# US 2019/0169616 A1 · Modulatory polynucleotides

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- Number: US 2019/0169616 A1
- Document ID: US20190169616A1
- Status: Published application
- Kind: A1
- Application number: 16/302,146
- Filed: 2017-05-18
- Published: 2019-06-06
- Earliest claimed: 2016-05-18
- Assignees: Voyager Therapeutics, Inc.
- Applicants: Voyager Therapeutics, Inc.
- Inventors: Dinah Wen-Yee Sah; Jinzhao Hou; Mathieu Nonnenmacher
- Web page: https://patentgrep.com/patent/US20190169616A1

## CPC

- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/85: for animal cells
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/141: MicroRNAs, miRNAs
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2310/533: having a mismatch or nick in at least one of the strands
- C12N 2320/52: modulating the physical stability, e.g. GC-content
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

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

## Claims

### Claim 1

A modulatory polynucleotide comprising
(a) a stem and loop to form a stem-loop structure, the sequence of said stem-loop structure from 5′ to 3′ comprising:
(i) a 5′ stem arm comprising a passenger strand;
(ii) a loop region, wherein the loop region comprises a sequence selected from the group consisting of SEQ ID NO: 16, 10, 11, 12, 13, 14, 15, 17, 18, and 19;
(iii) a 3′ stem arm comprising a guide strand, wherein a uridine is present at the 5′ end of the guide strand;
(b) a first flanking region located 5′ to said stem-loop structure, wherein the first flanking region comprises a sequence selected from the group consisting of SEQ ID NO: 5, 1, 2, 3, 4, 6, 7, 8, and 9; and
(c) a second flanking region located 3′ to said stem-loop structure and wherein said second flanking region comprises a sequence is selected from the group consisting of SEQ ID NO: 21, 20, 22, 23, 24, 25, 26 and 27.

### Claim 2

A modulatory polynucleotide comprising
(a) a stem and loop to form a stem-loop structure, the sequence of said stem-loop structure from 5′ to 3′ comprising:
(i) a 5′ stem arm comprising a guide strand, wherein a uridine is present at the 5′ end of the guide strand;
(ii) a loop region, wherein the loop region comprises a sequence selected from the group consisting of SEQ ID NO: 16, 10, 11, 12, 13, 14, 15, 17, 18, and 19;
(iii) a 3′ stem arm comprising a passenger strand;
(b) a first flanking region located 5′ to said stem-loop structure, wherein the first flanking region comprises a sequence selected from the group consisting of SEQ ID NO: 5, 1, 2, 3, 4, 6, 7, 8, and 9; and
(c) a second flanking region located 3′ to said stem-loop structure and wherein said second flanking region comprises a sequence is selected from the group consisting of SEQ ID NO: 21, 20, 22, 23, 24, 25, 26 and 27.

### Claim 3 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the loop region of said stem-loop structure is derived from a canonical microRNA.

### Claim 4 (depends on 3)

The modulatory polynucleotide of claim 3, wherein the canonical microRNA is miR-22.

### Claim 5 (depends on 4)

The modulatory polynucleotide of claim 4, wherein at least one of said first or second flanking regions are derived from let-7b.

### Claim 6 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the loop region comprises SEQ ID NO: 16.

### Claim 7 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the first flanking region comprises SEQ ID NO: 5.

### Claim 8

The modulatory polunucleotide of 1 or 2, wherein the second flanking region comprises SEQ ID NO: 21.

### Claim 9 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the stem-loop structure comprises a UG motif at or near the base of the 5′ stem of the stem-loop structure.

### Claim 10 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the loop region comprises a UGUG motif at the 5′ end of said loop region.

### Claim 11 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the second region comprises a CNNC motif.

### Claim 12 (depends on 9)

The modulatory polynucleotide of claim 9, wherein the 5′ stem arm comprises a 5′ spacer region located between the UG motif and the passenger strand or the guide strand.

### Claim 13 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the 3′ stem arm comprises a 3′ spacer region, and wherein the 3′ spacer region has a length sufficient to form one helical turn.

### Claim 14 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the guide strand comprises a microRNA seed sequence between positions 2 and 9.

### Claim 15 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the guide strand comprises a microRNA seed sequence between positions 2 and 8.

### Claim 16 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the guide strand comprises a microRNA seed sequence between positions 2 and 7.

### Claim 17 (depends on 1, 2)

The modulatory polynucleotide of claim 1 or 2, wherein the guide strand is between 15-30 nucleotides in length.

### Claim 18 (depends on 17)

The modulatory polynucleotide of claim 17, wherein the guide strand is from between 21-25 nucleotides in length.

### Claim 19 (depends on 18)

The modulatory polynucleotide of claim 18, wherein the guide strand is 22 nucleotides in length.

### Claim 20 (depends on 18)

The modulatory polynucleotide of claim 18, wherein the guide strand is 21 nucleotides in length.

### Claim 21 (depends on 17)

The modulatory polynucleotide of claim 17, wherein the passenger strand is at least 70% complementary to the guide strand.

### Claim 22 (depends on 17)

The modulatory polynucleotide of claim 17, wherein the guide strand is at least 60% complementary to a target RNA, said target RNA selected from the group consisting of mammalian coding mRNA and mammalian non-coding RNA.

### Claim 23 (depends on 22)

The modulatory polynucleotide of claim 22, wherein the target RNA is a mammalian coding mRNA.

### Claim 24 (depends on 23)

The modulatory polynucleotide of claim 23, wherein the coding mRNA is expressed in a neurologic cell, tissue or organ.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)

A plasmid or vector encoding the modulatory polynucleotide of any of claims 1-24.

### Claim 26 (depends on 25)

The vector of claim 25 comprising a recombinant adeno-associated viral (AAV) construct.

### Claim 27 (depends on 26)

A recombinant AAV virus comprising the AAV construct of claim 26.

### Claim 28 (depends on 27)

The recombinant AAV virus of claim 27, comprising a capsid serotype selected from the group consisting of AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48, AAV1-8/rh.49, AAV2-15/rh.62, AAV2-3/rh.61, AAV2-4/rh.50, AAV2-5/rh.51, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/rh.52, AAV3-11/rh.53, AAV4-8/r11.64, AAV4-9/rh.54, AAV4-19/rh.55, AAV5-3/rh.57, AAV5-22/rh.58, AAV7.3/hu.7, AAV16.8/hu.10, AAV16.12/hu.11, AAV29.3/bb.1, AAV29.5/bb.2, AAV106.1/hu.37, AAV114.3/hu.40, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV161.10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17, AAV33.4/hu.15, AAV33.8/hu.16, AAV52/hu.19, AAV52.1/hu.20, AAV58.2/hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVCS, AAV-DJ, AAV-DJ8, AAVF3, AAVFS, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1/hu.1, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVN721-8/rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVCy.5, AAVcy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, cy.AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101 , AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8/rh.64, AAVLG-9/hu.39, AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAV46.2/hu.28, AAV46.6/hu.29, AAV128.1/hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV C1v1-7, AAV C1v1-8, AAV C1v1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3, AAVF4/HSC4, AAVFS/HSCS, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, AAVF9/HSC9, AAVDJ, AAVDJ-8 PHP.B, PHP.A, G2B-26, G2B-13, TH1.1-32 and/or TH1.1-35.