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Compositions and Methods for Modulating Gene Expression

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Compositions and Methods for Modulating Gene Expression (19) TZZ¥____T (11) EP 3 511 416 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 17.07.2019 Bulletin 2019/29 C12N 15/113 (2010.01) A61P 7/06 (2006.01) A61K 31/7088 (2006.01) A61K 31/712 (2006.01) (2006.01) (21) Application number: 18204559.1 A61K 31/7125 (22) Date of filing: 16.05.2013 (84) Designated Contracting States: • The General Hospital Corporation d/b/a AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Massachusetts General Hospital GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Boston, MA 02114 (US) PL PT RO RS SE SI SK SM TR (72) Inventors: (30) Priority: 16.05.2012 US 201261647949 P • Krieg, Arthur M. 16.05.2012 US 201261648016 P Wellesley, MA 02482 (US) 16.05.2012 US 201261648021 P • Subramanian, Romesh 16.05.2012 US 201261647858 P Framingham, MA 01701 (US) 16.05.2012 US 201261647925 P • McSwiggen, James 16.05.2012 US 201261647886 P Arlington, MA 02474 (US) 16.05.2012 US 201261647901 P • Lee, Jeannie T. 16.05.2012 US 201261648041 P Boston, MA 02114 (US) 16.05.2012 US 201261648051 P 16.05.2012 US 201261648058 P (74) Representative: Hoffmann Eitle 27.10.2012 US 201261719394 P Patent- und Rechtsanwälte PartmbB 14.03.2013 US 201361785832 P Arabellastraße 30 14.03.2013 US 201361785778 P 81925 München (DE) 14.03.2013 US 201361785885 P 14.03.2013 US 201361786232 P Remarks: 14.03.2013 US 201361785956 P •Thecomplete document including Reference Tables 14.03.2013 US 201361785529 P and the Sequence Listing can be downloaded from the EPO website (62) Document number(s) of the earlier application(s) in •This application was filed on 06-11-2018 as a accordance with Art. 76 EPC: divisional application to the application mentioned 13791343.0 / 2 850 189 under INID code 62. •Claims filed after the date of receipt of the divisional (71) Applicants: application (Rule 68(4) EPC). • Translate Bio MA, Inc. Cambridge, MA 02139 (US) (54) COMPOSITIONS AND METHODS FOR MODULATING GENE EXPRESSION (57) Aspects of the invention provide methods for se- sion of a target gene. Further aspects provide single lecting a candidate oligonucleotide for activating expres- stranded oligonucleotides that modulate gene expres- sion of a target gene. Further aspects of the invention sion and compositions and kits comprising the same. provide methods of selecting a set of oligonucleotides Methods for modulating gene expression using the single that is enriched in oligonucleotides that activate expres- stranded oligonucleotides are also provided. EP 3 511 416 A1 Printed by Jouve, 75001 PARIS (FR) EP 3 511 416 A1 Description CROSS-REFERENCE TO RELATED APPLICATIONS 5 [0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/648,016, entitled, "COMPOSITIONS AND METHODS FOR MODULATING GENE EXPRESSION", filed on May 16, 2012, of U.S. Provisional Application No. 61/648,021, entitled, "COMPOSITIONS AND METHODS FOR MODULATING GENE EX- PRESSION", filed on May 16, 2012, of U.S. Provisional Application No. 61/786,232, entitled, "COMPOSITIONS AND METHODS FOR MODULATING GENE EXPRESSION", filed on March 14, 2013, of U.S. Provisional Application No. 10 61/647,858, entitled, "COMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILY EXPRESSION", filed on May 16, 2012, of U.S. Provisional Application No. 61/719,394, entitled, "COMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILY EXPRESSION", filed on October 27, 2012, of U.S. Provisional Application No. 61/785,529, entitled, "COMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILY EXPRESSION", filed on March 14, 2013, of U.S. Provisional Application No. 61/647,886, entitled, "COMPOSITIONS AND METHODS 15 FOR MODULATING UTRN EXPRESSION", filed on May 16, 2012, of U.S. Provisional Application No. 61/647,901, entitled, "COMPOSITIONS AND METHODS FOR MODULATING HEMOGLOBIN GENE FAMILY EXPRESSION", filed on May 16, 2012, of U.S. Provisional Application No. 61/785,956, entitled, "COMPOSITIONS AND METHODS FOR MODULATING HEMOGLOBIN GENE FAMILY EXPRESSION", filed on March 14, 2013, of U.S. Provisional Application No. 61/647,925, entitled, "COMPOSITIONS AND METHODS FOR MODULATING ATP2A2 EXPRESSION", filed on 20 May 16, 2012, of U.S. Provisional Application No. 61/785,832, entitled, "COMPOSITIONS AND METHODS FOR MOD- ULATING ATP2A2 EXPRESSION", filed on March 14, 2013, of U.S. Provisional Application No. 61/647,949, entitled, "COMPOSITIONS AND METHODS FOR MODULATING APOA1 AND ABCA1 EXPRESSION", filed on May 16, 2012, of U.S. Provisional Application No. 61/785,778, entitled, "COMPOSITIONS AND METHODS FOR MODULATING APOA1 AND ABCA1 EXPRESSION", filed on March 14, 2013, of U.S. Provisional Application No. 61/648,041, entitled, "COM- 25 POSITIONS AND METHODS FOR MODULATING PTEN EXPRESSION", filed on May 16, 2012, of U.S. Provisional Application No. 61/785,885, entitled, "COMPOSITIONS AND METHODS FOR MODULATING PTEN EXPRESSION", filed on March 14, 2013, of U.S. Provisional Application No. 61/648,058, entitled, "COMPOSITIONS AND METHODS FOR MODULATING BDNF EXPRESSION", filed on May 16, 2012, and of U.S. Provisional Application No. 61/648,051, entitled, "COMPOSITIONS AND METHODS FOR MODULATING MECP2 EXPRESSION", filed on May 16, 2012, the 30 contents of each of which are incorporated herein by reference in their entireties. FIELD OF THE INVENTION [0002] The invention relates to oligonucleotide based compositions, as well as methods of using oligonucleotide based 35 compositions for treating disease. BACKGROUND OF THE INVENTION [0003] Transcriptome analyses have suggested that, although only 1-2% of the mammalian genome is protein coding, 40 70-90% is transcriptionally active. Recent discoveries argue that a subset of these non-protein coding transcripts play crucial roles in epigenetic regulation. In spite of their ubiquity, the structure and function of many of such transcripts remains uncharacterized. Recent studies indicate that some long non-coding RNAs function as an epigenetic regula- tor/RNA cofactor in chromatin remodeling through interactions with Polycomb repressor complex 2 (PRC2) and thus function to regulate gene expression. 45 SUMMARY OF THE INVENTION [0004] Aspects of the invention provide methods for selecting oligonucleotides for activating or enhancing expression of target genes. The methods are particularly useful for identifying candidate oligonucleotides for activating or enhancing 50 expression of target genes for which reduced expression or activity results in, or contributes to, disease. Further aspects of the invention provide methods of selecting a set of oligonucleotides that is enriched in oligonucleotides ( e.g., compared with a random selection of oligonucleotides) that activate expression of a target gene. Accordingly, the methods may be used to establish large libraries of clinical candidates that are enriched in oligonucleotides that activate gene expres- sion. Such libraries may be utilized, for example, to identify lead oligonucleotides for therapeutic development. Thus, 55 the methods provided are useful for establishing a broad platform of candidate oligonucleotides for targeting the expres- sion of most known genes, including protein coding genes. Further aspects provide single stranded oligonucleotides that modulate gene expression, and compositions and kits comprising the same. Methods for modulating gene expression using the single stranded oligonucleotides are also provided. 2 EP 3 511 416 A1 [0005] In some aspects, the invention is a method for selecting a candidate oligonucleotide for activating expression of a target gene by selecting a PRC2-associated region within a first nucleotide sequence, wherein the first nucleotide sequence maps to a position in a first chromosome between 50 kilobases upstream of a 5’-end of the target gene and 50 kilobases downstream of a 3’-end of the target gene; determining a second nucleotide sequence that is complementary 5 with at least 8 consecutive nucleotides of the PRC2-associated region; and selecting as the candidate oligonucleotide, a single stranded oligonucleotide comprising the second nucleotide sequence, wherein the oligonucleotide has at least one of following features: a) a sequence comprising 5’-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, wherein X is anchored at the 5’ end of the oligonucleotide; b) a sequence that does not comprise 10 three or more consecutive guanosine nucleotides; c) a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length to the second nucleotide sequence, that is between 50 kilobases upstream of a 5’-end of an off-target gene and 50 kilobases downstream of a 3’-end of the off-target gene; d) a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops; and/or e) a sequence that has greater than 60% G-C content. 15 [0006] In some embodiments, the single stranded oligonucleotide has only one of features a), b), c), d), and e). In some embodiments, the single stranded oligonucleotide has at least two of features a), b), c), d), and e), each inde- pendently selected. In some embodiments, the single stranded oligonucleotide has at least three of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has at least four of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide 20 has each of features a), b), c), d), and e). In certain embodiments, the oligonucleotide has the sequence 5’X-Y-Z, in which the oligonucleotide is 8-50 nucleotides in length. In some embodiments, Y is a sequence selected from Table 3.
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