WO2021097155A1 - modRNAS THAT EXPRESS ANTI MULLERIAN HORMONE (AMH) AND USES THEREOF - Google Patents

Abstract

The present disclosure is directed to modRNAs encoding the anti-Mullerian hormone (AMH) methods for improving follicular health and natural conception using these modRNAs. The disclosure is also directed to methods for protecting the follicular reserve from gonadotoxic attacks from chemotherapy or radiotherapy using modRNAs encoding AMH.

Description

modRNAS THAT EXPRESS ANTI MULLERIAN HORMONE (AMH) AND USES

THEREOF

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 62/935,443, filed November 14, 2019, the entire contents of which are incorporated herein by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] The Sequence Listing in an ASCII text file, named as

37939WO_9222_02_PC_SequenceListing.txt of 6 KB, created on November 12, 2020, and submitted to the United States Patent and Trademark Office via EFS-Web, is incorporated herein by reference.

BACKGROUND

[0003] Maintenance of follicular reserve for a full reproductive lifespan requires a careful balance of inputs that activate or suppress primordial follicles (PF) mobilization. A prominent regulator of follicular growth is anti-Mullerian hormone (AMH), a secreted transforming growth factor beta (TGF ) superfamily member that was initially identified for its role in mediating regression of Mullerian ducts during male sexual development. In women, AMH is specifically expressed in granulosa cells (GC), the main accessory cell in developing follicles, and serum AMH is a common diagnostic indicator of follicular reserve. Studies of AMH function suggest that it suppresses follicular activation and/or growth, perhaps balancing positive inputs from other TGF ligands, bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9). In sheep, null mutations of BMP 15 or GDF9 result in infertility and follicle arrest at the primary stage.

In mice, GDF 9 mutants are infertile, exhibiting follicular arrest at the primary stage, and oocyte- specific overexpression of BMP15 results in accelerated folliculogenesis. In humans, mutations in BMP15 have been described in women with premature ovarian insufficiency (POI), with at least some mutant variants affecting the capacity of BMP15 to heterodimerize with GDF9.

[0004] Based on the expression pattern of AMH in the ovary, a model has emerged in which AMH produced by larger, growing follicles provides negative feedback to quiescent and/or smaller follicles to maintain a reservoir of oocytes over decades. Indeed, following transplantation of cryopreserved ovarian tissue, in which larger follicles are disproportionately lost to freeze/thaw and/or ischemia, the remaining pool of PFs is globally activated, further compounding loss of the follicular cohort as they are mobilized into a hypoxic and inflammatory milieu. To address this, the inventors have previously shown that paracrine overexpression of AMH attenuates premature activation of PFs in ovarian cortical xenografts. Kano et al. have utilized superphysiological administration of AMH to suppress follicle growth in mice for both contraception and mitigation of chemotherapy-induced gonadotoxicity. These approaches highlight the promise of therapeutic applications of AMH. Application of this suppressive input conferred by AMH could mitigate premature mobilization of follicles in the relatively scarce group of patients undergoing ovarian tissue auto-transplantation (OTAT) for the purpose of fertility preservation, as well as in the much broader population of patients undergoing alkylating agents’ chemotherapy regimen. Moreover, modulation of the local signaling milieu within the ovary could provide a means of controlling activation/growth of quiescent/early stage follicles.

[0005] ModRNA is well suited to therapeutic application in the ovary: expression of gene products is transient but sustained for about ten days, modRNA can be delivered repeatedly by either injection or topical application. The clinical administration of modRNAs can be achieved with minor modification to minimally invasive procedures that are routinely used for oocyte retrieval; and unlike other organs, in which a critical threshold of tissue function is required, preservation or activation of even a relatively small proportion of the follicular reserve can significantly improve outcomes.

SUMMARY OF THE DISCLOSURE

[0006] 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.

[0007] In some embodiments, the modRNA further comprises a 5' untranslated region (UTR). In some embodiments, the modRNA further comprises a 3' UTR.

[0008] In some embodiments, the modRNA is codon optimized. [0009] In some embodiments, the modRNA is modified to have an increased G/C content as compared to a native RNA encoding AMH.

[0010] In some embodiments, the modRNA comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] In some embodiments, the ovarian follicle cell is a granulosa cell (GC).

[0012] In some embodiments, the ovarian follicle cell is contacted by the modRNA in vitro.

[0013] In some embodiments, the ovarian follicle cell is contacted by the modRNA in vivo in a subject.

[0014] In some embodiments, the contacting is achieved by injection or by topical application of the modRNA to the subject.

[0015] In some embodiments, the subject is undergoing, will be undergoing or has undergone chemotherapy.

[0016] In some embodiments, the method further comprises performing conventional hormonal hyperstimulation and in vitro fertilization (IVF) on the subject.

[0017] Another aspect of the disclosure is directed to a modified RNA (modRNA) encoding anti-Mullerian hormone (AMH), wherein the modRNA comprises pseudouridine nucleotides and does not comprise uridine nucleotides.

[0018] In some embodiments, the modRNA further comprises a 5' untranslated region (UTR). In some embodiments, the modRNA further comprises a 3' UTR.

[0019] In some embodiments, the modRNA is codon optimized.

[0020] In some embodiments, the modRNA is modified to have an increased G/C content as compared to a native RNA encoding AMH.

[0021] In some embodiments, the modRNA comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0022] 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. [0023] In some embodiments, the modRNA further comprises a 5' untranslated region (UTR). In some embodiments, the modRNA further comprises a 3' UTR.

[0024] In some embodiments, the modRNA is codon optimized. [0025] In some embodiments, the modRNA is modified to have an increased G/C content as compared to a native RNA encoding AMH.

[0026] In some embodiments, the modRNA comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0027] In some embodiments, the administering is achieved by injecting the modRNA to the ovaries of the subject.

[0028] In some embodiments, the administering is achieved by topical application of the modRNA to the subject.

[0029] In some embodiments, the ovarian follicle cell is a granulosa cell (GC).

[0030] In some embodiments, the modRNA is expressed in the ovarian follicle cell.

[0031] In some embodiments, the gonadotoxicity is caused by chemotherapy.

[0032] In some embodiments, the gonadotoxicity is caused by radiotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0034] FIGS. 1A - II. (A) Overview of the experiment. Recombinant AMH was injected directly into mouse ovary while also introducing EdU intraperitoneally and at 24, 48, or 72 hours after injection, a second treatment of CldU was injected. (B) Specific labeling of actively proliferating cells in discrete temporal windows following AMH treatment. Upon recovery of ovaries 5 days after injection of AMH, while gross differences between conditions were not significant, ovary weight (C), primordial follicle count (D) and average follicle diameter (E) trended downward. Importantly, AMH treated ovaries showed a significantly increased frequency of proliferative cells in the 24 and 48 hour window following injection (F), and this difference was driven mostly by small follicles (<100 pm) (G), which showed significantly increased proliferation at all time periods. (H) shows follicles between 100 and 250 pm, and (I) shows follicles >250 pm. [0035] FIGS. 2A - 2B. (A) 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. (B) Principle component analysis at 24 hours following injection with recombinant or modRNA-encoded AMH. [0036] FIGS. 3A-3B. Administration of MOD RNA encoding AMH to mouse ovaries in advance of Cp markedly improved retention of primordial follicles. (A) Overview of the experiment. Mice were treated with intraovarian injection of Mod AMH 15pg-study group or saline-control, followed by either 60 mg/Kg IP Cp- chemotherapy or saline- control. The regimen was repeated a week later, and the ovary was harvested 2 weeks after the initial chemotherapy administration. Mice were euthanized, ovaries were serially sectioned and primordial follicles were counted. (B) Number of primordial follicles Average follicle counts were analyzed by Students t-test, P valve lower than 0.05 was considered statistically significant.

[0037] FIG. 4. Percent primordial follicles. Administration of MOD RNA encoding AMH to xenografted human ovarian tissue in advance of Cp retained the primordial follicle to a level similar to control. Two weeks after xenotransplantation of human ovarian frozen thawed cortical ovary fragment, a direct injection of either Mod AMH 50pg-study group, Rec AMH 50pg or saline-control, followed by either 60 mg/Kg IP Cp- chemotherapy or saline-control. The regimen was repeated a week later, and the graft was harvested 2 weeks after the initial chemotherapy administration. Mice were euthanized, grafts were serially sectioned and primordial follicles were counted. Median follicle counts with interquartile range were analyzed by Mann-Whitney U test, P valve lower than 0.05 was considered statistically significant.

[0038] 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. When comparing the subpopulations of follicles between the arm mentioned above the profile with MOD AMH+ Cp is similar to the saline+ Saline control. The Rec AMH confers a protection to a certain degree, but not to the same extent as the MOD RNA- AMH.

[0039] FIG. 6. Ratio between primordial to growing follicles in long term grafts. The ratio is higher in the groups with pretreatment of MPD-RNA. Results are in medians with interquartile range. DETAILED DESCRIPTION

[0040] Prior to the instant disclosure, a broad phase of the follicular life cycle has not been accessible to existing treatment paradigms. The instant methods and compositions provide a potential for improving endocrine function and natural conception by protecting the follicular reserve. Moreover, the instant approach is expected to decrease both expense and effort relative to ovarian tissue and/or oocyte cryopreservation as AMH modified RNA-based fertility protection (fertoprotection) is easy and has low risk. The importance of the disclosure relates to a broad scope of patients who may benefit from this approach. Local suppression of follicular activation may provide an inexpensive, minimally invasive, safe and effective alternative to more orthodox means of fertility preservation. Practically speaking, therapeutic delivery of modRNA can easily be incorporated into minimally invasive protocols used for oocyte retrieval and could address the unmet need of an increasing pool of patients who are affected by age-related or inatrogenic infertility.

[0041] As opposed to existing technologies that aim to exploit the bioactivity of AMH for therapeutic use, the unique approach for delivery of AMH to tissues described in this disclosure is based on a modified RNA (modRNA) technology.

Modified Ribonuceic acids (modRNAs) encoding AMH [0042] In connection with the present invention, a modified ribonucleic acid (a "modRNA" or a "base-modified RNA") of the instant disclosure comprises an RNA that encodes anti-Mullerian hormone (AMH).

[0043] In some embodiments, the modRNA comprises the coding region for AMH. In some embodiments, the modRNA further comprises a 5' untranslated region (UTR). A 5' UTR is transcribed, but is not translated and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide. In some embodiments, the modRNA further comprises a 3' UTR. A 3' UTR can be positioned between the translation termination codon and the end of the transcript. UTRs can have particular functions such as increasing mRNA stability or attenuating translation. Examples of 3' UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences, e.g., a nopaline synthase termination sequence.

[0044] In some embodiments, the modRNA comprises SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the modRNA comprises a sequence with at least 70% sequence identity, 80% sequence identity, 85% sequence identity , 90% sequence identity, 95% sequence identity, 99% sequence identity or more to SEQ ID NO: 1 or SEQ ID NO: 2. [0045] In some embodiments, the modRNA is a single-stranded RNA. In some embodiments, the modRNA is a messenger RNA (mRNA). In some embodiments, the modRNA of the instant disclosure is a double stranded RNA. In some embodiments, the modRNA of the instant disclosure is a linear RNA. In some embodiments, the modRNA of the instant disclosure is a circular RNA.

[0046] In some embodiments, a modRNA of the instant disclosure has a length between 50 to 15,000 nucleotides, between 50 to 10,000 nucleotides, between 500 to 10,000 nucleotides, between 500 to 5000 nucleotides, between 1000 to 2000 nucleotides.

[0047] In some embodiments, the modRNA comprises a coding RNA region that encodes an anti Mullerian Hormone (AMH).

[0048] In some embodiments, the modRNA comprises at least one structural element. In some embodiments 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.

[0049] The modRNA sequence of the instant disclosure typically contains at least one base modification, which is suitable for increasing the expression of the protein coded for by the RNA significantly as compared with the unaltered, i.e. natural (=native), RNA sequence. "A significant increase" refers to an increase in the expression of a protein by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more, as compared to expression of the native RNA sequence.

[0050] In some embodiments, the modRNA comprises a base-modified nucleotide selected from the group consisting of 2-amino-6-chloropurineriboside-5 '-triphosphate, 2- aminoadenosine-5 '-triphosphate, 2-thiocytidine-5 '-triphosphate, 2-thiouridine-5 '- triphosphate, 4-thiouridine-5 '-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5- aminoallyluridine-5 '-triphosphate, 5-bromocytidine-5 '-triphosphate, 5-bromouridine-5 '- triphosphate, 5-iodocytidine-5'-triphosphate, 5 -iodouridine-5 '-triphosphate, 5- methylcytidine-5 '-triphosphate, 5-methyluridine-5 '-triphosphate, 6-azacytidine-5 '- triphosphate, 6-azauridine-5 '-triphosphate, 6-chloropurineriboside-5'-triphosphate, 7- deazaadenosine-5 '-triphosphate, 7 -deazaguanosine-5 '-triphosphate, 8-azaadenosine-5 '- triphosphate, 8-azidoadenosine-5 '-triphosphate, benzimidazole-riboside-5 '-triphosphate,

N 1 -methyladenosine-5 '-triphosphate, N 1 -methylguanosine-5 '-triphosphate, N6- methyladenosine-5 '-triphosphate, 06-methylguanosine-5 '-triphosphate, pseudouridine- 5 '- triphosphate, puromycin-5 '-triphosphate, and xanthosine-5 '-triphosphate. [0051] In some embodiments, 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.

[0052] In a specific embodiment, all uridine nucleotides in a native RNA are substituted with pseudouridines to provide a modRNA.

[0053] Without being limited to a particular theory, the inventors attribute an increase in the expression of the protein coded for by the modRNA inter alia to the improvement in the stabilization of secondary structures and optionally to the resulting "more rigid" structure of the RNA and the increased "base stacking." For example, pseudouridine-5 '- triphosphate is known to occur naturally in structural RNAs (tRNA, rRNA and snRNA) in both eukaryotes and prokaryotes. It is assumed in this connection that pseudouridine is necessary in rRNA for stabilizing secondary structures. In the course of evolution, the amount of pseudouridine in the RNA has increased and it has been possible to show, surprisingly, that translation is dependent on the presence of pseudouridine in the tRNA and rRNA, the interaction between tRNA and mRNA presumably being increased thereby. It is also believed that increased pseudouridine content decreases the immunogenecity of a modRNA.

[0054] In some embodiments, the conversion of uridine to pseudouridine takes place posttranscriptionally by pseudouridine synthase. In some embodiments, methyl transferases catalyze the posttranscriptional modification of RNA to contain 5- methylcytidine-5'-triphosphate. A similar mechanism as for pseudouridine-5 '-triphosphate is therefore assumed for 5-methylcytidine-5'-triphosphate and the other base modifications mentioned herein, that is to say an improved stabilization of secondary structures and, based thereon, an improved translation efficiency. In addition to this structurally based increase in expression, however, a positive effect on translation is also supposed independently of the stabilization of secondary structures and a "more rigid" structure of the RNA. Further causes of increased expression are due to the lower rate of degradation of the modRNA sequences by RNAses in vitro or in vivo.

[0055] The base modification(s) of the RNA of the instant disclosure can be introduced into the RNA by means of methods known to a person skilled in the art. Suitable methods include synthesis methods using (automatic or semi-automatic) oligonucleotide synthesis devices, biochemical methods, such as in vitro transcription methods. [0056] Base modifications of RNA sequences of the instant disclosure typically occur on at least one base-modifiable nucleotide of an RNA sequence, on at least 2, 3, 4, 5, 6, 7, 8,

9 or 10 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. In other words, base modifications in an RNA sequence typically occur on at least one base-modifiable nucleotide of the modRNA sequence, on at least 10% of all base-modifiable nucleotides, more preferably on at least 25% of all base- modifiable nucleotides, on at least 50% of all base-modifiable nucleotides, on at least 75% of all base-modifiable nucleotides and on 100% of the nucleotides contained in the modRNA sequence of the instant disclosure. In some embodiments, 10%, 20%, 25%,

30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100% of the base-modifiable nucleotides of the coding region of the RNA may be substituted.

[0057] As used herein, a "base-modifiable nucleotide" refers to any nucleotide (e.g., naturally occurring (natural, native) and hence unmodified nucleotide) that is to be replaced by a base-modified nucleotide as described above. It is thereby possible for all the nucleotides of the RNA sequence to be base-modified, or only specific chosen nucleotides of the RNA sequence. If all the nucleotides of the RNA sequence are to be base-modified, then 100% of the "base-modifiable nucleotides" of the RNA sequence are all the nucleotides of the RNA sequence used. If, on the other hand, only particular chosen nucleotides of the RNA sequence are base-modified, then the chosen nucleotides are, for example, adenosine, cytidine, guanosine or uridine. Thus, for example, an adenosine of the native sequence can be replaced by a base-modified adenosine, a cytidine can be replaced by a base-modified cytidine, a uridine by a base-modified uridine and a guanosine by a base-modified guanosine. In some embodiments, 100% of the "base- modifiable nucleotides" of the RNA sequence are 100% of the adenosines, cytidines, guanosines or uridines contained in the RNA sequence used. In a specific embodiment, 100% of the "base-modifiable nucleotides" of the RNA sequence are 100% of the uridines. [0058] In some embodiments, modRNA sequences of the instant disclosure contain backbone modifications. A backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in the RNA are chemically modified. Such backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5'-0-(l- thiophosphate)).

[0059] In some embodiments, modRNA sequences of the instant disclosure contain sugar modifications. A sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides present and typically includes, without implying any limitation, sugar modifications selected from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine-5 '-triphosphate, 2'- fluoro-2'-deoxyuridine-5 '-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotide (2'- amino-2'-deoxycytidine-5 '-triphosphate, 2'-amino-2'-deoxyuridine-5 '-triphosphate), 2'-0- alkyl oligoribonucleotide, 2'-deoxy-2'-C-alkyl oligoribonucleotide (2'-0-methylcytidine- 5 '-triphosphate, 2'-methyluridine-5'-triphosphate), 2'-C-alkyl oligoribonucleotide, and isomers thereof (2'-aracytidine-5'-triphosphate, 2'-arauridine-5'-triphosphate), or azidotriphosphate (2'-azido-2'-deoxycytidine-5 '-triphosphate, 2'-azido-2'-deoxyuridine-5'- triphosphate).

[0060] In some embodiments, modRNA of the instant disclosure has a G/C content that has been changed as compared to a native sequence. In some embodiments, the G/C content for the coding region of the base-modified RNA is greater than the G/C content for the coding region of a native RNA sequence, the amino acid sequence that is coded for being unchanged as compared with the wild type, that is to say the amino acid sequence coded for by the native RNA sequence. The composition and the sequence of the various nucleotides play a large part here. In particular, sequences having an increased G(guanine)/C(cytosine) content are more stable than sequences having an increased A(adenine)/U(uracil) content. Therefore, in some embodiments, the codons are varied as compared with the wild type, while retaining the translated amino acid sequence, in such a manner that they contain an increased number of G/C nucleotides. Because several codons code for the same amino acid (due to the degeneracy of the genetic code), the codons advantageous for stability can be determined (alternative codon usage).

[0061] In the case of amino acids coded for by codons that contain solely G or C nucleotides, no modification of the codon is required. Accordingly, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) do not require any change because no A or U is present. [0062] In the following cases, the codons containing A and/or U nucleotides are changed by substitution with different codons that code for the same amino acids but do not contain A and/or U. Examples are: the codons for Pro can be changed from CCU or CCA to CCC or CCG; the codons for Arg can be changed from CGU or CGA or AGA or AGG to CGC or CGG; the codons for Ala can be changed from GCU or GCA to GCC or GCG; the codons for Gly can be changed from GGU or GGA to GGC or GGG.

[0063] In other cases, although A or U nucleotides cannot be eliminated from the codons, it is possible to reduce the A and U content by the use of codons containing fewer A and/or U nucleotides. For example: the codons for Phe can be changed from UUU to UUC; the codons for Leu can be changed from UUA, CUU or CUA to CUC or CUG; the codons for Ser can be changed from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr can be changed from UAU to UAC; the stop codon UAA can be changed to UAG or UGA; the codon for Cys can be changed from UGU to UGC; the codon His can be changed from CAU to CAC; the codon for Gin can be changed from CAA to CAG; the codons for lie can be changed from AUU or AUA to AUC; the codons for Thr can be changed from ACU or ACA to ACC or ACG; the codon for Asn can be changed from AAU to AAC; the codon for Lys can be changed from AAA to A AG; the codons for Val can be changed from GUU or GUA to GUC or GUG; the codon for Asp can be changed from GAU to GAC; the codon for Glu can be changed from GAA to GAG.

[0064] In the case of the codons for Met (AUG) and Trp (UGG), on the other hand, there is no possibility of sequence modification.

[0065] In some embodiments, the modRNAs of the instant disclosure are codon optimized for improved expression. As used herein, the term "codon optimized" refers to nucleic acid molecules that are modified based on the codon usage of the host species (e.g., human), but without altering the polypeptide sequence encoded by the nucleic acid. [0066] In some embodiments, modRNAs and modified bases described in US publication No: 2020/0047261, which is incorporated herein in its entirety, is used for the methods of the instant dsiclosure.

Subjects/Patients

[0067] In some embodiments, 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.

[0068] In some embodiments, the subject is undergoing, will be undergoing or has undergone chemotherapy. In some embodiments, the chemotherapy is a DNA damaging agent. In some embodiments, the chemotherapy is one or more drug including, but not limited to, rituximab (rituxan), carfilzomib, fludarabine, cyclophosphamide, vincristine, prednisalone. chlorambucil, ifosphamide, doxorubicin, mesalazine, thalidomide, revlimid, lenalidomide, temsirolimus, everolimus, fostamatinib, paclitaxel, docetaxel, ofatumumab, dexamethasone, bendamustine, prednisone, CAL- 101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, ritonavir, ketoconazole, an anti-VEGF antibody, herceptin, cetuximab, cisplatin, carboplatin, docetaxel, erlotinib, etopiside, 5-fluorouracil, gemcitabine, ifosphamide, imatinib mesylate (Gleevec), gefitinib, erlotinib, procarbazine, prednisone, irinotecan, leucovorin, mechlorethamine, methotrexate, oxaliplatin, paclitaxel, sorafenib, sunitinib, topotecan, and vinblastine.

[0069] In some embodiments, the subject is undergoing, will be undergoing or has undergone radiotherapy.

[0070] In some embodiments, the subject is a mammal. In a specific embodiment, the subject is a human. In some embodiments, the subject is selected is a horse, a camel, a dog, a pig, a cow, or a sheep.

Methods of expressing modRNA encoding anti-Mullerian hormone in an ovarian follicle cell

[0071] 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). In some embodiments, each uridine nucleotide of the modRNA is substituted with a pseudouridine nucleotide. In some embodiments, the modRNA comprises at least one modification or modified nucleotide as defined above. [0072] In some embodiments, the ovarian follicle cell is contacted by the modRNA in vitro. In some embodiments, the ovarian follicle cell which is contacted by the modRNA in vitro, is implanted into a subject. In some embodiments, 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).

[0073] In some embodiments, the ovarian follicle cell is contacted by the modRNA in vivo in a subject. In some embodiments, the modRNA is administered by injection. In some embodiments, the modRNA is administered systemically. In some embodiments, modRNA is delivered via the ovarian artery, the main blood supply to the ovary.

[0074] In some embodiments, the modRNA is administered locally. In some embodiments, the modRNA is administered locally using intra-ovarian injection guided by transvaginal ultrasound. Intra-ovarian injection results in sustained, local increase in bioavailability of AMH within the ovary, providing a more robust influence than recombinant protein injection, while avoiding use of integrative or immunogenic methods of delivery. As used herein, immunogenic methods of delivery refer to adeno and adeno- associated viruses is known to elicit an immune response in some cases. As used herein, integrative methods of delivery refer to methods which drive expression from transgenes that are integrated into the genome at random and can therefore result in oncogenesis. An example of integrative methods includes lentiviruses.

[0075] In some embodiments, the modRNA is injected into a follicle cell between about 1 pg/cell to about 100 pg/cell. As used herein, the term "about" refers to ±10% of a given value. In some embodiments, the modRNA is injected into a follicle cell at about 1 pg/cell, about 5 pg/cell, about 10 pg/cell, about 15 pg/cell, about 20 pg/cell, about 25 pg/cell, about 30 pg/cell, about 35 pg/cell, about 40 pg/cell, about 45 pg/cell, about 50 pg/cell, about 55 pg/cell, about 60 pg/cell, about 65 pg/cell, about 70 pg/cell, about 75 pg/cell, about 80 pg/cell, about 85 pg/cell, about 90 pg/cell, about 95 pg/cell, or about

100 pg/cell.

[0076] In some embodiments, the modRNA is administered to an ovarian follicle tissue (e.g., ovarian cortex, which is a tissue comprising at least one ovarian follicle) between about 0.1 pg/milligram (mg) of tissue to about 10 pg/mg of tissue. In some embodiments, the modRNA is administered to an ovarian follicle tissue at about 0.1 pg/mg of tissue, about 0.25 pg/mg of tissue, about 0.5 pg/mg of tissue, about 1 pg/mg of tissue, about 2 pg/mg of tissue, about 3 mg/mg of tissue, about 4 mg/mg of tissue, about 5 mg/mg of tissue, about 6 mg/mg of tissue, about 7 mg/mg of tissue, about 8 mg/mg of tissue, about 9 mg/mg of tissue, or about 10 mg/mg of tissue.

[0077] In some embodiments, the modRNA is delivered using topical application to the subject. “Topical administration” means the direct contact of a substance with tissue, such as skin or membrane, particularly the membranes surrounding the ovary.

[0078] In some embodiments, when modRNA is administered systemically or topically, the modRNA is administered at a dosage amount between 0.2 mg/kg and 20 mg/kg. In another embodiment an effective dosage of modRNA is 0.2mg/kg, 0.5mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 18 mg/kg, or 20mg/kg.

[0079] For topical administration to the skin or mucous membrane, the composition comprising the modRNA (active ingredient) is preferably prepared as ointments, tinctures, creams, gels, solution, lotions, sprays; aerosols and dry powder for inhalation, suspensions, shampoos, hair soaps, perfumes and the like. In fact, any conventional composition can be utilized in this invention. Among the preferred methods of applying the composition containing the agents of this invention is in the form of an ointment, gel, cream, lotion, spray; aerosol or dry powder for inhalation. The pharmaceutical preparation for topical administration to the skin can be prepared by mixing the aforementioned active ingredient with non-toxic, therapeutically inert, solid or liquid carriers customarily used in such preparation. These preparations generally contain 0.01 to 5.0 percent by weight, or 0.1 to 1.0 percent by weight, of the active ingredient, based on the total weight of the composition.

[0080] In preparing the topical preparations described above, additives such as preservatives, thickeners, perfumes and the like conventional in the art of pharmaceutical compounding of topical preparation can be used. In addition, conventional antioxidants or mixtures of conventional antioxidants can be incorporated into the topical preparations containing the aforementioned active agent. Among the conventional antioxidants which can be utilized in these preparations are included N-methyl-a-tocopherolamine, tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, ethoxyquin and the like. [0081] Cream-based pharmaceutical formulations containing the active agent, used in accordance with this invention, are composed of aqueous emulsions containing a fatty acid alcohol, semi-solid petroleum hydrocarbon, ethylene glycol and an emulsifying agent. [0082] Ointment formulations containing the active agent in accordance with this invention comprise admixtures of a semi-solid petroleum hydrocarbon with a solvent dispersion of the active material. Cream compositions containing the active ingredient for use in this invention preferably comprise emulsions formed from a water phase of a humectant, a viscosity stabilizer and water, an oil phase of a fatty acid alcohol, a semi solid petroleum hydrocarbon and an emulsifying agent and a phase containing the active agent dispersed in an aqueous stabilizer-buffer solution. Stabilizers may be added to the topical preparation. Any conventional stabilizer can be utilized in accordance with this invention. In the oil phase, fatty acid alcohol components function as a stabilizer. These fatty acid alcohol components function as a stabilizer. These fatty acid alcohol components are derived from the reduction of a long-chain saturated fatty acid containing at least- 14 carbon atoms.

[0083] In one embodiment, a composition comprising a modRNA is administered once to a subject in need. In one embodiment, a composition comprising a modRNA is administered more than once and intermittently. In a specific embodiment, intermittent administration is performed once every other day, every three days, every four days, every five days, or once a week. In another specific embodiment, intermittent administration is performed once every hour, every two hours, every three hours, every six hours, every ten hours, or every twelve hours.

[0084] In some embodiments, the method further comprises performing conventional hormonal hyperstimulation and in vitro fertilization (IVF) on the subject. Conventional (or "controlled") ovarian hyperstimulation is a technique used in assisted reproduction involving the use of fertility medications to induce ovulation by multiple ovarian follicles. These multiple follicles can be taken out by oocyte retrieval (egg collection) for use in in vitro fertilisation (IVF), or be given time to ovulate, resulting in superovulation which is the ovulation of a larger-than- normal number of eggs, generally at least two.

[0085] In a specific embodiment, the ovarian follicle cell is a granulosa cell (GC).

Methods for Improving Resistance of an Ovarian Follicle Cell to Gonado toxicity [0086] 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. In some embodiments, each uridine nucleotide of the modRNA is substituted with a pseudouridine nucleotide.

[0087] In some embodiments, the gonadotoxicity is caused by chemotherapy or radiotherapy.

[0088] In some embodiments, the ovarian follicle cell is contacted by the modRNA in vitro. In some embodiments, the ovarian follicle cell which is contacted by the modRNA in vitro, is implanted into a subject. In some embodiments, 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).

[0089] In some embodiments, the ovarian follicle cell is contacted by the modRNA in vivo in a subject. In some embodiments, the modRNA is administered by injection. In some embodiments, the modRNA is administered systemically.

[0090] In some embodiments, the modRNA is administered locally. In some embodiments, the modRNA is administered locally using intra-ovarian injection guided by transvaginal ultrasound.

[0091] In some embodiments, the modRNA is injected into a follicle cell between about 1 pg/cell to about 100 pg/cell. As used herein, the term "about" refers to ±10% of a given value. In some embodiments, the modRNA is injected into a follicle cell at about 1 pg/cell, about 5 pg/cell, about 10 pg/cell, about 15 pg/cell, about 20 pg/cell, about 25 pg/cell, about 30 pg/cell, about 35 pg/cell, about 40 pg/cell, about 45 pg/cell, about 50 pg/cell, about 55 pg/cell, about 60 pg/cell, about 65 pg/cell, about 70 pg/cell, about 75 pg/cell, about 80 pg/cell, about 85 pg/cell, about 90 pg/cell, about 95 pg/cell, or about

100 pg/cell.

[0092] In some embodiments, the modRNA is administered to an ovarian follicle tissue (e.g., ovarian cortex, which is a tissue comprising at least one ovarian follicle) between about 0.1 pg/milligram (mg) of tissue to about 10 pg/mg of tissue. In some embodiments, the modRNA is administered to an ovarian follicle tissue at about 0.1 pg/mg of tissue, about 0.25 pg/mg of tissue, about 0.5 pg/mg of tissue, about 1 pg/mg of tissue, about 2 pg/mg of tissue, about 3 pg/mg of tissue, about 4 pg/mg of tissue, about 5 pg/mg of tissue, about 6 pg/mg of tissue, about 7 pg/mg of tissue, about 8 pg/mg of tissue, about 9 pg/mg of tissue, or about 10 pg/mg of tissue. [0093] In some embodiments, the modRNA is delivered using topical application to the subject.

[0094] In some embodiments, when modRNA is administered systemically or topically, the modRNA is administered at a dosage amount between 0.2 mg/kg and 20 mg/kg. In another embodiment an effective dosage of modRNA is 0.2mg/kg, 0.5mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 18 mg/kg, or 20mg/kg.

[0095] For topical administration to the skin or mucous membrane, the composition comprising the modRNA (active ingredient) is preferably prepared as ointments, tinctures, creams, gels, solution, lotions, sprays; aerosols and dry powder for inhalation, suspensions, shampoos, hair soaps, perfumes and the like. In fact, any conventional composition can be utilized in this invention. Among the preferred methods of applying the composition containing the agents of this invention is in the form of an ointment, gel, cream, lotion, spray; aerosol or dry powder for inhalation. The pharmaceutical preparation for topical administration to the skin can be prepared by mixing the aforementioned active ingredient with non-toxic, therapeutically inert, solid or liquid carriers customarily used in such preparation. These preparations generally contain 0.01 to 5.0 percent by weight, or 0.1 to 1.0 percent by weight, of the active ingredient, based on the total weight of the composition.

[0096] In preparing the topical preparations described above, additives such as preservatives, thickeners, perfumes and the like conventional in the art of pharmaceutical compounding of topical preparation can be used. In addition, conventional antioxidants or mixtures of conventional antioxidants can be incorporated into the topical preparations containing the aforementioned active agent. Among the conventional antioxidants which can be utilized in these preparations are included N-methyl-a-tocopherolamine, tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, ethoxyquin and the like. [0097] Cream-based pharmaceutical formulations containing the active agent, used in accordance with this invention, are composed of aqueous emulsions containing a fatty acid alcohol, semi-solid petroleum hydrocarbon, ethylene glycol and an emulsifying agent. [0098] Ointment formulations containing the active agent in accordance with this invention comprise admixtures of a semi-solid petroleum hydrocarbon with a solvent dispersion of the active material. Cream compositions containing the active ingredient for use in this invention preferably comprise emulsions formed from a water phase of a humectant, a viscosity stabilizer and water, an oil phase of a fatty acid alcohol, a semi solid petroleum hydrocarbon and an emulsifying agent and a phase containing the active agent dispersed in an aqueous stabilizer-buffer solution. Stabilizers may be added to the topical preparation. Any conventional stabilizer can be utilized in accordance with this invention. In the oil phase, fatty acid alcohol components function as a stabilizer. These fatty acid alcohol components function as a stabilizer. These fatty acid alcohol components are derived from the reduction of a long-chain saturated fatty acid containing at least- 14 carbon atoms.

[0099] In some embodiments, the method further comprises performing conventional hormonal hyperstimulation and in vitro fertilization (IVF) on the subject. Conventional (or "controlled") ovarian hyperstimulation is a technique used in assisted reproduction involving the use of fertility medications to induce ovulation by multiple ovarian follicles. These multiple follicles can be taken out by oocyte retrieval (egg collection) for use in in vitro fertilisation (IVF), or be given time to ovulate, resulting in superovulation which is the ovulation of a larger-than- normal number of eggs, generally at least two.

[00100] In a specific embodiment, the ovarian follicle cell is a granulosa cell (GC). [00101] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs.

[00102] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The specific examples listed below are only illustrative and by no means limiting.

EXAMPLES

Example 1: Materials and Methods

[00103] Murine model: intraovarian injection (IO) (saline/murine ModRNA-AMH) followed by intraperitoneal (IP) injection 24 hours later (saline/cyclophosphamide ("Cp")). A total of 4 arms, 6 ovaries in each. Ovaries were harvested 2 weeks after the first chemotherapy injection.

[00104] 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. A total of 3 arms, 4 grafts in each group in the short term (2 weeks after first Cp injection), and 8 grafts in each arm in the long term (12 weeks after the first Cp injection).

[00105] In the murine model, 6-week old C57/B6 females were used. C57/B6 females aged 6 weeks were obtained from Jackson Laboratories, USA, and housed under specific pathogen-free conditions. Under general anesthesia, the dorsal wall was open, and injection was performed directly into the ovary under a stereoscope. After the injection, the peritoneum was sutured, and the dorsal wall skin closed with sutures.

[00106] In the xenograft model, human ovarian cortical tissue from 12- and 29-year old organ donors were co-transplanted with endothelial cells into NOD scid gamma (NSG) mice. Ovarian cortical tissue was xenotransplanted into immunocompromised mice. Human ovarian cortical tissue from 12- and 29-year-old organ donors was co-transplanted. Briefly, cryopreserved tissue was thawed rapidly, washed of cryoprotectant, and encapsulated in fibrin that was pre-mixed with a single-cell suspension of endothelial cells. Fibrin embedded tissue was then transplanted into oophorectomized mice bilaterally under the fascia of the Gluteus Maximus and the fascia, dorsal wall skin closed with sutures. Female mice were utilized to preclude the influence of male sex steroid hormones on the graft, and mice were oophorectomized to disrupt the endogenous hormonal axis and induce high system gonadotropins. Xenografted animals were maintained in sterile conditions before animals were euthanized and xenografts recovered for fixation, cryosectioning, and immunohistochemicai staining.

[00107] Cyclophosphamide (Cp) was administered at the dose of 60mg/Kg and the protocol was repeated a week later.

[00108] After harvest of the ovaries/xenografts, histologic sections using H&E staining and light microscopy were used for counting of follicles.

Example 2: Proliferative feedback response following acute treatment of ovaries with recombinant AMH

[00109] To assess the longevity of biological activity endowed by the recombinant form of AMH, the inventors monitored actively proliferating cells via intercalation of thymidine analogues EdU and CldU at defined windows. Recombinant AMH was injected directly into mouse ovary while also introducing EdU intraperitoneally and at 24, 48, or 72 hours after injection, a second treatment of CldU was injected (FIG. 1A). This labeling scheme enables specific labeling of actively proliferating cells in discrete temporal windows following AMH treatment (FIG. IB). Upon recovery of ovaries 5 days after injection of AMH, while gross differences between conditions were not significant, ovary weight (FIG. 1C), primordial follicle count (FIG. ID) and average follicle diameter (FIG. IE) trended downward. Importantly, AMH treated ovaries showed a significantly increased frequency of proliferative cells in the 24 and 48 hour window following injection (FIG. IF), and this difference was driven mostly by small follicles (<100 pm), which showed significantly increased proliferation at all time periods (FIG. 1G).

Example 3: RNA sequencing of mouse ovaries reveals sustained activity of modRNA- encoded AMH

[00110] 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

[00112] In xenografts, in the short term, retention of PF was markedly improved with ModRNA-AMH pre-treatment. Buffer-IG/Cp-IP 9.41+10.91% PF, vs 56.24+39.44% with IG- ModRNA- AMH/Cp. Notably, the percentage of PF was similar to grafts treated with buffer/saline 51.67+ 4.04% (FIG. 4 - FIG. 5). [00113] In the long-term xenografts, retention of primordial follicles was highest in grafts pretreated with MOD-RNA prior to Cp (FIG. 6).

[00114] The inventors evaluated the potential for ModRNA-AMH to confer a benefit to ovaries/ovarian tissue in the presence of CP and demonstrated retention of the PF pool in the short term and improved output in the long term. Administration of ModRNA-AMH provides an optimal mode of delivery due to its high efficiency and transient protein expression without eliciting a substantial innate immune response. Intra-ovarian administration before chemotherapy may confer a fertoprotective benefit with minimal expense and intervention.

Claims

WHAT IS CLAIMED IS:

1. 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.

2. The method of claim 1, wherein the modRNA further comprises a 5' untranslated region (UTR).

3. The method of claim 2, wherein the modRNA further comprises a 3' UTR.

4. The method of any one of claims 1-3, wherein the modRNA is codon optimized.

5. The method of any one of claims 1-4, wherein the modRNA is modified to have an increased G/C content as compared to a native RNA encoding AMH.

6. The method of claim 1, wherein the modRNA comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

7. The method of any one of claims 1-6, wherein the ovarian follicle cell is a granulosa cell (GC).

8. The method of any one of claims 1-7, wherein the cell is contacted by the modRNA in vitro.

9. The method of any one of claims 1-8, wherein the cell is contacted by the modRNA in vivo in a subject.

10. The method of claim 9, wherein the contacting is achieved by injection or by topical application of the modRNA to the subject.

11. The method of claim 9, wherein the subject is undergoing, will be undergoing or has undergone chemotherapy.

12. The method of claim 9 or claim 11, further comprising performing conventional hormonal hyperstimulation and in vitro fertilization (IVF) on the subject.

13. A modified RNA (modRNA) encoding anti-Mullerian hormone (AMH), wherein the modRNA comprises pseudouridine nucleotides and does not comprise uridine nucleotides.

14. The modRNA of claim 1, further comprising a 5' untranslated region (UTR).

15. The modRNA of claim 14, further comprising a 3' UTR.

16. The modRNA of any one of claims 13-15, wherein the modRNA is codon optimized.

17. The modRNA of any one of claims 13-16, wherein the modRNA is modified to have an increased G/C content as compared to a native RNA encoding AMH.

18. The modRNA of claim 13, wherein the modRNA comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

19. 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.

20. The method of claim 19, wherein the modRNA further comprises a 5' untranslated region (UTR).

21. The method of claim 20, wherein the modRNA further comprises a 3' UTR.

22. The method of any one of claims 19-21, wherein the modRNA is codon optimized.

23. The method of any one of claims 19-22, wherein the modRNA is modified to have an increased G/C content as compared to a native RNA encoding AMH.

24. The method of claim 19, wherein the modRNA comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

25. The method of any one of claims 19-24, wherein the administering is achieved by injecting the modRNA to the ovaries of the subject.

26. The method of any one of claims 19-24, wherein the administering is achieved by topical application of the modRNA to the subject.

27. The method of any one of claims 19-26, wherein the ovarian follicle cell is a granulosa cell (GC).

28. The method of any one of claims 19-27, wherein the modRNA is expressed in the ovarian follicle cell.

29. The method of any one of claims 19-28, wherein the gonadotoxicity is caused by chemotherapy.

30. The method of any one of claims 19-28, wherein the gonadotoxicity is caused by radiotherapy.