Multi-targeting stringed sirna for simultaneous knockdown of two or more targets

Abstract

Disclosed herein is a polynucleotide comprising two or more siRNAs linked together with a single stranded nucleic acid. The two or more siRNAs may bind the same or bind different genes and may comprise a long passenger strand that is annealed to two or more guide strands with gaps of single stranded nucleic acids in between each duplex. Also disclosed herein a method of treating a disease or disorder comprising administering to a subject in need thereof a polynucleotide that targets genes associated with the disorder. The disease may include Alzheimer's disease.

Description

MGB Ref No. 32286 / 70819MULTI-TARGETING STRINGED SIRNA FOR SIMULTANEOUS KNOCKDOWN OF TWO OR MORE TARGETS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U. S. Provisional Patent Application No.63 / 737,321, filed on December 20, 2024, the disclose of which is hereby incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF MATERIALS SUBMITTED ELECTRONICALLY

[0002] This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form having a Filename: 70819_SeqListing. xml; Size: 141,351 bytes; Created December 16, 2025, which is incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Small interfering RNAs (siRNAs) are double stranded RNA molecules which associate with the RNA-induced silencing complex (RISC) to degrade mRNA. The siRNA duplex consists of a passenger (sense) strand which is discarded by RISC and a guide (antisense) strand which associates with RISC to guide it to a complementary target mRNA. In recent years, chemically modified siRNAs have been clinically successful as a novel class of therapeutics. However, siRNAs are limited to single targets which hinders their use for polygenic disorders.SUMMARY OF THE INVENTION

[0004] Disclosed herein is a polynucleotide comprising two or more siRNAs linked together with a single stranded nucleic acid. In some embodiments, two or more siRNAs bind the same or bind different genes. In some embodiments, a long passenger strand may be annealed to two or more guide strands with gaps of single stranded nucleic acids in between each duplex.

[0005] In some embodiments, the passenger strand comprises sense RNA and the guide strands antisense RNA. In some embodiments, the guide strands anneal to the passenger strand. In some embodiments, the guide strands comprise 5’ and 3’ overhangs that do not anneal to the passenger strand.

[0006] In some embodiments, the single stranded gaps link the two or more siRNAs to combine multiple siRNAs into a single molecule. In some embodiments, the passenger strand is composed of 20-200 nucleotides In some embodiments, each guide strand is composed of 15-40IMGB Ref No. 32286 / 70819nucleotides. In some embodiments, the single stranded gaps of the passenger strand comprise 2-10 nucleotides. In some embodiments, the single stranded gaps comprise 6-8 thymidine residues.

[0007] In some embodiments, the siRNA is between 20-30 nucleotides in length. In some embodiments, the nucleotides are chemically modified. In some embodiments, the chemical modification is one or more of 2’0 methyl, 2’-fluoro, 2’-O-methoxyethyl, or locked nucleic acids.

[0008] In some embodiments, the chemical modification of the single stranded gaps is a phosphorothioate, or phosphodiester bond.

[0009] In some embodiments, the two or more siRNAs bind genes related to neurological genes. In some embodiments, the genes are amyloid precursor protein (APP) and microtubule-associated protein tau (MAPT).

[0010] Also disclosed herein is a composition comprising a polynucleotide of the disclosure. In some embodiments, the composite may also comprise a pharmaceutically acceptable excipient, carrier or diluent.

[0011] Also disclosed herein is a vector comprising a polynucleotide of the disclosure. In some embodiments, the vector is a lentiviral vector, an adeno-associated virus, a retrovirus vector, a recombinant viral vector, or an adenovirus-based vector.

[0012] Also disclosed herein is a method of treating Alzheimer’s disease, comprising administering to a subject in need thereof a polynucleotide, a composition, or a vector of the disclosure. In some embodiments, the polynucleotide is specific for APP and MAPT. In some embodiments, the polynucleotide reduces both MAPT and APP mRNA expression. In some embodiments, the polynucleotide reduces both phosphorylated tau and amyloid beta expression.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1. depicts a schematic of simultaneous knockdown of APP and MAPT to reduce amyloid beta and phosphor-Tau, as knocking down both APP and MAPT as a therapeutic strategy to reduce both AB and p-Tau.

[0014] FIG. 2 depicts a table of exemplary APP siRNA sequences.

[0015] FIG. 3 depicts a graph of the reduction in APP mRNA levels in SK-N-AS cells after transfection with some of the exemplary APP siRNA sequences of FIG. 2.iMGB Ref No. 32286 / 70819

[0016] FIG. 4 depicts a graph of the reduction in APP protein levels in SK-N-AS cells after transfection with some of the exemplary APP siRNA sequences of FIG. 2.

[0017] FIG. 5 depict a graph of the reduction in APP mRNA levels in African green monkey Vero-76 cells

[0018] FIG. 6 depicts a table of exemplary MAPT siRNA sequences.

[0019] FIG. 7 depicts a graph of the reduction in MAPT mRNA levels in SK-N-AS cells after transfection with some of the exemplary MAPT siRNA sequences of FIG. 6.

[0020] FIG. 8 depicts a graph of the reduction in MAPT protein levels in SK-N-AS cells after transfection with some of the exemplary MAPT siRNA sequences of FIG. 6.

[0021] FIG. 9A depicts a graph of the reduction in MAPT mRNA in nonhuman primate cells after transfection with some exemplary MAPT siRNA sequences using qPCR analysis.

[0022] FIG. 9B depicts a graph of the reduction in Tau protein in nonhuman primate cells after transfection with some exemplary MAPT siRNA sequences using ELISA analysis.

[0023] FIG. 10 depicts a graph of the reduction in protein levels of APP, Tau, pTau 181, pTau 217, and amyloid beta 42 in SK-N-AS cells after cotransfection with a APP siRNA and a MAPT siRNA.

[0024] FIGS. 11A-11G depict schematics of exemplary designs of stringed siRNAs.

[0025] FIG. 12 depicts a table of exemplary APT / MAPT stringed siRNA sequences.

[0026] FIG. 13 depicts a representative image of an agarose gel demonstrating the size separation of individual strands and the annealed product of exemplary stringed siRNAs.

[0027] FIG. 14 depicts a graph of the simultaneous reduction in mRNA of APP and MAPT in cells transfected with a stringed siRNA targeting APP and MAPT.

[0028] FIG. 15 depicts a graph of the simultaneous reduction in protein levels of APP, Tau, amyloid beta, and phosphor-Tau in cells transfected with a stringed siRNA targeting APP and MAPT.

[0029] FIG. 16 depicts a graph of cell viability of cells transfected with four different exemplary stringed siRNAs.MGB Ref No. 32286 / 70819

[0030] FIG. 17 depicts a representative image of an agarose gel demonstrating a size difference for siRNAs generated with a cleavable or non-cleavable linker.

[0031] FIG. 18 depicts a schematic of an exemplary design of an APP / MAPT-targeting stringed siRNA comprising chemically modified nucleotides.DETAILED DESCRIPTION OF THE INVENTION

[0032] Disclosed herein are compositions and methods of use that utilize stringed siRNAs that comprise two or more siRNAs linked together. The siRNAs have multiple copies directed to the same target gene or are directed to multiple target gene, including targets within the same biological pathway or targets in different pathways that may be associated with a disease or disorder.

[0033] It is to be understood that this application is not limited to particular formulations or process parameters, as these may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Further, it is understood that a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure.

[0034] In accordance with the present application, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques as explained fully in the art. The definitions contained herein supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0035] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0036] The terms “and / or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.”IMGB Ref No. 32286 / 70819

[0037] The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0038] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0039] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0040] As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.

[0041] As used herein the term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0042] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosure.IMGB Ref No. 32286 / 70819

[0043] “Polynucleotide,” or “nucleic acid as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Nucleic acid can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S(“thioate”), P(S)S (“dithioate”), “(O)NR2 (“amidate”), P(O)R, P(O)OR', CO or CH2 (“formacetal”), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether ( — O — ) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including isolated nucleic acid, RNA and DNA.iMGB Ref No. 32286 / 70819

[0044] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. In some embodiments, the nucleic acid molecule comprises an isolated nucleic acid.

[0045] “Treating” and “treatment” refers to any reduction in the severity and / or onset of symptoms associated with a disease or disorder. Accordingly, “treating” and “treatment” includes therapeutic and prophylactic measures. One of ordinary skill in the art will appreciate that any degree of protection from, or amelioration of, the onset and / or progression of a disease or disorder is beneficial to a subject, such as a human patient. The quality of life of a patient is improved by reducing to any degree the severity of symptoms in a subject and / or delaying the appearance of symptoms.siRNAs

[0046] Disclosed herein are polynucleotides comprising two or more siRNAs linked together with a single strand nucleic acid, wherein the two or more siRNAs are configured to bind to the same or different genes. The two or more siRNAs may be configured to reduce the gene expression of the targeted gene(s).

[0047] In some aspects, the polynucleotide may comprise two or more duplexes, wherein each duplex may comprise a sense strand or a passenger strand and a corresponding antisense strand or a guide strand. As used herein, the term “sense strand” may be used interchangeably with the term “passenger strand” and the term “antisense strand” may be used interchangeably with the term “guide strand”.

[0048] In some aspects, the two or more duplexes may be linked together with a single strand nucleic acid to form a single molecule, forming a stringed siRNA. The two or more duplexes and the single strand nucleic acid may comprise a long passenger strand. The long passenger strand may comprise the passenger strand from the first duplex coupled to the single strand nucleic acid coupled to the passenger strand from the second duplex.

[0049] In some aspects, the passenger strand from each of the duplexes may comprise sense RNA and the guide strand from each of the duplexes may comprise antisense RNA. In some aspects, the guide strands may anneal to their corresponding passenger strands to form a duplex.

[0050] In some aspects, the stringed siRNA comprising the two or more duplexes enters the cell as a single molecule which is then processed into individual siRNAs which target complementaryiMGB Ref No. 32286 / 70819mRNA independently or the stringed siRNA may be cleaved extracellularly into two siRNAs. In some aspects, a passenger (sense) strand may be discarded by RISC and two guide (antisense) strands may associate with RISC, where each guide strand associates with RISC to guide it to a complementary target mRNA.Passenger strand

[0051] In some aspects, the long passenger strand comprising the passenger strand from the first duplex, the passenger strand from the second duplex and the single stranded nucleic acid may comprise about 20 nucleotides to about 200 nucleotides. For example, the long passenger strand may comprise about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides, about 180 nucleotides, about 190 nucleotides, or about 200 nucleotides.

[0052] In some aspects, the long passenger strand may be generated as a single strand or may be generated as a first passenger strand, a linker, and a second passenger strand that may be coupled together to form the long passenger strand.

[0053] In some aspects, the nucleotides that comprise the passenger strand may be chemically modified. In some aspects, the chemical modifications may include 2’-O-methyl (OMe), 2’-fluoro (F), 2’-O-methoxyethyl (MOE), locked nucleic acids (LNA), or any other chemical modification. In some aspects, about 10% to about 100% of nucleotides of the passenger strand may be chemically modified. For example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% of the passenger strand may be chemically modified.

[0054] In some aspects, the bonds connecting any one or more of the nucleotides within the passenger strand may be chemically modified. In some aspects, the chemically modified bonds may include phosphodiester, phosphorothioate, or any other chemical modification.Guide strands

[0055] In some aspects, the guide strands may comprise about 10 to about 40 nucleotides. In some aspects, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 iMGB Ref No. 32286 / 70819nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, or about 40 nucleotides. In some aspects, the guide strand may be about 27 nucleotides (27-mer). Advantageously, having a guide strand having greater than a shorter 20 nucleotide guide strand may allow the guide strand to be more potent and more durable.

[0056] In some aspects, the guide strands may comprise the same length. For example, the first guide strand may comprise a length of about 27 nucleotides and the second guide strand may comprise a length of about 27 nucleotides. In some aspects, the first guide strand and the second guide strand may each comprise a length of about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, or about 35 nucleotides.

[0057] It can be appreciated that in some aspects, the length of the guide strands may be different and may be engineered for specific targets. For example, a first guide strand may comprise a length of 25 nucleotides, and a second guide strand may comprise a length of 29 nucleotides, or the first guide strand may comprise a length of 27 nucleotides and the second guide strand may comprise a length of 35 nucleotides.

[0058] In some aspects, the second guide strand may have a length greater than the first guide strand. In some aspects, the first guide strand may comprise a length of about 27 nucleotides and the second strand may comprise a length of about 29 nucleotides. In some aspects, the first guide strand may comprise a length of about 25 nucleotides and the second guide strand may comprise a length of about 30 nucleotides. It can be appreciated that second guide strand may comprise any number of nucleotides between about 11 nucleotides to about 40 nucleotides and still have a length greater than the first guide strand.

[0059] In some aspects, the first guide strand may have a greater length than the second guide strand. In some aspects, the first guide strand may comprise a length of about 29 nucleotides and the second guide strand may comprise a length of about 27 nucleotides. In some aspects, theMGB Ref No. 32286 / 70819first guide strand may be about 35 nucleotides and the second guide strand may be about 30 nucleotides. It can be appreciated that the first guide strand may comprise number of nucleotides between about 11 nucleotides to about 40 nucleotides and still have a length greater than the first guide strand.

[0060] In some aspects, the nucleotides of each guide strand may be chemically modified. In some aspects, the chemical modifications may include 2’-O-methyl (OMe), 2’-fluoro (F), 2’-O-methoxyethyl (MOE), locked nucleic acids (LNA), or any other chemical modification. In some aspects, about 10% to about 100% of nucleotides of each guide strand may be chemically modified. For example, about 10 %, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% of each guide strand may be chemically modified.

[0061] In some aspects, a first guide strand and a second guide strand may comprise the same chemical modifications. For example, the first guide strand may comprise the same chemical modifications as the second guide strand. In some aspects, the first guide strand and the second guide may comprise different chemical modifications. For example, the first guide strand may comprise only OMe chemical modifications and the second guide strand may comprise only MOE chemical modifications.

[0062] In some aspects, any nucleotide within each of the guide strand may be chemically modified. It can be appreciated that the same nucleotides within each guide strand may be modified. For example, each of the same type of nucleotide within each guide strand may be chemically modified. In some aspects, the same position of the nucleotide within each guide strand may be modified. For example, the 3rdnucleotide from the 5’ end of the guide strand may be chemically modified, regardless of the identity of the nucleotide.

[0063] In some aspects, the first guide strand and the second guide strand may comprise the same chemical modification pattern. For example, in some aspects, each of the first three 5’ nucleotides may be chemically modified.

[0064] In some aspects, the first guide strand and the second guide strand may comprise different chemical modification patterns. For example, in some aspects, each of the chemical modification patterns of the first guide strand and the second guide strand may be independently engineered and may be different in makeup of the specific type of chemical modification and in chemical modification pattern.ioMGB Ref No. 32286 / 70819

[0065] In some aspects, the bonds connected any one or more of the nucleotides within each of the guide strands may be chemically modified. In some aspects, the chemically modified bonds may include phosphodiester, phosphorothioate, or any other chemical modification.

[0066] For example, in some aspects, when the passenger strand comprises 21 nucleotides, nucleotides 7, 9,10, and 11 from the 5’ end of the passenger strand may be modified with 2’-fluoro. In some aspects, nucleotides 7, 9, 10, and 11 from the 5’ end of the passenger strand comprise 2’-fluoro chemical modifications. In some aspects, nucleotides 7, 9, 10, and 11 from the 5’ end of the passenger strand consist of 2’-fluoro chemical modifications.

[0067] In some aspects, when the guide strand comprises 23 nucleotides, nucleotides 2, 6, 14, and 16 from the 5’ end of the guide strand may be modified with 2’-fluoro. In some aspects, nucleotides 2, 6, 14, and 16 from the 5’ end of the guide strand comprise 2’-fluoro chemical modifications. In some aspects, nucleotides 2, 6, 14, and 16 from the 5’ end of the guide strand consist of 2’-fluoro chemical modifications. In some aspects, 7, 9,10, and 11 from the 5’ end of the passenger strand and nucleotides 2, 6, 14, and 16 from the 5’ end of the guide strand may be modified with 2’-fluoro. In some aspects, 7, 9,10, and 11 from the 5’ end of the passenger strand and nucleotides 2, 6, 14, and 16 from the 5’ end of the guide strand comprise 2’-fluoro chemical modifications. In some aspects, 7, 9,10, and 11 from the 5’ end of the passenger strand and nucleotides 2, 6, 14, and 16 from the 5’ end of the guide strand consist of 2’-fluoro chemical modifications.

[0068] In some aspects, the first two internucleotide bonds at the 5’ end of the passenger strand may be phosphorothioate and all others may be phosphodiester. In some aspects, the first two internucleotide bonds at the 5’ end of the passenger strand comprise phosphorothioate bonds and all other bonds of the passenger strand comprise phosphodiester bonds. In some aspects, the first two internucleotide bonds at the 5’ end of the passenger strand consist of phosphorothioate bonds and all other bonds of the passenger strand consist of phosphodiester bonds.

[0069] In some aspects, the first two bonds at the 5’ end and first two bonds at the 3’ end of the guide strand may be phosphorothioate while all others may be phosphodiester. In some aspects, the first two bonds at the 5’ end and the first two bonds at the 3’ end of the guide strand comprise phosphorothioate bonds while all other bonds of the guide strand comprise phosphodiester bonds. In some aspects, the first two bonds at the 5’ end and the first two bonds at the 3’ end ofMGB Ref No. 32286 / 70819the guide strand consist of phosphorothioate bonds while all other bonds of the guide strand consist of phosphodiester bonds.

[0070] It can be appreciated that the location of the chemically modified bonds connecting any of the nucleotides within the guide strands may be the same. For example, the first three bonds of the 5’ end of the guide strands may all be chemically modified. It can also be appreciated that the location of the chemically modified bonds connecting any of the nucleotides within the guide strands may be difference between the first guide strand and the second guide strand.

[0071] In some aspects, the guide strands may comprise nucleotides at the 5’ end or the 3’ end that do not form a complementary base pair with the passenger strand (e.g., overhangs). In some aspects, the overhangs may bias the RNA-induced silencing complex to select the strand(s) with the overhang to be selected as the guide strand.Single stranded nucleic acid

[0072] In some aspects, the passenger strand of the two siRNAs may be physically linked together by a single stranded nucleic acid or a linker, to form the long passenger strand. In some aspects the linker may comprise about 2 to about 10 nucleotides. For example, in some aspects, the linker may comprise about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, or about 10 nucleotides. In some aspects, the linker may comprise no more than about 10 nucleotides, about 9 nucleotides, about 8 nucleotides, about 7 nucleotides, about 6 nucleotides, about 5 nucleotides, about 4 nucleotides, about 3 nucleotides, or about 2 nucleotides.

[0073] In some aspects, the linker may comprise about 2 to about 10 thymidine residues. For example, the linker may comprise about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 thymidine residues. In some aspects, the linker may comprise between about 6 to about 8 thymidine residues.

[0074] In some aspects, the linker may be engineered to be cleavable. The stringed siRNA may be delivered as a single molecule, wherein the linker may be cleaved extracellularly, to generate two duplexes that are delivered to a cell. As single stranded nucleic acid can be cleaved by nuclease enzymes, stringed siRNAs with a cleavable linker uniquely have the potential to be administered as a single molecule and cleaved extracellularly for improved cellular uptake and RISC loading.MGB Ref No. 32286 / 70819

[0075] However, if enhanced tissue retention is desirable for a given indication, stringed siRNAs comprising a non-cleavable linker can be modified to be non-cleavable by using phosphorothioate bonds in the single stranded region. For example, in a non-cleavable linker, the linker may comprise phosphorothiate bonds. The stringed siRNA may be delivered as a single molecule, wherein the linker is not cleaved, and the stringed siRNA may be delivered to a cell.

[0076] In some aspects, the nucleotides of the linker may be chemically modified. In some aspects, the bonds connecting the nucleotides (e.g., internucleotide) may be chemically modified. In some aspects, the nucleotides of the linker and / or the bonds connecting the nucleotides within the linker may both be chemically modified. In some aspects, the ribose modifications (e.g., chemical modifications of the nucleotides) may comprise 2’-O-methyl (OMe), 2’-fluoro (F), 2’-O-methoxyethyl (MOE), locked nucleic acids (LNA), or any other chemical modification. In some aspects, the chemically modified internucleotide bonds may comprise either phosphodiester bonds, phosphorothioate bonds, or any other chemical modification.Synthesis of stringed siRNA

[0077] The stringed siRNA may be synthetized in any manner. In some aspects, each individual guide strand may be synthesized and then annealed to the long passenger strand to form the stringed siRNA. In some aspects, the long passenger strand may be synthesized separately and then combined with the individual guide strands to form the stringed siRNA. In some aspects, the stringed siRNA may be synthesized by annealing together equal molar amounts of each of the guide strands with the corresponding long passenger strand and heated to a temperature to anneal the passenger strand and guide strands together into the stringed siRNA.siRNA targets

[0078] In some aspects, the stringed siRNA may target or bind to one or more genes. In some aspects, the guide strands may target or bind to one or more genes. The one or more genes may be the same or may be different. In some aspects, the one or more genes may be genes identified in a disease or disorder.

[0079] In some aspects, the one or more genes may include genes in the same biological pathway or in different biological pathways. The one or more genes may include genes related to neurological genes. For example, in some aspects, the one or more genes may include APP or MAPT. In some aspects, wherein the stringed siRNA targets APP, the sense strand or the antisense strand may comprise a nucleotide sequence selected from the group consisting of SEQIBMGB Ref No. 32286 / 70819ID NOS: 1 -32. In some aspects, wherein the stringed siRNA targets APR, the sense strand or the antisense strand may comprise a nucleotide sequence as disclosed in Table 1.Table 1: Exemplary APP siRNAsName Sense sequence Antisense sequencesiAPP 5' 5'-1 rUrArGrUrGrCrArUrGrArArUrArGrArllr rC r ArG rG r ArG r ArG r Ar Ar U rC r U r Ar U r U rC r Ar UrCrUrCrUrCrCTG 3' (SEQ ID NO: 1) UrGrCrArCrUrArGrU 3' (SEQ ID NO: 17) siAPP 5' 5'-2 rCrCrUrGrArUrCrArCrllrArllrGrCrArUr rArArCrUrUrUrArArArArUrGrCrArllrArGrllr UrUrUrArArArGTT 3' (SEQ ID NO: 2) GrArUrCrArGrGrArA 3' (SEQ ID NO: 18) siAPP 5' 5'-3 rGrArArUrArGrArUrUrCrUrCrUrCrCrllr rU r Ar Ar Aril r Ar ArU rCrArG rG r ArG rArG rAr Ar GrArUrUrArUrUTA 3' (SEQ ID NO: 3) UrCrUrArUrUrCrArU 3' (SEQ ID NO: 19) siAPP 5' 5'-4 rArUrArGrArUrUrCrUrCrUrCrCrUrGrAr rG rArU r ArArArU r ArArU rCrArG rG rArG rArG r UrUrArUrUrUrATC 3' (SEQ ID NO: 4) ArArUrCrUrArUrUrC 3' (SEQ ID NO: 20) siAPP 5' 5'-5 rG rC r ArG rG r Ar U rG r Ar U r II rG r U r ArC r Ar rC rAr Ar U rG r Ar U rU rC r U rG r U r ArC r Ar Ar U rC r GrArArUrCrArUTG 3' (SEQ ID NO: 5) ArUrCrCrUrGrCrArG 3' (SEQ ID NO: 21) siAPP 5' 5'-6 rUrUrGrUrArCrArGrArArUrCrArllrUrGr rUrGrUrCrArUrArArGrCrArArUrGrArUrUrCr CrUrUrArUrGrACA 3' (SEQ ID NO: 6) UrGrUrArCrArArUrC 3' (SEQ ID NO: 22) siAPP 5' 5'-7 rUrCrUrGrCrArGrGrArUrGrArllrllrGrUr rUrGrArUrUrCrUrGrUrArCrArArUrCrArUrCr ArCrArGrArArUCA 3' (SEQ ID NO: 7) CrUrGrCrArGrArArA 3' (SEQ ID NO: 23) siAPP 5' 5'-8 rArllrUrGrArGrArCrUrUrCrArArGrCrUr rArArArArArGrArArArArGrCrUrUrGrArArGr UrUrUrCrUrUrUTT 3' (SEQ ID NO: 8) UrCrUrCrArArUrGrC 3' (SEQ ID NO: 24) siAPP 5' 5'-9 rCrArGrUrArArUrGrUrArUrUrCrUrArUr rU rAr Ar ArG rArG rArG r ArU rArG rAr Ar Ur ArC r CrUrOrUrCrUrUTA 3' (SEQ ID NO: 9) ArUrUrArCrUrGrArU 3' (SEQ ID NO: 25)MGB Ref No. 32286 / 70819siAPP 5' 5'-10 r Aril rCr ArG rU r Ar ArUrG rU r Ar U rU rCrU r rArArG rArGr ArG r ArU r ArG r Ar ArU r ArCr ArU r ArUrCrUrCrUrCTT 3' (SEQ ID NO: 10) UrArCrUrGrArUrGrU 3' (SEQ ID NO: 26) siAPP 5' 5'-11 rUrCrArGrUrArArUrGrUrArUrUrGrUrAr rArArArG rArG r ArG rArU rArG r ArArUr ArCrAr UrCrUrCrUrGrUTT 3' (SEQ ID NO: 11) UrUrArCrUrGrArUrG 3' (SEQ ID NO: 27) siAPP 5' 5'-12 rGrCrUrGrUrArUrCrArArArCrUrArGrUr rUrArUrUrCrArUrGrCrArCrUrArGrUrUrUrG GrCrArUrGrArATA 3' (SEQ ID NO: 12) rArUrArCrArGrCrUrA 3' (SEQ ID NO: 28) siAPP 5' 5'-13 rUrArGrUrGrCrArUrGrArArUrArGrArUr rC rArG rG r ArG r ArG r Ar Ar U rC r U r Ar U r U rG r Ar UrCrUrCrUrCrCTG 3' (SEQ ID NO: 13) UrGrCrArCrUrArGrU 3' (SEQ ID NO: 29) siAPP 5' 5'-14 rCrGrGrUrGrUrCrCrArUrUrUrArUrArGr rC r ArC r Ar U r U r ArU r U rC r U r Ar U r Ar Ar Ar U rG r ArArUrArArUrGTG 3' (SEQ ID NO: 14) GrArCrArCrCrGrArU 3’ (SEQ ID NO: 30) siAPP 5’ 5’-15 rCrArGrUrArArUrGrUrArUrUrCrUrArUr rG rArG rArG r Ar U rArG r ArArU r ArCrAr U rU r Ar CrUrCrUrC 3’ (SEQ ID NO: 15) CrUrGrArU 3' (SEQ ID NO: 31)siAPP 5' 5’-16 rGrUrArArUrGrUrArUrUrCrUrArUrCrUr rArArG rArGr ArG r ArU rArG r Ar ArU r ArCr ArU r CrUrGrUrU 3' (SEQ ID NO: 16) UrArCrUrG 3' (SEQ ID NO: 32)RNA: rA, rC, rG, rU DNA: A, C, G, T

[0080] In some aspects, wherein the stringed siRNA targets MAPT, the sense strand or the antisense strand may comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS: 33-72. In some aspects, wherein the stringed siRNA targets MAPT, the sense strand or the antisense strand may comprise a nucleotide sequence as disclosed in Table 2.Table 2: Exemplary MAPT siRNAsName Sense sequence Antisense sequencesiMAP 5' rArArArArUrCrUrGrArG rArArG rCrUr 5’ rCrUrUrGrArArGrUrCrArArGrCrUrUrCrU T-1 UrGrArCrUrUrCrAAG 3' (SEQ ID NO: rCrArGrArUrUrUrUrArC 3' (SEQ ID NO:33) 53)MGB Ref No. 32286 / 70819siMAP 5' rGrUrGrUrGrGrCrUrCrArArArGrGrAr 5' rUrUrUrGrArUrArUrUrArUrCrCrUrUrUrG T-2 UrArArUrArUrCrAAA 3' (SEQ ID NO: rArGrCrCrArCrArCrUrU 3' (SEQ ID NO:34) 54)siMAP 5' rC r ArG r II rG r II rG rC r Ar Ar Ar II r ArG r II r 5' TUrGrGrUrUrUrGrUrArGrArCrUrArUrUrU T-3 CrUrArCrArArArCCA 3' (SEQ ID NO: rGrCrArCrArCrUrGrCrC 3' (SEQ ID NO:35) 55)siMAP 5' rCrllrArCrArArArCrCrArGrllrUrGrAr 5’ rCrUrUrGrCrUrCrArGrGrUrCrArArCrUrG T-4 CrCrUrGrArGrCrAAG 3' (SEQ ID NO: rGrUrUrUrGrUrArGrArC 3' (SEQ ID NO:36) 56)siMAP 5' rGrUrGrGrCrGrArGrGrUrGrGrArArG 5' rUrCrArGrArUrUrUrUrArCrUrUrCrCrArCr T-5 rUrArArArArUrCrUGA 3' (SEQ ID NO: CrUrGrGrCrCrArCrCrU 3' (SEQ ID NO: 57) 37)siMAP 5' rGrUrGrUrGrCrArArArUrArGrllrCrUr 5’ TArCrUrGrGrUrUrUrGrUrArGrArCrUrArU T-6 ArCrArArArCrCrAGT 3' (SEQ ID NO: rUrUrGrCrArCrArCrUrG 3' (SEQ ID NO:38) 58)siMAP 5' 5'T-7 rGrArCrCrCrArArGrCrllrCrGrCrArUrGr rUrUrUrArCrUrGrArCrCrArUrGrCrGrArGrC GrUrCrArGrUrAAA 3' (SEQ ID NO: 39) rUrUrGrGrGrUrCrArC 3' (SEQ ID NO: 59) siMAP 5' 5' rCrCrGrUrCrUrUrUrGrCrUrUrUrUrArCrU T-8 rGrCrArllrGrGrUrCrArGrllrArArArArGr rGrArCrCrArUrGrCrGrA 3' (SEQ ID NO:CrArArArGrArCGG 3' (SEQ ID NO: 40) 60)siMAP 5' 5' rUrGrGrUrUrUrGrUrArGrArCrUrArUrUrU T-9 rGrArArGrGrllrGrCrArArArUrArGrllrCr rGrCrArCrCrUrUrCrCrC 3' (SEQ ID NO:UrArCrArArArCCA 3' (SEQ ID NO: 41) 61 )siMAP 5' rArG rG r U rG rCr Ar ArArll rArG r U rCrll r 5' TArCrUrGrGrUrUrUrGrUrArGrArCrUrArU T-10 ArCrArArArCrCrAGT 3' (SEQ ID NO: rUrUrGrCrArCrCrUrUrC 3' (SEQ ID NO:42) 62)siMAP 5' rGrUrCrUrArCrArArArCrCrArGrUrUr 5’ rUrGrCrUrCrArGrGrUrCrArArCrUrGrGrU T-11 GrArCrCrUrGrArGCA 3' (SEQ ID NO: rUrUrGrUrArGrArCrUrA 3' (SEQ ID NO:43) 63)siMAP 5' rCrUrArCrArArArCrCrArGrUrUrGrAr 5' rCrUrUrGrCrUrCrArGrGrUrCrArArCrUrG T-12 CrCrUrGrArGrCrAAG 3' (SEQ ID NO: rGrUrUrUrGrUrArGrArC 3' (SEQ ID NO:44) 64)MGB Ref No. 32286 / 70819siMAP 5' rGrGrArCrGrCrArUrGrUrArllrCrUrUr 5' rC r Ar ArG rC r Ar U r U r U rC r Ar ArG r Ar U r ArCr T-13 GrArArArUrGrCrUTG 3' (SEQ ID NO: ArUrGrCrGrUrCrCrUrU 3' (SEQ ID NO: 65) 45)siMAP 5' 5' rUrCrArArUrUrUrArUrCrUrGrCrCrArGrC T-14 rUrGrArllrCrArGrUrGrCrUrGrGrCrArGr rArCrUrGrArUrCrArCrC 3' (SEQ ID NO:ArUrArArArUrUGA 3' (SEQ ID NO: 46) 66)siMAP 5' rGrUrGrUrGrCrArArArUrArGrllrCrUr 5' rGrUrUrUrGrUrArGrArCrUrArUrUrUrGrC T-15 ArCrArArArC 3' (SEQ ID NO: 47) rArCrArCrUrG 3' (SEQ ID NO: 67) siMAP 5' rGrCrArArArUrArGrllrCrllrArCrArAr 5' rArCrUrGrGrUrUrUrGrUrArGrArCrUrArU T-16 ArCrOrArGrU 3' (SEQ ID NO: 48) rUrUrGrCrArC 3' (SEQ ID NO: 68) siMAP 5' rG r II rG rC r Ar Ar Ar U r ArG r U rC rll r ArCr 5' rUrGrGrUrUrUrGrUrArGrArCrUrArUrUrU T-17 ArArArCrCrA 3' (SEQ ID NO: 49) rGrCrArCrArC 3' (SEQ ID NO: 69) siMAP 5' rArG rG r U rG rC r Ar ArArll rArG rll rCrU r 5’ rGrUrUrUrGrUrArGrArCrUrArUrUrUrGrC T-18 ArCrArArArC 3' (SEQ ID NO: 50) rArCrCrUrUrC 3' (SEQ ID NO: 70) siMAP 5' rGrCrArArArUrArGrUrCrUrArCrArAr 5' rArCrUrGrGrUrUrUrGrUrArGrArCrUrArU T-19 ArCrCrArGrU 3' (SEQ ID NO: 51) rUrUrGrCrArC 3' (SEQ ID NO: 71 ) siMAP 5' rG r U rG rC r Ar Ar Ar U r ArG r U rC rU r ArCr 5' rUrGrGrUrUrUrGrUrArGrArCrUrArUrUrU T-20 ArArArCrCrA 3' (SEQ ID NO: 52) rGrCrArCrCrU 3' (SEQ ID NO: 72) RNA: rA, rC, rG, rU DNA: A, C, G, T

[0081] In some aspects, wherein the stringed siRNA targets APP and MAPT, the sense strand (long passenger strand) or either of the antisense strands may comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS: 73-84. In some aspects, wherein the stringed siRNA targets APP and MAPT, the sense strand (long passenger strand) or either of the antisense strands may comprise a nucleotide sequence disclosed in Table 3Table 3: Exemplary stringed siRNAs targeting APP and MAPTMGB Ref No. 32286 / 70819 Name Sense sequence Antisense Antisense sequence 1 sequence 2 String 5’ 5’ 5’-1 rCrArGrUrArArUrGrUrArUrUrCrllrArUr rUrArArArGrArGrAr rArCrUrGrGrUrUrUr CrUrCrUrCrUrUTA I I l l i I rGrUrGrUrG GrArUrArGrArArUr GrUrArGrArCrUrAr rCrArArArUrArGrUrGrUrArOrArArArCrG ArCrArUrUrArCrUr UrUrUrGrCrArCrAr rAGT 3’ (SEQ ID NO: 73) GrArU 3’ (SEQ ID CrUrG 3’ (SEQ ID NO: 74) NO: 75)String 5’ 5’ 5’-2 rGrUrGrUrGrCrArArArUrArGrllrGrllrAr rUrArArArGrArGrAr rArCrUrGrGrUrUrUr CrArArArCrCrAG I I I I I I I rCrArGrUrAr GrArUrArGrArArUr GrUrArGrArCrUrAr ArUrGrUrArUrUrCrUrArUrCrUrCrUrCrU ArCrArUrUrArCrUr UrUrUrGrCrArCrAr rUTA 3’ (SEQ ID NO: 76) GrArU 3’ (SEQ ID CrUrG 3’ (SEQ ID NO: 77) NO: 78)String 5’ 5’ 5’-3 rCrArGrUrArArUrGrUrArUrUrCrllrArUr rUrArArArGrArGrAr rArCrUrGrGrUrUrUr CrUrCrUrCrUrUTA I I I I I I I I rGrUrGrU GrArUrArGrArArUr GrUrArGrArCrUrAr rG rC r Ar Ar Ar U r ArG r U rC r U r ArC r Ar Ar ArC ArCrArUrUrArCrUr UrUrUrGrCrArCrAr rCrAGT 3’ (SEQ ID NO: 79) GrArU 3’ (SEQ ID CrUrG 3’ (SEQ ID NO: 80) NO: 81)String 5’ 5’ 5’-4 rGrUrGrUrGrCrArArArUrArGrUrCrUrAr rUrArArArGrArGrAr rArCrUrGrGrUrUrUr CrArArArCrCrAG I I I I I I I I I rCrArGrUr GrArUrArGrArArUr GrUrArGrArCrUrAr ArArUrGrUrArUrUrCrUrArUrCrUrCrUrC ArCrArUrUrArCrUr UrUrUrGrCrArCrAr rUrUTA 3’ (SEQ ID NO: 82) GrArU 3’ (SEQ ID CrUrG 3’ (SEQ ID NO: 83) NO: 84)RNA: rA, rC, rG, rU DNA: A, C, G, TMethods of Treating a Disorder or Disease using stringed siRNAs

[0082] Also disclosed herein are methods of treating a disorder or disease in a subject in need thereof using stringed siRNAs. In some aspects, the methods may comprise administering to a subject in need thereof a polynucleotide comprising a stringed siRNA of the disclosure. In some aspects, administering a polynucleotide to a subject in need thereof may include administration of the polynucleotide using any standard mechanism or method. In some aspects, administeringI SMGB Ref No. 32286 / 70819a polynucleotide to a subject in need thereof may include administering the polynucleotide intrathecally to the subject in need thereof.

[0083] The present approach may be useful for the targeted reduction of protein levels in a disease or disorder. In some embodiments, the disease or disorder may include Alzheimer's disease.

[0084] Alzheimer’s Disease (AD) is an age-related neurological disorder affecting 7 million people worldwide and is driven by deposition of amyloid beta plaques and phosphorylated Tau protein, which lead to neurodegeneration and cognitive decline1. The only disease-modifying therapeutics currently for AD target amyloid beta alone and provide little to no clinical benefit for AD patients with moderately high Tau burden, resulting in exclusion of patients with high Tau burden from clinical trials2. While there are early-stage clinical trials for oligonucleotide therapeutics targeting APP or MAPT separately, there are no therapeutic strategies to reduce both APP and MAPT simultaneously.

[0085] The accumulation of amyloid beta, derived from the gene amyloid-beta precursor protein (APP), has been linked to Alzheimer's disease. As seen in the schematic of FIG. 1, APP is cleaved by beta secretase and gamma secretase and can form plaques in the brain of subjects with Alzheimer's disease. Likewise, Tau protein, derived from the gene microtubule-binding protein Tau (MAPT), can be phosphorylated and accumulate in the brain to form neurofibrillary tangles in subjects with Alzheimer's disease. Sequentially, amyloid beta (A|3) is cleaved from APP and accumulation of amyloid beta results in hyperphosphorylation of microtubule associated protein Tau (MAPT), which is neurotoxic. Knocking down both APP and MAPT could be a therapeutic strategy to reduce both Ap and p-Tau.

[0086] In various embodiments, a stringed siRNA comprising a APP siRNA and a MAPT siRNA simultaneously reduces both amyloid beta mRNA levels and Tau mRNA levels, In various embodiments, a stringed siRNA comprising a APP siRNA and a MAPT siRNA may further reduce APP and Tau protein levels in subjects.

[0087] In various embodiments, the stringed siRNA, reduces mRNA levels of a target gene by 10% to 100%. In various embodiments, the stringed siRNA reduces mRNA levels of each target gene in the stringed siRNA by 10% to 100%. In various embodiments, the stringed siRNA reduces mRNA levels of the target by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.ioMGB Ref No. 32286 / 70819

[0088] In some embodiments, the stringed siRNA reduces mRNA levels of the target by about 10% to about 20%, about 20% to about 30%, about 30% to about 50%, about 40% to about 60%, about 50% to about 70%, or about 80% to about 100%.

[0089] In various embodiments, the stringed siRNA, reduces protein levels of a target protein by 10% to 100%. In various embodiments, the stringed siRNA reduces protein levels of each target protein targeted by the stringed siRNA by 10% to 100%. In various embodiments, the stringed siRNA reduces protein levels of the target protein by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0090] In some embodiments, the stringed siRNA reduces protein levels of the target by about 10% to about 20%, about 20% to about 30%, about 30% to about 50%, about 40% to about 60%, about 50% to about 70%, or about 80% to about 100%.

[0091] In various embodiments, the siRNA is provided as a composition. In various embodiments, the composition comprises a pharmaceutically acceptable carrier or diluent.

[0092] The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, media, encapsulating material, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in maintaining the stability, solubility, or activity of, an antibody or antigen binding fragment thereof of the present disclosure. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like, and may include other proteins for enhanced stability, such as albumin, lipoprotein, globulin, etc., subjected to mild chemical modifications or the like. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. The terms “excipient,” “carrier,” “pharmaceutically acceptable carrier,” and the like are used interchangeably herein. The compositions of the present disclosure may further comprise one or more pharmaceutically acceptable carriers, excipients, and other agents10MGB Ref No. 32286 / 70819that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like (herein collectively referred to as “pharmaceutically acceptable carriers or diluents”).

[0093] The compositions may also comprise other ingredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).

[0094] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

[0095] In various embodiments, the siRNA is administered to a subject in a vector, liposome, lipid nanaoparticles, or other delivery vehicle. In some aspects, the delivery vehicle may comprise a peptide, lipid, or other macromolecule conjugated to the siRNA.

[0096] In various embodiments, the vector is a lentiviral vector, an adeno-associated virus, a retrovirus vector, a recombinant viral vector, or an adenovirus-based vector. In some aspects, retro virus based vectors may include any one or more of: murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency vims (SIV), human immuno deficiency11MGB Ref No. 32286 / 70819vims (HIV), or combinations thereof. Exemplary recombinant viral vectors may include a lentiviral vector genome, poxvirus vector genome, vaccinia vims vector genome, adenovirus vector genome, adenovirus-associated virus vector genome, herpes virus vector genome, or alpha vims vector genome.

[0097] In various embodiments, the siRNA is administered intravenously, intramuscularly, intrathecally, intracerebroventricularly, subcutaneously, intracerebroventricularly, intranasally, intracranially, intracelially, or intracerebellar.

[0098] In some embodiments, stringed siRNAs may be useful in treating a disorder or disease in a subject in need thereof. In some embodiments, the disease or disorder may include but is not limited to any one or more of the following: immune disorders, autoimmunity, a cell proliferative disease or disorder, cancer, inflammation, graft vs host, transplantation, gastrointestinal disorder, rheumatoid arthritis, systemic lupus, cachexia, neurodegenerative disease or disorders, neurological diseases or disorders, cardiac dysfunction, or microbial infection (e.g., viral, bacterial, and / or fungi infection, parasitic, or infection caused by other microorganism).

[0099] In some aspects, the disease or disorder may include a proliferative disease or disorder and the proliferative disease or disorder may be non-cancerous. In some aspects, the non-cancerous disease or disorder includes, but is not limited to, rheumatoid arthritis; inflammation; autoimmune disease; lymphoproliferative conditions; acromegaly; rheumatoid spondylitis; osteoarthritis; gout; other arthritic conditions; sepsis; septic shock; endotoxic shock; gramnegative sepsis; toxic shock syndrome; asthma; adult respiratory distress syndrome; chronic obstructive pulmonary disease; chronic pulmonary inflammation; inflammatory bowel disease; Crohn’s disease; skin-related hyperproliferative disorders; psoriasis; eczema; atopic dermatitis; hyperpigmentation disorders; eye-related hyperproliferative disorders; age-related macular degeneration; ulcerative colitis; pancreatic fibrosis; hepatic fibrosis; acute and chronic renal disease; irritable bowel syndrome; pyresis; restenosis; cerebral malaria; stroke and ischemic injury; neural trauma; Alzheimer’s disease; Huntington’s disease; Parkinson’s disease; acute and chronic pain; allergic rhinitis; allergic conjunctivitis; chronic heart failure; acute coronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter’s syndrome; acute synovitis; muscle degeneration, bursitis; tendonitis; tenosynovitis; herniated, ruptures, or prolapsed intervertebral disk syndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonary sarcosis; bone resorption diseases, such as osteoporosis; graft- versus-host reaction; fibroadipose hyperplasia; spinocerebullar ataxia type 1; CLOVES syndrome; Harlequin ichthyosis; macrodactyly syndrome; Proteus syndrome (Wiedemann syndrome); LEOPARD11MGB Ref No. 32286 / 70819syndrome; systemic sclerosis; Multiple Sclerosis; lupus; fibromyalgia; AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I or II, influenza virus and cytomegalovirus; diabetes mellitus; hemihyperplasia-multiple lipomatosis syndrome; megalencephaly; rare hypoglycemia, Klippel-Trenaunay syndrome; harmatoma; Cowden syndrome; or overgrowthhyperglycemia.

[0100] In some embodiments, the proliferative disease or disorder is a cancer. In some aspects, the cancer may comprise any one or more of the following: lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemias, lymphomas, myelomas, or solid tumors.

[0101] The term ‘cancer’ includes, but is not limited to, the following cancers: breast, myeloma, lymphoma, ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma, bone, colon, colorectal, adenoma, pancreas, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, liver carcinoma and biliary passages, kidney carcinoma, myeloid disorders, lymphoid disorders, Hodgkin's, hairy cells, buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine, rectum, large intestine, brain and central nervous system, chronic myeloid leukemia (CML), or a cancer selected from gastric, renal, head and neck, oropharangeal, non-small cell lung cancer (NSGLC), endometrial, hepatocarcinoma, NonHodgkins lymphoma, and pulmonary, epidermoid Oral: buccal cavity, Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma, Lung: bronchogenic carcinoma (squamous cell or epidermoid, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma, Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or small intestines (adenocarcinoma, lymphoma, leiomyoma, hemangioma, lipoma, neurofibroma), large bowel or large intestines (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), rectum, Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma,MGB Ref No. 32286 / 70819transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroadenoma, adenomatoid tumors, lipoma), Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, biliary passages, Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors, Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, glioma, sarcoma), Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), Hematologic: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, myelodysplastic syndrome), Hodgkin's disease, nonHodgkin's lymphoma (malignant lymphoma) hairy cell, lymphoid disorders, Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis, Thyroid gland: papillary thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma, undifferentiated thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma; and adrenal glands: neuroblastoma, leukemias and lymphomas such as cutaneous T-cell lymphomas (CTGL), noncutaneous peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotrophic virus (HTLV) such as adult T-cell leukemia / lymphoma (ATLL), B-cell lymphoma, acute nonlymphocytic leukemias, chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), or hepatocellular carcinoma. Further examples include myelodisplastic11MGB Ref No. 32286 / 70819syndrome, childhood solid tumors such as brain tumors, retinoblastoma, Wilms' tumor, and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular), lung cancer (e.g., small-cell and non-small cell), pancreatic cancer, and other skin cancers, stomach cancer, tumors related to Gorlin's syndrome (e.g., medulloblastoma, meningioma, etc.), and liver cancer. Additional exemplary forms of cancer which may be treated include, but are not limited to, cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, rectum carcinoma, cancer of the salivary gland, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer. Cancer may also include colon carcinoma, familial adenomatous polyposis carcinoma or hereditary non-polyposis colorectal cancer. Further, cancers include, but are not limited to, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, brain tumors such as glioblastoma, astrocytoma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, myosarcoma, liposarcoma, fibrosarcoma, and plasmocytoma.

[0102] Cancer may also include thyroid, breast, and lung cancer; and myeloproliferative disorders, such as polycythemia vera, thrombocythemia, myeloid metaplasia with myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilic syndrome, juvenile myelomonocytic leukemia, and systemic mast cell disease. In one embodiment, the compounds of this application are useful for treating hematopoietic disorders, in particular, acute-myelogenous leukemia (AML), chronic-myelogenous leukemia (CML), acute- promyelocytic leukemia, and acute lymphocytic leukemia (ALL).

[0103] Exemplary cancers may also include, but are not limited to, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma,MGB Ref No. 32286 / 70819supratentorial primitive neuroectodeimal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas / carcinoids, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, ocular cancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non- Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma, Merkel cell carcinoma, mesothelioma malignant, mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer of the tongue, multiple endocrine neoplasia syndrome, Mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing family of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, and Wilm’s Tumor. Thus,BiMGB Ref No. 32286 / 70819the term ‘cancerous cell’ as provided herein, includes a cell afflicted by any one of the aboveidentified conditions.EXAMPLES

[0104] The following Examples describe the methods and materials.Example 1 -APP siRNAs are active at picomolar concentrations

[0105] Only an estimated 10% of the genome is druggable with small molecules. However, an estimated 99.2% of the genome may contain siRNA target sites. Advantageously, the stringed siRNA platform allows for combination of more than two siRNAs into a single molecule, which may target a plethora of other disorders with unmet therapeutic needs.

[0106] As a proof of concept for the stringed siRNA technology, a single molecule was developed which reduces both APP and MAPT. The stringed siRNA demonstrated a reduction of both phosphorylated tau and amyloid beta, which has not been accomplished by any other AD therapeutic. The combination of APP siRNA and MAPT siRNA into a single molecule yields a synergistic therapeutic effect for AD pathology.

[0107] 16 custom APP siRNAs (sense and antisense sequences seen in FIG. 2 or Table 1) were designed that were unique to other sequences found in the literature or published patents. All oligonucleotides were synthesized by Integrated DNA Technologies (IDT, USA) or ChemGenes (USA).

[0108] The 16 custom APP siRNAs were screened in human SK-N-AS cells from a concentration range of 0.01 -10nM. SK-N-AS cells were cultured in DMEM (Gibco, USA) with 10% FBS (Gibco, USA), 0.1 mM NEAA (Gibco, USA), and 1x Pen / Strep (Gibco, USA) at 37 °C and 5% CO2. Cells were split upon reaching 80-90% confluency.

[0109] 2.5 x 105cells in 1 mL culture media were plated per well in 6 well plates and incubated at 37 °C and 5% CO2 for 24 hours prior to transfection. On the day of transfection, oligonucleotides were diluted in ULTRAPURETMDistilled Water (Invitrogen, USA) to desired concentrations. For transfecting siRNA, 1.5 pL of diluted oligonucleotide were incubated with 300 pL of Opti-MEM media (Gibco, USA) and 9 pL Lipofectamine RNAiMAX (Invitrogen, USA) for five minutes at room temperature. For each mixture, 250 pL were added to appropriate wells harboring the cells in the 6 well plates. The cells were further cultured under normal growth conditions for another 48 hours for downstream RNA analysis. Following the incubation, the media was discarded, the cells were washed with PBS and processed for RNA.11MGB Ref No. 32286 / 70819

[0110] Cultured cells were lysed with 300pl of TRIzol™ Reagent (ThermoFisher, USA) per well. RNA was extracted from the samples using Direct-zol RNA Miniprep kit (Zymo Research, USA) following manufacturer’s protocol. The quality and concentration of extracted RNA was determined using Nanodrop 2000 (ThermoFisher, USA) as described by the manufacturer prior to reverse transcription. cDNA was synthesized with qScript™ cDNA synthesis kit (Quantabio, USA) using 1000ng of total RNA based on manufacturer’s protocol. 2 pl of RT reaction containing cDNA were mixed with TaqMan® Fast Advanced MasterMix and TaqMan® Gene Expression Assays labeled with FAM for detection of APP (Hs00169098_m1) in Quantstudio 6 Flex (Applied Biosystems).

[0111] Human SK-N-AS cells were transfected with 0.01 -10nM siRNA for 48 hours prior to collecting total cellular RNA for qPCR analysis. Error bars reflect SEM. “NTC” is Origene nontargeting control (SR30004). Figure 3 demonstrates that APP siRNA reduces APP mRNA levels at picomolar concentrations. While each of the APP siRNAs demonstrated a reduction in APP mRNA at 10 pM compared to control in SK-N-AS cells, siAPP-9 was identified as the most potent of the initial 14 siRNAs tested, reducing APP mRNA levels by 90% at.01 nM, or 10pM (as seen in Figure 3).

[0112] siAPP-9 was evaluated for knockdown of APP protein levels. SK-N-AS cells were transfected with 0.01 or 10nM siRNA for 72 hours prior to collecting total cellular protein for western blot analysis. Error bars reflect SEM. NTC is Origene non-targeting control (SR30004). Statistical results obtained using a two-way ANOVA. p < 0.05(*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (**“). n = 3.

[0113] Knockdown of APP protein with siAPP-9 was confirmed in SK-N-AS cells, observing a 60% reduction of APP protein with 10pM of siAPP-9 and a 95% reduction of APP protein with 10nM of siAPP-9, as seen in Figure 4. siAPP-9 reduces APP protein levels at picomolar concentrations.

[0114] With an interest in advancing siAPP-9 to nonhuman primate studies, African Green Monkey Vero-76 cells were cultured in DMEM (Gibco, USA) with 10% FBS (Gibco, USA) and 1x Pen / Strep (Gibco, USA) at 37 °C and 5% CO2. Cells were split upon reaching 80-90% confluency. African green monkey Vero-76 cells were transfected with 0.01 or 10nM siRNA for 48 hours prior to collecting total cellular RNA for qPCR analysis. Error bars reflect SEM. NTC: Origene nontargeting control (SR30004). Statistical results obtained using a two-way ANOVA. p < 0.05(*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). n = 3.IBMGB Ref No. 32286 / 70819

[0115] Knockdown efficiency in Vero-76 cells was tested and observed a 15% reduction of APP mRNA with 10pM siAPP-9 and a 60% reduction of APP mRNA with 10nM siAPP-9 (as seen in Figure 5). siAPP-9 reduces APP mRNA in nonhuman primate cells.Example 2 - MAPT siRNAs are active at picomolar concentrations

[0116] 20 custom MAPT siRNAs were designed unique to other sequences found in the literature or published patents (as seen in Figure 6 or in Table 2). All oligonucleotides were synthesized by Integrated DNA Technologies (IDT, USA) or ChemGenes (USA).

[0117] Six of the MAPT siRNAs were screened in human SK-N-AS cells from a concentration range of 0.1 -1 OnM. 2.5 x 105SK-N-AS cells in 1 mL culture media were plated per well in 6 well plates and incubated at 37 °C and 5% CO2 for 24 hours prior to transfection. On the day of transfection, MAPT oligonucleotides were diluted in ULTRAPURE ™ Distilled Water (Invitrogen, USA) to desired concentrations. For transfecting MAPT siRNA, 1.5 pL of diluted oligonucleotide were incubated with 300 pL of Opti-MEM media (Gibco, USA) and 9 pL Lipofectamine RNAiMAX (Invitrogen, USA) for five minutes at room temperature. For each mixture, 250 pL were added to appropriate wells harboring the cells in the 6 well plates. The cells were further cultured under normal growth conditions for another 48 hours for downstream RNA analysis. Following the incubation, the media was discarded, the cells were washed with PBS and processed for RNA. Human SK-N-AS cells were transfected with 0.1 or 10nM siRNA for 48 hours prior to collecting total cellular RNA for qPCR analysis. Error bars reflect SEM. NTC is Origene non-targeting control (SR30004).

[0118] Cultured cells were lysed with 300pl of TRIzol™ Reagent (ThermoFisher, USA) per well. RNA was extracted from the samples using Direct-zol RNA Miniprep kit (Zymo Research, USA) following manufacturer’s protocol. The quality and concentration of extracted RNA was determined using Nanodrop 2000 (ThermoFisher, USA) as described by the manufacturer prior to reverse transcription. cDNA was synthesized with qScript™ cDNA synthesis kit (Quantabio, USA) using 1000ng of total RNA based on manufacturer’s protocol. 2 pl of RT reaction containing cDNA were mixed with TaqMan® Fast Advanced MasterMix and TaqMan® Gene Expression Assays labeled with FAM for detection of MAPT (Hs00902194_m1) in Quantstudio 6 Flex (Applied Biosystems).

[0119] siMAPT-6 was identified at the most potent of the tested siRNAs, reducing MAPT mRNA levels by 63% at 0.1 nM, or 100pM (as seen in Figure 7).MGB Ref No. 32286 / 70819

[0120] siMAPT-6 was also evaluated for knockdown of Tau protein. Human SK-N-AS cells were transfected with 0.01 or 1 OnM siRNA for 72 hours prior to collecting total cellular protein for ELISA analysis. Error bars reflect SEM. NTC: Origene non-targeting control (SR30004). Statistical results obtained using a two-way ANOVA. p < 0.05(*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). n = 3.

[0121] siMAPT-6 reduces Tau protein levels at picomolar concentrations as knockdown of Tau protein with siMAPT-6 was confirmed, observing a 50% reduction of Tau protein with 100pM siMAPT-6 (as seen in Figure 8).

[0122] Similar for the evaluation of siAPP for future nonhuman primate studies, siMAPT-5 and siMAPT-6 were evaluated in African Green Monkey Vero-76 cells. African Green Monkey Vero-76 cells were cultured in DMEM (Gibco, USA) with 10% FBS (Gibco, USA) and 1x Pen / Strep (Gibco, USA) at 37 °C and 5% CO2. Cells were split upon reaching 80-90% confluency and were transfected with 0.1 or 10nM siRNA of either siMAPT-5, siMAPT-5, or control for 48 hours prior to collecting total cellular RNA for qPCR analysis (FIG. 9A) or for 72 hours prior to collecting protein for ELISA analysis (FIG. 9B).

[0123] The results of knockdown efficiency of MART mRNA are plotted in FIG. 9A and the results of knockdown efficiency of Tau protein are plotted in FIG. 9B. siMAPT-6 reduces MAPT mRNA and Tau protein in nonhuman primate cells. Transfection with siMAPT-6 demonstrated a 78% reduction of MAPT mRNA, as seen in FIG. 9A. Transfection with siMAPT-6 also demonstrated a 55% reduction of Tau protein as seen in FIG. 9B.Example 3: Co-transfection of siAPP-9 and siMAPT-6 simultaneously reduces protein levels of APP, Tau, phosphor-Tau and amyloid beta 42

[0124] While target engagement and potency of each individual siRNA are important, a priority was placed on developing a single condition that can reduce not only APP and Tau protein, but phospho-Tau and amyloid beta, which are the main drivers of AD. Importantly, phospho-Tau181 (pTau181) and phospho-Tau217 have been found to be the phosphorylated Tau residues most predictive of AD pathology.

[0125] To determine if using two different siRNAs to knock down two different targets is effective, 100pM of siAPP-9 and siMAPT-6 were co-transfected in human SK-N-AS cells for five days prior to collecting total cellular protein and media for ELISA analysis.MGB Ref No. 32286 / 70819

[0126] Transfected cells were rinsed with PBS and lysed with MPER buffer (Thermo Scientific, USA) containing protease and phosphatase inhibitor (Thermo Scientific, USA). Proteins from MPER extracts are loaded into 96-well ELISA microplates for APP (KHB0051, Thermo Scientific, USA), total Tau (KHB0041, Thermo Scientific, USA), phospho-Tau181 (KHO0631, Thermo Scientific, USA), phospho-Tau217 (TBS3293, Tribioscience, USA), and Amyloid Beta 42 (KHB3544) and processed according to manufacturer’s protocol.

[0127] Error bars reflect SEM. NTC1 is Origene non-targeting control (SR30004). NTC2: Ambion non-targeting control (AM4635). Statistical results obtained using a two-way ANOVA. p < 0.05(*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (“"). n = 4.

[0128] Protein levels of APP, Tau, pTau181, pTau217, and extracellular amyloid beta 42 were measured and the results are depicted in FIG. 10. Co-transfection of siAPP-9 and siMAPT-6 demonstrates a 77% reduction of APP protein, 61% reduction of Tau protein, 62% reduction in pTau181, 43% reduction in pTau217, and a 17% reduction was observed in extracellular amyloid beta 42. As amyloid beta is cleaved off the APP protein and accumulates extracellularly, the minimal reduction is believed to be due to accumulation of amyloid beta prior to APP protein knockdown as there was no media renewal during this five-day assay.Example 4: Design of Stringed siRNA

[0129] siRNAs consist of a sense / passenger strand that can be 15-30 nucleotides long and an antisense / guide strand that can be 10-30 nucleotides long that form a duplex as seen in the schematic of FIG. 11 A. Stringed siRNAs can be designed to use single stranded nucleotides to link two or more duplexes together. Nucleotide linked siRNAs can be synthesized by annealing two separate guide strands (seen in the schematics of FIGS. 11D-11E) to a single passenger strand (as seen in the schematic of FIG. 11 B or FIG. 11 C) containing both complementary sense sequences linked together by six or eight thymidine nucleotides. After annealing, the product is a single oligonucleotide containing two duplexed structures (siRNAs) combined by a single stranded region of DNA (as seen in the schematics of FIG. 11 F or FIG. 11 G). Each nucleotide in the final linked siRNAs (stringed siRNA) can be RNA (A, G, G, U, as depicted in the Tables herein as rA, rC, rG, or rU), DNA (A, C, G, T), and / or chemically modified nucleotides (2’-0me, 2’-Fluoro, 2’-O-MOE, etc.). Each internucleotide bond can be phosphodiester or phosphorothioate. The passenger strand can be composed of 20-200 nucleotides, each guide strand can be composed of 15-40 nucleotides, and the single stranded gaps of the passenger strand which serve as the11MGB Ref No. 32286 / 70819siRNA linkers can be composed of 2-10 nucleotides. These single stranded nucleotides that serve as the siRNA linker can be designed to be cleavable or non-cleavable.Example 5: Annealing two separate RNA guide strands to DNA / RNA chimeric passenger strands yields a single molecule

[0130] Single passenger strands were synthesized containing the passenger strand sequences for both siAPP-9 and siMAPT-6 separate by six or eight thymidine nucleotides (as seen in schematics of FIG. 11F or FIG. 11G). Exemplary sequences are identified in FIG. 12 or Table 3 above. Four stringed siRNAs were generated (e.g., String-1, String-2, String-3, and String-4). The stringed siRNAs differ in the number of thymidine nucleotides that comprise the linker.

[0131] Stringed siRNAs were synthesized by mixing equal molar amounts of a single passenger / sense strand with two separate guide / antisense strands and annealing by heating at 95°C for ten minutes prior to cooling at room temperature for one hour. Oligonucleotides were separated by size by loading 9pg of oligonucleotide mixed with Gel Loading Dye (6X), No SDS (New England BioLabs) into a 3% agarose gel containing SYBR Safe (ThermoFisher, USA) and running at 120V for two hours prior to imaging (Azure).

[0132] A representative photo of the agarose gel is depicted in FIG. 13. The annealed products are larger in size than the individual passenger and guide strands with no other visible bands in the lane, suggesting high annealing efficiency. Lane 1 is 6T-1 Passenger, Lane 2 is 6T-2 Passenger, Lane 3 is 8T-1 Passenger, Lane 4 is 8T-2 Passenger, Lane 5 is Guide 1, Lane 6 is Guide 2, Lane 7 is 6T-1 Annealed (String-1), Lane 8 is 6T-2 Annealed (String-2), Lane 9 is 8T-1 Annealed (String-3), Lane 10 is 8T-2 Annealed (String-4).

[0133] Passenger strands were synthesized with the passenger sequence for siAPP-9 beginning at the 5’ end and the passenger sequence for siMAPT-6 at the 3’ end and vice versa for a total of four separate passenger strands (as seen in Figure 13). Guide strands for siAPP-9 and siMAPT-6 were annealed to each individual passenger strand and ran on a 3% agarose gel for size separation. Imaging confirmed the annealed products (String-1, String-2, String-3, and String-4) were larger in size than the individual passenger strands with no other visible bands, suggesting the annealing was efficient and yielded single molecules containing two duplexes separated by a single stranded region of DNA, called stringed siRNAs (as seen in Figure 13).Example 6: APP / MAPT stringed siRNAs simultaneously knock down APP and MAPTMGB Ref No. 32286 / 70819

[0134] To test the effectiveness of the four stringed siRNAs in their knockdown efficiency, SK-N-AS cells were transfected with 10nM of each of the stringed siRNAs (e.g., strings 1 -4) for 48 hours and included a 10nM co-transfection group for comparison. After 48 hours, RNA was collected for qPCR analysis and the results are depicted in FIG. 14. Statistical results obtained using a two-way ANOVA. p < 0.050, P < 0.01 (**), p < 0.001 (*“), P < 0.0001 (““). n = 3.

[0135] Co-transfection of siRNAs targeting APR and MAPT results in simultaneous knockdown of APP and MAPT mRNA. Transfection of Stringed siRNAs knocks down APP and MAPT mRNA to the same degree as co-transfection of separate siRNAs, whereas transfection of guide strands only does not affect mRNA levels of either APP or MAPT.

[0136] For example, following transfection with stringed siRNAs, a simultaneous 95% reduction of APP mRNA and 82% reduction of MAPT mRNA was observed (as seen in Figure 14). To confirm that unannealed single stranded guide strands were not responsible for the knockdown, groups with siAPP-9 guide strand, siMAPT-6 guide strand, and a combination of each were included but observed no knockdown of either target, suggesting the final structure of stringed siRNAs is essential for dual-targeting activity.Example 7: APP / MAPT stringed siRNAs simultaneously reduce protein levels of APP, Tau, amyloid beta, and phosphor-Tau

[0137] While transfection with the stringed siRNAs reduced mRNA levels, it was not yet determined if the stringed siRNAs could reduce protein levels. SK-N-AS cells were transfected with 100pM of individual or stringed siRNAs and included a 100pM co-transfection group for comparison for five days prior to collecting protein for quantification. Statistical results obtained using a two-way ANOVA. p < 0.05(*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). n = 4.

[0138] Transfected cells are rinsed with PBS and lysed with MPER buffer (ThermoFisher, USA) containing protease and phosphatase inhibitor (ThermoFisher, USA). Proteins from MPER extracts will be size fractionated in precast 4-12% SDS-PAGE (Criterion gels, Bio-Rad) and transferred onto PVDF membranes. Primary antibody from Millipore Sigma was used to detect APP (171610) as well as GAPDH as an endogenous control (BioLegend, 607902).

[0139] All four stringed siRNAs simultaneously reduce APP, Tau, AB42, and pTau181. Following a five day incubation post-transfection with stringed siRNAs, a simultaneous reduction of protein levels of APP (-85%), Tau (-65%), AB42 (-80%), and pTau181 (-75%) was observed with all four stringed siRNAs (as seen in Figure 15).MGB Ref No. 32286 / 70819 Example 8: APP / MAPT stringed siRNAs are less toxic than individual siRNAs

[0140] The four synthetized stringed siRNAs were evaluated for toxicity in SK-N-AS cells. 10,000 cells in 100 pL culture media were plated per well in 96 well plates and incubated at 37 °C and 5% GO2 for 24 hours prior to transfection. On the day of transfection, oligonucleotides were diluted in ULTRAPURETMDistilled Water (Invitrogen, USA) to desired concentrations. For transfecting siRNA, 15 pL of diluted oligonucleotide were incubated with 15 pL of Opti-MEM media (Gibco, USA) and 0.45 pL Lipofectamine RNAiMAX (Invitrogen, USA) for five minutes at room temperature. For each mixture, 25 pL were added to appropriate wells harboring the cells in the 96 well plates. The cells were further cultured under normal growth conditions for five days prior to performing the CellTiter Gio assay (Promega) according to manufacturer’s protocol.

[0141] SK-N-AS cells were transfected with up to 1uM siRNA for five days prior to performing CellTiter Gio assay for cellular viability. While String-1 and String-3 reduce cellular viability at high concentrations, String-2 and String-4 do not (as seen in Figure 16). Furthermore, String-2 and String-4 are less toxic than the individual siRNAs alone or co-transfected, suggesting stringed siRNAs confer a safety benefit relative to traditional siRNAs.Example 9: Stringed siRNAs can be engineered to be cleavable or non-cleavable

[0142] As described above, the linker joining the two siRNAs can be engineered to be cleavable or non-cleavable. A cleavable linker would allow the stringed siRNA to be cleaved extracellularly, allowing each siRNA to be delivered to a cell. A non-cleavable linker would allow the entire stringed siRNA to be delivered to a cell where it may be cleaved within the cell.

[0143] To test this, stringed oligonucleotides with either a phosphodiester linker (cleavable) or a phosphorothioate linker (non-cleavable) were incubated with DNase at 37°C for 30 mins prior to running on a 2.5% agarose gel for size separation. Figure 17 depicts a representative image of the agarose gel.

[0144] The stringed oligonucleotide comprising the cleavable linker (phosphodiester linker) shows the stringed oligonucleotide was digested into individual oligonucleotides, as evidenced by the cleavable linker running at approximately the same size as the individual oligonucleotides. The stringed oligonucleotide comprising the non-cleavable linker (phosphorothioate linker) was not digested and ran at a higher size on the agarose gel.Example 10: Design of APP / MAPT-targeting chemically optimized Stringed siRNAsMGB Ref No. 32286 / 70819

[0145] The stringed siRNAs may be chemically optimized for efficient delivery to cells. Chemically optimized Stringed siRNAs may comprise of two or more chemically modified siRNAs linked together with single stranded chemically modified nucleic acids or any other chemical matter. Figure 18 depicts an exemplary schematic of a chemical optimized stringed siRNA for APP and MAPT.

[0146] This molecule is therefore composed of a long passenger strand that is annealed to two or more guide strands with gaps of single stranded nucleic acids or other chemical matter in between each duplex. The single stranded gaps serve as a linker of two or more siRNAs to combine multiple siRNAs into a single molecule. The passenger strand is composed of 20-200 nucleotides which can be chemically modified and the bonds connecting the nucleotides can be chemically modified as well. Each guide strand is composed of 10-40 nucleotides which can be chemically modified and the bonds connecting the nucleotides can be chemically modified as well. The single stranded gaps of the passenger strand which serve as the siRNA linkers can be composed of 2-10 nucleotides which can be chemically modified and the bonds connecting the nucleotides can be chemically modified as well. The ribose modifications can be 2’-O-methyl (OMe), 2’-fluoro (F), 2’-O-methoxyethyl (MOE), locked nucleic acids (LNA), or any other chemical modification while the internucleotide bonds can be either phosphodiester, phosphorothioate, or any other chemical modification. For example, in the schematic of Figure 18, the phosphorothioate bonds have been indicated with arrows. In some aspects, the phosphorothioate bonds may be located towards the 5’end, the 3’ end, or both the 5’ and 3’ end of the passenger strand. In some aspects, the phosphonothioate bonds in the passenger strand may be located adjacent to the single stranded nucleotide linker. In some aspects, the phosphonothioate bonds may be located towards the 5’ ends, the 3’ ends, or both the 5’ and 3’ ends at one or both of the guide strands. In some aspects, the phosphorothioate bonds may be located in the overhang regions of the guide strands.Additional Considerations

[0147] The various embodiments described above can be combined to provide further embodiments. All U. S. patents, U. S. patent application publications, U. S. patent application, foreign patents, foreign patent application and non-patent publications referred to in this specification and / or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments.MGB Ref No. 32286 / 70819

[0148] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

MGB Ref No. 32286 / 70819CLAIMSWhat is claimed is:

1. A polynucleotide comprising two or more siRNAs linked together with a single stranded nucleic acid.

2. The polynucleotide of claim 1 wherein the two or more siRNAs bind the same or bind different genes.

3. The polynucleotide of claim 1 or 2 comprising a long passenger strand that is annealed to two or more guide strands with gaps of single stranded nucleic acids in between each duplex.

4. The polynucleotide of claim 3 wherein the passenger strand comprises sense RNA and the guide strands antisense RNA.

5. The polynucleotide of claim 4 wherein the guide strands anneal to the passenger strand.

6. The polynucleotide of any one of claims 3-5, wherein the guide strands comprise 5’ and 3’ overhangs that do not anneal to the passenger strand.

7. The polynucleotide of any one of claims 4 or 5 wherein the single stranded gaps link the two or more siRNAs to combine multiple siRNAs into a single molecule.

8. The polynucleotide of any one of claims 4 or 5 wherein the passenger strand is composed of 20-200 nucleotides9. The polynucleotide of any one of claims 3-8, wherein each guide strand is composed of 15-40 nucleotides.

10. The polynucleotide of any one of claims 3-9, wherein the single stranded gaps of the passenger strand comprise 2-10 nucleotides.

11. The polynucleotide of claim 10, wherein the single stranded gaps comprise 6-8 thymidine residues.

12. The polynucleotide of any one of claims 1-11, wherein the siRNA is between 20-30 nucleotides in length.MGB Ref No. 32286 / 7081913. The polynucleotide of any one of claims 1-12 wherein the nucleotides are chemically modified.

14. The polynucleotide of claim 13, wherein the chemical modification is one or more of 2’0 methyl, 2’-fluoro, 2’-O-methoxyethyl, or locked nucleic acids.

15. The polynucleotide of any one of claims 3-9, wherein the chemical modification of the single stranded gaps is a phosphorothioate, or phosphodiester bond.

16. The polynucleotide of any one of claims 1-15 wherein the two or more siRNAs bind genes related to neurological genes.

17. The polynucleotide of claim 16, wherein the genes are amyloid precursor protein (APP) and microtubule-associated protein tau (MAPT).

18. The polynucleotide of any claims of claims 4-17, the passenger strand or guide strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1-84.

19. A composition comprising a polynucleotide of any one of claims 1-18.

20. The composition of claim 19 comprising a pharmaceutically acceptable excipient, carrier or diluent.

21. A vector comprising a polynucleotide of any one of claims 1-18.

22. The vector of claim 21, wherein the vector is a lentiviral vector, a adeno-associated virus, a retrovirus vector, a recombinant viral vector, or an adenovirus-based vector.

23. A method of treating Alzheimer’s disease, comprising administering to a subject in need thereof a polynucleotide of any one of claims 1 -18, a composition of claims 19-20 or a vector of claims 21-22.

24. The method of claim 23, wherein the polynucleotide is specific for APP and MAPT25. The method of any one of claims 23 or 24, wherein the polynucleotide reduces both MAPT and APP mRNA expression.

26. The method of any one of claims 23-25, wherein the polynucleotide reduces both phosphorylated tau and amyloid beta expression.ii

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