a substance or entity that has been separated from at least some of the components with which it is associated (whether in nature or in an experimental environment). The separated material may have a different level of purity relative to the material with which it is associated. The isolated substance and/or entity may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which it is associated initially. In some embodiments, the purity of the isolated material is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99%. As used herein, a substance is "pure" if it is substantially free of other components. Substantially isolated (substantially isolated): by "substantially isolated" is meant that the compound is substantially separated from the environment in which the compound is formed or detected. Partial isolation may include, for example, a composition enriched in a compound of the present disclosure. Substantial separation may include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of a compound of the present disclosure or a salt thereof. Methods for isolating compounds and salts thereof are conventional in the art.

Label (label): the term "label" refers to a substance or compound incorporated into a subject such that the substance, compound, or subject can be detected.

Joint (linker): as used herein, a linker refers to a group of atoms, e.g., 10-1,000 atoms, and may contain atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker may be attached at a first end to a modified nucleoside or nucleotide on the nucleobase or sugar moiety and at a second end to a load, such as a detectable substance or therapeutic agent. The linker may be of sufficient length so as not to interfere with incorporation into the nucleic acid sequence. The linker can be used for any useful purpose, such as forming a saRNA conjugate and administering a cargo, as described herein.

Examples of chemical groups that may be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which may be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene glycol or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol or tetraethylene glycol), and dextran polymers and derivatives thereof. Other examples include, but are not limited to, cleavable moieties within the linker, such as disulfide linkages (-S-) or azo linkages (-n=n-), which can be cleaved using a reducing agent or photolytic action. Non-limiting examples of selectively cleavable bonds include amide bonds cleavable, e.g., by use of tris (2-carboxyethyl) phosphine (TCEP) or other reducing agents and/or photolysis, and ester bonds cleavable, e.g., by acidic or basic hydrolysis.

Transfer (metatasis): as used herein, the term "metastasis" refers to the process by which cancer invades and spreads from the site where it originally emerged as a primary tumor to distant locations in the body. Metastasis also refers to cancer caused by the spread of the primary tumor. For example, a person with breast cancer may develop metastases in their lymphatic system, liver, bone or lung.

Modified (modified): as used herein, "modified" refers to an altered state or structure of a molecule of the present disclosure. The molecules may be modified in a variety of ways, including chemically, structurally, and functionally. In one embodiment, the saRNA of the present disclosure is modified by the introduction of non-natural nucleosides and/or nucleotides.

Naturally occurring (naturally occurring): as used herein, "naturally occurring" refers to the presence in nature without artificial assistance.

Nucleic acid): as used herein, the term "nucleic acid" refers to a molecule comprising one or more nucleotides, i.e., ribonucleotides, deoxyribonucleotides, or both. The term includes monomers and polymers of ribonucleotides and deoxyribonucleotides, in the case of polymers, the ribonucleotides and/or deoxyribonucleotides are joined together by 5 'to 3' linkages. Ribonucleotides and polymers of deoxyribonucleotides can be single-stranded or double-stranded. However, the linkage may include any linkage known in the art, including, for example, a nucleic acid comprising a 5 'to 3' linkage. The nucleotide may be naturally occurring or may be a synthetically produced analogue capable of forming a base pairing relationship with a naturally occurring base pair. Examples of non-naturally occurring bases capable of forming base pairing relationships include, but are not limited to, aza (aza) and deaza (deaza) pyrimidine analogs, aza and deaza purine analogs, and other heterocyclic base analogs in which one or more carbon and nitrogen atoms of the pyrimidine ring have been replaced with a heteroatom, such as oxygen, sulfur, selenium, phosphorus, and the like.

Patient (patient): as used herein, "patient" refers to a subject who may seek or need treatment, require treatment, be receiving treatment, or be receiving care from a trained professional for a particular disease or condition.

Peptide (peptide): as used herein, a "peptide" is less than or equal to 50 amino acids in length, for example about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

Pharmaceutically acceptable (pharmaceutically acceptable): the phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable excipients (pharmaceutically acceptable excipient): as used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) and having substantially non-toxic and non-inflammatory properties in the patient. Excipients may include, for example: anti-tackifiers, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (pigments), softeners, emulsifiers, fillers (diluents), film or coating agents, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners and hydration water. Exemplary excipients include, but are not limited to: butylhydroxytoluene (BHT), calcium carbonate, calcium phosphate (di) hydrate, calcium stearate, croscarmellose, crospovidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac (shellac), silica, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin a, vitamin E, vitamin C and xylitol.

Pharmaceutically acceptable salts (pharmaceutically acceptable salts): the present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting the existing acid or base moiety to its salt form (e.g., by reacting the free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; basic salts or organic salts of acidic residues such as carboxylic acids; etc. Representative acid addition salts include acetates, adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, bisulphates, borates, butyrates, camphorites, camphorsulphonates, citrates, cyclopentanepropionates, digluconates, dodecylsulphates, ethanesulphonates, fumarates, glucoheptonates, glycerophosphate, hemisulphates, heptonates, caprates, hydrobromites, hydrochlorides, iodides, 2-hydroxyethanesulphonates, lactates, laurates, lauryl sulphates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulphonates, nicotinates, nitrates, oleates, oxalates, palmates, pamonates, pectinates, persulphates, 3-phenylpropionates, phosphates, bittering salts, pivalates, propionates, stearates, succinates, sulphates, tartrates, thiocyanates, tosylates, undecanoates, valerates, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound, which are formed, for example, from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, these salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of water and an organic solvent; in general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 th edition, mack Publishing Company, easton, pa.,1985,p.1418,Pharmaceutical Salts:Properties,Selection,and Use,P.H.Stahl and c.g. wermput (eds.), wiley-VCH,2008, and Berge et al, journal of Pharmaceutical Science,66,1-19 (1977), each of which is incorporated herein by reference in its entirety.

Pharmaceutically acceptable solvate (pharmaceutically acceptable solvate): as used herein, the term "pharmaceutically acceptable solvate" refers to a compound of the present disclosure in which a molecule of a suitable solvent is introduced into the crystal lattice. Suitable solvents are physiologically tolerable at the doses administered. For example, solvates may be prepared by crystallization, recrystallization or precipitation from solutions comprising organic solvents, water or mixtures thereof. Examples of suitable solvents are ethanol, water (e.g. monohydrate, dihydrate and trihydrate), N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), N '-Dimethylformamide (DMF), N' -Dimethylacetamide (DMAC), 1, 3-dimethyl-2-imidazolidinone (DMEU), 1, 3-dimethyl-3, 4,5, 6-tetrahydro-2- (1H) -pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a "hydrate".

Pharmacological effect (pharmacologic effect): as used herein, a "pharmacological effect" is a measurable biological phenomenon in an organism or system that occurs after the organism or system has been contacted or exposed to an exogenous substance. The pharmacological effect may produce a therapeutically effective result, such as treating, ameliorating one or more symptoms, diagnosing, preventing, and delaying the onset of a disease, disorder, condition, or infection. The measurement of such a biological phenomenon may be quantitative, qualitative or relative to another biological phenomenon. Quantitative measurements may be statistically significant. Qualitative measurements may be made to a degree or type and may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more different. They may be observed as present or absent, better or worse, more or less. Exogenous substances, when referring to pharmacological effects, are those substances that are wholly or partially foreign to the organism or system. For example, modifications to wild-type biomolecules, both structural and chemical, can result in exogenous materials. Similarly, the introduction or combination of compounds, molecules or substances in wild-type molecules that do not naturally occur in an organism or system also produces exogenous substances.

The saRNA of the present disclosure comprises exogenous material. Examples of pharmacological effects include, but are not limited to, changes in cell count, such as increases or decreases in neutrophils, reticulocytes, granulocytes, erythrocytes (red blood cells), megakaryocytes, platelets, monocytes, connective tissue macrophages, epidermal langerhans cells, osteoclasts, dendritic cells, microglia, neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, or reticulocytes. Pharmacological effects also include changes in blood chemistry, pH, hemoglobin, hematocrit, changes in enzyme (such as but not limited to liver enzymes AST and ALT) levels, changes in lipid profiles, electrolytes, metabolic markers, hormones, or other markers or profiles known to those skilled in the art.

Physicochemical (physiochemical): as used herein, "physicochemical" means or refers to physical and/or chemical properties.

Prevention (presntation): as used herein, the term "preventing" refers to partially or completely delaying the onset of an infection, disease, disorder, and/or condition; partially or completely delay the onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delay the onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delay progression of an infection, a particular disease, disorder, and/or condition; and/or reduce the risk of developing lesions associated with infections, diseases, disorders and/or conditions.

Prodrugs (pro drug): the present disclosure also includes prodrugs of the compounds described herein. As used herein, "prodrug" refers to any substance, molecule, or entity in a form that is expected to function as a therapeutic agent upon chemical or physical modification of the substance, molecule, or entity. The prodrug may be covalently bound or sequestered in some manner and released or converted into the active drug moiety prior to, simultaneously with, or after administration to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compound such that the modification can be cleaved to the parent compound in conventional procedures or in vivo. Prodrugs include compounds wherein a hydroxy, amino, sulfhydryl, or carboxyl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, amino, sulfhydryl, or carboxyl group, respectively. The preparation and use of prodrugs is discussed below: higuchi and V.stilla, "Pro-drugs as Novel Delivery Systems," Vol.14of the A.C.S. symposium Series and Bioreversible Carriers in Drug Design, ed.Edward B.Roche, american Pharmaceutical Association and Pergamon Press,1987, both of which are incorporated herein by reference in their entirety.

Prognosis (prognosing): as used herein, the term "prognosis" refers to a statement or claim that a particular biological event will occur or is highly likely to occur in the future.

Progress (progress): as used herein, the term "progression" or "cancer progression" refers to progression or worsening of a disease or condition, or progression or worsening toward a disease or condition.

Proliferation (proliferate): as used herein, the term "proliferation" refers to growth, amplification or increase or causes rapid growth, amplification or increase. "proliferative" means having proliferative capacity. "antiproliferative" means having properties that are opposite or opposite to the properties of proliferation.

Protein (protein): "protein" refers to a polymer of amino acid residues joined together by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, however, the protein will be at least 50 amino acids in length. In some cases, the encoded protein is less than about 50 amino acids. In this case, the polypeptide is referred to as a peptide. If the protein is a short peptide, it will be at least about 10 amino acid residues in length. The protein may be naturally occurring, recombinant or synthetic, or any combination of these. A protein may also comprise a fragment of a naturally occurring protein or peptide. The protein may be a single molecule, or may be a multi-molecular complex. The term protein may also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acid.

Protein expression (protein expression): the term "protein expression" refers to the process by which a nucleic acid sequence is translated to express a detectable level of an amino acid sequence or protein.

Purified (purified): as used herein, "purified" refers to substantially pure or free of unwanted components, contaminants, mixtures or impurities.

Regression (regression): as used herein, the term "regression" or "extent of regression" refers to the reversal of cancer progression in phenotype or genotype. Slowing or stopping cancer progression may be considered regression.

Sample (sample): as used herein, the term "sample" or "biological sample" refers to a subset of its tissue, cell, or component parts (e.g., body fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). The sample may also include homogenates, lysates or extracts prepared from whole organisms or a subset of tissues, cells or component parts thereof or fractions or parts thereof (including, but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, external parts of the skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs). A sample also refers to a culture medium, e.g., a nutrient broth or gel, which may contain cellular components such as proteins or nucleic acid molecules.

Signal sequence (signal sequence): as used herein, the phrase "signal sequence" refers to a sequence that can direct the transport or localization of a protein.

Single unit dose (single unit dose): as used herein, "single unit dose" refers to the dose of any therapeutic agent administered in one dose/at one time/in a single route/at a single point of contact (i.e., a single administration event).

Similarity (similarity): as used herein, the term "similarity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. The percent similarity of polymer molecules to each other can be calculated in the same manner as the percent identity is calculated, except that the percent similarity is calculated taking into account conservative substitutions, as is understood in the art.

Split dose (split dose): as used herein, a "split dose" is a single unit dose or total daily dose divided into two or more doses.

Stable (stable): as used herein, "stable" refers to a compound that is robust enough to withstand separation from a reaction mixture to a usable purity, and in one embodiment, capable of being formulated into an effective therapeutic agent.

Stabilized (stabilized): as used herein, the terms "stabilized", "stabilized region" refer to being or becoming stabilized.

Subject (subject): as used herein, the term "subject" or "patient" refers to any organism to which a composition of the present disclosure may be administered, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Common subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

Basically (subtotally): as used herein, the term "substantially" refers to a qualitative case of a feature or characteristic of interest that exhibits all or nearly all of the degree or breadth. Those of ordinary skill in the biological arts will appreciate that little, if any, biological and chemical phenomena reach completion and/or advance to completion, or that little or no absolute results are achieved or avoided. Thus, the term "substantially" is used herein to represent a potential lack of integrity inherent in many biological and chemical phenomena.

Substantially equal to (substantially equal): as used herein, the term refers to plus/minus 2% when it relates to a fold difference between doses.

Substantially simultaneously (substantially simultaneously): as used herein and when referring to multiple doses, the term refers to within 2 seconds.

Suffers from (buffering from): an individual who is "suffering from" a disease, disorder, and/or condition has been diagnosed as suffering from or exhibiting one or more symptoms of the disease, disorder, and/or condition.

Susceptibility (to): an individual who is "susceptible to" a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition and/or may not exhibit symptoms of the disease, disorder, and/or condition, but has a propensity to develop the disease or symptoms thereof. In some embodiments, an individual susceptible to a disease, disorder, and/or condition (e.g., cancer) can be characterized by one or more of the following: (1) Mutations in genes associated with the development of diseases, disorders and/or conditions; (2) Genetic polymorphisms associated with the development of diseases, disorders and/or conditions; (3) An increase and/or decrease in expression and/or activity of a protein and/or nucleic acid associated with a disease, disorder, and/or condition; (4) Habit and/or lifestyle associated with developing diseases, disorders and/or conditions; (5) a family history of a disease, disorder, and/or condition; and (6) exposure to and/or infection with microorganisms associated with the development of diseases, disorders and/or conditions. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

Sustained release (sustained release): as used herein, the term "sustained release" refers to a pharmaceutical composition or compound release profile that conforms to the release rate over a particular period of time.

Synthetic (synthetic): the term "synthetic" refers to being produced, prepared and/or manufactured manually by a person. The synthesis of polynucleotides or polypeptides or other molecules of the present disclosure may be chemical or enzymatic.

Targeting cells (targeted cells): as used herein, "targeted cells" refers to any one or more target cells. Cells may be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism may be an animal, in one embodiment a mammal or a human, and in one embodiment a patient.

Therapeutic agent (therapeutic agent): the term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic, and/or prophylactic effect and/or that causes a desired biological and/or pharmacological effect when administered to a subject.

Therapeutically effective amount (therapeutically effective amount): as used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) to be delivered that is sufficient to treat an infection, disease, disorder, and/or condition, ameliorate a symptom of the infection, disease, disorder, and/or condition, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition when administered to a subject suffering from or susceptible to the infection, disease, disorder, and/or condition.

Therapeutically effective results (therapeutically effective outcome): as used herein, the term "therapeutically effective result" refers to a result of diagnosing, preventing, and/or delaying the onset of an infection, disease, disorder, and/or condition in a subject suffering from or susceptible to the infection, disease, disorder, and/or condition sufficient to treat the infection, disease, disorder, and/or condition, ameliorate symptoms of the infection, disease, disorder, and/or condition.

Total daily dose (total day dose): as used herein, a "total daily dose" is the amount administered or prescribed over a 24 hour period. It may be administered in a single unit dose.

Transcription factor (transcription factor): as used herein, the term "transcription factor" refers to a DNA binding protein that modulates transcription of DNA into RNA by, for example, activating or repressing transcription. Some transcription factors alone achieve regulation of transcription, while others act synergistically with other proteins. Under certain conditions, certain transcription factors can both activate and repress transcription. In general, transcription factors bind to specific target sequences or sequences that are highly similar to specific consensus sequences in the regulatory region of the target gene. Transcription factors can regulate transcription of a target gene either alone or in complex with themselves (as homodimers) or with other molecules (as heterodimers). Each of these complex formations is capable of inducing multiple regulatory functions from a single transcription factor.

Treatment (treatment): as used herein, the term "treatment" refers to partially or fully alleviating (activating), improving (improving), ameliorating (improving), alleviating (reliving) one or more symptoms or features of a particular infection, disease, disorder, and/or condition, delaying its onset, inhibiting its progression, reducing its severity, and/or reducing its incidence. For example, "treating" cancer may refer to inhibiting the survival, growth, and/or spread of a tumor. The treatment may be administered to a subject that does not exhibit signs of a disease, disorder, and/or condition and/or to a subject that exhibits only early signs of a disease, disorder, and/or condition in order to reduce the risk of developing a pathology associated with the disease, disorder, and/or condition.

The phrase "treatment (a method of treating)" or equivalent terms, when applied to, for example, cancer, refers to a process or regimen of action designed to reduce, eliminate, or prevent the number of cancer cells or reduce symptoms of cancer in an individual. A "treatment" of cancer or other proliferative disorder does not necessarily mean that the cancer cells or other disorder will be virtually completely eliminated, that the number of cells or disorder will be virtually reduced, or that the symptoms of the cancer or other disorder will be virtually alleviated. In general, cancer treatment methods will be performed even with little likelihood of success, but may still be considered as an overall beneficial course of action considering the individual's medical history and estimated survival expectancy.

Tumor growth (tumor growth): as used herein, unless otherwise indicated, the term "tumor growth" or "tumor metastasis growth" is used as commonly used in oncology, wherein the term is primarily associated with an increase in mass or volume of a tumor or tumor metastasis, primarily as a result of tumor cell growth.

Tumor burden (tumor burden): as used herein, the term "tumor burden" refers to the total tumor volume of all tumor nodules carried by a subject that are greater than 3mm in diameter.

Tumor volume (tumor volume): as used herein, the term "tumor volume" refers to the size of a tumor. Calculated in mm by the following formula ³ Tumor volume in units: volume= (width) ² x length/2.

Unmodified (unmodified): as used herein, "unmodified" refers to any substance, compound, or molecule prior to being altered in any way. Unmodified may refer to the wild-type or native form of the biomolecule, but this is not always the case. The molecules may undergo a series of modifications such that each modified molecule may serve as a "unmodified" starting molecule for subsequent modification.

Equivalent and scope

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the present disclosure is not intended to be limited by the foregoing description, but rather is as set forth in the appended claims.

In the claims, articles such as "a" and "an" may mean one or more unless indicated to the contrary or apparent from the context. Unless indicated to the contrary or apparent from the context, claims or descriptions that include "or" between one or more members of a group are deemed to be satisfied if one, more than one, or all of the members of the group are present, are used in a given product or process, or are related thereto. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or associated with a given product or process. The present disclosure includes embodiments in which multiple or all of the group members are present in, used in, or associated with a given product or process.

It should also be noted that the term "comprising" is intended to be open-ended and to allow for the inclusion of additional elements or steps.

When ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or apparent from the context and understanding of one of ordinary skill in the art, in various embodiments of the disclosure, a value expressed as a range may be considered to be any particular value or subrange within the range, to one tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

In addition, it should be understood that any particular embodiment of the present disclosure that falls within the scope of the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if not explicitly set forth herein. Any particular embodiment of a composition of the present disclosure (e.g., any nucleic acid or protein encoded thereby; any method of manufacture; any method of use; etc.) may be excluded from any one or more claims for any reason, regardless of the presence or absence of prior art.

All cited sources, such as references, publications, databases, database entries, and technologies cited herein, are incorporated by reference into this application, even if not explicitly indicated in the citation. If a source of a reference conflicts with a statement of this application, the statement in this application shall govern.

The disclosure is further illustrated by the following non-limiting examples.

Examples

Materials and procedures:

transfection of saRNA

The sense and antisense strands of saRNA were synthesized. They were first annealed in Tris-EDTA-based buffer, then subjected to a denaturation step at 90 ℃, then to a gradual annealing step to room temperature. Cells were plated at 0.25 to 1x10 per well ⁵ Individual cells were seeded into 24-well plates and transfected using Lipofectamine2000 (Life Technologies). Transfection was performed immediately after inoculation of the oligonucleotide concentration using 1uL Lipofectamine 2000. Cells were then harvested for analysis at 48 hours and 72 hours post-inoculation 24 hours after cell transfection.

RT-PCR

Total RNA was harvested 48 to 96 hours post inoculation as shown in each experiment. RNA was recovered using the RNeasy Mini Kit (QIAGEN) following the manufacturer's recommended methods and quantified using the QIAxpert machine (QIAGEN). RNA samples were normalized and reverse transcribed using the Quantitech reverse transcription kit (Qiagen) following the manufacturer's recommended method. The relative expression levels were determined by real-time PCR using PowerUp SYBR Green Master Mix (QIAGEN) and verified QuantiTech SYBR primers from QIAGEN.

Western blot

Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors (Fisher Scientific), and the lysates were incubated on ice for 10 min and sonicated 3 times for 30 seconds. Total protein levels were determined by Rapid Gold BCA protein assay kit (Pierce) and lysates were normalized for total protein content prior to loading. The expression level of the target protein was determined by Wes system (Biotechne) using 12-230kDa Wes separation module and anti-rabbit detection module. The target was detected using TMEM173 specific antibody (Cell Signaling). Total protein content was confirmed using a total protein detection module.

Example 1 upregulation of TMEM173 mRNA expression with saRNA in vitro

In this study, human HepG2 cells (hepatocellular carcinoma) were transfected with 10nM control FLuc oligonucleotide (oligo) or different saRNA variants targeting TMEM173 (STING). RNA was extracted at 72 hours and the expression level of TMEM173 mRNA was measured by RT-qPCR (FIGS. 2 and 3).

As shown in FIG. 2, TMEM173-Pr-1, TMEM173-Pr-32, TMEM173-Pr-48, TMEM173-Pr-70, TMEM173-Pr-89, TMEM173-Pr-121, TMEM173-Pr-161 and TMEM173-Pr-164 upregulate TMEM173 mRNA.

As shown in FIG. 3, TMEM173-Pr-70-invAb-Se-m2, TMEM173-Pr-70-invAb-Se-m1, TMEM173-Pr-70-invAb-Se-0, TMEM173-Pr-70-invAb-Se-p1 and TMEM173-Pr-70-invAb-Se-p2 all up-regulated TMEM173 mRNA.

In another study, human a549 cells (lung adenocarcinoma) were transfected with 10nM control FLuc oligonucleotides or different saRNA variants targeted to TMEM173 (STING). RNA was extracted at 72 hours and the expression level of TMEM173 mRNA was measured by RT-qPCR (FIG. 4). The sequence of the control saRNA is shown in table 5.

As shown in FIG. 4, TMEM173-Pr-70-invAb-Se-m1 upregulates TMEM173 mRNA, and TMEM173-Pr-70-invAb-As-m1 has less activity. The negative control TMEM173-Pr-70-invAb-Se-m 1-setmut did not up-regulate TMEM173 mRNA.

TABLE 5 sequence of control saRNA

In another study, the cells were untreated a549 cells (UNTR), a549 cells transfected with 10nM control FLuc oligonucleotides or TMEM173 (STING) -targeted saRNA. RNA and protein were extracted at 72 hours and expression levels of TMEM173 mRNA and TMEM173 protein were measured by RT-qPCR and Wes Protein Simple assays, respectively (fig. 5).

As shown in FIG. 5, TMEM173-Pr-70-invAb-Se-m1 upregulates TMEM173 mRNA (FIG. 5A) and TMEM173 protein (FIG. 5B). Total protein quantification showed that the loading was equal for all 3 samples for Wes analysis.

In another study, human a549 cells were transfected with 10nM control FLuc oligonucleotides or different saRNA variants targeted to TMEM173 (STING). RNA was extracted at 72 hours and the expression level of TMEM173 mRNA was measured by RT-qPCR (FIG. 6).

As shown in FIG. 6, TMEM173-Pr-70-invAb-Se-m1 and TMEM173-Pr-70-m1-emod51 both up-regulated TMEM173 mRNA.

In another study, human a549 cells were transfected with different concentrations (0.37-50 nM) of control FLuc oligonucleotides or different saRNA variants targeted to TMEM173 (STING). RNA was extracted at the indicated time points and the expression level of TMEM173 mRNA was measured by RT-qPCR (FIG. 7).

As shown in FIG. 7, TMEM173-Pr-70-invAb-Se-m1 and TMEM173-Pr-70-m1-emod51 upregulated TMEM173 mRNA in a dose-dependent manner at all time points.

Other embodiments

It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence listing

<110> Mi Na treatment Co., ltd (MINA THERAPEUTICS LIMITED)

<120> TMEM173 saRNA compositions and methods of use

<130> 2058.1034PCT

<140>

<141>

<150> US63/166,390

<151> 2021-03-26

<150> US63/318,927

<151> 2022-03-11

<160> 85

<170> PatentIn version 3.5

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gtcacagagg ccttcagaat gaagacccaa agacagaggg caaattgtct atttttacgt 60

ttaggttcat taaagtatgg acacccttgt tgaaatataa ttggacagaa agggtatgct 120

ctaatgctaa tagaatgagt ggggaaaccc agcctcgccc gtctgtctag attcttcttg 180

gcctctctga gcgtgtgttc cttctttctg agtgtggggc agggccctct ctggaagggg 240

gattttataa tctacactca aacaatgtag gtcagacctt cccttccctt cccttccctt 300

cccttccttc ctttctctct ctctcttttt ctttctttct tctctttttt ttttttttga 360

gacggagttt tgctcttgtc gcccaggctg gagtgcaatg gcacgatctc ggctcagtgc 420

aacctccgcc tcccaggttc aagcagttct cctgcctcag cctcccaaga agctgggatt 480

acaggcatgc gccatcacgc ccagctaatt ttttgtattt ttagtagaga tggggtttct 540

ccatgttggt caggatggtc ttgaactcct gacctcaggt gatctgcctg ccttggcctc 600

ccaaattgct gggattatag gagtgagcca ctgcacccgg cctttttttt tttttttttt 660

gagatggagt ttgactcttg ttgccgaggc tggagtgcag tggcatgatc tcggctcacc 720

gcaagctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcctga gtagctggga 780

ttataggcac ccgccaccac gcccagcgaa ttttttgtac ttttagtaga gatggggttt 840

cactatgttg gccaggctgg tctcgaactc ctgacctcaa gtgatccatt tgccttggcc 900

tcccaaagtg cgaggattac aaggatgagc cattgcgcct ggcctatttt tttttttttt 960

tttttgagat agggtatcac tctgtcactt aggctggagt gcagtggtgc catcacaact 1020

cactgcaacc tctacctcca ggggtcaagt gatgctccca cctcagcctc ccaagtagct 1080

gggactatag gcgtgttccg tcatgcttgg ctaatttttt tttttttttt ttgtagagat 1140

gggatctccc tgtgttgctt aggctggtct caaacttctg ggctcaagtg atcctcctgc 1200

cttggcctcc caaagtgctg ggattactgg aatgaaatca aggcacagag caagctgggc 1260

tttggagcaa cccaccaggc ttcaagtccc cactctcaat tacttaaacc agttatttca 1320

cctccctgag cctcggatta tccatctata aaatggggct agaattatac ctacctgaca 1380

gggtggctgg tgaaatgata tacaagtgaa gtgatatatg caacacttgg cataatgtct 1440

ggaacaaggt aaacacttta ttattattat tattattata atttaggttg atgcatgggg 1500

attttataac ctacactcaa acaatgtagg tcagatcatt tttcttttct tttcttttct 1560

tttcttttga gacagtctcg ctcttgttgc ccaggctgga gtgcagtggc ctaatctctg 1620

ctcactgcaa cttccacctc tcaggttcca gcgattctcc tgcctcagcc tcccaagtag 1680

ctaagattac aagcgcccac caccacgcct ggctaatttt tgtttttagt agagatgggg 1740

tttcaccatg ttgctcaggc tggtcttgaa ctcctgacct caagtgatcc acccatctcg 1800

gcctcccaaa gcgctgggat tacaggcatg agccactgtg ccaggcctgc aattactttt 1860

gctcctacct aatatcatcc ccacaaccgc cttctgggca gaaaccggca ggctctcttg 1920

gagaagtcac aggcgtggcc atttcctgca aagagccaaa cccccattcc tctgtgcccc 1980

tcctctccca ccaagtgctt tataaaaata gctcttgtta ccggaaataa ctgttcattt 2040

ttcactcctc cctcctaggt cacacttttc agaaaaagaa tctgcatcct ggaaaccaga 2100

agaaaaatat gagacgggga atcatcgtgt gatgtgtgtg ctgcctttgg ctgagtgtgt 2160

ggagtcctgc tcaggtgtta ggtacagtgt gtttgatcgt ggtggcttga ggggaacccg 2220

ctgttcagag ctgtgactgc ggtgagtgtt tctaaacacc cttggtttgg gggtagcaaa 2280

gggcaatgga atggaggctt tctcaaacct cacccctgac cccaggactc aggcccagct 2340

catcagggct ttgagggaag gttcctacct cccttcctga ggaacaggaa ataccctcct 2400

tcccagcacc agtaaagctg cggtttggag aaacgccagg gctagagtgt tgtggagaaa 2460

ccaatcgttg ttaacatctc attttcaggc tgcactcaga gaagctgccc ttggctgctc 2520

gtagcgccgg gccttctctc ctcgtcatca tccagagcag ccagtgtccg ggaggcagaa 2580

ggtaggctca agatcagcct ggcagaacgc caaacctagg gcccctggca cccagaggcg 2640

agggggtgcc tgctggctgc cctgtcccca ctccctgagc tctgttttcc actttgttga 2700

ctaaggtcct ccctggggtg ggttccgggg acaggggaac ccaggtcccc aagggttctt 2760

ggttgggtac ggctgcacag gacagcttca agtctgggtc tgggatagtt gctgccttct 2820

ttcttcacca cacctgtggt ctccctgggt cttggtgggg cgtgtatgtg caggccctgc 2880

tctgttttca gcaaacctcg ctgagacagg agctttgggg tgacttattc ccagcctgcc 2940

tcctagaggt gtctctaaga gcagcctctg ggagtggctg ggcaccaggg aaaggggaac 3000

tgggaggaag tgcccagcca gagcctcagt cccagaaggg caggagggca aggggagaat 3060

ggtcatggat ttcttggtgc ccacagatgc cccactccag cctgcatcca tccatcccgt 3120

gtcccagggg tcacggggcc cagaaggcag ccttggttct gctgagtgcc tgcctggtga 3180

ccctttgggg gctaggagag ccaccagagc acactctccg gtacctggtg ctccacctag 3240

cctccctgca gctgggactg ctgttaaacg gggtctgcag cctggctgag gagctgcgcc 3300

acatccactc caggtgactc actgcagtac ccagggacgg ggtatccaac gtgtgtcact 3360

cccttgatgc ctagccctgc ccctccttga acctctctgg ctgagctggg ctgggggctg 3420

gggtctgggg tctggctgtc actcacaggt accggggcag ctactggagg actgtgcggg 3480

cctgcctggg ctgccccctc cgccgtgggg ccctgttgct gctgtccatc tatttctact 3540

actccctccc aaatgcggtc ggcccgccct tcacttggat gcttgccctc ctgggcctct 3600

cgcaggcact gaacatcctc ctgggcctca aggtatgaca cagggggagg tagaagctct 3660

ggccaagtgg tggctgtggc tggtgtgacc tgccctgagc tgagtactgg gagtgggact 3720

ggtttaaagg ctggagtcca tggagtagaa cctataatgt cctggaacag tgggtttggc 3780

aatggcaaaa gagggatcaa gtcaggagca ggttgggaag ccttggagga ggaggaggag 3840

ttctctgggt gtccttgatg gaggcccccc agccacatcc tgctgtccac agggcctggc 3900

cccagctgag atctctgcag tgtgtgaaaa agggaatttc aacgtggccc atgggctggc 3960

atggtcatat tacatcggat atctgcggct gatcctgcca g 4001

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<213> Homo sapiens (Homo sapiens)

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<212> DNA

<213> Homo sapiens (Homo sapiens)

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<212> DNA

<213> Homo sapiens (Homo sapiens)

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<212> DNA

<213> Homo sapiens (Homo sapiens)

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<212> DNA

<213> Homo sapiens (Homo sapiens)

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<212> DNA

<213> Homo sapiens (Homo sapiens)

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<212> DNA

<213> Homo sapiens (Homo sapiens)

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<212> DNA

<213> Homo sapiens (Homo sapiens)

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<212> DNA

<213> Homo sapiens (Homo sapiens)

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<220>

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<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

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<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

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<210> 19

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<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 19

augagauguu aacaacgau 19

<210> 20

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 20

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<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

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<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

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guaggaaccu ucccucaaa 19

<210> 23

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

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caggaucagc cgcagauau 19

<210> 24

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

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<210> 25

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 25

uccugucuca gcgagguuu 19

<210> 26

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 26

aacgauuggu uucuccaca 19

<210> 27

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 27

caacgauugg uuucuccac 19

<210> 28

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 28

cgauugguuu cuccacaac 19

<210> 29

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 29

gauugguuuc uccacaaca 19

<210> 30

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 30

uauuacaucg gauaucugc 19

<210> 31

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 31

ugauauaugc aacacuugg 19

<210> 32

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 32

ucuggaacaa gguaaacac 19

<210> 33

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 33

aucguuguua acaucucau 19

<210> 34

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 34

uuguggagaa accaaucgu 19

<210> 35

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 35

uuucuaaaca cccuugguu 19

<210> 36

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 36

uuugagggaa gguuccuac 19

<210> 37

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 37

auaucugcgg cugauccug 19

<210> 38

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 38

auuucuacua cucccuccc 19

<210> 39

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 39

aaaccucgcu gagacagga 19

<210> 40

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 40

uguggagaaa ccaaucguu 19

<210> 41

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 41

guggagaaac caaucguug 19

<210> 42

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 42

guuguggaga aaccaaucg 19

<210> 43

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<400> 43

uguuguggag aaaccaauc 19

<210> 44

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 44

gcagauaucc gauguaauau u 21

<210> 45

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 45

ccaaguguug cauauaucau u 21

<210> 46

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 46

guguuuaccu uguuccagau u 21

<210> 47

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 47

augagauguu aacaacgauu u 21

<210> 48

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 48

acgauugguu ucuccacaau u 21

<210> 49

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 49

aaccaagggu guuuagaaau u 21

<210> 50

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 50

guaggaaccu ucccucaaau u 21

<210> 51

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 51

caggaucagc cgcagauauu u 21

<210> 52

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 52

gggagggagu aguagaaauu u 21

<210> 53

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 53

uccugucuca gcgagguuuu u 21

<210> 54

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 54

aacgauuggu uucuccacau u 21

<210> 55

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 55

caacgauugg uuucuccacu u 21

<210> 56

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 56

cgauugguuu cuccacaacu u 21

<210> 57

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 57

gauugguuuc uccacaacau u 21

<210> 58

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> reverse abasic nucleotide

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 58

nacgauuggu uucuccacaa uu 22

<210> 59

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> reverse abasic nucleotide

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 59

naacgauugg uuucuccaca uu 22

<210> 60

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> reverse abasic nucleotide

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 60

ncaacgauug guuucuccac uu 22

<210> 61

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> reverse abasic nucleotide

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 61

ncgauugguu ucuccacaac uu 22

<210> 62

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> reverse abasic nucleotide

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 62

ngauugguuu cuccacaaca uu 22

<210> 63

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<223> description of combined DNA/RNA molecules: synthetic

Oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> Inv ddT

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 63

tacgauuggu uucuccacaa uu 22

<210> 64

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<223> description of combined DNA/RNA molecules: synthetic

Oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> Inv ddT

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 64

taacgauugg uuucuccaca uu 22

<210> 65

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<223> description of combined DNA/RNA molecules: synthetic

Oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> Inv ddT

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 65

tcaacgauug guuucuccac uu 22

<210> 66

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<223> description of combined DNA/RNA molecules: synthetic

Oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> Inv ddT

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 66

tcgauugguu ucuccacaac uu 22

<210> 67

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<223> description of combined DNA/RNA molecules: synthetic

Oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> Inv ddT

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 67

tgauugguuu cuccacaaca uu 22

<210> 68

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<223> description of combined DNA/RNA molecules: synthetic

Oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> Inv ddT

<220>

<221> modified base (modified_base)

<222> (2)..(3)

<223> 2' -OMe modified nucleotide

<220>

<221> modified base (modified_base)

<222> (5)..(5)

<223> 2' -OMe modified nucleotide

<220>

<221> modified base (modified_base)

<222> (11)..(11)

<223> 2' -OMe modified nucleotide

<220>

<221> modified base (modified_base)

<222> (15)..(15)

<223> 2' -OMe modified nucleotide

<220>

<221> modified base (modified_base)

<222> (21)..(22)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 68

tcgauugguu ucuccacaac uu 22

<210> 69

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 69

uauuacaucg gauaucugcu u 21

<210> 70

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 70

ugauauaugc aacacuuggu u 21

<210> 71

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 71

ucuggaacaa gguaaacacu u 21

<210> 72

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 72

aucguuguua acaucucauu u 21

<210> 73

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 73

uuguggagaa accaaucguu u 21

<210> 74

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 74

uuucuaaaca cccuugguuu u 21

<210> 75

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 75

uuugagggaa gguuccuacu u 21

<210> 76

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 76

auaucugcgg cugauccugu u 21

<210> 77

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 77

auuucuacua cucccucccu u 21

<210> 78

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 78

aaaccucgcu gagacaggau u 21

<210> 79

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 79

uguggagaaa ccaaucguuu u 21

<210> 80

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 80

guggagaaac caaucguugu u 21

<210> 81

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 81

guuguggaga aaccaaucgu u 21

<210> 82

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 82

uguuguggag aaaccaaucu u 21

<210> 83

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> reverse abasic nucleotide

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 83

guuguggaga aaccaaucgu u 21

<210> 84

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (1)..(1)

<223> reverse abasic nucleotide

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 84

cgauugguuu cacgagaucu u 21

<210> 85

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of Artificial sequence synthetic oligonucleotides

<220>

<221> modified base (modified_base)

<222> (20)..(21)

<223> 2' -OMe modified nucleotide

<220>

<223> see detailed description of substitution in the specification filed

And preferred embodiments

<400> 85

gaucucguga aaccaaucgu u 21

Claim (modification according to treaty 19)

1. A synthetic isolated small activating RNA (saRNA) that upregulates expression of a target gene, wherein the target gene is TMEM173,

wherein the saRNA comprises an antisense strand that is at least 80% complementary to a targeting sequence of the target gene, wherein the targeting sequence is selected from the group consisting of SEQ ID NOs 2-15, and

wherein the antisense strand has 14-30 nucleotides.

2. The saRNA of claim 1, wherein the antisense strand comprises a sequence selected from SEQ ID NOs 30-43.

3. The saRNA of claim 2, wherein the antisense strand comprises a 3' overhang.

4. The saRNA of claim 3, wherein the 3' overhang is UU, or UUU.

5. The saRNA of claim 4, wherein the antisense strand comprises a sequence selected from SEQ ID NOs 69-82.

6. The saRNA of any one of claims 1-5, wherein the saRNA is double stranded and further comprises a sense strand.

7. The saRNA of claim 6, wherein the sense strand comprises a sequence selected from SEQ ID NOs 16-29.

8. The saRNA of claim 7, wherein the sense strand comprises a 3 'overhang and/or a 5' overhang.

9. The saRNA of claim 8, wherein the 3' overhang is UU, or UUU.

10. The saRNA of claim 8, wherein the 5' overhang is dT, ddT, invddt, or invAb.

11. The saRNA of claim 8, wherein the sense strand comprises a sequence selected from SEQ ID NOs 44-68.

12. The saRNA of any one of claims 1-11, wherein the saRNA comprises at least one modification.

13. The saRNA of claim 1, wherein the saRNA is TMEM173-Pr-70-invAb-Se-m1 or TMEM173-Pr-70-m1-emod51.

14. A pharmaceutical composition comprising the saRNA of any one of claims 1-13 and at least one pharmaceutically acceptable excipient.

15. A method of up-regulating expression of a target gene, wherein the target gene is TMEM173, comprising administering the saRNA of any one of claims 1-13.

16. The method of claim 15, wherein the expression of the target gene is increased by at least 30%, 40% or 50%.

17. A method of treating cancer in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of the saRNA of any one of claims 1-13 or the pharmaceutical composition of claim 14.

18. The method of claim 17, wherein the cancer is a solid tumor.

19. The method of claim 18, wherein the cancer is hepatocellular carcinoma, pancreatic cancer, or ovarian cancer.

Claims (19)

1. A synthetic isolated small activating RNA (saRNA) that upregulates expression of a target gene, wherein the target gene is TMEM173,

wherein the saRNA comprises an antisense strand that is at least 80% complementary to a targeting sequence of the target gene, wherein the targeting sequence is selected from the group consisting of SEQ ID NOs 2-15, and

wherein the antisense strand has 14-30 nucleotides.

2. The saRNA of claim 1, wherein the antisense strand comprises a sequence selected from SEQ ID NOs 30-43.

3. The saRNA of claim 2, wherein the antisense strand comprises a 3' overhang.

4. The saRNA of claim 3, wherein the 3' overhang is UU, or UUU.

5. The saRNA of claim 4, wherein the antisense strand comprises a sequence selected from SEQ ID NOs 69-82.

6. The saRNA of any one of claims 1-5, wherein the saRNA is double stranded and further comprises a sense strand.

7. The saRNA of claim 6, wherein the sense strand comprises a sequence selected from SEQ ID NOs 16-29.

8. The saRNA of claim 7, wherein the sense strand comprises a 3 'overhang and/or a 5' overhang.

9. The saRNA of claim 8, wherein the 3' overhang is UU, or UUU.

10. The saRNA of claim 8, wherein the 5' overhang is dT, ddT, invddt, or invAb.

11. The saRNA of claim 8, wherein the sense strand comprises a sequence selected from SEQ ID NOs 44-68.

12. The saRNA of any one of claims 1-11, wherein the saRNA comprises at least one modification.

13. The saRNA of claim 1, wherein the saRNA is TMEM173-Pr-70-invAb-Se-m1 or TMEM173-Pr-70-m1-emod51.

14. A pharmaceutical composition comprising the saRNA of any one of claims 1-13 and at least one pharmaceutically acceptable excipient.

15. A method of up-regulating expression of a target gene, wherein the target gene is TMEM173, comprising administering the saRNA of any one of claims 1-13.

16. The method of claim 15, wherein the expression of the target gene is increased by at least 30%, 40% or 50%.

17. A method of treating cancer in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of the saRNA of any one of claims 1-13 or the pharmaceutical composition of claim 13.

18. The method of claim 17, wherein the cancer is a solid tumor.

19. The method of claim 18, wherein the cancer is hepatocellular carcinoma, pancreatic cancer, or ovarian cancer.