WO2021097155A1 - Modarn exprimant l'hormone anti-müllérienne (amh) et utilisations associées - Google Patents

Modarn exprimant l'hormone anti-müllérienne (amh) et utilisations associées Download PDF

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Publication number
WO2021097155A1
WO2021097155A1 PCT/US2020/060335 US2020060335W WO2021097155A1 WO 2021097155 A1 WO2021097155 A1 WO 2021097155A1 US 2020060335 W US2020060335 W US 2020060335W WO 2021097155 A1 WO2021097155 A1 WO 2021097155A1
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WO
WIPO (PCT)
Prior art keywords
modrna
amh
cell
subject
nucleotides
Prior art date
Application number
PCT/US2020/060335
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English (en)
Inventor
Daylon James
Limor MAN
Lior ZANGI
Original Assignee
Cornell University
Mount Sinai Innovation Partners
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cornell University, Mount Sinai Innovation Partners filed Critical Cornell University
Publication of WO2021097155A1 publication Critical patent/WO2021097155A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links

Definitions

  • An aspect of the disclosure is directed to a method, comprising subjecting an ovarian follicle cell to a modified RNA (modRNA) encoding anti-Mullerian hormone (AMH), wherein the modRNA comprises pseudouridine nucleotides and does not comprise uridine nucleotides.
  • modRNA modified RNA
  • AH anti-Mullerian hormone
  • the ovarian follicle cell is a granulosa cell (GC).
  • modified RNA encoding anti-Mullerian hormone (AMH)
  • AMH anti-Mullerian hormone
  • the modRNA is codon optimized.
  • the modRNA is modified to have an increased G/C content as compared to a native RNA encoding AMH.
  • the modRNA comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • Another aspect of the disclosure is directed to a method for improving resistance of an ovarian follicle cell to gonadotoxicity in a subject, comprising administering a modified RNA (modRNA) encoding anti-Mullerian hormone (AMH) to the subject, wherein the modRNA comprises pseudouridine nucleotides and does not comprise uridine nucleotides.
  • modRNA modified RNA
  • the modRNA further comprises a 5' untranslated region (UTR).
  • the modRNA further comprises a 3' UTR.
  • the modRNA is codon optimized.
  • the modRNA is modified to have an increased G/C content as compared to a native RNA encoding AMH.
  • the modRNA comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • FIGS. 5A-5D Administration of MOD AMH to xenografted human ovarian tissue in advance of Cp confers a higher retention rate of follicles, at a comparable level to control.
  • A Saline treatment (control for modRNA) followed by saline treatment (control for chemotherapy);
  • B Saline + cyclophosphamide (Cp);
  • C MOD-AMH + Cp;
  • D Recombinant AMH (Rec- AMH) + Cp.
  • the Rec AMH confers a protection to a certain degree, but not to the same extent as the MOD RNA- AMH.
  • the modRNA comprises at least one structural element.
  • the at least one structural element is selected from the group consisting of a 5'-UTR region, an upstream positioned ribosomal binding site, a 3'-UTR region, a poly-A tail and a poly-C-tail.
  • nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5 -methylcytidine-5 '-triphosphate, 7- deazaguanosine-5 '-triphosphate, 5 -bromocytidine-5 '-triphosphate, and pseudouridine-5'- triphosphate.
  • base-modifiable nucleotides on at least 10 to 20 base-modifiable nucleotides, on at least 10 to 100 base-modifiable nucleotides and on at least 10 to 200 or more base- modifiable nucleotides.
  • the subject of the instant methods is a female mammal who would benefit from AMH expression in her ovarian follicle cells. In some embodiments, the subject suffers from, or is in risk of, infertility due to low follicle reserve.
  • the ovarian follicle cell is autologous to the subject (i.e., the ovarian cell is from the same subject). In some embodiments, the ovarian follicle cell is heterologous to the subject (i.e., the ovarian cell is from a compatible donor).
  • Another aspect of the disclosure is directed to improving resistance of an ovarian follicle cell to gonadotoxicity in a subject, comprising administering a modified RNA (modRNA) encoding anti- Mullerian hormone (AMH) to the subject.
  • modRNA modified RNA
  • AMH anti- Mullerian hormone
  • each uridine nucleotide of the modRNA is substituted with a pseudouridine nucleotide.
  • the modRNA is administered locally. In some embodiments, the modRNA is administered locally using intra-ovarian injection guided by transvaginal ultrasound.
  • the ovarian follicle cell is a granulosa cell (GC).
  • GC granulosa cell
  • Murine model intraovarian injection (IO) (saline/murine ModRNA-AMH) followed by intraperitoneal (IP) injection 24 hours later (saline/cyclophosphamide (“Cp”)).
  • IO intraovarian injection
  • IP intraperitoneal
  • Cp saline/cyclophosphamide
  • Xenotransplantation model human ovarian tissue transplanted into immunocompromised mice for two weeks, followed by: intra-graft (IG) injection (buffer) followed by an IP injection 24 hours later (saline/Cp), or IG injection of human ModRNA- AMH followed by IP Cp.
  • IG intra-graft
  • IP injection 24 hours later
  • IP Cp IG injection of human ModRNA- AMH followed by IP Cp.
  • Cyclophosphamide (Cp) was administered at the dose of 60mg/Kg and the protocol was repeated a week later.
  • the inventors directly compared the biological potency of modRNA-encoded AMH to the recombinant form of the protein generated by Pepin.
  • Mouse were treated with recombinant of modRNA forms of AMH injected directly into ovaries, followed by recovery of ovaries and RNA sequencing at 24 or 72 hours following injection (FIG. 2A). While principle component analysis (FIG. 2B) and gene expression were similar at 24 hours following injection with recombinant or modRNA-encoded AMH, the expression of factors representing granulosa cell identity, steroidogenesis, proliferation, and transcriptional response were increased at 72 hours in the recombinant group, but remained suppressed in the modRNA treated ovaries.
  • Example 4 ModRNA-AMH protects mouse ovaries from cyclophosphamide [00111] In WT mice, the inventors found retention of PF similar to the controls, Saline - IO /saline -IP; 49+12.10, ModRNA- AMH/saline 53.33+7.81 compared to ModRNA- AMH/Cp 47.17+13.61. A 50% decreased was noted with saline/Cp 26.5+9.01. (FIGs. 3A- 3B)
  • Example 5 ModRNA-AMH provides superior fertoprotection relative to recombinant protein

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente divulgation concerne des modARN codant pour l'hormone anti-müllérienne (AMH), des procédés permettant d'améliorer la santé folliculaire et la conception naturelle à l'aide de ces modARN. La divulgation concerne également des procédés de protection de la réserve folliculaire contre des attaques gonadotoxiques de la chimiothérapie ou de la radiothérapie à l'aide de modARN codant pou l'AMH.
PCT/US2020/060335 2019-11-14 2020-11-13 Modarn exprimant l'hormone anti-müllérienne (amh) et utilisations associées WO2021097155A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962935443P 2019-11-14 2019-11-14
US62/935,443 2019-11-14

Publications (1)

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WO2021097155A1 true WO2021097155A1 (fr) 2021-05-20

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100047261A1 (en) * 2006-10-31 2010-02-25 Curevac Gmbh Base-modified rna for increasing the expression of a protein
US20160310574A1 (en) * 2013-12-11 2016-10-27 The General Hospital Corporation Use of mullerian inhibiting substance (mis) proteins for contraception and ovarian reserve preservation
US20190100752A1 (en) * 2016-03-10 2019-04-04 Novartis Ag Chemically modified messenger rna's
US20190321490A1 (en) * 2011-12-30 2019-10-24 Cellscript, Llc MAKING AND USING IN VITRO-SYNTHESIZED ssRNA FOR INTRODUCING INTO MAMMALIAN CELLS TO INDUCE A BIOLOGICAL OR BIOCHEMICAL EFFECT

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100047261A1 (en) * 2006-10-31 2010-02-25 Curevac Gmbh Base-modified rna for increasing the expression of a protein
US20190321490A1 (en) * 2011-12-30 2019-10-24 Cellscript, Llc MAKING AND USING IN VITRO-SYNTHESIZED ssRNA FOR INTRODUCING INTO MAMMALIAN CELLS TO INDUCE A BIOLOGICAL OR BIOCHEMICAL EFFECT
US20160310574A1 (en) * 2013-12-11 2016-10-27 The General Hospital Corporation Use of mullerian inhibiting substance (mis) proteins for contraception and ovarian reserve preservation
US20190100752A1 (en) * 2016-03-10 2019-04-04 Novartis Ag Chemically modified messenger rna's

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KANO ET AL.: "AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy", PROC NATL ACAD SCI USA, vol. 114, 30 January 2017 (2017-01-30), pages 1688 - 1697, XP055369838 *

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