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US-12618065-B2 - MiRNA detargeting system for tissue specific interference

US12618065B2US 12618065 B2US12618065 B2US 12618065B2US-12618065-B2

Abstract

The present disclosure relates to a tissue-specific promoter system for expressing microRNA (miRNA) for RNA interference-based methods of gene therapy. In these systems, the miRNA will inhibit gene expression or replace natural miRNA expression using microRNA.

Inventors

  • Scott Quenton HARPER

Assignees

  • RESEARCH INSTITUTE AT NATIONWIDE CHILDREN'S HOSPITAL

Dates

Publication Date
20260505
Application Date
20181002

Claims (20)

  1. 1 . A method of inhibiting expression of the double homeobox 4 (DUX4) gene in a cell comprising contacting the cell with a recombinant adeno-associated virus comprising a nucleic acid comprising (a) a nucleotide sequence encoding a U6 promoter; (b) a nucleotide sequence encoding a mature guide strand of a miRNA targeting DUX4 mRNA, wherein the mature guide strand of the miRNA comprises the nucleotide sequence of SEQ ID NO: 7397 or 8147; (c) a nucleotide sequence comprising at least one copy of a detargeting sequence comprising the mir-122 binding site having the nucleotide sequence of SEQ ID NO: 5 or 66, and/or at least one copy of a detargeting sequence comprising the miR-208 binding site having the nucleotide sequence of SEQ ID NO: 6 or 67; and (d) 5-6 thymidines at the 5′ end.
  2. 2 . A method of delivering double homeobox 4 (DUX4) miRNA-encoding DNA to the skeletal muscle of an animal in need thereof, comprising contacting the skeletal muscle with a recombinant adeno-associated virus comprising a nucleic acid comprising (a) a nucleotide sequence encoding a U6 promoter; (b) a nucleotide sequence encoding a mature guide strand of a miRNA targeting DUX4 mRNA, wherein the mature guide strand of the miRNA comprises the nucleotide sequence of SEQ ID NO: 7397 or 8147; (c) a nucleotide sequence comprising at least one copy of a detargeting sequence comprising the mir-122 binding site having the nucleotide sequence of SEQ ID NO: 5 or 66, and/or at least one copy of a detargeting sequence comprising the miR-208 binding site having the nucleotide sequence of SEQ ID NO: 6 or 67; and (d) 5-6 thymidines at the 5′ end.
  3. 3 . A method of treating facioscapulohumeral muscular dystrophy in a subject comprising administering to the subject an effective amount of a recombinant adeno-associated virus comprising (a) a nucleotide sequence encoding a U6 promoter; (b) a nucleotide sequence encoding a mature guide strand of a miRNA targeting DUX4 mRNA, wherein the mature guide strand of the miRNA comprises the nucleotide sequence of SEQ ID NO: 7397 or 8147; (c) a nucleotide sequence comprising at least one copy of a detargeting sequence comprising the mir-122 binding site having the nucleotide sequence of SEQ ID NO: 5 or 66, and/or at least one copy of a detargeting sequence comprising the miR-208 binding site having the nucleotide sequence of SEQ ID NO: 6 or 67; and (d) 5-6 thymidines at the 5′ end.
  4. 4 . The method of claim 2 , wherein the recombinant adeno-associated virus is administered by intramuscular injection, transdermal transport, injection into the blood stream or injection into the liver.
  5. 5 . The method of claim 3 , wherein the recombinant adeno- associated virus is administered by intramuscular injection, transdermal transport, injection into the blood stream or injection into the liver.
  6. 6 . The method of claim 1 , wherein the U6 promoter comprises the nucleotide sequence of SEQ ID NO: 3.
  7. 7 . The method of claim 1 , wherein the nucleic acid comprises the polynucleotide sequence of any one of SEQ ID NOS: 1, 2, and 10913-10968.
  8. 8 . The method of claim 1 , wherein the adeno-associated virus (AAV) is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh.74 or AAV-B1.
  9. 9 . The method of claim 1 , wherein the adeno-associated virus (AAV) is AAV6, AAV rh.74 or AAV-B1.
  10. 10 . The method of claim 2 , wherein the U6 promoter comprises the nucleotide sequence of SEQ ID NO: 3.
  11. 11 . The method of claim 2 , wherein the nucleic acid comprises the polynucleotide sequence of any one of SEQ ID NOS: 1, 2, and 10913-10968.
  12. 12 . The method of claim 2 , wherein the adeno-associated virus (AAV) is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh.74 or AAV-B1.
  13. 13 . The method of claim 2 , wherein the adeno-associated virus (AAV) is AAV6, AAV rh.74 or AAV-B1.
  14. 14 . The method of claim 3 , wherein the U6 promoter comprises the nucleotide sequence of SEQ ID NO: 3.
  15. 15 . The method of claim 3 , wherein the nucleic acid comprises the polynucleotide sequence of any one of SEQ ID NOS: 1, 2, and 10913-10968.
  16. 16 . The method of claim 3 , wherein the adeno-associated virus (AAV) is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh.74 or AAV-B1.
  17. 17 . The method of claim 3 , wherein the adeno-associated virus (AAV) is AAV6, AAV rh.74 or AAV-B1.
  18. 18 . The method of claim 1 , wherein the U6 promoter comprises the nucleotide sequence of SEQ ID NO: 4.
  19. 19 . The method of claim 2 , wherein the U6 promoter comprises the nucleotide sequence of SEQ ID NO: 4.
  20. 20 . The method of claim 3 , wherein the U6 promoter comprises the nucleotide sequence of SEQ ID NO: 4.

Description

This application claims priority benefit of U.S. Provisional Patent Application No. 62/566,966, filed Oct. 2, 2017, which is incorporated by reference herein in its entirety. FIELD OF THE INVENTION The present disclosure relates to a tissue-specific promoter system for expressing microRNA (miRNA) for RNA interference-based methods of gene therapy. In these systems, the miRNA will inhibit gene expression or replace natural miRNA expression using microRNA. INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING This application contains, as a separate part of disclosure, a Sequence Listing in computer-readable form (Filename: 52375A_SeqListing.txt; 1,684,382 bytes—ASCII text file, created Oct. 1, 2018) which is incorporated by reference herein in its entirety. BACKGROUND RNA interference (RNAi) is a mechanism of gene regulation in eukaryotic cells that has been considered for the treatment of various diseases. RNAi refers to post-transcriptional control of gene expression mediated by microRNAs (miRNAs). Natural miRNAs are small (21-25 nucleotides), noncoding RNAs that share sequence homology and base-pair with 3′ untranslated regions of cognate messenger RNAs (mRNAs), although regulation in coding regions may also occur. The interaction between the miRNAs and mRNAs directs cellular gene silencing machinery to degrade target mRNA and/or prevent the translation of the mRNAs. The RNAi pathway is summarized in Duan (Ed.), Section 7.3 of Chapter 7 in Muscle Gene Therapy, Springer Science+Business Media, LLC (2010). As an understanding of natural RNAi pathways has developed, researchers have designed artificial miRNAs for use in regulating expression of target genes for treating disease. As described in Section 7.4 of Duan, supra, artificial miRNAs can be transcribed from DNA expression cassettes. The miRNA sequence specific for a target gene is transcribed along with sequences required to direct processing of the miRNA in a cell. Viral vectors such as adeno-associated virus have been used to deliver miRNAs to muscle [Fechner et al., J. Mol. Med., 86: 987-997 (2008)]. Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including two 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 {1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV -9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). Cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of Translational Medicine 5, 45 (2007). Isolation of the AAV-B 1 serotype is described in Choudhury et al., Mol. Therap. 24(7): 1247-57, 2016. Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992). AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV