Granted Patent
B2
US 12,644,123 · App. 18/778,981 · Granted Jun 2, 2026
Modulating expression of ALAS1 (5′-aminolevulinate synthase 1) gene
Inventors:
Mary-Lee Dequeant (South Boston, MA); Heidi Heath (South Boston, MA); Rachel Yuen (South Boston, MA)
Assignee:
CRISPR THERAPEUTICS AG
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Abstract
The present disclosure relates to methods, compositions and kits for treating conditions that are related to the modulation of expression of 5′-Aminolevulinate Synthase 1 (ALAS1) by gene editing.
Claims (10)
1 . A guide RNA (gRNA) for targeting a 5′-Aminolevulinate Synthase 1 (ALAS1) genomic locus, comprising a spacer sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NOs: 25-47, 83-87, and 89-112, and wherein the gRNA is capable of inducing a cutting efficiency of at least 40% for targeting the ALAS1 genomic locus.
2 . The gRNA of claim 1 , comprising a spacer sequence comprising any one of the sequences of SEQ ID NOs: 25-47, 83-87, and 89-112.
3 . The gRNA of claim 1 , wherein the gRNA comprises a spacer sequence comprising any one of the sequences of SEQ ID NOs: 25-37 and 100-112.
4 . The gRNA of claim 1 , wherein the gRNA is capable of inducing a cutting efficiency of at least 50% for targeting the ALAS1 genomic locus.
5 . The gRNA of claim 1 , wherein the gRNA is a chemically-modified gRNA.
6 . A composition, comprising:
(a) a guide RNA (gRNA) that targets a 5′-Aminolevulinate Synthase 1 (ALAS1) genomic locus, or a nucleic acid encoding the gRNA, wherein the gRNA comprises a spacer sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NOs: 25-47, 83-87, and 89-112, and is capable of inducing a cutting efficiency of at least 40% for targeting the ALAS1 genomic locus; and
(b) an endonuclease or a nucleic acid encoding an endonuclease.
7 . The composition of claim 6 , wherein the gRNA is a chemically-modified gRNA comprising one or more phosphorothioate linkages and/or one or more 2′-O-methyl nucleotides at the 3′ end, the 5′ end, or both.
8 . The composition of claim 6 , wherein the composition comprises (a) the ALAS1 gRNA and (b) the Cas9 endonuclease, and the ALAS1 gRNA and Cas9 endonuclease are formulated as a ribonucleoprotein particle (RNP).
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jun 19, 2025
From: DEQUEANT, MARY-LEE; HEATH, HEIDI; YUEN, RACHEL
To: CRISPR THERAPEUTICS AG
Reel/Frame 071459/0148 →
Continuity (4)
Provisional Application
63624610
· Jan 24, 2024
Provisional Application
63598408
· Nov 13, 2023
Provisional Application
63515045
· Jul 21, 2023
References Cited (103)
US 5173414A
· Lebkowski et al.
· 1992
[cited by applicant]
US 5658776A
· Flotte et al.
· 1997
[cited by applicant]
US 5786211A
· Johnson
· 1998
[cited by applicant]
US 5871982A
· Wilson et al.
· 1999
[cited by applicant]
US 6140081A
· Barbas
· 2000
[cited by applicant]
US 6258595B1
· Gao et al.
· 2001
[cited by applicant]
US 6453242B1
· Eisenberg et al.
· 2002
[cited by applicant]
US 6534261B1
· Cox et al.
· 2003
[cited by applicant]
US 7427396B2
· Arbetman et al.
· 2008
[cited by applicant]
US 7588772B2
· Kay et al.
· 2009
[cited by applicant]
US 7888121B2
· Urnov et al.
· 2011
[cited by applicant]
US 7972854B2
· Miller et al.
· 2011
[cited by applicant]
US 9193769B2
· Chiorini et al.
· 2015
[cited by applicant]
US 9238800B2
· Bossis et al.
· 2016
[cited by applicant]
WO WO1995013365
· 1995
[cited by applicant]
WO WO1995013392
· 1995
[cited by applicant]
WO WO1996017947
· 1996
[cited by applicant]
WO WO1997006243
· 1997
[cited by applicant]
WO WO1997008298
· 1997
[cited by applicant]
WO WO1997009441
· 1997
[cited by applicant]
WO WO1997021825
· 1997
[cited by applicant]
WO WO1998053058
· 1998
[cited by applicant]
WO WO1998053059
· 1998
[cited by applicant]
WO WO1998053060
· 1998
[cited by applicant]
WO WO1999011764
· 1999
[cited by applicant]
WO WO2001083692
· 2001
[cited by applicant]
WO WO2002016536
· 2002
[cited by applicant]
WO WO2003016496
· 2003
[cited by applicant]
WO WO2008016473
· 2008
[cited by applicant]
WO WO2008157688
· 2008
[cited by applicant]
WO WO2009149253
· 2009
[cited by applicant]
WO WO2011015347
· 2011
[cited by applicant]
WO WO2013052523
· 2013
[cited by applicant]
WO WO2013059475
· 2013
[cited by applicant]
WO WO2013151666
· 2013
[cited by applicant]
WO WO2015121501
· 2015
[cited by applicant]
WO WO2015188933
· 2015
[cited by applicant]
WO WO2016061487
· 2016
[cited by applicant]
WO WO2016193226
· 2016
[cited by applicant]
WO WO2017048843
· 2017
[cited by applicant]
WO WO2017053297
· 2017
[cited by applicant]
WO WO2017066781
· 2017
[cited by applicant]
WO WO2017066782
· 2017
[cited by applicant]
WO WO2017066789
· 2017
[cited by applicant]
WO WO2017066791
· 2017
[cited by applicant]
WO WO2017066793
· 2017
[cited by applicant]
WO WO2017066797
· 2017
[cited by applicant]
WO WO2017070632
· 2017
[cited by applicant]
WO WO2018027078
· 2018
[cited by applicant]
WO WO2018075827
· 2018
[cited by applicant]
WO WO2019108559
· 2019
[cited by applicant]
WO WO2019217942
· 2019
[cited by applicant]
WO WO2020191153
· 2020
[cited by applicant]
WO WO2020191171
· 2020
[cited by applicant]
WO WO2020191233
· 2020
[cited by applicant]
WO WO2020191234
· 2020
[cited by applicant]
WO WO2020191239
· 2020
[cited by applicant]
WO WO2020191241
· 2020
[cited by applicant]
WO WO2020191242
· 2020
[cited by applicant]
WO WO2020191243
· 2020
[cited by applicant]
WO WO2020191245
· 2020
[cited by applicant]
WO WO2020191246
· 2020
[cited by applicant]
WO WO2020191248
· 2020
[cited by applicant]
WO WO2020191249
· 2020
[cited by applicant]
WO WO2022067130
· 2022
[cited by applicant]
WO WO2022150790
· 2022
[cited by applicant]
WO WO2023015309
· 2023
[cited by applicant]
Anderson, “Acute hepatic porphyrias: current diagnosis & management,” Molecular genetics and metabolism 2019, 128(3), 219-227.
[cited by applicant]
Carter, “Adeno-associated virus vectors,” Current opinion in biotechnology 1992, 3(5), 533-539.
[cited by applicant]
Chan et al., “Preclinical development of a subcutaneous ALAS1 RNAi therapeutic for treatment of hepatic porphyrias using circulating RNA quantification,” Molecular Therapy-Nucleic Acids 2015, 4, in 9 pages.
[cited by applicant]
Chen et al., “Characterization of the hepatic transcriptome following phenobarbital induction in mice with AIP,” Molecular genetics and metabolism 2019, 128(3), 382-390.
[cited by applicant]
Clark et al., “A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors,” Gene Therapy 1996, 3(12), 1124-1132.
[cited by applicant]
Deverman et al., “Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain,” Nature biotechnology 2016, 34(2), 204-209.
[cited by applicant]
Gaudelli et al., “Programmable base editing of A⋅ T to G⋅ C in genomic DNA without DNA cleavage,” Nature 2017, 551(7681), 464-471.
[cited by applicant]
Grimm et al., “In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses,” Journal of virology 2008, 82(12), 5887-5911.
[cited by applicant]
Hermonat & Muzyczka, “Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells,” Proceedings of the National Academy of Sciences 1984, 81(2…
[cited by applicant]
Invitation to Pay Additional Fees and, Where Applicable, Protest Fee dated Nov. 20, 2024 in PCT Patent Application No. PCT/182024/057065.
[cited by applicant]
Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature 2016, 533(7603), 420-424.
[cited by applicant]
Kothadia, et al., “Acute Hepatic Porphyria,” National Library of Medicine 2024, in 8 pages. https://www.ncbi.nlm.nih.gov/books/NBK537178/.
[cited by applicant]
Lebkowski et al., “Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types,” Molecular and cellular biology 1988, in 9 pages.
[cited by applicant]
Lian et al., “Alas1 is essential for neutrophil maturation in zebrafish,” haematologica 2018, 103(11), in 11 pages.
[cited by applicant]
Mclaughlin et al., “Adeno-associated virus general transduction vectors: analysis of proviral structures,” Journal of virology 1988, 62(6), 1963-1973.
[cited by applicant]
Muzyczka, “Use of adeno-associated virus as a general transduction vector for mammalian cells,” Viral expression 1992, vectors, 97-129.
[cited by applicant]
National Institutes of Health, “Porphyria,” nih.gov 2024, in 10 pages. https://www.niddk.nih.gov/health-information/liver-disease/porphyria.
[cited by applicant]
Paul et al., “Increased viral titer through concentration of viral harvests from retroviral packaging lines,” Human gene therapy 1993, 4(5), 609-615.
[cited by applicant]
Perrin et al., “An experimental rabies vaccine produced with a new BHK-21 suspension cell culture process: use of serum-free medium and perfusion-reactor system,” Vaccine 1995, 13(13), 1244-1250.
[cited by applicant]
Pulicherla et al., “Engineering liver-detargeted AAV9 vectors for cardiac and musculoskeletal gene transfer,” Molecular Therapy 2011, 19(6), 1070-1078.
[cited by applicant]
Saitoh et al., “5-aminolevulinic acid (ALA) deficiency causes impaired glucose tolerance and insulin resistance coincident with an attenuation of mitochondrial function in aged mice,” PLoS One 2018, 13(1), in 20 pages.
[cited by applicant]
Samulski et al., “Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression,” Journal of virology 1989, 63(9), 3822-3828.
[cited by applicant]
Sardh & Harper, “RNAi therapy with givosiran significantly reduces attack rates in acute intermittent porphyria,” Journal of Internal Medicine 2022, 291(5), 593-610.
[cited by applicant]
Sato et al., “Different kinetics for the hepatic uptake of lipid nanoparticles between the apolipoprotein E/low density lipoprotein receptor and the N-acetyl-d-galactosamine/asialoglycoprotein receptor pathway,” Journal…
[cited by applicant]
Tratschin et al., “A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase,” Molecular and cellular 1984, …
[cited by applicant]
Tratschin et al., “Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells,” Molecular and cellular biology 1985, 5(11), 3251-3260.
[cited by applicant]
Van Wijk et al., “Heterozygous disruption of ALAS1 in mice causes an accelerated age-dependent reduction in free heme, but not total heme, in skeletal muscle and liver,” Archives of Biochemistry and Biophysics 2021, 697…
[cited by applicant]
Verma & Eckstein, “Modified oligonucleotides: synthesis and strategy for users,” Annual review of biochemistry 1998, 67(1), 99-134.
[cited by applicant]
Yasuda et al., “Homozygous hydroxymethylbilane synthase knock-in mice provide pathogenic insights into the severe neurological impairments present in human homozygous dominant acute intermittent porphyria,” Human molecu…
[cited by applicant]
Yasuda et al., “RNAi-mediated silencing of hepatic Alas1 effectively prevents and treats the induced acute attacks in acute intermittent porphyria mice,” Proceedings of the National Academy of Sciences 2014, 111(21), 77…
[cited by applicant]
International Search Report and Written Opinion dated Jan. 14, 2025 in PCT Application No. PCT/IB2024/057065.
[cited by applicant]