WO2024165876A2 - Compositions and methods of using c/ebp alpha sarna - Google Patents

Abstract

The invention relates to methods of using saRNAs targeting C/EBPα and pharmaceutical compositions comprising the saRNAs to treat diseases such as lysosomal storage disorders.

Description

COMPOSITIONS AND METHODS OF USING C/EBP ALPHA SARNA

RELATED APPLICATION

[00011 This application claims priority to US Provisional Application No. 63/484,276 filed February 10, 2023, entitled “Compositions and Methods of Using C/EBP Alpha SARNA,” the contents of which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed with a Sequence Listing in electronic format. 'Hie sequence listing filed, entitled 0l 337-0044-00PCT_ SL.xml was created on January 25, 2024, and is 10,759 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0003] The invention relates to polynucleotide, specifically saRNA, compositions for the modulating C/EBPa and C/EBPa pathways and to the methods of using the compositions in therapeutic applications .

BACKGROUND OF THE DISCLOSURE

[0004] CCAAT/enhancer-binding protein a (C/EBPa, C/EBP alpha, C/EBPA or CEBPA) is a leucine zipper protein that is conserved across humans and rats. This nuclear transcription factor is enriched in hepatocytes, myelomonocytes, adipocytes, as well as other types of mammary epithelial cells [Lekstrom- Himes et al., 7. Bio. Chem, vol. 273, 28545-28548 (1998)], It is composed of two transactivation domains in the N-tenninal part, and a leucine zipper region mediating dimerization with other C/EBP family members and a DNA-b inding domain in the C- terminal part. The binding sites for the family of C/EBP transcription factors are present in the promoter regions of numerous genes that are involved in the maintenance of normal hepatocyte function and response to injury'. The CCAAT/enhancer-binding protein alpha (CEBPA) gene encodes C/EBPa, a transcription factor that plays a fundamental role in controlling maturation of the myeloid lineage. C/EBPa is a myeloid specific transcription factor. It has been shown that myeloid lineage-specific deletion of C/EBP a results in significantly enhanced myeloid-derived suppressor cell (MDSC) proliferation and expansion and in an increase in myeloid progenitors and a decrease in mature cells.

[0005] Thus, there is a need for targeted modulation of C/EBPa for therapeutic purposes with saRNA. SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides methods of using CEBPA-saRNA molecules and compositions comprising CEBPA-saRNA molecules to treat mucopolysaccharides type I (M.PS1 ) of a subject. The present disclosure also provides methods of using CEBPA-saRNA molecules and compositions comprising CEBPA-saRNA molecules to increase IDUA gene expression, IDUA protein level, or IDUA protein activity in a subject. The present disclosure also provides methods of using CEBPA-saRNA molecules and compositions comprising CEBPA-saRNA molecules to reduce glycosaminoglycans (G AG) levels and/or TN Fa levels in .a subject. The subject may also receive bone marrow transplantation (BMT) before saRNA administration.

[0007] The present disclosure further provides methods of using CEBPA-saRNA molecules and compositions comprising CEBPA-saRNA molecules to treat a lysosomal storage disorder (LSD) of a subject. The LSD may be Fabry, MPSIIIA or Sanfilippo syndrome. Sialic acid storage disease, MPS1 or Hurler disease, M PSI I or Hunter syndrome, CLN5 Batten disease, Metachromatic leukodystrophy, Fucosidosis, Hermansky-Pudlak disease type 1 , Sialidosis, Beta- Mannosidosis, G.M2 activator deficiency, Farber disease, Multiple Sulfatase deficiency, Batten disease type I, MPS1VB or Morquio disease, MPSIVA or Morquio disease, MPSHIB or Sanfilippo syndrome, MPSHID or Sanfilippo syndrome, Aspartylglucosaminuria, Schindler disease, Cystinosis, Mucolipidosis IL Lysosomal Acid Lipase deficiency, Sandhoff disease, MPS VI or Maroteaux-Lamy syndrome, Hermansky-Pudlak disease type 5, Gaucher disease, CLN8 Batten disease, CLN10 Batten disease, CLN 1 1 Batten disease, or CLN 13 Batten disease. The subject may also receive bone marrow transplantation (BMT) before saRNA administration.

[0008] The present disclosure further provides methods of using CEBPA-saRNA molecules and compositions comprising CEBPA-saRNA molecules to increase the expression of a target gene in a cell, wherein the target gene is GLA, SGSFI, SLCL7A5, IDU A, IDS, CLN5, ARSA, FUCAI, HPSl, NEU1 , MANBA, GM2A, ASAHI, SUMFL PPT1, GLB 1, GALNS, NAGLU, GNS, AGA, NAGA, CTNS, GNPTAB, LIPA, HEXB, ARSB, FIPS5, GBA, MFSD8, CTSD, GRN, or CTSF.

[0009] The details of various embodiments of the in vention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0010 ] Fig. I A and Fig. IB shows CEBPA-51 induces a sustained 7DL/A expression vitro in MSCs.

[0011| Fig. 2A-2D show MTL-CEBPA increases CEBPA and ZDLM gene expressions b? vitro in human primary bone marrow CD34+- cells.

[0012] Fig. 3A and Fig. 3B show CEBPA-51 induces a dose specific IDUA expression in vitro in IMR90 cells.

[0013] Fig. 4A-4C show CEBPA-51 increases IDUA protein expression and intracellular IDUA enzymatic activity vitro in IMR90 cells. ( 0014 ] Fig. 5A-5B shows pharmacodynamic data in the bone marrow compartment.

[0015] Fig. 6A-6C show MTL-CEBPA-treated PBMCs (treated ex vivo) leads to an upregulation of CEBPA mRNA, IDUA mRNA and protein at 48h.

[0016] Fig. 7 Shows MTL-CEBPA treated whole blood leads to an increase in Alpha L iduronidase (IDUA) enzyme activity by at least two-fold. [0017] Fig. 8A-8B shows changes in MTL-CEBPA treated whole blood.

[0018] Fig. 9A shows genetic upregulation IDUA in bone marrow with MTL-CEBPA treatment as a bone marrow transplant adjuvant. Fig. 9B shows an increase in systemic IDUA enzyme with MTL-CEBPA treatment as a bone marrow transplant adjuvant. Fig. 9C shows MTL-CEBPA treatment as a bone marrow transplant adjuvant improved bone phenotypes in a mouse model of MPS 1 H. Fig. 9D shows MTL-CEBPA treatment as a bone marrow transplant adjuvant reduced chronic inflammation.

[0019] Fig. 10A-10C show CEBPA mRNA levels, IDUA mRNA levels, and IDUA enzymatic activities in bone marrow tissues after MTL-CEBPA treatment.

[0020] Fig. I LA and 1 1 B show 2mg/kg (2mpk) MTL-CEBPA significantly reduced plasma TNFd levels.

[002] 1 Fig. 12 A- 12B show IDUA protein levels in the monocytes and IDUA enzyme activities in the plasma of patients who received MTL-CEBPA treatments.

[0022] Fig. 13 A- 13B show upregulation of CEBPA and GALNS in A549 cells treated with CEBPA-51. [0023 | Fig. 14A-14B show upregulation of CEBPA and CMZ.A'5 in mouse bone marrow cells after 1.5 months and 3 months of dosing animals with MTL-CEBPA. [0024] Fig. I SA-15B show upregulation of CEBPA and A SAJI/ in A549 cells treated with CEBPA-51 .

[0025] Fig. 16A- 16B show upregulation of CEBPA and SG.S77 in A549 cells treated with CEBPA-51.

[0026] Fig. 17A-17B show upregulation of CEBPA and SLC17A5 in A549 cells treated with CEBPA-51 .

[0027] Fig. 18A-18B show upregulation of CEBPA and NEU/ in A549 cells treated with CEBPA-51.

[0028] Fig. 19A-19B show upregulation of CEBPA and SUMFI in A549 cells treated with CEBPA-51.

DETAILED DESCRIPTION

[0029] The present invention provides compositions, methods and kits for modulating C/EBPa gene expression and/or function for therapeutic purposes. These compositions, methods and kits comprise nucleic acid constructs that target a C/EBPa transcript.

[0030 j CZEBPa protein is known as a criticafregulator of metabolic processes and cell proliferation. Modulating C/EBPa gene has great potentials for therapeutic purposes. The present invention addresses this need by providing nucleic acid constructs targeting a C/EBPa transcript, wherein the nucleic acid constructs may include single or double stranded DNA or RN A with or without modifications,

[0031] C/EBPa gene as used herein is a double-stranded DN A comprising a coding strand and a template strand. It may also be referred to the target gene in the present application.

[0032] The terms “C/EBPa transcript’ \ “C/EBPa target transcript’* or "target transcript” in the context may be C/EBPa m.RN A encoding C/EBPa protein. C/EBPa mRNA is transcribed from the template strand of C/EBPo, gene and may exist in the mitochondria.

[0033] The ant isen se RNA of the C/EBPa gene transcribed from the coding strand of the

C/EBPa gene is called a target antisense RNA transcript herein after. The target antisense RNA transcript may be a long non-coding antisense RNA transcript.

[0034i The terms “small activating RNA”, “short activating RNA”, or “saRNA” in the context of the present invention means a single-stranded or double-stranded RN A that upregulates dr has a positive effect on the expression of a specific gene. The saRNA may be single-stranded of 14 to 30 nucleotides. The saRNA may also be double-stranded, each strand comprising 14 to 30 nucleotides. The gene is called the target gene of the saRNA. A saRNA that upregulates the expression of the C/'EB Pa gene is called a "C/EBPa-saRNA” and the C/EBPa gene is the target gene of the C/EBPu-saRNA.

[0035] The terms '‘target" or “targeting” in the context mean having an effect on a C/EBPa gene. The effect may be direct or indirect. Direct effect may be caused by complete or partial hybridization with the C/EBPa target antisense RN A transcript. Indirect effect may be upstream or downstream.

[0036] C/EBPa-saRNA may have a downstream effect on a biological process or activity. In such embodiments, C/EBPa-saRNA may have an effect (either upregulating or downregulating) on a second, non-target transcript.

[0037] The term “gene expression” in the context may include the transcription step of generating C/EBPa mRNA from C/EBPa gene or the translation step generating C/EBPa protein from C/EBPa mRNA. An increase of C/EBPa mRNA and an increase of C/EBPa protein both indicate an increase or a positive effect of C/EBPa gene expression.

[0038] By “upregulation” or “acti vation” of a gene is meant an increase in the level of expression of a gene, or levels of the polypeptidefs) encoded by a gene or the activity thereof, or levels of the RN A transcript(s) transcribed from the template strand of a gene above that observed in the absence of the saRNA of the present invention. The saRNA of the present invention may have a direct or indirect upregulating effect on the expression of the target gene. [0039] In one embodiment, the saRNA of the present invention may show efficacy in proliferating cells. As used herein with respect to cells, “proliferating” means cells which: are gro wing and/or reproducing rapidly.

I. Composition of the In vention

[0040] One aspect of the present invention provides phannaceutical compositions comprising a saRNA that upregulates CEB PA gene, and at least one pharmaceutically acceptable carrier. Such a saRN A is referred herein after as “C/EBPa-saRNA”, or “saRNA of the present invention”, used interchangeably in this application.

[0041] The C/EBPa-saRNA has 14-30 nucleotides and comprises a sequence that is at least 80%, 90%, 95%, 98%, 99% or 100% complementary to a targeted sequence on the template strand of the C/EBPa gene. The targeted sequence may have the same length, i.e.. the same number of nucleotides, as the saRNA and/or the reverse complement of the saRN A. [0042] In some embodiments, the targeted sequence comprises at least 14 and less than 30 nucleotides.

[0043] Ift some embodiments, the targeted sequence has 19, 20, 21, 22, or 23 nucleotides, [0044] In some embodiments, the location of the targeted sequence is situated within a promoter area of the template strand.

[0045] i n some embodiments, the targeted sequence of the CZEBP«-saRNA is located wi thin a TSS (transcription start site) core of the template stand of the C/EBPo gene. A "TSS core” or “TSS core sequence” as used herein, refers to a region between 2000 nucleotides upstream and

2000 nucleotides downstream of the TSS (transcription start site). Therefore, the TSS core comprises 4001 nucleotides and the TSS is located at position 2001 from the 5’ end of the TSS core sequence. CEBPA TSS core sequence is show in the table below:

> > > > tgCCaggtecgagcaeg^acagggagnactcigcct.agtggucgccgggKgctgUgwcccgggatcctagggaccgaggcg.gccaggccctg^gccicct i tgagtgcggcagCtaatgvtctcaccgvggvgggggAjggagcttgccaecgagaccvccagccacgtgcgtccclcgcartcWaecggggecggggtggcg | gctacggaecgtcag€igggcceaEafg2agtetiggg<igix\'K'aagtgictcctgux'tigcccgcgccgccecteg<;eac(ggcgc(gaggcctgacgccgc i j cigcgtcccggctagagg<. gcgctigcetacaggigaggj<!3g«Rce<.citc.KegjcagtggccttaggccJggcaaggegicc?tcgacccgcccaggagccc I | ^:cggagggggcacagctaajaacaecgv'tgga_J.’agecc<.«agetkc.kg.K^,g:Ui'ucagtaaagaageagttcaidgggca^cgcsJcaetgcgcttaatcaa I | gttcciatteaacaiagteccagtgaUaatagcccaactgcticgntteggtccagagetcataaac'aagatattttiagcngacgetittggacgggagggagtaaaa | | aceagaiaegtiaaa)Matatcccgatgtgag<.x'ggagagc{gcugc(gageeaa3tg<agga>.xcattcatatagcaucacc<gtggagggagacctggacgg i | aaatcaaaaagcai'caagagcgantgigtuttiugcggtgctaauactaatggctttcvta^aggaacaaagaaacgccactgtac j | t gctgttgcc(gc€cagagaK'gggaac(£tg€C(;cgtaggactg j I

_ _

|

[0046] In some embodiments, the targeted sequence is located between 1000 nucleotides upstream and 1000 nucleotides downstream of t he TSS. In some embodiments, the targeted sequence is located between 500 nucleotides upstream and 500 nucleotides .downstream of the TSS. In some embodiments, the targeted sequence is located between 250 nucleotides upstream and 250 nucleotides downstream of the TSS. In some embodiments, the targeted sequence is located between 100 nucleotides upstream and 100 nucleotides downstream of the TSS. In some embodiments, the targeted sequence is located upstream of the TSS in the TSS core. The targeted 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 targeted sequence is located downstream of the TSS in the TSS core. The targeted sequence may be less than 2000, less than 1000, less than 500, less than 250, or less than 100 nucleotides downstream of the TSS. [0047] In some embodiments, the targeted sequence is located +/- 50 nucleotides surrounding the TSS of the TSS core. In some embodiments, the targeted sequence substantially overlaps the TSS of the TSS core. In some embodiments, the targeted sequence begins or ends at the TSS of the TSS core. In some emlxxl intents, the targeted sequence overlaps the TSS of the TSS core by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in either the upstream or downstream direction. [0048] The l ocation of the targeted sequence on the templ ate strand is defined by the location of the 5’ end of the targeted sequence. The 5’ end of the targeted sequence may be at any position of the TSS core and the targeted sequence may start at any position selected from position 1 to position 4001 of the TSS core. For reference herein, when the 5' most end of t he targeted sequence from position I to position 2000 of the TSS core, the targeted sequence is considered upstream of the TSS and when the 5’ most eiid of the targeted sequence is from position 2002 to 4001, the targeted sequence is considered downstream of the TSS. When the 5’ most end of the targeted sequence is at nucleotide 200 I , the targeted sequence is considered to be a TSS centric sequence and is neither upstream nor downstream of the TSS.

[0049] For further reference, for example, when the 5’ end of the targeted sequence is at position 1600 of the TSS core, i,e., it is the 1600^th nucleotide of the TSS core, the targeted sequence starts at position 1600 of the TSS core and is considered to be upstream of the TSS. [0050] In one embodiment, the saRNA of the present invention may have two strands that form a duplex, one strand being a guide strand. The saRNA duplex is also called a doublestranded saRNA. A double-stranded saRNA or saRNA duplex, as used herein, is a saRNA that includes more than one, and preferably, two, strands in which interstrand hybridization can form a region of duplex structure. The two strands of a double-stranded saRNA are referred to as an antisense strand or a guide strand, and a sense strand or a passenger strand. fOOSlJ hi some embodiments, the C/EBPa-saRNA may comprising any C/EBPa-saRNA disclosed in WO2015/O75557 or WO2016/ 170349 to MiNA Therapeutics Limited, the contents of each of which are incorporated herein by reference in their entirety, such as saRNAs in Table 1 , Table 1 A, Table 3-1 and Table 3-2, AW51 , and CEBPA-51 disclosed in WO2016/170349.

[0052] In some embodiments, the C/EBPa-saRNA may be modified and may comprising any modification disclosed in WO2016/ 170349 to MiNA Therapeutics Limited.

[0053] In one embodiment, the C/EBPa-saRNA is CEBPA-51 (or CEBPA51 ), which is an saRNA duplex that upregulates C/EBPa, Its design, sequences, and compositionsZformulations are disclosed in the Detailed Description and Examples of W02O16/17O349 to MiNA Therapeutics Li mited. The sequences of the sense and antisense strands of CEBPA-51 are shown in Table 1.

Table 1. CEBPA-51 (CEBPA51) Sequences

mU, mG, and mC mean 2’-O-methyl modified U, G, and C. invabasic = inverted abasic sugar cap.

[0054] The alignment of the strands is shown in the Table 2.

Table 2. CEBPA-51 Alignment of Strands

[0055] CEBPA-51 is encapsulated into liposomes (NOV340 SM ARTICLES* "technology owned by "Marina Biotech) to make MTL-CEBPA. The lipid components of the NOV340 SM ARTICLES* are comprised of 1 -palmitoyl-2-oleoyl~sn-glycero-3-phosphochoIine (POPC), l,2-dioleoyl-sn-glycero~3-phosphoethanolamine (DOPE), cholesteryl-hemisuecinate (CHEMS), and 4<2-amirioethyl)-morpholino-cholesterdl hemisuccinate (MOCFIOL), NOV340 SMARTICLES* consists of POPC, DOPE, CHE-MS and MOCHOL in the molar ratio of 6:24:23:47, These nanoparticles are anionic at physiological pH, and their specific lipid ratio imparts a “pH-ftmable” character and a charge to the liposomes, which changes depending upon the surrounding pH of the naicroenvironment to facilitate movement across physiologic membranes, SM ARTICLES* nanoparticles are sized to avoid extensive immediate hepatic sequestration, with an average diameter of approximately about 50 - about 150 nm, or about 100 - about 120 nm, facilitating more prolonged systemic distribution and improved serum stability after i.v. injection leading to broader tissue distribution with high levels in liver, spleen and bone marrow reported,

[0056] MTL-CEBPA also comprises the buffer forming excipients such as sucrose and phosphate-salts. The concentration CEBPA-51 in MTL-CEBPA is 2.5x0.5 mg/mL, and the ratio of CEBPA-51 to the liposome is approximately 1 :25 (by weight). Qualitative and quantitative composition of MTL-CEBPA (2.5 nigftnl) are shown In Table 3,

Table 3. MTL-CEBPA Composition

Adminisfralion

[0057] C/EBPa-saRNAs or C/EBPa-saRNA compositions, such as CEBPA-5 i and/or MTL- CEBPA, may be administered by any route which results in a therapeutically effective Outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intraeerebro ventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a 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), intracavemous injection, ( into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), or in ear drops. In specific embodiments, compositions may be administered in away which allows them to cross the bloodbrain barrier, vascular hairier, or other epithelial barrier. Routes of administration disclosed in International Publication WO 2013/090648 filed December 14, 2012, the contents of which are incorporated herein by reference in their entirety, may be used to administer the saRNA of the present invention.

Dosing

[0058] In some embodiments, C/EBPa-saRNAs or C/EBPu-saRNA compositions, such as CEBPA-51 and/or MTL-CEBPA, are administered once every day, once every 2 days, once every 3 days, once every 4 days, or once every 5 days. [0059] In some embodiments, at least two doses of C/EBPa-saRNAs or C/EBPa-saRNA compositions, such as CEBPA-51 or MTL-CEBPA, are administered to a subject The doses are less than 7 days apart. In one embodiment, CEBPA-51 or MTL-CEBPA is administered every 24 hours. In one embodiment, CEBPA-51 or MTL-CEBPA is administered every 48 hours. In one embodiment, CEBPA-51 or MTL-CEBPA is administered every week. In one embodiment, CEBPA-51 Or MTL-CEBPA is administered evety week for 3 weeks, followed by 1 week of rest. In one embodiment, CEBPA-51 or MTL-CEBPA is administered every 3 weeks. In some embodiments, CEBPA-51 or MTL-CEBPA is administered once every 4 weeks (Q4W) or monthly or every 2 weeks (Q2W). In some cases, patients receive chronic dosing of CEBPA-51 or MTL-CEBPA treatment for life.

[0060] In some embodiments, the patient receives at least 2 doses, e,g, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, or 10 doses, of C/EBPa-saRNAs or C/EBPa-saRNA compositions, such as CEBPA-51 and/or .MTL-CEBPA,

[006] 1 In some embodiments, C/EBPa-saRNAs or C/EBPa-saRNA compositions, such as CEBPA-51 and/or MTL-CEBPA, are administered for a period of at least 2 days, such as 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks,

[0062[ In some embodiments, CZEBPa-saRNAs or C/EBPa-saRNA compositions, such as CEBPA-51 and/or MTL-CEBPA, are administered via intravenous infusion over 60 minutes. Doses are between about 15 to about 160 mg/riA

[0063] The dosing regimen disclosed in the present application may apply to any indication or disorder that can be treated with C/EBPa-saRNAs or C/EBPa-saRNA compositions.

IL Methods of Use

[0064] One aspect of the present invention provides methods of using C/EBPa-saRNA and pharmaceutical compositions comprising said C/EBPa-saRNA and at least one pharmaceutically acceptable carrier. CZEBPa-saRNA modulates C/EBPa gene expression. In one embodiment, the expression of C/EBPa gene is increased by at least 20, 30, 40%, at least 45, 50, 55, 60, 65, 70, 75%, or at least 80% in the presence of the saRNA of the present invention compared to theexpression of C/EBPa gene in the absence of the saRNA of the present invention. In a further embodiment, the expression of C/EBPu gene is increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10, in the presence of the saRN A of the present invention compared to the expression of C/EBPa gene in the absence of the saRNA of the present invention.

[0065] In some embodiments, saRNAs o f the present disclosure and/or its pharmaceutical compositions may be used to increase the expressi on of a target gene, such as but not limi ted to IDUA, GLA, 4SJ777. G7U GLBI, HEXA, HEXB, GM2A, GALC. ARSA, PSAP, SMPDI, LIPA, IDS. SGSH, NAGLU, HGSNAT, GNS, GALES, GLBI, ARSB, GUSB, HYALI. SUMFL GNPIAB, GNPTAB. GNPTG, MAN2BL MANBA, FUCAL AGA, EAGA, NEU I, CTSA, CTNS, LAMPS. SCARB2. SLCI7AS, NPCL NPC2. MCOLNI, HPSL AP3BI, BLOCI53. MYOSA, RAB27A. LYST, GAA, PPTL TPPI, CLN3, DNAJC5, CLN5, CLN6, MFSDS, CLN& CTRL). GRN, ATPI3A2, CTSF, or KCTD7. In some embodiments, the target gene is IDUA. GLA, SGSH SLCI7A5, IDS, CLN5. /I7?M FUCAL HPSI, NEU/, MANBA. GM2A, ASAHI, SLW7, PPTI, GLBI, GAINS. NAGLU, GNS, AGA, NAGA, CTNS. GNPTAB, LIPA, HEXB, ARSB, HPS3, GBA, MFSD8, CTSD. GRN, or CTSF. In some embodiments, the target gene is 7/JtM

JSJ777, SGSH, SLCI7A3, NEU/, or SUMF I. In one embodiment, the expression of the target gene is increased by at least 20, 30, or 40%, after treatment with the saRNA of the present invention compared to the expression of target gene in the absence of the saRNA of the present invention, In a further embodiment, the expression of target gene is increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10, after treatment with the saRNA of the present invention compared to the expression of the target gene in the absence of the saRNA of the present invention.

LSDs

[0066] In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat lysosomal storage disorders (LSDs). Lysosomal storage diseases, as used herein, refers to metabolic diseases that are characterized by an abnormal build- up of various toxic materials in the body ’s cells as a result of enzyme deficiencies. Non-limiting examples of LSDs include mucopolysaccharides type i (MPS I), mucopolysaccharides type 2 (MPSII), MPS 1I1A, mucopolysaccharides type IVa (MPS IVa), and mucopolysaccharides type VI (MPS VI), Gaucher disease. Lysosomal acid lipase deficiency, Fabry disease, Aspartylglucosaminuria, Schindler disease, Sialidosis, Sialic acid storage disease. Batten disease, Multiple Sulfatase deficiency. Cystinosis, Farber disease and Mucolipidosis type IL [0067] In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat Sphinogolipidoses, such as but not limited to Fabry disease (associated with GLA), Farber lipogranulomatosis (associated with ASAHI), Gaucher disease (associated with G73.4), GM1 gangliosidosis (associated with GO/), GM2 gangliosidosis or Tay-Sachs disease (associated with ZZEXt), GM2 gangliosidosis or Sandhoff disease (associated with HEXB), GM2 gangliosidosis or GM2 activator deficiency (associated with GMX4), Krabbe disease (associated with GALC), Metachromatic leukodystropliy (associated with

and/or RSzlP), or Nieman-Pick (A/B) (associated with SMPEM). In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to a lipid storage disease, such as but not limited to acid lipase deficiency, such as Wolman disease and cholesterol ester storage disease (associated with LIPA).

[0068] In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat Mucopolysacharidoses, such as but not limited to MPS1 or Hurler Syndrome (associated with ZZXZ4), MPS II or Hunter Syndrome (associated with /DS), MPS IIIA (associated with 5’GSZ/), MPS IIIB (associated vvith AMGZ.IZ), MPS IIIC (associated with HGSNAI), MPS HID (associated with GM MPS IVA (associated with G.42JVS), MPS IVB (associated with GLB1), MPS VI (associated with .-4RSB), MPS VII (associated with GUSB), or MPS IX (associated with HYALI).

[0069] In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat post-translational modification defects, such as but not limited to Multiple sulfatase deficiency (associated with MZMF/), Mucolipidosis II a/]l I -cell disease (associated with GNPTAB). Mucolipodosis II a/0. pseudo- Hurler polydystrophy (associated with GNPTAB). Mucolipidosis II y, or variant pseudo-Hurler polydystrophy (associated with GNPTG).

[0070] In some embodiments, saRN As of the present disclosure and/or their pharmaceutical compositions may be used to treat Glycoproteinoses, such as but not limited to u-Mannosidosis (associated with MAN2B1). p-Mannosidosis (associated with AZHA7Z4), Fueosidosis (associated with FVCA /), Aspartygiucosaminuria (associated with ,4G,4), Schindler disease (associated with AM GM), Sialidosis type I (associated with NEUI), Sialidosis type II (associated with NEUI ), or

Galactosialidosis (associated with CFS/i),

I 3 [0071] In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat Integral membrane protein disorders, such as but not limited to Cystinosis (associated with CTTVS), Danon disease (associated with £dMP2), Action myoclonus-renal failure syndrome (associated with SCAPP2), Sialic acid storage disease (associated with SLC17.4 S), Nieman-Pick disease type Cl (NPC1 ) (associated with NPCJ), or Mucolipidosis IV (associated with MCOLNI).

[0072] In some embodiments, saRN As of the present disclosure and/or their pharmaceutical compositions may be used to treat intra luminal protein disorders, such as but not limited to Nieman-Pick disease type C2 (associated with NPG2).

[00731 In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat lysosome-related organelles (LRO) disorders, such as but not limited to Hermansky-Pudlak disease type 1 (associated with HPS1), I IPS2 (associated with ,4P.W), HPS3, HPS4, HPS5, HPS6, HPS7, HPS8 (associated with M CO 53), HPS9, Griscelli syndrome 1 (associated with A-O'OS'.t), Griscelli syndrome 2 (associated with: /7.4F27,4), or Chediak- Higashi disease (associated with LYST).

[0074] In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat a Glycogen storage disease, such as Pompe disease (associated with GAAp'

]0075| In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat Neuronal ceroid lipofuscinoses, such as CLN 1 (associated with PP77), CLN2 (associated with TPPP)^ CLN3 (associated with CZJV3), CLN4 (associated with DNAJC5), CLN5 (associated with CZ.N5), CLN6 (associated with CXW), CLN7 (associated with MFSZM), CLNS (associated with CLN8), CLN9, CLN 10 (associated with CTSD), CLN LI (associated with G7?/V), CLN12 (associated with ,477V 3,42), CLN 13 (associated with C7NF), or CLN 14 (associated with KCTD7),

[0076] In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat inherited fronto temporal dementia (associated with haploinsufficiency of G/hV).

[0077] In some embodiments, saRN As of the present disclosure and/or their pharmaceutical compositions may be used to treat Parkinson's disease and Dementia with Lewy Bodies, Parkinson’s disease may be linked to genes such as but not limited to GBA1, SMPDl, TMEMI75. ATP13A2, SCARB2, CTSD. GLA, CTSB, GALC, ATP6VQA I, GUSB, NEU1 , SLC17A5, ASAHI , LAMP I, ARSA, NPC1, or NAGLU.

[0078] In some embodiments. saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat Fabry disease, Farber lipogranulomatosis, GM2 gangliosidosis or Tay-Sachs disease, Metachromatic leukodystrophy, Neiman-Pick (A/B), MPS III A, MPS II IB,

MPS IUD, MPS IVA, Mucolipidosis: III y. variant pseudo-Hurler polydystrophy, Fucosidosis,

Sialidosis type I, Sialidosis type IL Galactosial idosis, Danon disease, Sialic acid storage disease, HPS2, HPS6, Pompe disease, CLN1 , CLN3, or CLN5.

[0079] In some embodiments, saRNAs of the present disclosure and/or their pharmaceutical compositions may be used to treat any disease in the table below.

Table 4. Nou and associated genes

MPSI

[0080] Mutations in both copies of IDUA are implicated in a rare genetic disease called mucopolysaccharides type 1 (MPSI), which may also be known as Hurler syndrome, Hurler- Scheie syndrome or Scheie syndrome dependent upon the loss of enzyme activity and severity of disease in patients. The IDUA gene encodes the enzyme alpha- L iduronidase which is required for the lysosomal degradation of the glycosaminoglycans, dermatan and heparan sulfate. Mutation of the IDUA gene leads to deficiency of alpha-L-iduronidase enzyme activity. Inefficient production of alpha-L-iduronidase in MPSI -affected individuals causes an accumulation of glycosaminoglycans (GAGs) in cells throughout their bodies, ul timately leading to systematic cell death. Most individuals with MPSI die in the first 10 years, primarily due to toxic GAG accumulation in all organs, including the brain.

[0081] The current standard of care is to perform a hematopoietic stem cell transplant

(HSCT). This involves chemotherapy and removal of the patient’s mutated bone marrow cells, which are subsequently replaced with donor cells— preferably with intact human leukocyte antigen (HLA)-matched related donors who have wild-type alleles tor IDUA, During the pretransplant period enzyme replacement therapy (ERT) is administered. ERT is not continued in Europe post transplant as it can lead to antibody formation and rejection of the donor cells.

[0082] HSCT is an important therapy that significantly protects brain development. However, as outlined above, there are important deficits in HSCT and alpha-L-iduronidase ERT.

Specifically, when measuring ZDIZ4 enzyme activity in leukocytes of transplanted children affected by Hurler’s disease, 26% of children have less than the normal range of serum alpha-L- iduronidase activity levels. It is accepted by key opinion leaders that doubling the alpha-L- iduronidase enzyme level can lead to significant improvements in quality of life tor these children.

[0083] The present disclosure provides a method of treating MPSI with CEBPA-saRNAs or pharmaceutical compositions comprising CEBPA-saRNAs. In some embodiments, the pharmaceutical composition comprising CEBPA-saRNAs comprises liposomes, such as but not limited to liposomes comprising POPC, DOPE, CH EMS and MOCHOL in the molar ratio of 6:24:23:47. In some embodiments, the ratio ofC'EBPA-saRNAs to the liposome is approximately 1:25 (by weight). In some embodiments, the concentration CEBPA-saRNAs in the pharmaceutical composition is about 2.5±0.5 mg/mL. In some embodiments, the CEBPA- saRNA is CEBPA-51 ,

[0084] In some embodiments, CEBPA-saRNAs or pharmaceutical compositions comprising CEBPA-saRNAs are used to increase I DU A gene expression, IDUA protein level, or IDUA protein activity in a subject in need thereof. In some embodiments, the subject has MPS1. In some embodiments, IDUA gene expression in bone marrow is increased.

](M)85J In some embodiments, CEBPA-saRNAs or pharmaceutical compositions comprising CEBPA-saRNAs are used to reduce glycosaminoglycans (GAG) levels in a subject in need thereof. In some embodiments, the subject has MPS 1 ,

[0086] In some embodiments, CEBPA-saRNAs or pharmaceutical compositions comprising CEBPA-saRNAs are used to reduce TNFa levels in a subject in need thereof. In some embodiments, the subject has MPS 1 ,

(0()87( In some embodiments, CEBPA-saRNAs or pharmaceutical compositions comprising CEBPA-saRNAs are used to reduce chronic inflammation in a subject, in need thereof. In some embodiments, the subject has MPS 1. [0088] In some embodiments, CEBPA-saRNAs or pharmaceutical compositions comprising CEBPA-saRNAs are used to improve bone phenotypes, reduce the musculoskeletal-related symptoms, reduce skull width and/or slow skeletal deterioration in a subject in need thereof. In some embodiments, the subject has MPS1,

[0089] In some embodiments, CEBPA-saRNAs or pharmaceutical compositions comprising CEBPA-saRNAs are used to improve mobility and pain in a subject in need thereof. In some embodiments, the subject has MPS 1 .

[0090] In some embodiments, patients in need thereof receive an intravenous (i.v.) infusion of a composition comprising CEBPA-saRNAs (such as MTL-CEBPA) monthly or once every 4 weeks (Q4W), [0091] In some embodiments, patients in need thereof receive an intravenous (i.v.) infusion of a composition comprising CEBPA-saRNAs (such as MTL-CEBPA) ones (QW) a week for 3 weeks followed by a rest period of I week [3 plus 1, week = 4 weeks ~ one cycle]

[0092] In some embodiments, patients in need thereof receive an intravenous (i,v.) infusion of a composition comprising CEBPA-SaRNAs (such as MTL-CEBPA) once every 3 weeks.

[0093] In some embodiments, patients in need thereof receive an intravenous (i.v.) infusion of a composition comprising CEBPA-saRNAs ( such as .MTL-CEBPA) once every 2 weeks (Q2W), [0094] In some embodiments, patients in need thereof receive CEBP A-saRNAs (such as CEBPA-51) at a dose between 15-160 mg/m2, such as but not limited to about 18 mg/m2, about 70 mg/m2, about 98 mg/m2, or about 130 mg/m2. In some embodiments, patients in need thereof receive CEBPA-saRNAs (such as CEBPA-51 ) at a dose between about 0.5 mg/kg to about 5 mg/kg, such as but not limited to about 0.5 mg/kg, 2.0 mg/kg, or 3.5 mg/kg.

TNFa

[0095] Children and adults with the lysosomal storage diseases MPS types 1, 11 and VI live shortened lives permeated by chronic pain and physical disability. Higher tumour necrosis factor « (TNFa) levels are implicated in the pain and decreased physical function present in individuals with .MPS despite treatment with enzyme replacement therapy (ERT) and/or HSCT. Current treatments do not alleviate these problems. The present disclosure provides methods of using saRNAs of the present disclosure and/or their pharmaceutical compositions to reduce TNFa levels and/or TNFa gene expressions. In some embodiments, TNFa levels and/or TNFa gene expressions in a subject are reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% after treatment with the saRNA of the present invention compared to the levels and/or expressions in the absence of the saRNA of the present invention. In some embodiments, the subject has MPS 1.

[0096] The saRNA of the present invention may be provided in combination with additional active agents or therapies known to have an effect in the particular method being considered. For example, the combination therapy comprising saRNA and additional active agents or therapies may be given to any patient in need thereof to treat any disorder described herein, including metabolics regulation, surgical care, hyperproliferative disorders, and/or stem cell regulation. [0097] The additional active agents may be administered simultaneously or sequentially with the saRNA. The additional active agents may be administered in a mixture with, the saRNA or be administered separately from the saRNA.

[0098] The term “administered simultaneously'’ as used herein is not specifically restricted and means that the components of the combination therapy, Le., saRNA of the present invention and the additional active agents, are substantially administered at the same time, e.g. as a mixture or in immediate subsequent sequence.

[0099] The term “administered sequentially'’ as used herein is not specifically restricted and means that the components of the combination therapy, i.e., saRNA of the present invention and the additional active agents, are not administered at the same time but one after the other, or in groups, with a specific time interval between administrations. The time interval nuiy be the same or different between the respective admi nistrations of the components of the combination therapy and may be selected, for example, from the range of 2 minutes to 96 hours, 1 to 7 days or one, two or three weeks. Generally, the time interval between the administrations may be in the range of a few minutes to hours, such as in the range of 2 minutes to 72 hours, 30 minutes to 24 hours, or I to 12 hours. Further examples include time intervals in the range of 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12. hours. In some embodiments, the saRN A of the present invention is administered before the additional active agents. In some embodiments, the additional active agents are administered before the saRNA of the present invention. (0100 ] The molar ratio of the saRNA of the present invention and the additional active agents is not particularly restricted. For example, when two components are combined in a composition, the molar ratio between the two components may be in the range of 1 :500 to 500:1, or of 1 : 100 to 100:1, or of 1 :50 to 50:1, or of 1:20 to 20:1, or of 1 :5 to 5: I , or 1 :1. Similar molar ratios apply when more than two components are combined in a composition. Each componentmay comprise, independently, a predetermined molar weight percentage from about 1% to 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to 40%, or about 40% to 50%, or about 50% to 60%. or about 60%: to 70%, or about 70% to 80%, or about 80% to 90%, or about 90% to 99% of the composition.

[010] ] In some embodiments, the C/EBPa-saRNAs and/or compositions of the present application may be combined with at least one additional therapy, such as hematopoietic stem cell transplantation (e.g., bone marrow transplantation (BMT)), where the wild-type enzyme from the donor cells replenishes enzyme levels. The C/EBPa-saRNAs and/or compositions of the present application may be administered after the BMT treatment. Other non-limiting examples of an additional therapy include enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). 1 he C/EBPa-saRNAs and/or compositions of the present application may be administered after the ERT or SRT treatment. In some embodiments, C/EBPa-saRNAs and/or compositions of the present application may be administered after AAV and/or lentivirus (LV) gene therapies.

[0102] In some embodiments, the C/E BPa-saRNAs and/or compositions of the present application may be administered after BMT to enhance BMT therapy across numerous monogenic rare diseases and LSDs. Monogenic rare diseases, or rare monogenic disorders, as used herein, refers to a group of single-gene-mutated diseases that have a low incidence rate (e.g.. less than 1 in 2000 people: or less than 200,000 people in the United States).

III. Kits and Devices

Kits

[0103] The invention provides a variety of kits for conveniently and/or effectively carrying out methods of the present invention. Typically, kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.

[0104] In one embodiment, the kits comprising saRN A described herein may be used with proliferating cells to show efficacy.

[0105] In one embodiment, the present invention provides kits for regulate the expression of genes -in vitro or tn vivo, comprising C/EBPa-saRNA of the present invention or a combination of C/EBPa-saRN A, saRNA modulating other genes, siRNAs, or miRNAs. The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, a lipidoid, a dendrimer or any delivery agent disclosed herein. Non-limiting examples of genes include C/E BPa. other members of C/EBP family, albumin gene, alphafectoprotein gene, liver speci fic factor genes, growth factors, nuclear factor genes, tumor suppressing genes, pluripotency factor genes.

[0106] 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, saline with 2m M calcium, 5% sucrose, 5% sucrose with 2mM calcium, 5% Mannitol, 5% Mannitol with 2mM calcium. Ringer's lactate, sodium chloride, sodium chloride with 2mM calcium and mannose (See U.S. Pub. No. 20120258046; herein incorporated by reference in its entirety). In yet another non-limiting example, the buffer solutions may be precipitated, or it may be lyophilized. The amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations. The components may also be varied in order to increase the stability of saRNA in the buffer solution over a period of time and/or under a variety of conditions.

[0107] In another embodiment, the present invention provides kits to regulate the proliferation of cells, comprising C/EBPa-saRNA of the present invention, provided in an amount effective to inhibit the proliferation of cells when introduced into said cells', optionally siRNAs and miRNAs to further regulate the proliferation of target cells; and packaging and instructions and/or a delivery agent to form a formulation composition.

[0108] In another embodiment, the present invention provides kits for reducing LDL levels in cells, comprising saRNA molecules of the present invention; optionally LDL reducing drugs; and packaging and instructions and/or a delivery agent to form a formulation composition.

JOI 09] In another embodiment, the present invention provides kits for regulating miRNA expression levels in cells, comprising C/lvBPu-saRNA of the present invention; optionally siRNAs, eRNAs and IncRNAs; and packaging and instructions and/or a delivery agent to form a formulation composition,

[0110] In another embodiment, the present invention provides kits for combinational therapies comprising C/EBPa-saRNA of the present invention and at least one other acti ve ingredient or therapy.

Devices

[0111] The present invention provides for devices which may incorporate C/EBPa-saRNA of the present invention. These devices contain in a stable formulation available to be immediately delivered to a subject in need thereof, such as a human patient.

[0112] hi some embodiments, the device contains ingredients in combinational therapies comprising C/EBPa-saRNA of the present invention and at least one other active- ingredient or therapy.

[0113] Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, iontophoresis devices, multi-layered microfluidic devices. The devices may be employed to deliver C/EBPa-saRNA. of the present invention according to single, multi- or split-dosing regiments. The devices may be employed to deliver C/EBPft-saRNA of the present invention across biological tissue, intradermal, subcutaneously, or intramuscularly. More examples of devices suitable for delivering oligonucleotides are disclosed in International Publication WO 2013/090648 filed December 14, 2012, the contents of which are incorporated herein by reference in their entirety.

Definitions

10 ] 14] For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this sect ion, the definition in this section shall prevail.

|0I 15| zlhouf: As used herein, the term “about” means W- 10% of the recited value.

[0116] Administered in combination.^ As used herein, the term “administered in combination” or “'combined administration” means that two or more agents, e.g., saRNA, are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 1.5, 10. 5, or 1 m inute of one another. In some embodiments, the administrations of the agents are spaced sufficiently close together such that a combinatorial (e.g., a synergistic) effect is achieved.

[0117] Amino add: As used herein, the terms "amino acid" and "amino acids" refer to all naturally occurring L-aipha-ammo acids. The amino acids are identified by either the one-letter or three-letter designations as follows: aspartic acid (Asp:D), isoleucine ( lle:I), threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (GlurE), phenylalanine (Phe:F), praline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (VakV), glutamine (GlruQ) methionine (Met:M), asparagines (AsntN), where the amino acid is listed first followed parenthetically by the three arid one letter codes, respectively.

[0118] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g. , a rodent, a mouse, a rat, a rabbit. a monkey, a dog, a cat, a sheep, cattle, a primate, or a 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, genetically-engineered animal, or a clone, [0119] Approximately: As used herein, the term “approximatelv” or “about/’ as appl ied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that tall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value),

[0120] Associated wdh: As used herein, the terms “associated with,” “conjugated,” “linked,”

“attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g,, physiological conditions. An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity' sufficiently stable such that the “associated” entities remain physical ly associa ted . [012] ] Bifunctwnal: As used herein, the term “bifunctiona!” refers to any substance, molecule or moiety which is capable of or maintains at least two functions. The functions may affect the same outcome or a different outcome. The structure that produces the function may be the same or different .

[0122] Biocompatible: As used herein, the term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.

[0123] Biodegradable: As used herein, the term “biodegradable” means capable of being broken down into innocuous products by the action of living things.

[0124] .Biologically active: As used herein, the phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, the saRNA of the present i nvention may be considered biologically active if even a portion of the saRNA is biologically active or mimics an activity considered biologically relevant.

[0125] Cell type: As used herein, the term "cell type" refers to a cell from a given source (e.g., a tissue, organ) or a cell in a gi ven state of differentiation, or a cell associated with a gi ven pathology or genetic makeup.

[0126] C Chromosome: As used herein, the term “chromosome” refers to an organized structure ofDNA and protein found in cells.

[0127] Camplementary: As used herein, the term ‘ complementary” as it relates to nuclei c acids refers to hybridization or base pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double-stranded DN A molecule or between an oligonucleotide probe and a target are complementary.

[0128] Condition: As used herein, the term “condition” refers to the status of any cell, organ, organ system or organism. Conditions may reflect a disease state or simply the physiologic presentation or situation of an entity. Conditions may be characterized as phenotypic conditions such as the macroscopic presentation of a disease or genotypic conditions such as the underlying gene or protein expression profiles associated with the condition. Conditions may be benign or malignant.

]0129] C 'ontroiled Release: As used herein, the term '‘controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.

[0130] Delivery: As used herein, “delivery” refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload,

[0131] Delivery Agent: As used herein, “delivery agent” refers to any substance which facilitates, at least in part, the in viva delivery of a saRNA of the present invention to targeted cells.

[0132] Desteihilized: As used herein, the term “destable,” “destabilize,” or “destabilizing region” means a region or molecule that is less stable than a starting, wild-type or native form of the same region or molecule.

[0133] Delectable label: As used herein, “detectable label” refers to one or more markers, signals, or moi eties which are attached, incorporated or associated with another entity that is readily detected 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. Detectable labels may be located at any position in the peptides, proteins or polynucleotides, e.g, saRNA, disclosed herein. They may be within the amino acids, the peptides, proteins, or polynucleotides located at the N- or C- termini or 5’ or 3’ termini as the case may be,

[0134] Encapsiilaie: As used herein, the term ‘encapsulate” means to enclose, surround or encase,

[0135] Engineered: As used herein, embodiments of the invention are “engineered" when they are designed to have a feature or property, whether structural or chemical , that varies from a starting point, wild type or native molecule,

[0136] Equivalent subject: As used herein, “equivalent subject'* may be e.g. a subject of similar age, sex and health such as liver health or cancer stage, or the same subject prior to treatment according to the invention. The equivalent subject is "untreated" in that he does not receive treatment with a saRNA according to the invention. However, he may receive a conventional anti-cancer treatment, provided that the subject who is treated with the saRN A of the invention receives the same or equivalent conventional anti-cancer treatment.

|0137[ Exosome: As used herein, “exosome” is a vesicle secreted by mammalian cells.

[0138] Expression'. As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1 ) production of an RNA template from a DN A sequence (e.g,, by transcription): (2) processing of an RNA transcript (e.g,, by splicing, editing, .5’ cap fbnuation, and/or 3’ end processing); ( 3) translation of an RN A into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0139] Feature: As used herein, a “feature” refers to a characteristic, a property, or a distinctive element.

|0I40[ Formulation:. As used herein, a “formulation” includes at least a saRNA of the present invention and a delivery agent,

[0141] Fragment: A “fragment,” as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells. [0142] Functional: As used herein, a ^funetional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

[0143] Gene: As used herein, the term "gene" refers to a nucleic acid sequence that comprises control and most often coding sequences necessary for producing a polypeptide or precursor. Genes, however, may not be translated and instead code for regulatory or structural RNA molecules,

[0144] A gene may be derived in whole or in part from any source known to the art. including a plant, a fungus, an animal, a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cD NA, viral DNA, or chemically synthesized DNA. A gene may contain one or more modifications in either the coding or the untranslated regions that could affect the biological activity or the chemical structure of the expression product, the rate of expression, or the manner of expression control. 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 include one or more introns, bound by the appropriate splice junctions,

[0145] Gene egression: As used herein, the term "gene expression" refers to the process by which a nucleic acid sequence undergoes successful transcription and in most instances translation to produce a protein or peptide. For clarity, when reference is made to measurement of ‘'gene expression"’, this should be understood to mean that measurements may be of the nucleic acid product of transcription, e.g., RNA or mRNA or of the amino acid product of translation, e.g., polypeptides or peptides. Methods of measuring the amount or levels of RN A, mRNA, polypeptides and peptides are well known in the art.

[0146] Genome: The term "genome¹’ is intended to include the entire DNA complement of an organism, including the nuclear DNA component, chromosomal or extrachromosomal DNA, as well as the cytoplasmic domain (e.g., mitochondrial DNA).

[0147] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another 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). In accordance with the invention, two polynucleotide seq uences are considered to be homologo us if the polypeptides they encode are at least about 50%, 60%. 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch 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 a stretch of at least 4-5 uniquely specified amino acids. In accordance with the invention, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical -tor at least one stretch of at least about 20 amino acids.

[0148] Identity', As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g,, between oligonucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the lengt h of a sequence 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. The nucleotides at corresponding nucleotide positions are then 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 the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be detennined using methods such as those described 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 Stockton Press, New York, 1991 ; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CAB IOS, 1989, 4: 1 147), which has been incorporated into the ALIGN program (version 2,0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, 11., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are hot limited to, GCG program package, Devereux. J., ei aL, Nucleic Acids Research, 12(1 ), 387 (1984)). BLASTP, BLASTN, and FASTA Altschul, S. F. et aL J. Mo/ec. Rio/., 215, 403 (1990)).

[0149] inhibit expression of a gene: As used herein, the phrase “inhibit expression of a gene” means to cause a reduction in the amount of an expression product of the gene. The expression product can be an RNA transcribed from the gene (<?.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene. Typically a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom. The level of expression may be determined using standard techniques for measuring mRN A or protein.

[0150] hi vitro: As used herein, the term

vitro* refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish. etc., rather than within an organism (e.g., animal, plant, or microbe). [0151] In vivo: As used herein, the term ‘'in vivo* refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0152] 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 was associated (whether in nature or in an experimental setting). Isolated substances may have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and/or entities 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 they were initially associated. In some embodiments, isolated agents are more 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 more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components, Sufe/«n/Z«Z/p isoiaf&k By “substantially isolated” is meant that the compound is substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure. Substantial separation can inchide 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 the compound of the present di sclosure, or salt thereof Methods for isolating compounds and their salts are routine in the art.

|01531 Labe/: The term “label” refers to a subst ance or a compound which is incorporated into an object so that the substance, compound or object may be detectable, [0154] Linker: As used herein, a linker refers to a group of atoms, e.g.. 10-1 ,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be attached to a modified nucleoside or nucleotide on the nucleobase or sugar moiety at a first end, and to a payload, e.g., a detectable or therapeutic agent, at a second end . The linker may be of sufficient length as to not interfere with incorporation into a nucleic acid sequence. The linker can be used for any useful purpose, such as to form saRNA conjugates, as well as to administer a payload, as described herein. Examples of chemical groups that can 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 can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomeric units, e.g,, diethylene giycoL dipropylene glycol, triethylene glycol, tripropylene glycol, tetraefhylene 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, for example, a disulfide bond (-S-S-) or an azo bond (-N=N-), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of a selectively cleavable bond include an amido bond can be cleaved for example by the use of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/or photolysis, as well as an ester bond can be cleaved for example by acidic or basic hydrolysis.

[0155] Modified: As used herein “modified*' refers to a changed state or structure of a molecule of the invention. Molecules may be modified in many ways includi ng chemically, structurally, and functionally, hi one embodiment, the saRNA molecules of the present invention are modified by the introduction of non-natural nucleosides and/or nucleotides.

[0156] Naturally occurring: As used herein, “naturally occurring" means existing In nature without artificial aid.

]0157| Abic/eic acid: The term ’’nucleic acid" as used herein, refers to a molecule comprised of one or more nucleotides, i.e., ribonucleotides, deoxyribonucleotides, or both. The term includes monomers and polymers of ribonucleotides and deoxyribonucleotides, wi th the ribonucleotides and/or deoxyribonucleotides being bound together, in the case of the polymers, via 5’ to 3’ linkages. The ribonucleotide and deoxyribonucleotide polymers may be single or double-stranded. However, linkages may include any of the linkages known in the art including, for example, nucleic acids comprising 5' to 3' l inkages. The nucleotides may be naturally Occurring or may be synthetically produced analogs that are capable of forming base-pair relationships with naturally occurring base pairs. Examples of non-naturally occurring bases that are capable of forming base-pairing relationships include, but are not limited to, aza and deaza pyrimidine analogs, aza and deaza purine analogs, and other heterocyclic base analogs, wherein one or more of the carbon and nitrogen atoms of the pyrimidine rings have been substituted by heteroatoms, e,g., oxygen, sulfur, selenium, phosphorus, and the like.

[01.58] Patient: As used herein, “patient" refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.

[0159] Peptide: As used herein, “peptide’’ is less than or equal to 50 amino acids long, e.g„ about 5, 10, 15. 20, 25. 30, 35, 40, 45, or 50 amino acids long.

[0160] Pharmaceutically- acceptable: The phrase “pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which arc, 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 benefitZrisk ratio. |01611 Pharmaceutically acceptable excipients: The phrase “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and noninflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butyl ated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycol ate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0162] Preventing: As used herein, the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition: partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition: and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.

[0163] Prognofing: As used herein, the term “prognosing” means a statement or claim that a particular biologic event will, or is very likely to, occur in the future.

[0164] Progression: As used herein, the term “progression” or “cancer progression” means the advancement or worsening of or toward a disease or condition.

[0165] Proliferate; As used herein, the term “proliferate” means to grow, expand or increase or cause to grow, expand or increase rapidly. “Proliferative” means having the ability to proliferate. ‘“Anti-proliferative” means having properties counter to or inapposite to proliferative properties.

[0166] Protein: A ’’protein** means a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, however, a protein will be at least 50 amino acids long. In some instances the protein encoded is smaller than about 50 amino acids. In this case, the polypeptide is termed a peptide. If the protein is a short peptide, it will be at least about 10 amino acid residues long, A 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. A protein may be a single molecule or may be a multi-molecular complex. The term protein may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0167] Protein expression: The term ’’protein expression" refers to the process by which a nucleic acid sequence undergoes translation such that detectable levels of the amino acid sequence or protein are expressed.

[0168] Purified: As used herein, “purify,” “purified,” “purification” means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.

[0169] degression: As used herein, the term “regression” or “degree of regression” refers to the reversal, either phenotypically or genotypically, of a cancer progression. Slowing or stopping cancer progression may be considered regression.

[0170] Sample: As used herein, the term “sample” or “biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, pells or component parts, or a fraction or portion thereof including but not limited io, lor example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule. [017] j Signal Sequences: As used herein, the phrase “signal sequences” refers to a sequence which can direct the tran sport or localization of a protein.

[0172] Single unit dose: As used herein, a“single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.

[0173] Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RN A molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.

[0.174] Split dose: A.s used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses.

[0175] Stable: As used herein “stable” refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable o f formulation into an efficacious therapeutic agent.

[0176] Stabilised: As used herein, the term “stabilize” “stabilized,” “stabilized region” means to make or become stable.

[0177] Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the invention may be administered, eg., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

[0178]

As used herein, the term “substantially” refers to the qualitati ve condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary' skil l in the biological arts will understand that biological and chemical phenomena rarely , i f ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially’’ is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. [0179] Substantially equal: As used herein as it relates to time differences between doses, the term means plus/minus 2%. [0180] Substantially sinntltanepusly: As used herein and as it relates to plurality of doses, the term means within 2 seconds.

[018] 1 Sufferingfconi; An individual who is ‘“suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition.

[0182] Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and.for condition but harbors a propensity to develop a disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (for example, cancer) may be characterized by one or more of the following: ( 1 ) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition wilt not develop the disease, disorder, and/or condition.

[0183] Sustained release: As used herein, the term “sustained release” refers to a pharmaceutical composition or compound release profile that conforms to a release rate over a specific period of time.

[0184] Synthetic: The term “synthetic” means produced, prepared, and/or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present invention may be chemical or enzymatic.

[0185] Targeted Cells: As used herein, “targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism . The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient. [0186] Therapeutic Agent: The term ‘therapeutic agent” refers to any agent that when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or el icits a desired biological and/or pharmacological effect.

[0187] Therapeutically effective amoirnt: As used herein, the term “therapeutically effective amount*’ means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms off diagnose, prevent, and/or delay the onset of the infection, d isease, disorder, and/or condition. [0188] Therapeudcalfy effective Outcome; As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition,

[0189] Total daily dose: As used herein, a “total daily dose’’ is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose,

[0190] Transcription factor: As used herein, the term “transcription factor” refers to a DNA- binding protein that regulates transcription of DNA into RNA, for example, by activation or repression of transcription. Some transcription factors effect regulation of transcription alone, while others act in concert with other proteins. Some transcription factor can both activate and repress transcription under certain conditions. In general, transcription factors bind a specifictarget sequence or sequences highly similar to a specific consensus sequence in a regulatory region of a target gene. Transcription factors may regulate transcription of a target gene alone or in a complex with other molecules.

[019] 1 'Treating; As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression off reducing severity off and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. For example* “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condi tion and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition,

[0192] The phrase "a method of treating" or its equivalent, when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce, eliminate or prevent the number of cancer ceils in an individual, or to alleviate the symptoms of a cancer. "A method of treating" cancer or another proliferative disorder does not necessarily mean that the cancer cells or o ther disorder will, in fact, be completely el iminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will , in fact, be alleviated. Often, a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of an individual, is nevertheless deemed an overall beneficial course of action.

[0193] l/mnodified: As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification,

[0194] lysosomal Storage Disease: As used herein, “lysosomal storage disease'’ or lysosomal storage disorder, refers to metabolic diseases that are characterized by an abnormal build-up of various toxic materials in the body’s cells as a result of lysosomal protein deficiencies.

[0195] Rare Disease: As used herein, “rare disease’' refers to a group of diseases that have a low incidence rate (e.g., less than 1 in 2000 people; or less than 200,000 people in the United States).

[0196] Mucopolysaccharidosis: As used herein, mucopolysaccharidosis refers to a metabolic disease caused by the absence or malfunctioning of certain enzymes the body needs to break down glycosaminoglycans. It is a kind of lysosomal storage disorder.

[0197] Sphbtgolipidosis: As used herein, sphingplipidosis refers to a metabolic disorder characterized by the accumulation of harmful quantities of glycosphingolipids and phosphosphingolipids. It is a kind of lysosomal storage disorder,

[0198] Neuronal Ceroid Lipofuscinoses: As used herein, neuronal ceroid lipofuscinoses refers to a metabolic disorder characterized by the accumulation of lipofuscin. It is a kind of lysosomal storage disorder. f(H99] Giycopwteinosist As used herein, glycoproteinoses refers to a metabolic disorder characterized by the accumulation of harmful quantities of glycoproteins. It is a kind of lysosomal storage disorder.

Equivalents and Scope (021)0] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the in vention described herein. The scope of the present invention is not intended to be limi ted to the above Description, but rather is as set forth in the appended claims.

[0201] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a gi ven product or process.

[0202] It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. ]0203| Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated of otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges i n different embodiments of the i nvention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise, [0204] In addi tion, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g„ any nucleic acid or protein encoded thereby; any method of production ; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art. |02<>5] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control. [0206] The invention is further illustrated by the following non-limiting examples,

EXAMPLES

Example 1. Preparations of CEBPA-51 and MTL-CEBPA

[0207] Materials and Procedures of preparing CEBPA-saRNAs have been disclosed in

WO20 15/075557 and WO2016/T70349 to MiNA Therapeutics Limited. The preparations of CEBPA-51 and MTL-CEBPA have been disclosed in Examples of WQ2016/170349,

[0208] In brief, each strand of CEBPA-51 was synthesized on a solid support by coupling phosphoramidite monomers sequentially. The synthesis was performed on an automatic synthesizer such as an Akta Oligopilot 100 (GE Healthcare) and a Technikrom synthesizer (Asahi Kasei Bio) that delivers specified volumes of reagents and solvents to and from the synthesis reactor (column type) packed with solid support. The process began with charging reagents to the designated reservoirs connected to the reactor and packing of the reactor vessel with the appropriate solid support; The flow of reagent and solvents was regulated by a series of computer-controlled val ves and pumps with automatic recording of flow rate and pressure. The solid-phase approach enabled efficient separation of reaction products as coupled to the solid phase from reagents in solution phase at each step in the synthesis by washing of the solid support with sol vent.

[0209] CEBPA-51 was dissolved at ambient temperature in sodium acetate/ sucrose buffer pH 4.0 and lipids were dissolved in absolute ethanol at 55 °C. Liposomes were prepared by crossflow ethanol injection technology. Immediately after liposome formation, the suspension was diluted with sodium chloride / phosphate buffer pH 9.0. The collected intermediate product was extruded through polycarbonate membranes with a pore size of 0.2 pm. The target saRNA concentration was achieved by ultrafiltration. Nou-encapsulated drug substance and residual ethanol were removed by subsequent diafiltration with sucrose / phosphate buffer pH 7.5, Thereafter, the concentrated liposome suspension was 0.2 gm filtrated and stored at 5 ± 3 °C. Finally, the bulk product was formulated, 0.2 pm filtrated and filled in 20 ml vials. [0210] MTL-CEBPA was presented as a concentrate solution tor infusion. Each vial contains

50 mg of CEBPA-51 (saRNA) in 20 ml of sucrose / phosphate buffer pH about 7,5.

Example 2. bt Vitro Studies on the Effect of CEBPA-51 oft IDUA Expressions [0211] To investigate the effect of CEBPA-51 on IDUA RNA upregulation in vitro across diverse cell types, mesenchymal stem cell (MSC), human fetal lung fibroblasts (IMR9Q) and primary bone marrow CD34⁺ cells were tested. MSCs were transfected with CEBPA-51 or the non-specific control FLUC using RNAiMAX at a final saRNA concentration of 10 nM< Due to the intrinsic propagation characteristics of MSCs in culture, niRNA expression levels for both CEBPA and its downstream target IDUA were assessed at extended timepdints. Fig, 1 shows quantification of RNA transcripts relative to POLR2A expression for (A) CEBPA, and (B) IDUA at 72, 192 and 240 h after initial transfection of MSC with CEBPA-51 treatment by qRT- PCR. Data are expressed as mean ± SEM of three independent experiments with black circles representing biological replicates. * p < 0.05, ** p < 0,01. *** p < 0.001 (two-tailed Student’s t- test). IDUA expression increased after C/EBP-a upregulation and exhibited a sustained duration of IDUA expression in response to CEBPA-51 , where expression was detected even after 192 h (8 days) post-transfection.

[0212] Human primary bone marrow was also assessed, where CD34* cells were treated with

35 ng/pL of MTL-CEBPA or 35 ng/pL NOV340-FLUC as a non-specific control and were incubated for 7 days. At 24h, 48h, 72h, 144h, and 168h, RNA samples were collected for analysis of CEBPA and IDUA gene expression. Quantification of RNA transcripts relative to GAPDFI expression for CEBPA (Fig, 2A and Fig. 2B) and IDUA (Fig. 2C and Fig. 2D) at 24h and 168h after initial treatment of human primary bone 'marrow CD34+ with 35 ng/pL of MTL- CEBPA treatment by qRT-PCR. Data were expressed as mean £ SD of four independent experiments and analysed using an unpaired Student's t test (*p<0,05). Analysis showed upregulation of both CEBPA and alpha-L-iduronidase at 24h, 144h and 168h following a single treatment with MTL-CEBPA when compared to MTL-FLUC controls.

[0213] In another study, IMR90 lung fibroblasts were transfected with increasing doses of

CEBPA-51 (1 nM, 5 nM, 10 nM and 20 nM of CEBPA-51) using Hiperfect. FLUC control was added at a concentration of 20 nM. Gene expression was assessed by RT-PCR at 72h for both

CEBPA and IDUA. Quantification of RNA transcripts relative to GAPDH expression for CEBPA (Fig. 3A) and IDUA (Fig. 3B) at 72h after initial transfection of IMR90 -cells with CEBPA-51 treatment by qRT-PCR. N~3 biological replicates. Error bars arc -/+ SEM and statistics are. performed rising impaired Student’s t test (*p<0.05). It was observed that C£BR4 gene expression was significantly upregulated with 10 nM and 20 nM CEBPA-51 and IDUA gene expression was statistically upregulated at the 20 nM dose.

[0214] To determine if the increases in RNA translated into protein and enzyme activity upregulation in IMR90 cells, samples were analysed using an anti-alpha-L-iduronidase mouse monoclonal antibody on an automated capillary Western blot (WES) digital platform (ProteinSimple, USA). To assess intracellular alpha-L-iduronidase enzyme activity, samples were collected and mixed with a fluorescently-tagged substrate of alpha-L-iduronidase. The amount of fluorescent signal cleaved from the substrate was then used as an indication of alpha- L-iduronidase enzyme activity levels,

[0215] Fig. 4A shows quantification of protein levels normalized to total protein analyzed and relative to imtransfected samples for alpha-L-iduronidase (WfZ4,J at 96h after initial transfection of 1MR90 cells with CEBPA-51. N^:::3 biological replicates. Fig. 4B shows quantification of intracellular alpha-L-iduronidase enzymatic activity relative to untransfected samples, analyzed at 96h after initial transfection of IMR90 cells. Fig. 4C shows representati ve WES analysis for alpha-L-iduronidase with loading protein control. Data were analysed using unpaired Student’s t test (*p<Q.O5, **p<0,01). IMR90 cells transfected with CEBPA-51 showed increased alpha-L-iduronidase protein expression followed by increased intracellular alpha-L-iduronidase activity at 96h when compared to FLUC negative control with CEBPA-51. These data therefore again demonstrate the ability of CEBP A-51 to increase IDUA gene expression and subsequently alpha-L-iduronidase enzyme activity in a diverse range of cell types.

Example 3. In Vivo Phaimiacodvimniic Study Showing that MTL-CEBPA Increases IDUA Expression and alpha-L-iduronidase Activity

]0216| To evaluate the in viva pharmacodynamic activity of MTL-CEBPA on upregulation of IDUA gene expression and alpha-L-iduronidase enzyme activity, normal C57/BL6 female mice were administered with MTL-CEBPA via tail vein IV infusion at 0.5, 1, 2, 3, and 4 mg/kg CEBPA-51 (n -^:: 6 for each dose). Each concentration level was administered in two doses 24 hours apart. To evaluate the duration of efficacy, animals were sacrificed one, two. and three weeks after the first dose (n = 6). Animals dosed with PBS and NOV340-FLUC at 3 mg/kg were used as controls and sacrificed at Week 1.

|0217] mRNA analyses of CE8PA in the bone marrow at Weeks 1 to 4 post initial dose show a control led activation of the CPPPzl gene with an approximate doubling ofmRNA across Weeks 1 to 3. At Week 4, GEBPA expression increased over 3-fold difference at 0.5 and

1,0 mg/kg doses when compared with mice at Week 1 treated with PBS (Fig. 5A). Interestingly, a two-fold or greater upregulation of CEBP/1 mRNA was achieved at all doses tested, even at the lower doses of 0.5 mg/kg and 1 mg/kg. Upregulation of IDU4 mRN A correlates with CEff/>A expression level as JPUfmRNA levels were highest at Week 4, similar to those of CEBPA (Fig. 513).

[0218] In summary, consistent with the in vitro findings, these in vivo studies demonstrate that MTL-CEBPA significantly upregulates both IDUA expression and activity following the upregulation of C/EBP-a.

[02191 To determine if the increase in LDUA RNA could improve alpha-L.-iduronidase enzyme activity a fluorometric assay using 4-methylumbelljfeiyl alpha-L-iduronide (4-MU iduronide) as substrate was used on pl amsa obtained from MTL-CEBPA treated mice. As presented in Table 5, MTL-CEBPA activated alpha-L-iduronidase enzyme activity across all doses tested, with 2 mg/kg resulting in seven-fold upregulation of JDU4 lasting up to three weeks. Alpha-L-iduronidase enzyme activity was increased vs untreated control (Week 1 ) at Week 2 and Week 3. This suggests that onset of activity by two weeks with duration of increased enzyme activity of at least one week in these normal mice.

Table 5 Summary of Fold Change Increase in IDUA Activity Relative to Pre-treatment

Example 4. Ex

Evidence that MTL-CEBPA Increases IDlfA Expression and AIpha-L- Enzyme Activity

[0220] In this study, further evidence was generated that MTL-CEBPA increases I£)K4 expression and alpha-l...-iduronidase enzyme activity in a human-relevant experimental system. (02211 Whole blood was obtained from eight healthy human donors. An ex w'ra assay was developed where whole blood was incubated with 35 ng/pL of MTL-CEBPA and MTL-FLUC (i.e., NOV340 FLUC) at 37*C with 5% CQ» in vented tubes placed on a rotating chamber. [02221 Fig. 6A-B show quantification of RNA transcripts relative to the geometric mean expression of GAPDEL 18s, and PRLPO genes for CESPA (Fig. 6A) and /D K4 (Fig. 6B) at 24h and 48h after initial e.v

treatment of healthy human donor blood with 17.5 ng/uL of

MTL-CEBPA treatment by qRT-PCR. Fig. 6C shows quantification of protein levels normalized to total protein analyzed and relative to MTL-FLUC treated sample tor alpha-L-iduronidase (7Z)K1) at 48h after initial ex viva treatment of healthy volunteer blood with CEBPA-51 . N=6 biological replicates. Mixed effect analysis with multiple comparisons (*p<0.05). [0223] As shown in Fig. 6A-C, an increase in CEBPA RNA expression in peripheral blood mononuclear cells (PBMCs) in 6 out of 8 volunteer bloods was observed following treatment with MTL-CEBPA. Furthermore, among those donors, an increase in /DU4 RNA in 5 of those 6 volunteer samples was observed where an increase in CEffPA expression had been demonstrated. An upregulation of alpha-L-iduronidase protein was also observed. [0224] As the goal of leveraging MTL-CEBPA for patients with Hurler’s disease is to increase 1DUA gene expression, and subsequently protein expression and alpha-L-iduronidase enzymatic activity, the ability of MTL-CEBPA treated PBMCs to boost alpha-L-iduronidase enzyme activity in fibroblasts from MPS1 patients (cell line GM00798) was tested. The fibroblasts were exposed to media which the PBMCs secreted enzyme into. As shown in Fig. 7, MTL-CEBPA boosted PBMCs led to a significant increase in alpha-L-iduronidase enzyme activity in these cells, with a median told increase of 2.6.

|0225[ It was then sought to establish whether this two-fold increase in alpha-L-iduronidase enzyme activity would reflect higher clearance of naturally accumulated heparan and dermatan sulphated GAG residues. A spectrophotometric assay was performed that relies on absorption spectrum changes of the dye 1 J-dimethyhn ethylene blue (DMMB) when bound to sulphated GAGs with an additional nitrous acid treatment to distinguish between the content of the N- sulfated GAGs (heparan and dermatan sulfate) from the O-sulfated GAGs (chondroitin sulfate). As shown in Fig, 8A and 8B. levels ofN-sulfated GAGs were significantly reduced by an average of 30% in the PBMCs of the three volunteer blood samples that responded to MTL-CEBPA treatment. Example 5, Vivo Studies of MTL-CEBPA Adjuvant to BMT in MPS1 Animals

[0226] This study was conducted to determine if MTL-CEBPA treatment of M PS I mice post bone marrow transplant can improve the disease related symptoms beyond bone marrow transplant alone. The following animals were studied in this in vivo study: heterozygotes (mice carrying a single copy of the mutation but do not develop pathology) and homozygotes (BMT) (mice carrying two disease-causing MPS I genes and are treated with bone marrow transplanted (BMT)). I'hese animals were divided into five groups: 1 ). heterozygotes; 2). homozygotes (BMT): 3). homozygotes (BMT) treated with FLUC; 4). homozygotes (BMT) treated with 0.5mg.''kg MTL-CEBPA; and 5). homozygotes (BMT) treated with 2,0mg/kg MTL-CEBPA. The homozygotes (BMT) animals were 5 weeks old when they received BMT. They were 10 weeks old when MTL-CEBPA or FLUC treatment started (5 weeks post BMT). Treatment was administered biweekly for either 1.5 or 3 months.

[0227] Radiography was conducted for bone measures (such as Femur Width, Femur Length, Zygomatic arch width. Inter-zygomatic distance, and Norma superior skull length). Dual Energy X-ray Absorptiometry (DEXA) was conducted for body composition (such as Bone mineral content and density; Percent fat and fat mass; and Body weight). Gait analysis was conducted for abnormal and normal step. Regularity index. Stride length and Speed.

[0228] The radiography data show a reduction in zygomatic bone thickness and a lower inter- zygomatic bone distance with MTL-CEBPA treatment. It was observed from the Gait analysis that there i s no increase in abnormal step front baseline with treatment while Flue shows sig increase. There is an increase in normal step count from baseline (significant) and regularity index at end of treatment (trend to dose-dependent). DEXA data show a lower bone mineral content and a lower fat mass/perccntage with MTL-CEBPA treatment. Body weight is equivalent across all groups. Therefore, MTL-CEBPA treatment of MP SI mice post bone marrow transplant may slow skeletal deterioration. [0229] IDUA gene expression, circulating IDU A enzyme levels, skull width and circulating

TNFa levels were also measured. The data in Fig. 9A demonstrated an upregulated expression of the IDUA gene is observed after 3 months of 0,5 mgZkg MTL-CEBPA treatment (dotted line is the mRN A level in a non-diseased animal). The data in Fig, 98 demonstrated circulating enzyme levels are increased after 3 months of 0.5 mg/kg MTL-CEBPA treatment (dotted line is the enzyme level in a non-diseased animal). The data in Fig. 9C demonstrated skull width (interzygoniatic distance), which increases as a pathological consequence of M PS-1 H, is reduced in 0.5 mg/kg MTL-CEBPA treated animals (dotted line is the skull width in a non-diseased animal). The data in Fig. 9D demonstrated circulating TNFa levels decreased within 1.5months following 2 mg/kg MTL-CEBPA treatment (dotted line is the TNFa level in non-diseased animal). Example 6. In Fn’o Studies of MTL-CEBPA in MPS1 Animal Model

[0230] In this study, a MPS1 transgenie animal model (Homozygote MPS-ldua^/w/JJ<W;J) was used. Mice were divided into the following treatment groups: BMT, Flue, MTL-CEBPA (0,5mg/kg) and MTL-CEBPA (2.0mg/kg). CEBPA mRNA levels, IDUA mRNA- levels, and IDUA enzymatic activities in bone marrow tissues at end of study (3 months) were measured. As shown in Fig. 10A-10C. MTL-CEBPA significantly unregulated IDUA at both gene expression and enzymatic activity level Plasma TNFa levels were also measured at 1.5 months and at 3 months. As shown in Fig. 1 1 A and 1 IB. 2mg/kg (2mpk) MTL-CEBPA significantly reduced plasma TNFa levels.

Example 7. MTL-CEBPA Clinical Pharmacodynamics in Cancer Patients [0231] Monocytes and plasma from cancer patients who received MTL-CEBPA treatment in a Phl b clinical trial were obtained. IDUA protein levels in the monocytes and IDUA enzyme activities in the plasma were measured. As shown in Fig. 12 A, there is a correlation between CEB P A protein increase and IDUA upregulation in MTL-CEBPA treated patients. The data in Fig. 12B showed Increased IDUA enzyme activity in patients (more pronounced in subgroup with low activity at baseline).

Example 8, In Vitro Studies on the Effect of CEBPA-51 on expression of other disease related genes

[0232] Since CEBPA is a transcription factor known to influence the expression of many downstream genes, RNA-seq was performed upon A549 cells (lung cancer) untransfected or transfected with saRNA FLUC as control and saRN A CEBPA-51 , In brief, A549 cells were plated on a 12-weft plate and 10 nM of FLUC and CEBPA-51 or an equivalent volume of QptiMEM was mixed with 1 ul lipofectamipe RNAiMax reagent (Thermofisher) diluted with OptiMEM. The mixture was added in a dropwise manner to the cells and incubated overnight at 37C. The next day the media was changed and cells were left to grow for a total of 72 hours. After this time, cells were lysed using Qiagen RLT buffer and pushed through a Qiashredder column. RNA was then extracted using the Qiagen RNeasy kit and DNase treated. 3 independent biological replicates were performed. For experiment 1, RNA was sent to the Imperial BRC Genomic facility where they prepared the libraries using the TruSeq Stranded m'RNA kit (Rumina) and sequenced them using NextSeq 2000 (Rumina) with 3' paired end sequencing. Lysosomal storage disease genes were filtered out along with their corresponding fold changes and p adjusted value (padj). Experiment 2 was performed independently, sequenced and analysed at another feciiity .

[0233] In Table 6, the data in the first two columns refer to the fold change after CEBPA51 treatment vs FLUC (control) and the p adjusted value (padj) for an n=3 experiment (Experiment I) and the next two columns refer to an independent n~3 experiment (Experiment 2). Treatment of A549 cells with CEBPA-51 leads to increased expression of various genes related to lysosomal disease when analysed by RNAseq,

Table 6 - Changes in disease related genes in response to treatment with CEBPA-51

[0234] To confirm the results obtained in the RNA-seq, RT-qPCR was performed. Samples for RT~qPCR were obtained and processed the same way as the RN A-seq samples. Once obtained the RNA, reverse transcription was performed using the QuantiTect Reverse Transcription kit using 250 ng of RNA and oligo (d'T). qPCR was performed using SYBR green and Qiagen primers corresponding to the gene of interest relative to GAPDH in a QuantStudio 5 Real-Time PCR system. Three technical replicates were performed. Results were plotted using GraphPad using -/+ SEM and statistics were performed using paired Student's t test (*p<0,O5, **p<0.01 ), GALNS Z MP SIVA or Morquio disease

102351 To determine if the observed upregulation of LSD genes could be replicated using other analytical approaches A549 cells were transfected with CEBPA-51 at 10 nM using RNAiMax. FLUC control was added at a concentration of 10 nM. Gene expression was assessed by RT-PCR using oligo (dT) for both CEBPA and GAINS. Quantification of RN A transcripts relative to GAPDH expression for CEBPA (Fig. 13 A) and GALNS (Fig, 13B) at 72h after initial transfection of A549 cells with CEBPA-51 . N~3 biological replicates. Error bars are -/t- SEM and statistics are performed using paired Student’s t test (*p<0.05, **p<0.01 ). It was observed that CEBPA and (7TZ.2VS gene expression was significantly upregulated with 10 nM CEBPA-51 compared to FLUC control. [0236] To further examine the effects of CEBPA on LSD genes in an in vivo study, mice into which healthy bone marrow cells had been transplanted were treated with bi weekly injections of MTL-CEBPA for 1.5 or 3 months at a dose of either 0.5 mg/kg or 2 mg/kg. Upon sacrifice bone marrow cells were extracted from animals and RNA was extracted and quantified by RT-PCR for expression of both CEBPA and GALNS. Error bars are

SEM and statistics are performed using a ordinary one-way ANOVA (*p<0.05, **p<0.01, ****p<0.0001). As shown in Fig. 14 A- I4B, it was observed that CEBPA gene expression was significantly upregulated and G/ILAS gene expression was statistically significantly upregulated alongside this. HETMieterozygons mice without any treatment (control).

ASAHI / Farber disease

[0237] To determine if the observed upregulation of LSD genes could be replicated using other analytical approaches A 549 cells were transfected with CEBPA-51 at 10 nM using RNAiMax. FLUC control was added at a concentration of 10 nM. Gene expression was assessed by RT-PCR using oligo (dT) for both CEBPA and ASAHI . Quantification of RNA transcripts relative to GAPDH expression for CEBPA (Fig. 15 A) and ASAHI (Fig. 15B) at 72h after initial transfection of A549 cells with CEBPA-51 . N~3 biological replicates. Error bars are -/+ SEM and statistics are performed using paired Student’s t test (**p<0.0l ). It- was observed that CEBPA and ASAHI gene expression was significantly upregulated with 10 nM CEBPA-51 compared to FLUC control.

SQSH / MPSIilA or Sanfilippo syndrome

[0238] To determine if the observed upregulation of LSD genes- could be replicated using other analytical approaches A549 cells were transfected with CEBPA-51 at 10 nM using RNAiMax. FLUC control was added at a concentration of 10 nM, Gene expression was assessed by RT-PCR using oligo (dT) for both CEBPA and SGSH. Quantification of RNA transcripts relative to GAPDH expression for CEBPA (Fig. 16A) and SGSH (Fig. 16B) at 72h after initial transfection of A549 cells with CEBPA-51. FUG biological replicates. Error bars are -/+ SEM and statistics are performed using paired Student’s t test (**p<0.01). It was observed that CEBPA and SGSH gene expression was significantly upregulated with 10 n.M CEBPA-51 compared to FLUC control. SLC17A5 / Sialic acid storage disease

[0239 J T o determine if the observed upregulation of LSD genes could be replicated using other analytical approaches A 549 cells were transfected with CEBPA-51 at 10 n.M using RNAiMax, FLUC control was added at a concentration of 10 nM. Gene expression was assessed by RT-PCR using oligo (dT) for both CEBPA and 5£C7 A15. Quantification of RNA transcripts relative to GAPDH expression for CEBPA (Fig. 17A) and SLC 17A5 (Fig. 17B) at 72h after initial transfection of A549 cells with CEBPA-5L N~3 biological replicates. Error bars are -/A SEM and statistics are performed using paired Student’s t test (*p<0,05, **p<0.01). I t was observed that CEBPA and S£C77.4.5 gene expression was significantly upregulated with 10 nM CEBPA-51 compared to FLUC control.

NEU I / Sialtdosis

[0240] To determine if the observed upregul ation of LSD genes could be replicated using other analytical approaches A549 cells were transfected with CEBPA-51 at 10 nM using RNAiMax. FLUC control was added at a concentration of 10 nM. Gene expression was assessed by RT-PCR using oligo (dT) for both CEBPA and NEUL Quantification of RNA transcripts relative to GAPDH expression for CEBPA (Fig. ISA) and NEU1 (Fig. 18B) at 72h after initial transfection of A549 cells with CEBPA-51 , N--3 biological replicates. Error bars are -/*• SEM and statistics are performed using paired Student’s t test (*p<0.05, **p<0.0T). It was observed that CEBPA and NEU1 gene expression was significantly upregulated with 10 nM CEBPA-51 compared to FLUC control.

SUMFi / Multiple Sulfatase deficiency

[0241] To determine if the observed upregulation of LSD genes could be replicated using other analytical approaches A549 cells were transfected with CEBPA-51 at 10 nM using RNAiMax. FLUC control was added at a concentration of 10 nM. Gene expression was assessed by RT-PCR using oligo (dT) for both CEBPA and SUMFL Quantification of RN A transcripts relative to GAPDH expression tor CEBPA (Fig. 19A) and SUMF1 (Fig. 19B) at 72h after initial transfection of A549 cells with CEBPA-51 . .N~3 biological replicates. Error bars are -/+ SEM and statistics are performed using paired Student’s t test QpMJ.OS, **p<0.01 ). It was observed that CEBPA and SUMF1 gene expression was significantly upregulated with 10 nM CEBPA-51 compared to FLUC control.

Claims

Claims

1. A method of treating mucopolysaccharides type I (MPS. I ) of a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a synthetic isolated small activating RNA (saRNA), wherein the saRNA up~regulates the expression of the C/EBPct gene.

2. The method of claim 1 , wherein the saRNA is at least 80% complementary to a region on the transcription start site core of the C/EBPa gene (SEQ ID NO: 3).

3. The method of claim 1 or claim 2, wherein the saRNA comprises an antisense strand with

14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1.

4. The method of any one of claims I -3, wherein the saRNA is double-stranded and comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1, and a sense strand with 14-30 nucleotides, wherein the sense strand comprises a sequence of SEQ ID NO: 2,

5. The method of any one of claims 1-4, wherei n the pharmaceutical composition further comprises a liposome,

6. The method of claim 5, wherein the liposome comprises I -palmitoyl-2-oleoy l-sn-gIycero- 3 -phosphocholine (POPC), 1 ,2~dioleoyl-sn"glycero-3~phosphoethanolamine (DOPE), chol estery I-hemi succinate (CH E M S ), and 4-( 2-ami noethyl )-morpholino- cholestero I hemisuccinate (MOCHOL).

7. The method of any one of claims 1 -6, wherein the pharmaceutical composi tion is administered once a month, once every three weeks, or once every two weeks.

8. The method of any one of claims 1-7, wherein the pharmaceutical composition is administered through intravenous infusion.

9. The method of any one of claims I -8, wherein the amount of saRNA is betweep about

15-160 mg/nr,

10; The method of any one of claims I -9, wherein the subject recei ved bone marrow transplantation (BMT) before administration of the pharmaceutical composition.

1 1. A method of increasing 1DU A gene expression, IDUA protein level, or IDUA protein activity in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a synthetic isolated small activating RNA (saRNA), wherein the saRNA up-regulates the expression of the C/EBPa gene.

12, The method of claim 11 , wherein the saRNA is at least 80% complementary to a region on the transcription start site core of the C'/EBPa gene (SEQ ID NO: 3),

13, The method of claim 1 1 or claim 12, wherein the saRNA. comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1 .

14. The method of any one of claims 1 1-13, wherein the saRNA is double-stranded and comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1 , and a sense strand with 14-30 nucleotides, wherein the sense strand comprises a sequence of SEQ ID NO: 2.

15. The method of any one of claims 11-14, wherein the pharmaceutical composition further comprises a Liposome.

16. The method of claim 15, wherein the liposome comprises 1 -palmitoyl-2-oleoyl-sn- glycero-3-phosphochoiine (POPC), 1 ,2 -dioleoyl-sii-glycero~3 -phosphoethanolamine (DOPE), cholesteiyl-hemisuccinate (CH EMS), and 4-(2-aminoethyI)’morpholino-cholesterol hemisuccinate (MOCHOL).

17. The method of any one of claims 1 1 - 16, wherein the subject received bone marrow transplantation (BMT) before admi nistration of the pharmaceutical composition.

18. A method of reducing glycosaminoglycans (GAG) levels and/or TN Fa levels in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a synthetic isolated small activating RNA (saRNA), wherein the saRNA up-regulates the expression of the C/EBPa gene.

19. The method of claim 18, wherein the saRNA is at least 80% complementary to a region on the transcription start site core of the C/EBPa gene (SEQ ID NO: 3).

20. The method of claim IS or claim 19, wherein the saRNA comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID No. 1,

21. The method of any one of claims 18-20, wherein the saRNA is double-stranded and comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1, and a sense strand with 14-30 nucleotides, wherein the sense strand comprises a sequence of SEQ ID NO: 2.

22. The method of any one o f clai ms 18-21 , wherein the pharmaceutical composi tion further comprises a liposome.

23. The method of claim 22, wherein the liposome comprises 1 -palmitoyl-2-oleoyi~sn- glycero-3-phosphocholine (PQPCj. l,2-dioleoyLsn-gIycero-3-phosphoethanolamine (DOPE), cholesteryl-hemisuccinate (CH EMS), and 4-(2-aminoethyl)-m0rpholino-cholesterol hemisuccinate (MOCHOL).

24. The method of any one of claims 18-23, wherein the pharmaceutical composition is administered once a month, once every three weeks, or once every two weeks,

25. The method of any one of claims 18-24, wherein the pharmaceutical composition is administered through intravenous infusion.

26. The method of any one of claims 18-25, wherein the therapeutically effective amount of saRNA is between about 15-160 mg/nr.

27. The method of any one of claims 18-26, wherein the subject received bone marrow transplantation (BMT) before administration of the pharmaceutical composition,

28. A method of treating a lysosomal storage disorder (LSD) of a subject in need thereof comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a synthetic isolated small activating RNA (saRNA), wherein the saRNA up-regulates the expression of the CZEBPa gene.

29. The method of 'claim 28, wherein the saRNA is at least 80% complementary to a region on the transcription start site core of the CZEBPa gene (SEQ ID NO: 3).

30. The method of claim 28 or claim 29, wherein the saRN A comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ® No, L

31 . The method of any one of claims 28-30, wherein the saRN A is double-stranded and comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1, and a sense strand with 14-30 nucleotides, wherein the sense strand comprises a sequence of SEQ ID NO: 2.

32. The method of any one of claims 28-31 , wherein the pharmaceutical composition further comprises a liposome.

33. The method of claim 32, wherein the liposome comprises l -pahnitoyl-2-oleoyl-sn- glycero-3 -phosphocholine (POPC), l ,2-di01eoyl-sn-glyeero-3-pliosphoethanolamine (DOPE), cholesteryl-hemisuccinate (CHEMS), and 4-(2-aminoethyl)-moq>holino-'Cholesterol hemisuccinate (MOCHOL).

34. The method of any one of claims 28-33, wherein the subject received bone marrow transplantation (BMT) before administration of the pharmaceutical composition.

35. The method of any one of claims 28-34, wherein the LSD is Fabry, MPSIIIA or Sanfilippo syndrome, Sialic acid storage disease, MPS I or Hurler disease, MPSII or Hunter syndrome, CLN5 Baten disease, Metaehromatic leukodystrophy, Fueosidosis, Hefmansky- Pudlak disease type 1, Sialidosis, Beta-Mannosidosis, GM2 activator deficiency, Farber disease, Multiple Sulfatase deficiency. Batten disease type 1, MPSIVB or Morquio disease, MPSIVA or Morquio disease, MPS1IIB or Sanfilippo syndrome, MPSIIID or Sanfilippo syndrome, Aspartylgiueosaminuria. Schindler disease, Cystinosis, Mucolipidosis 11, Lysosomal Acid Lipase deficiency. Sandhoff disease, MPS VI or Marotcaux-Lamy syndrome, Hermansky-Pudlak disease type 5, Gaucher disease, CLN8 Batten disease, CLN10 Batten disease, CLNLI Baten disease, or CLN13 Baten disease.

36. The method of claim 35, wherein the LSD is MPS1 , MPSIVA, Farber disease, MPSIIIA, Sialic acid storage disease, Sialidosis, or multiple sulfatase deficiency.

37. A method of treating a lysosomal storage disorder (LSD) of a subject in need thereof, comprising the steps of: conducting bone marrow transplantation on the subject, and then administering a therapeutically effective amount of a pharmaceutical composition comprising a synthetic isolated small activating RNA (saRNA), wherein the saRNA up-regulates the expression of the C/EBPa gene.

38. The method of claim 37, wherein the saRNA is at least 80% complementary to a region on the transcription start site core of the C/EBPrx gene (SEQ ID NO: 3).

39. The method of claim 37 or claim 38, wherein the saRNA comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: I.

40. The method of any one of claims 37-39, wherein the saRNA is double-stranded and comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1 , and a sense strand with 14-30 nucleotides, wherein the sense strand comprises a sequence of SEQ ID NO: 2.

41 . The method of any one of claims 37-40, wherein the pharmaceutical composition further comprises a liposome.

42. The method of claim 41, wherein the liposome comprises 1 -palmitoyl-2"Oleoyl-sn- glycero-3-phosphocholine (POPO), 1,2“dioleoyl-sn-glyeero«3-ph6sphdethariolamme (DOPE), cholesteryl-hemisuccinate (CH EMS), and 4-(2-aminoethyl)-morpholino-cholesterol hemi succinate ( MOC HOL).

43. The method of any one of claims 37-42, wherein the LSD is Fabry, MPS1HA or

Sanfilippo syndrome. Sialic acid storage disease, MPSI or Hurler disease, MPSII or Hunter syndrome, CLN5 Batten disease, Metachromatic leukodystrophy, Fucosidosis, Hermansky- Pudlak disease type I , Sialidosis, Beta-Mannosidosis, GM2 activator deficiency, Farber disease, Multiple Sulfatase deficiency, Batten disease type I, MPSIVB or Morquio disease, MPSIVA or Morquio disease, MPSIIIB or Sanfilippo syndrome, MPSHID or Sanfilippo syndrome, Aspartylglucosamin.uria, Schindler disease, Cystinosis, Mucolipidosis 11, Lysosomal Acid Lipase deficiency, Sandhoff disease, MPS VI or Maroteaux-Lamy syndrome, Hermansky-Pudlak disease type 5, Gaucher disease, CLN8 Batten disease, CLN 10 Batten disease, CLNl 1 Batten disease, or CLNl 3 Batten disease.

44. The method of any one of claims 37-43, wherein the LSD is MPS 1 , MPSIVA, Farber disease, MPSIIIA, Sialic acid storage disease, Sialidosis or multiple sulfatase deficiency.

45. A method of increasing the expression of at least one target gene in a cell, wherein the target gene is GLA, SGSH. SLC/7A5. /DIM. IDS, CLAP. ARSA. FUCA I, HPS1, NEU/. 3L4A7M, GMM ASAHE SUMFI. PPT1, GLB1. GALNS. NAGLU, GNS. AGA. NAGA. CTNS, GNPTAB. LlPA, HEXB, ARSB. HPS5. GBA. MFSDS. CTSD, GRN. and/or

comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a synthetic isolated small activating RNA (saRNA), wherein the saRNA up-regulates the expression of the C/EBPa gene.

46. The method of claim 45, wherein the saRN A is at least 80% complementaiy' to a region on the transcription start site core of the C/EBPa gene (SEQ ID NO: 3).

47. The method of claim 45 or claim 46, wherein the saRNA comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID No. 1.

48. The method of any one of claims 45-47. wherein the saRNA is double-stranded and comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ I D NO: 1 , and a sense strand with 14-30 nucleotides, wherein the sense strand comprises a sequence of SEQ ID NO: 2,

49. The method of any one of claims 45-48, wherein expression of the target gene is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, dr 50%.

50. A pharmaceutical composition comprising a synthetic isolated small activating RNA (saRNA) for use in treating a lysosomal storage disorder (LSD) in a subject, wherein the saRNA up-regul at es the expression of the CZEBPo gene.

51 . The pharmaceutical composition for use of claim 50, wherein the LSD is Fabry, MPSIIIA or Sanfilippo syndrome, Sialic acid storage disease, mucopolysaccharides type 1 (MPSI) or Hurler disease, MPSII or Hunter syndrome, CL N5 Batten disease, Metachromatic leukodystrophy, Fucosidosis, Hermansky-Pudlak disease type 1, Sialldosis, Beta-Mannosidosis, GM2 activator deficiency, Farber disease. Multiple Sulfatase deficiency. Baten disease type 1, MPSIVB or Morquio disease, MPSIVA or Morquio disease, MPSIIIB or Sanfilippo syndrome, MPSHID or Sanfilippo syndrome, Aspartylglucosaminuria, Schindler disease, Cystinosis, Mucolipidosis 11, Lysosomal Acid. Lipase deficiency, Sandhoff disease, MPS VI or Maroteaux- Lamy syndrome, Hermansky-Pudlak disease type 5, Gaucher disease, CLN8 Batten disease, CLN10 Batten disease, CLN11 Batten disease, or CLN13 Batten disease.

52. The pharmaceutical composition for use of claim 50, wherein the LSD is MPS 1 , MPSIVA, Farber disease, MPSIIIA, Sialic acid storage disease, Sialidosis, or multiple sulfatase deficiency.

53. The pharmaceutical composition for use of claim 50, wherein the LSD is MPSI.

54. The pharmaceutical composition tor use of any one of claims 50-53 , wherein the saRNA is at least 80% complementary to a region on the transcription start site core of the CZEBPu gene (SEQ ID NO: 3).

55. The pharmaceutical composition for use of any one of claims 50-54, wherein the saRNA comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1.

56. The pharmaceutical composition for- use of any one of claims 50-55. wherein the saRN A is double-stranded and comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1 , and a sense strand with 14-30 nucleotides, wherein the sense strand comprises a sequence of SEQ ID NO: 2.

57. The pharmaceutical composition tor use of any one of claims 50-56, further comprising a liposome.

58. The pharmaceutical composition for use of claim 57, wherein the liposome comprises l- pahnitoyL2-ole0yl-sn-glycero-3-phosphoeho!ine (POPO), l,2-diolec.iy!-sn~gIyeero-3- phosphoethanolamine (DOPE), cholesteryl-hemisuccinate (CHEMS), and 4-(2-arninoethyI)~ morpholino-cholesterol hemisuccinate (MOCHOL).

59. The pharmaceutical composition for use of any one of claims 50-58, wherein the pharmaceutical composition is administered once a month, once every 3 weeks, or once every 2 weeks.

60. The pharmaceutical composition for use of any one of claims 50-59, wherein the pharmaceutical composition is administered through intravenous infusion.

61. The pharmaceutical composition for use of any one of Claims 50-60, wherein the amount of saRNA administered is between about 15-160 mg/m².

62. The pharmaceutical composition for use of any one of claims 50-61 , wherein the subject received bone marrow transplantation (BMT) before administration of the pharmaceutical composition.

63. Use of a synthetic isolated small activating RNA (saRNA) for the preparation of a medicament for treating a lysosomal storage disorder (LSD) in a subject, wherein the saRN A up- regulates the expression o f the C/EBPa gene.

64. The use of claim 63, wherein the LSD is Fabry, MPSIIIA Or Sanfilippo syndrome, Sialic acid storage disease, mucopolysaccharides type 1 ( MPSI) or Hurler disease, MPSII or Hunter syndrome, CLN 5 Batten disease, Metachromatic leukodystrophy, Fucosidosis, Hermansky- Pudlak disease type 1, Sialidosis, Bcta-Mannosidosis, GM2 activator deficiency, Farber disease, Multiple Sulfatase deficiency, Batten disease type L MPSIVB or Morquio disease, MPSI VA or Morquio disease, MPSUIB or Sanfilippo syndrome, MPS! HD or Sanfilippo syndrome.

Aspartylglucosaminuria, Schindler disease, Cystinosis, Mucolipidosis II, Lysosomal Acid Lipase deficiency, Sandhoff disease, MPS VI or Maroteaux-Lamy syndrome, Hermansky-Pudlak disease type 5, Gaucher disease, CLN8 Batten disease, CLN 10 Baten disease, CLN 1 1 Batten disease, or CLN 13 Batten disease.

65. The use of claim 63, wherein the LSD is MPS 1. MPSI VA, Farber disease, M PS I HA, Sialic acid storage disease, Sialidosis, of multiple sulfatase deficiency.

66. The use of claim 63, wherein the LSD is MPSL

67. The use of any one of clai ms 63-66, wherein the saR'NA is at l east 80% complementary to a region on the transcription start site core of the C/EBPagene (SEQ ID NO: 3).

68. The use of any one of claims 63-67, wherein the saRN A comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequence of SEQ ID NO: 1.

69. The use of any one of claims 63-68, wherein the saRNA is double-stranded and comprises an antisense strand with 14-30 nucleotides, wherein the antisense strand comprises a sequpnee of SEQ ID NO: 1 , and a sense strand with 14-30 nucleotides, wherein the sense strand comprises a sequence of SEQ ID NO: 2,

70. The use of any one of claims 63-69, wherein the medicament further comprises a liposome.

71. The use of claim 70, wherein the liposome comprises 1 -pahnitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), I ,2-dioleoyl~sn-glyeero-3-phosphoetIianolamine (DOPE), eholesteryl- hemisuccinate (CHEMS), and 4-(2-aniinoethyl)-morpholino-choIesteroI hemisuccinate

(MOCHOL).

72. The use of any one of claims 63-71, wherein the medicament is tor administration once a month, once every three weeks, or once every two weeks.

73. The use of any one of claims 63-72, wherein the medicament is for administration through intravenous infusion.

74. The use of any one of claims 63-73, wherein the medicament is for administration to a subject that received bone marrow transplantation (BMT).