The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of modulatory polynucleotides.

Provided herein are double strand DNA molecules comprising inverted repeats, expression cassette and one or more restriction sites for nicking endonucleases, the methods of use thereof, and the methods of making therefor.

The invention provides compositions and methods for reducing expression of a target gene in a cell, involving contacting a cell with an isolated double stranded nucleic acid (dsNA) in an amount effective to reduce expression of a target gene in a cell. The dsNAs of the invention possess a single stranded extension (in most embodiments, the single stranded extension comprises at least one modified nucleotide and/or phosphate back bone modification). Such single stranded extended Dicer-substrate siRNAs (DsiRNAs) were demonstrated to be effective RNA inhibitory agents compared to corresponding double stranded DsiRNAs.

The present invention relates to miRNA interference technology. More specifically the invention relates to circular miRNA sponges that carry a plurality of binding sites directed to at least two types of miRNA and separated by random, non-identical spacers, allowing for the inhibition of functional classes of m1RNAs. Preferably, the binding sites are bulged binding sites wherein each bulge is created by a one base deletion and two base mismatch at positions 9-11 nt from the 3′ end of each binding site. Preferably, each spacer is 6 to 24 nucleotides in length. Preferably, the binding sites are against miR-132 and miR-212, miR-17-5p and miR-18a-5p, or miR-20b-5p and miR-106a-5p. Construction vectors and uses of said miRNA sponges for the treatment of diseases, such as cardiomyopathy and cancer, are also disclosed.

The present invention relates to an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising engineered dual guide nucleic acids (e.g., RNAs) capable of activating a CRISPR-Associated (Cas) nuclease, such as a type V-A Cas nuclease. Also provided are methods of targeting, editing, and/or modifying a nucleic acid using the engineered CRISPR system, and compositions and cells comprising the engineered CRISPR system.

This disclosure relates to oligonucleotides, compositions and methods useful for reducing PCSK9 expression, particularly in hepatocytes. Disclosed oligonucleotides for the reduction of PCSK9 expression may be double-stranded or single-stranded, and may be modified for improved characteristics such as stronger resistance to nucleases and lower immunogenicity. Disclosed oligonucleotides for the reduction of PCSK9 expression may also include targeting ligands to target a particular cell or organ, such as the hepatocytes of the liver, and may be used to treat hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.

The invention relates to editing oligonucleotides (EONs) for binding to a target nucleic acid and recruiting an enzyme with nucleotide deamination activity to edit the target nucleic acid. The EONs carry phosphonoacetate internucleotide linkage modifications and/or unlocked nucleic acid (UNA) ribose modifications at specified positions and do not carry such modifications on positions that would lower nucleic acid editing efficiency. The selection of positions that should or should not carry a modification is based on computational modelling that revealed incompatibilities of the modifications with the enzyme with nucleotide deamination activity.

An isolated RNA molecule includes an artificial microRNA precursor comprising in the 5′.fwdarw.3′ direction: a first terminal oligonucleotide consisting of AGGCCR (SEQ ID NO: 1) or a nucleotide sequence in which one to three nucleotides in SEQ ID NO: 1 are substituted; a passenger strand oligonucleotide; a first central oligonucleotide consisting of CYG (SEQ ID NO: 2); a second central oligonucleotide consisting of a nucleotide sequence having at least 70% homology with UUGAAUAKAAAU (SEQ ID NO: 3); a third central oligonucleotide consisting of YGG (SEQ ID NO: 4); a guide strand oligonucleotide; and a second terminal oligonucleotide consisting of UGGAYYK (SEQ ID NO: 5) or a nucleotide sequence in which one to three nucleotides in SEQ ID NO: 5 are substituted.

This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof.

One aspect of the present invention relates to double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. The sense strand of the dsRNA agent comprises at least one thermally destabilizing nucleotide, and at least one said thermally destabilizing nucleotide occurring at a site opposite to the seed region (positions 2-8) of the antisense strand; and the antisense strand of the dsRNA agent comprises. at least two modified nucleotides that provide the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2′-OMe modification, wherein said modified nucleotides are separated by 11 nucleotides in length. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.