Methods and compositions for augmenting oocyte health

Abstract

The present disclosure provides methods, compositions, and cell lines for augmenting oocyte health. The method may include introducing into the oocyte one or more of a nucleic acid comprising a coding sequence of a gene listed in any one of Tables 1-6 or 9.

Description

Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190METHODS AND COMPOSITIONS FOR AUGMENTING OOCYTE HEALTH CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(e) of provisional application 63 / 754,461, filed February 5, 2025, which application is hereby incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF XML SEQUENCE LISTING

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, "UCSF-839WO_SEQLIST.XML," created on February 4, 2026 and having a size of 137,576 bytes. The contents of the text file are incorporated by reference herein in their entirety.INTRODUCTION

[0003] Mammals are born with a limited supply of immature and dormant oocytes that are gradually recruited for growth over time. Mouse oocytes accumulate stockpiles of biosynthetic components as they increase in volume approximately 300-fold (Liu et al 2006). Many of these molecules are the components of general metabolic processes but others are transcripts encoded by maternal effect genes and the corresponding protein products that are specifically necessary for the oocyte-to-embryo transition (Yurttas 2010, Li et al 2010, Mitchell 2022). Identifying the minimal components necessary for proper oocyte maturation and the mechanism by which oocytes accumulate and organize these components is key to improving fertility outcomes.

[0004] Across species, a mature oocyte is the largest single cell by volume, growing between 300 to 1,000-fold larger than other diploid cells in the same organism (Hirohisa and Kitajima 2023). How oocytes grow so large, and on what timescales, differs across organisms. Yet one common feature is conserved - oocytes receive biosynthetic products from neighboring support cells (Doherty et al. 2021).

[0005] Following their differentiation in the fetal ovary, immature mouse oocytes become surrounded by a squamous layer of epithelial cells called the granulosa cells. The unit of an oocyte enclosed in granulosa cells is called a follicle. As subsets of immature follicles, called primordial follicles, are recruited to grow the oocyte, the oocyte begins secreting a layer of glycoproteins called the zona pellucida. The zona pellucida separatesAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 the oocyte from the granulosa cells. As the follicle and oocyte grow, the granulosa cells become cuboidal (primary follicle) and then proliferate to become multilayered (secondary follicle) (Fig 1A). Despite being physically separated from the oocyte by the zona pellucida, granulosa cells grow thin filopodia-like projections, called transzonal projections (TZPs), that penetrate the glycoprotein matrix and contact the oocyte (Fig IB). TZPs are conserved across mammals, from mice to primates (Fig 5 A and FIG. 5B). The follicle then grows a fluid-filled cavity called an antrum (antral follicle) and is eventually ovulated (Clarke 2017, Clarke 2022, Doherty et al. 2021) (Fig. 1A).

[0006] It is currently thought that mammalian granulosa cells provide only small molecules, like metabolic precursors, to the oocyte via gap junction connections at the tips of TZPs. Therefore, all macromolecules larger than 1 kDa, the upper size limit of a gap junction pore (Giepmans 2004), present in the oocyte cytoplasm are synthesized by the oocyte itself. For example, GFP protein is approximately 27 kDa and GFP mRNA is approximately 234,000 kDa, both too large to fit through a gap junction (Uckert et al. 2000, ThermoFisher DNA and RNA Molecular Weights and Conversions). However, the demonstration that GFP-tagged proteins expressed in cultured granulosa cells can be detected in bovine oocytes raises the possibility that transcripts or protein are transferred to the oocyte (Macaulay et al. 2014, Neonene et al. 2023).

[0007] In Drosophila, oocytes rely on support cells to synthesize many of the components and maternal effect transcripts present in oocytes. The Drosophila oocyte requires this support because it is paused in diplotene of prophase I and is largely transcriptionally quiescent. Mouse oocytes are also paused in the diplotene stage of prophase I from primordial stages until germinal vesicle breakdown (Eppig and O'Brien 1996).SUMMARY

[0008] The present disclosure provides methods and compositions for augmenting oocyte health. The method may include introducing into the oocyte one or more of a nucleic acid comprising a coding sequence of a gene listed in any one of Tables 1-6 or 9.

[0009] The method may include introducing into the oocyte one or more of: i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of aAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6; ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4; iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9; iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1; v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5.

[0010] The oocyte may be an isolated ex vivo oocyte, an ex vivo oocyte surrounded by granulosa cells, present in an ex vivo follicle, present in an ex vivo ovarian tissue, or an ex vivo oocyte present in an ovarian organoid, wherein the oocyte is from a human subject or derived in vitro from a pluripotent cell such as an embryonic stem cell or induced pluripotent stem cell.

[0011] In the method of the present disclosure, the introducing may comprise injecting, electroporating, transfecting, transducing, or using a gene gun for introducing the nucleic acid or protein into an ovary comprising the oocyte, into a follicle comprising the oocyte, into granulosa cells surrounding the oocyte, or into the oocyte of a subject, wherein the oocyte is in vivo. In the method of the present disclosure, the subject may be an adult human female suffering from infertility. The subject may be an adult human female older than 30 years or 35 years, e.g., up to 40 years old, up to 45 years old, up to 50 years old, orAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 up to 55 years old. Alternatively, the subject may be an younger adult human female suffering from subfertility or infertility, e.g., 35 years or younger.

[0012] In the method of the present disclosure, the introducing may comprise injecting the nucleic acid or protein into an isolated follicle comprising the oocyte, injecting the nucleic acid or protein into granulosa cells in an isolated follicle comprising the oocyte, or into an isolated oocyte. In the method of the present disclosure, the follicle may be a pre-antral follicle or an antral follicle. The oocyte in the follicle may be immature or mature. The oocyte or follicle may be recovered after priming with hormones such as gonadotropins or without hormone priming. The follicle may be a human follicle. The oocyte may be a human oocyte.

[0013] The method of the present disclosure may involve introducing the nucleic acid(s) or protein(s) disclosed herein into an oocyte (e.g., a human oocyte) via microinjection, electroporation, or injection (e.g., injecting an nnRNA or a mixture of different mRNAs).

[0014] The method of the present disclosure may involve introducing the nucleic acid(s) or protein(s) disclosed herein into granulosa cells surrounding an oocyte (e.g., a human oocyte) via electroporation, lipofection, or viral transduction.

[0015] In the method of the present disclosure, the nucleic acid of the present disclosure may be RNA or DNA. The nucleic acid may be present in a composition comprising carriers or vehicles, e.g., liposomes.

[0016] In the method of the present disclosure, the method may further comprise introducing one or more RNA-binding proteins (RBPs) into the oocyte, where the RBPs are introduced priorto, simultaneously, or after introducing the nucleic acid of protein. In some cases, the RBPs may be selected from RBPs listed in Table 7. The RBPs may be an RBP that stabilizes RNA and regulates RNA translation and trafficking.

[0017] In the method of the present disclosure, the method may further comprise introducing a GTPase into the oocyte, where the GTPase is introduced prior to, simultaneously, or after introducing the nucleic acid of protein. The GTPase may be Dynamin 2.

[0018] In the method of the present disclosure, the nucleic acid or protein described herein may be present in a lipid nanoparticle (LNP), a micelle, a liposome, or a vesicle.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0019] In the method of the present disclosure, the nucleic acid described herein may be present in a viral expression vector. The viral expression vector may be lentiviral or adenoviral vector.

[0020] Augmenting oocyte health may comprise increased fertility of the female subject as compared to fertility in absence of the introducing described herein. Increased fertility may include improved oocyte maturation, increase in number of mature oocytes, increased rate of fertilization, improved embryo development upon fertilization of the oocyte, and / or increase in live birth. For example, augmenting oocyte health may comprise improved oocyte maturation as compared oocyte maturation in absence of the introducing described herein. Augmenting oocyte health may comprise improved embryo development upon fertilization of the oocyte in absence of the introducing described herein. Increased oocyte health may be measured by observing in vitro maturation of oocytes that have been contacted with the nucleic acid(s) (e.g., RNA(s)) and / or protein(s) as provided herein and fertilized in vitro. Maturation may be measured by cell division, e.g., number of oocytes in a two-cell stage or blastocyst stage. An increased number of oocytes in a two-cell stage or blastocyst stage as compared to control oocytes (e.g., oocytes contacted with GFP or polyA) indicates improved oocyte health. The increased number may be an increase of at least 5%, at least 10%, at least 15%, or at least 20% relative to the number of control oocytes in a two-cell stage. The increased number may be an increase of at least 5%, at least 10%, at least 15%, or at least 20% relative to the number of control oocytes in a blastocyst stage.

[0021] Aspects of the present disclosure also provide a composition for augmenting oocyte's health. The composition may include: : i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6; ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4; iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9; iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a codingAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1; v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5. The nucleic acid may be present in a lipid nanoparticle (LNP), a vesicle, or a viral expression vector, the protein may be present in a LNP or a vesicle or is conjugated to a heterologous amino acid sequence. Optionally the composition further comprises an oocyte, an ex vivo follicle comprising an oocyte, ovarian tissue comprising an oocyte, or an ovarian organoid comprising an oocyte. In the composition of the present disclosure, the viral expression vector may be lentivirus or adenovirus. In the composition of the present disclosure, the nucleic acid may be RNA or DNA. The composition of present disclosure may further comprise one or more RNA-binding proteins (RBPs). The RBPs may stabilize RNA and regulate RNA translation and trafficking. The RBPs may be selected from RBPs listed in Table 7. The composition of present disclosure may further comprise GTPase enzyme. The GTPase enzyme may be Dynamin 2.

[0022] The nucleic acid(s) and / or protein(s) for increasing oocyte health as provided herein may be introduced directly into the oocyte and / or may be introduced indirectly into the oocyte via surrounding support cells, such as granulosa cells. The granulosa cells and / or oocyte may be contacted with the nucleic acid(s) and / or protein(s). The granulosa cells may be cells of a granulosa cell line. The granulosa cell line may be genetically modified to overexpress one or more of the nucleic acid(s) and / or protein(s) for increasing oocyte health as provided herein. In one particular example, the granulosa cell line may be genetically modified to overexpress one, two, three, or four of the genes: Zp2, Rpsa, Npml and Srd5a3, thereby providing the corresponding mRNAs to the oocyte.BRIEF DESCRIPTION OF THE FIGURESAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0023] FIGS. 1A-1E d epict follicle structure and oocyte reaggregation.

[0024] FIG. 1A depicts primordial follicles, primary follicles, secondary follicles, and antral follicles. Primordial follicles are quiescent oocytes present from birth that are surrounded by a layer of squamous granulosa cells. Primary follicles are growing follicles that have begun secreting a layer of glycoprotein, the zona pellucida, that separates the oocyte from the granulosa cells. To maintain contact with oocytes across the zona pellucida, granulosa cells grow thin, filapodia-like projections, called transzonal projections (TZPs). Secondary follicles have multiple layers of granulosa cells. Both inner and outer granulosa cells grow TZPs that contact the oocyte. Antral follicles grow a large fluid-filled sac and are the final stage of oogenesis prior to ovulation.

[0025] FIG. IB shows NSPARC z-projection of a mouse multilayered secondary follicle. Granulosa cells nuclei marked with DAPI in cyan and transzonal projections (TZPs) marked with pha I loidin in grayscale. A magnification ( B') highlights that TZPs grow to contact the oocyte from both the inner and outer layers of granulosa cells. Scale bars 10 um.

[0026] FIG. 1C shows wholemount confocal z-projection of secondary follicles isolated 16 hours after IP injection with ethynyl uridine (EU). DNA labelled with DAPI and nascent RNA labelled with EU. Nascent RNA does not appear in the oocyte nucleus (outlined in white dashes) however it does appear in small puncta at the oocyte cortex seen in the magnification (C') noted with arrowheads. Scale bars = 5 um.

[0027] FIG. ID shows re-aggregation scheme whereby unlabeled oocytes are reaggregated with granulosa cells labelled with EU. The granulosa cells were isolated 16 hours after IP injection of EU and reaggregated with naive oocytes for six days before detection with Click-iT kit.

[0028] FIG. IE shows wholemount z-projection of granulosa-oocyte reaggregates. EU -labelled puncta appears in the oocytes, indicating transfer of RNA from granulosa cells to oocytes. Scale bar = 5 pm.

[0029] FIGS.2A-2C show identification of specific transferred transcripts. FIG.2A shows schematic of chimeric follicle reaggregation combining B6 oocytes with cast granulosa cells. FIG.2B shows out of the top 500 genes with most abundant reads, 273 were present in all three replicates.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0030] FIG. 3 shows RNA-Binding Proteins that may be involved in RNA transfer. FIG. 3A shows TARDBP and FIG. 3B shows FMRP-TZP co-localization.

[0031] FIG. 4. Vesicles co-localize with TZPs. Wholemount confocal slice and magnification (A') of a follicle expressing the transgene mTmG, labeling membranes and vesicles with tdTomato in orange. TZPs labeled with phalloidin in grayscale and DNA labelled with DRAQ5 in cyan. Magnified image demonstrates co-localization of vesicles with TZPs. Scale bars = 5 um.

[0032] FIGS. 5A-5E show primate follicles and reaggregation experimental protocol.FIG. 5A shows confocal z-projection of a secondary baboon follicle. FIG. 5B shows confocal z-projection of a secondary human follicle. FIG. 5C shows mouse oocytes undergo the largest increase in volume between primary and antral stages. Data adapted from (Griffin et al. 2006). FIG. 5D shows brightfield image of granulosa cell-oocyte reaggregation in culture. Scale bar. FIG. 5E shows confocal z-projection of granulosa cells that have re-grown TZPs that contact oocytes. Scale bar = 10 um.

[0033] FIG. 6. Schematic of selection of genes for introduction into oocytes.

[0034] FIG. 7. Dynamics of GFP expression after mRNA delivery to cumulus-oocyte complexes (COCs).

[0035] FIG. 8. Effect of mRNA delivery to COCs on oocyte health.

[0036] FIG. 9. Effect of mRNA delivery to COCs on oocyte competence to the blastocyst stage.

[0037] FIG. 10. Delivery of Zp2 mRNA, Rpsa mRNA, Npml mRNA, and Srd5a3 mRNA increases survival of fertilized oocyte to 2-cell stage and to blastocyst stage as compared to delivery of GFP mRNA or polyA.DETAILED DESCRIPTIONOverview

[0038] As described in the Examples section, chimeric oocyte-granulosa reaggregates provide evidence that mouse oocytes receive RNAs from granulosa cells. Infertility-associated RNA binding proteins appear to facilitate the transfer across the zona pell ucida. These results suggest that oocytes and early embryos that fail to mature may not be intrinsically defective. Instead, they may have been insufficiently supported by granulosa cells during the oocyte growth process. Specific transcripts that are transferred into theAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 oocyte during maturation were identified by the inventors. Many of these identified transcripts decrease with age and correlate with reductions in transzonal projections, the protrusions that bridge the connection between encapsulating granulosa cells and the oocyte, which impairs transport to the oocyte. Therefore, the transported transcripts and their protein products can augment oocyte health.

[0039] The present disclosure provides methods and compositions for augmenting oocyte health. The method may include introducing into the oocyte one or more of: i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6; ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4; iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9; iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1; v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5.

[0040] Aspects of the present disclosure also provide a composition for augmenting oocyte's health. The composition may include: i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein theAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 protein comprises a sequence encoded by a gene listed in Table 6; ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4; iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9; iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1; v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5.

[0041] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding eitheror both of those included limits are also included in the invention.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0043] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace subject matter that are, for example, compounds that are stable compounds (i.e., compounds that can be made, isolated, characterized, and tested for biological activity). In addition, all sub-combinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. 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.

[0045] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes a plurality of such nucleic acids and reference to "a composition comprising a nucleic acid" includes reference to a composition comprising the nucleic acid as well as one or more additional nucleic acids that have different sequences. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0046] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination inAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0047] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.1. Definitions

[0048] As used herein, the terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to polymers of nucleotides of any length and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. When a nucleic acid sequence is provided, it may include thymine for a DNA sequence and that same sequence is understood to include uracil instead of thymine when it is an RNA sequence.

[0049] As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.

[0050] As used herein, the term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or RNA (e.g., mRNA) from a transcribed gene.

[0051] As used herein, the term "isolated" refers to a polypeptide, peptide, protein, polynucleotide, vector, cell, follicle, or ovary that is in a form not found in nature. An "isolated follicle" is an in vitro or ex vivo follicle. An "isolated ovary" is an in vitro or ex vivo ovary. In some aspects, isolated polypeptides, peptides, soluble proteins, polynucleotides, vectors, cells, follicle, or ovary are those which have been purified to a degree that they areAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 no longer in a form in which they are found in nature. A cell, follicle, or ovary may be isolated from a natural source or from a source that is genetically engineered.

[0052] By "an effective amount" is meant the amount of a required agent (e.g., a nucleic acid of the present disclosure) or a pharmaceutical composition of the present disclosure, comprising the agent to ameliorate a condition in subject relative, to an untreated subject. The effective amount of an agent or a composition varies depending upon the manner of administration, the age, body weight, and general health of the subject. A physician or veterinarian may decide the appropriate amount and dosage regimen.

[0053] As used herein, the term "pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in a composition and that causes no significant adverse toxicological effects to the patient.

[0054] As used herein, the term "chemically modified nucleic acid" or "chemically modified RNA" refers to nucleic acid that includes non-naturally occurring molecules, e. g., non-naturally occurring nucleotides or chemically synthesized nucleotides, non-naturally occurring phosphate moieties, etc. Accordingly, a nucleic acid or RNA comprising a modification can be chemically modified.

[0055] As used herein, the term "modified protein" or "protein comprising" a modification refers to protein that has been chemically modified and / or conjugated to another moiety. Chemical modifications include side chain modifications or non-peptide linkage. Conjugated to another moiety encompasses conjugation to a non-protein moiety (e.g., a nucleic acid) and fusion to another protein. Fusion proteins include conjugation to polypeptides that increase stability, provide a targeting moiety, increase serum half-life, etc.

[0056] As used herein, the term "combination" or "combining" refers to including therapies that can be administered separately, for example, formulated separately for separate administration and therapies that can be administered together in a single formulation (i.e., a "co-formulation").

[0057] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines,Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 bovines, ovines, mammalian farm animals, mammalian sport animals, and mammalian pets. In some cases, an "individual" is a human.

[0058] The use of the terms "a," "an," and "the," and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0059] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the embodiments of the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the embodiments of the disclosure.

[0060] As used herein, the term "about" used in connection with an amount indicates that the amount can vary by 10% of the stated amount. For example, "about 100" means an amount of from 90-110. Where about is used in the context of a range, the "about" used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and "about" used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.

[0061] The term "and / or" as used herein a phrase such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used herein a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0062] It is understood that aspects and embodiments of the present disclosure described herein include "comprising / ' "consisting," and "consisting essentially of" aspects and embodiments.

[0063] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of this disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to this disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0064] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.2. Methods and Compositions for augmenting oocyte health

[0065] The present disclosure provides methods and compositions for augmenting oocyte health. The method may include introducing into the oocyte one or more of: : i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6; ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4; iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9; iv) aAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1; v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5.

[0066] Methods for augmenting oocyte health may include introducing into the oocyte one, two, three, or four of: i) a nucleic acid encoding Zp2, ii) a nucleic acid encoding Rpsa, iii) a nucleic acid encoding Npml, iv) a nucleic acid encoding Srd5a3 or one, two, three, or four of the proteins, Zp2, Rpsa, Npml and Srd5a3. The nucleic acid may be RNA, e.g., an mRNA. The nucleic acid encoding Zp2 may comprise a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO:1 or 22. The nucleic acid encoding Npml may comprise a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO:2 or 23. The nucleic acid encoding Srd5a3 may comprise a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO:3 or 24. The nucleic acid encoding Rpsa may comprise a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO:4 or 25. The nucleic acid may be an mRNA with native 5'UTR and / or native 3' UTR or a heterologous 5'UTR and / or a heterologous 3'UTR. Any suitable UTRs that increase stability and / or translation of the mRNA may be used. The mRNA(s) may include a polyA tail, e.g., a 120 nucleotides long polyA tail (SEQ ID NO:43).

[0067] Methods for augmenting oocyte health may include introducing into the oocyte one or more of a nucleic acid comprising the coding sequence of the genes listed in Table 9. The nucleic acid may be RNA, e.g., a mRNA. The nucleic acid encoding a gene listed in Table 9 may comprise a a nucleotide sequence having at least 70%, at least 80%, at leastAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs:l-42. The nucleic acid may be an mRNA with native 5'UTR and / or native 3' UTR or a heterologous 5'UTR and / or a heterologous 3'UTR. Any suitable UTRs that increase stability and / or translation of the mRNA may be used. The mRNA(s) may include a polyA tail, e.g., a 120 nucleotides long polyA tail (SEQ ID NO:43).

[0068] Aspects of the present disclosure also provide a composition for augmenting oocyte's health. The composition may include: i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6; ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4; iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9; iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1; v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5.

[0069] The term, "a coding sequence" in the context of the nucleic acid encoding a protein and comprising a coding sequence of a gene listed in any one of Tables 1-6 or 9 or the protein encoded by a coding sequence of a gene listed in any one of Tables 1-6 or 9 refers to a coding sequence that encodes a functional protein having a sequence at leastAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 85% identical, at least 90% identical, at least 95% identical, or 100% identical to the amino acid sequence of a wild-type protein encoded by the gene. The encoded protein may also be a functional fragment of the wild-type protein.

[0070] The method of the present disclosure may comprise introducing into the oocyte one or more of a nucleic acid encoding a protein that supports pyruvate metabolism or the protein. The nucleic acid may include a coding sequence of a gene listed in Table 1. The protein may include a sequence encoded by a gene listed in Table 1. For example, the method may comprise contacting the oocyte with one or more RNAs encoding one or more proteins that supports pyruvate metabolism or with one or more proteins that supports pyruvate metabolism.

[0071] Table 1. Genes encoding proteins that support pyruvate metabolism in oocyte

[0072] The method of the present disclosure may comprise introducing into the oocyte a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein. The nucleic acid may include a coding sequence of a gene listed in Table 2. The protein present in SCMC may include a sequence encoded by a gene listed in Table 2.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 For example, the method may comprise contacting the oocyte with one or more RNAs encoding one or more proteins found in SCMC or with one or more proteins found in SCMC.

[0073] Table 2: Genes Encoding Subcortical Maternal Complex (SCMC)oocyte one or more of a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3. For example, the method may comprise contacting the oocyte with one or more RNAs encoding one or more proteins encoded by maternal effect genes (MEGs) or with one or more proteins MEGs.

[0075] Table 3: Maternal Effect Genes (excluding SCMC)Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0076] The method of the present disclosure may comprise contacting the oocyte with a nucleic acid encoding a protein associated with primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4. For example, the method may comprise contacting the oocyte with one or more RNAs encoding one or more proteins associated with POI or with one or more proteins associated with POI.

[0077] Table 4: Genes Associated with subfertility or infertility or Primary Ovarian InsufficiencyAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0078] The method of the present disclosure may comprise contacting the oocyte with a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5. For example, the method may comprise contacting the oocyte with one or more RNAs encoding one or more proteins associated with meiosis or with one or more proteins associated with meiosis.

[0079] Table 5: Genes Associated with meiosisAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0080] The method of the present disclosure may comprise contacting the oocyte with a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6. For example, the method may comprise contacting the oocyte with one or more RNAs encoding one or more proteins associated with embryonic lethality before or after implantation or with one or more proteins associated with meiosis.

[0081] Table 6: Genes Associated with Embryonic Lethality Before or After ImplantationAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0082] The method of the present disclosure may comprise introducing into the oocyte two or more, three or more, four or more, five or more, ten or more, or twenty or more of the nucleic acids or proteins as described above. In some cases, the method may comprise contacting the oocyte with a composition comprising two or more, three or more, four or more, five or more, ten or more, or twenty or more of the nucleic acids or proteins as described above, wherein the contacting is direct and / or indirect. Examples of indirect contacting includes delivering the nucleic acids or proteins to the oocyte via surrounding support cells.

[0083] In the method of the present disclosure, the introducing may include contacting the oocyte with the nucleic acid or protein, a composition comprising the nucleic acid or protein and LNP, a micelle or vesicle comprising the nucleic acid or protein, or a vector comprising the nucleic acid. The contacting may comprise injecting the nucleic acid or protein or the composition or vector into an ex vivo ovary comprising the oocyte, into an ex vivo follicle comprising the oocyte, or into ex vivo granulosa cells surrounding the oocyte. In the method of the present disclosure, the contacting may comprise injecting the composition into the oocyte, granulosa cells, follicle, or ovary of a subject.

[0084] In certain cases, the nucleic acid or protein can be injected to preantral follicle or antral-stage follicles during in vitro maturation (IVM) of the oocyte. In certain cases, the nucleic acid or protein can be introduced into preantral follicle or antral-stage follicles during in vivo using vehicles such as AAV or LNPs.

[0085] The composition may be pharmaceutically acceptable composition of the present disclosure. Generally, the pharmaceutically acceptable composition will contain about 0.1% to 95%, preferably about 0.5% to 50%, by weight of the subject one or more nucleic acids of the present disclosure, the remainder being suitable pharmaceuticalAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 excipients, carriers, etc. Dosage forms or compositions containing active ingredient in the range of 0.005% to 95% with the balance made up from non-toxic carrier can be prepared.

[0086] In the method of the present disclosure, the contacting may comprise injecting the composition into an isolated follicle comprising the oocyte, injecting the composition into granulosa cells in an isolated follicle comprising the oocyte, or into an isolated oocyte. In the method of the present disclosure, the follicle may be a pre-antral follicle. In some cases, the follicle may be an antral-stage follicle. In some cases, the follicle may be a primordial follicle. In other cases, the follicle may be a primary follicle. In still other cases, the follicle may be a secondary follicle.

[0087] In the method of the present disclosure, the subject may be an adult human female suffering from infertility. The adult human female may be a young person, e.g., younger than 35 years old or a human female older than 35 years. For example, the human female is older than 35 years, older than 36 years, older than 37 years, older than 38 years, older than 39 years, older than 40 years, older than 41 years, older than 42 years, older than 43 years, older than 44 years, older than 45 years, older than 46 years, older than 47 years, older than 48 years, older than 49 years, or older than 50 years. A female is defined as any person with ovaries or capable of producing an oocyte.

[0088] In the method of the present disclosure, the follicle may be a human follicle. In the method of the present disclosure, the follicle may be a non-human mammal follicle such as a mammalian follicle, exemplary mammal includes, but not limited to, a cow, a pig, a chicken, a horse, a dog, and a cat.

[0089] In the method of the present disclosure, the oocyte may be a human oocyte. The oocyte may be a non-human mammal oocyte, and the mammal includes, but not limited to, a cow, a pig, a chicken, a horse, a dog, and a cat.

[0090] In the method of the present disclosure, the nucleic acids of the present disclosure may be RNAs or DNA. The nucleic acids of the present disclosure may be transcripts, pre-mRNA or mRNA. In other cases, the nucleic acids of the present disclosure may be DNA comprising a promoter to control its expression in the oocyte.

[0091] In the method of the present disclosure, the method may further comprise contacting the oocyte with the nucleic acid(s) as disclosed herein and one or more RNA-binding proteins (RBPs). The RBPs may be present in a composition comprise a nucleic acid of the present disclosure, e.g., RNA and one or more RNA-binding proteins (RBPs). The RNAAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 comprising a coding sequence of a gene listed in Tables 1-5 may be in a complex with one or more RBPs. The method may comprise contacting the oocyte with ribonucleoprotein particles (RNPs) comprising one or more RNAs (e.g., comprising a coding sequence of a gene listed in Tables 1-5) and RNA-binding proteins (RBPs). The RBPs may be proteins that stabilize RNAs in addition to regulating their translation and trafficking. In some cases, the RBPs may be selected from RBPs listed in Table 7.

[0092] Table 7. RBPsAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0093] In the method of the present disclosure, a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte, a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs), a nucleic acid encoding an RNA or protein encoded by a maternal effect gene (MEG), a nucleic acid encoding an RNA or protein associated with subfertility or infertility not limited to primary ovarian insufficiency (POI), a nucleic acid encoding an RNA or protein associated with meiosis or embryonic lethality before or after implantation may be present in a lipid nanoparticle (LNP). LNP is a drug delivery vehicle for RNA therapeutics. In some cases, the nucleic acid of the present disclosure may be present in a liposome. In other cases, the nucleic acids of the present disclosure may be present in a vesicle as well as extracellular vesicles, a vector, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc.

[0094] Suitable lipid nanoparticles can include, e.g., one or more cationic lipids, lipids modified with poly(ethylene glycol) ("PEGylated lipids"), and the like. Suitable cationic lipids include, but are not limited to, XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((92,12Z)-octadeca-9,12-dienyl)tetrahydr- o-3aH-cyclopenta[d] [l,3]dioxol-5-amine)), NC98-5 (4,7,13-Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 tris(3-oxo-3-(undecylamino)propyl)-Nl,N16-diundecyl-4,7,10,13-tet- raazahexadecane-1,16-diamide), DODAP (l,2-dioleyl-3-dimethylammonium propane), HGT4003, ICE, HGT5000, cis or trans HGT5001, DOTAP (l,2-dioleyl-3-trirnethylammonium propane), DOTMA (l,2-di-0-octadecenyl-3-trimethylamrrionium propane), DLinDMA, DLin-KC2-DMA, and C12-200. Other suitable lipids that can be included in a lipid nanoparticle include, but are not limited to, DSPC (l,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (l,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol)), and cholesterol. Suitable PEGylated lipids include, e.g., PEG-DSG (l,2-Distearoyl-rac-glycero-3-methoxypolyethylene glycol conjugated to, e.g., PEG-1000, PEG-2000, PEG-5000, and the like), PEG-DMG (1,2-Dimyristoyl-rac-glycerol conjugated to PEG), and PEG-ceramides.

[0095] Vesicles comprising a nucleic acid or protein of the present disclosure may be any suitable vesicle. Vesicles may be prepared from a mammalian cell, e.g., human cells. Suitable human cells from which the vesicles can be generated include granulosa cells, cell lines made from granulosa cells, HEK 293 cells, etc. The granulosa cells may be differentiated from a pluripotent stem cell, e.g., an induced pluripotent stem cell or an embryonic stem cell. Cells may be recombinant cells genetically engineered to express an RNA or protein disclosed herein. Vesicles may be prepared from human granulosa cells genetically engineered to express an RNA or protein disclosed herein. Vesicles may be exosomes shed from cells, e.g., cells in culture, e.g., genetically engineered human granulosa cells. Vesicles may be prepared from membrane sheets generated from cells, where the membrane sheets are mixed with the DNA, RNA, or protein for augmenting oocyte health and sonicated to generate vesicles.

[0096] In the method of the present disclosure, the contacting comprises contacting with a viral expression vector that expresses the nucleic acid of the present disclosure.

[0097] The vector may be designed for delivering a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte, a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs), a nucleic acid encoding an RNA or protein encoded by a maternal effect gene (MEG), a nucleic acid encoding an RNA or protein associated with primary ovarian insufficiency (POI), and / or a nucleic acid encoding an RNAAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 or protein associated with meiosis to a cell for therapeutic purposes, such as augmenting oocyte health. Some examples of suitable viral vectors include, but are not limited to, herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. In certain cases, the viral expression vector of the present disclosure is lentivirus or adenovirus. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome.Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

[0098] In the method of the present disclosure, the vectors may be used in vitro, for example to produce RNA or DNA lacking introns or used to transfect or transform a host cell, for example, a follicle comprising the oocyte, granulosa cells surrounding the oocyte, or the oocyte of a subject. The vectors may also be adapted to be used in vivo, for example as a gene therapy vector.

[0099] In some embodiments, the expression vector comprises a nucleic acid sequence that encodes a protein that supports pyruvate metabolism in the oocyte, a protein found in a subcortical maternal complex (SCMCs), an RNA or protein encoded by a maternal effect gene (MEG), an RNA or protein associated with infertility or subfertility including primary ovarian insufficiency (POI), and / or a nucleic acid encoding an RNA or protein associated with meiosis or embryonic lethality before or after implantation of the present disclosure. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Other suitable vectors would be apparent to a person skilled in the art.

[0100] Expression vectors may be transformed into a suitable host cell to provide for expression of a polypeptide of the present disclosure. The host cell, which is transformed or transfected with an expression vector, is cultivated under conditions to allow forexpression of the polypeptide, and the expressed polypeptide is recovered by, e.g., purification. In the present disclosure, the host cell is a follicle comprising the oocyte, granulosa cells surrounding the oocyte, or the oocyte of a subject.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 [OO1O1] In the method of the present disclosure, contacting the oocyte may include contacting with a GTPase. The GTPase may be Dynamin 2. Endocytosis of vesicles requires pinching off by the membrane-remodeling GTPase, Dynamin 2. For example, the method for augmenting oocyte health may include contacting the oocyte (ex vivo or in a subject) with vesicles comprising a nucleic acid or protein as described herein and with a GTPase to enhance vesicle internalization.

[0102] In the method of the present disclosure, the augmenting oocyte health may comprise improved oocyte maturation. Also, the augmenting oocyte health may comprise improved embryo development upon fertilization of the oocyte. Improved oocyte maturation may improve outcomes from in vitro fertilization (IVF).

[0103] The method for augmenting oocyte maturation may further incubating the oocyte in the presence of a growth factor (e.g., EGF) after the introducing. The method for augmenting oocyte maturation may further include cryopreserving the oocyte.3. Composition for augmenting oocyte health

[0104] Aspects of the present disclosure also provide a composition for augmenting oocyte's health. The composition may comprise: i) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1; ii) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; iii) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; iv) a nucleic acid encoding a protein associated with primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4; v) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5; and / or vi) a nucleic acid encoding a protein associatedAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6.

[0105] The nucleic acid may be present in a lipid nanoparticle (LN P), a vesicle, or a viral expression vector. The protein may be present in a LNP or a vesicle or is conjugated to a heterologous amino acid sequence.

[0106] The composition may further comprise an oocyte, an ex vivo follicle comprising an oocyte, ovarian tissue comprising an oocyte, or an ovarian organoid comprising an oocyte.

[0107] The composition of present disclosure may comprise two or more of the nucleic acids or proteins listed in i)-v). The composition of present disclosure may comprise a nucleic acid and a protein listed in i)-v).

[0108] The composition of present disclosure may comprise two or more, three or more, four or more, five or more, ten or more, or twenty or more of the nucleic acids listed in i)-iv).

[0109] In the composition of the present disclosure, one or more nucleic acids selected from the nucleic acids encoding a protein that supports pyruvate metabolism in the oocyte, encoding a protein found in a subcortical maternal complex (SCMCs), encoding an RNA or protein encoded by a maternal effect gene (MEG), encoding an RNA or protein associated with primary ovarian insufficiency (POI), encoding an RNA or protein associated with meiosis and / or associated with embryonic lethality before or after implantation may be present in a lipid nanoparticle (LNP). LNP is a drug delivery vehicle for RNA therapeutics. In some cases, the nucleic acid of the present disclosure may be present in a liposome. In other cases, the nucleic acids of the present disclosure may be present in a vesicle as well as a vector, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc.

[0110] The composition may comprise a viral expression vector that expresses a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte, a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs), a nucleic acid encoding an RNA or protein encoded by a maternal effect gene (MEG), a nucleic acid encoding an RNA or protein associated with primary ovarian insufficiency (POI), and / or encoding an RNA or protein associated with meiosis and / or associated with embryonicAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 lethality before or after implantation. In the composition of the present disclosure, the viral expression vector may be lentivirus or adenovirus.

[0111] The vector may be designed for delivering a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte, a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs), a nucleic acid encoding an RNA or protein encoded by a maternal effect gene (MEG), a nucleic acid encoding an RNA or protein associated with primary ovarian insufficiency (POI), and / or encoding an RNA or protein associated with meiosis to a cell for therapeutic purposes, such as augmenting oocyte health. Some examples of suitable viral vectors include, but are not limited to, herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. In certain cases, the viral expression vector of the present disclosure is lentivirus or adenovirus. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome.Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

[0112] In some embodiments, the expression vector of the present disclosure comprises a nucleic acid sequence that encodes a protein that supports pyruvate metabolism in the oocyte, a protein found in a subcortical maternal complex (SCMCs), an RNA or protein encoded by a maternal effect gene (MEG), an RNA or protein associated with primary ovarian insufficiency (POI), encoding an RNA or protein associated with meiosis and / or associated with embryonic lethality before or after implantation of the present disclosure. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Other suitable vectors would be apparent to a person skilled in the art.

[0113] Expression vectors may be transformed into a suitable host cell to provide for expression of a polypeptide of the present disclosure. The host cell, which is transformed or transfected with an expression vector, is cultivated under conditions to allow forexpression of the polypeptide, and the expressed polypeptide is recovered by,Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 e.g., purification. In the present disclosure, the host cell is a follicle comprising the oocyte, granulosa cells surrounding the oocyte, or the oocyte of a subject.

[0114] The oocyte may be a human oocyte. The oocyte may be a non-human mammal oocyte, and the mammal includes, but not limited to, a cow, a pig, a chicken, a horse, a dog, camel and a cat. The oocyte may be a human oocyte isolated from a human female, e.g., an infertile female or a female older than 35 years old. The oocyte may be an oocyte derived from a human pluripotent stem cell (hPS), e.g., induced pluripotent stem cell or embryonic stem cell. The oocyte may be a freshly isolated (e.g., from a human female) or freshly generated oocyte (e.g., from in vitro differentiation of a hPS. The oocyte may be a cryopreserved oocyte.

[0115] In the composition of the present disclosure, the nucleic acids of the present disclosure may be RNAs. The nucleic acids of the present disclosure may be mRNA. The nucleic acids of the present disclosure may be granulosa-derived transcripts. In other cases, the nucleic acids of the present disclosure may be DNAs lacking introns.

[0116] The composition of present disclosure may further comprise one or more RNA-binding proteins (RBPs). When one or more nucleic acids of the present disclosure, in particular RNAs, are trafficked, they associate with RNA-binding proteins (RBPs) in phase-separated structures called ribonucleoprotein particles (RNPs). RNA-binding proteins (RBPs) stabilize RNAs in addition to regulating their translation and trafficking. The RBPs may be selected from RBPs listed in Table 7.

[0117] The composition of present disclosure may further comprise GTPase enzyme. The GTPase enzyme may be Dynamin 2. Endocytosis of vesicles requires pinching off by the membrane-remodeling GTPase, Dynamin 2. Herein, the term "Dynamin 2" refers to a large GTPase enzyme primarily responsible for the process of membrane scission, specifically during the formation of vesicles from the plasma membrane during endocytosis.

[0118] The composition of the present disclosure contributes to improved oocyte maturation. Also, the composition of the present disclosure contributes to improved embryo development upon fertilization of the oocyte.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 4. Pharmaceutical composition

[0119] Aspects of the present disclosure further provide a pharmaceutical composition comprising one or more of the compositions of the present disclosure and a pharmaceutically acceptable excipient. The one or more of compositions are described in section 3 ("3. Composition for augmenting oocyte health") above and incorporated into this section.

[0120] The terms "pharmaceutical composition" and "pharmaceutically acceptable composition" are interchangeably used herein. The pharmaceutical composition refers to a preparation that is in such form as to permit the biological activity of the agent (e.g., an antibody) to be effective. Generally, the pharmaceutically acceptable composition will contain about 0.1% to 95%, preferably about 0.5% to 50%, by weight of the subject one or more nucleic acids of the present disclosure, the remainder being suitable pharmaceutical excipients, carriers, adjuvant, buffers, etc. Dosage forms or compositions containing active ingredient in the range of 0.005% to 95% with the balance made up from non-toxic carrier can be prepared.

[0121] The terms "pharmaceutically acceptable excipient, carrier, or adjuvant" or "acceptable pharmaceutical carrier" as used herein refer to an excipient, carrier, or adjuvant that can be administered to a subject, together with at least one agent, and which does not have an effect on the pharmacological activity of the agent. In general, those of skill in the art and the U.S. FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[0122] In certain aspects, the composition comprising the nucleic acids of the present disclosure is present in a therapeutically effective amount in the pharmaceutical composition. A therapeutically effective amount can be determined based on an observed effectiveness of the composition. A therapeutically effective amount can be determined using assays that measure the desired effect in a cell, e.g., in a reporter cell line in which expression of a reporter is modulated in response to the polypeptides of the present disclosure. The pharmaceutical compositions can be administered ex vivo or in vivo to a mammal in order to practice the methods and uses described herein.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0123] The pharmaceutical compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein. Suitable pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients include, but are not limited to, nuclease inhibitors, protease inhibitors, a suitable vehicle such as physiological saline solution or citrate buffered saline.

[0124] Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).

[0125] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0126] Nucleic acid molecules selected from i) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte; ii) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs); iii) a nucleic acid encoding an RNA or protein encoded by a maternal effect gene (MEG); iv) a nucleic acid encoding an RNA or protein associated with primary ovarian insufficiency (POI); v) a nucleic acid encoding an RNA or protein associated with meiosis and / or a nucleic acid encoding an RNA or protein associated with embryonic lethality before or after implantation may also be inserted into vectors which can be used as gene therapy vectors. In general, gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration, or by stereotactic injection. The pharmaceutical compositions comprising a gene therapy vector can include an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can beAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 produced intact from recombinant cells, e.g., retroviral or lentiviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0127] Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.5. Examples of Non-Limiting Aspects of the Disclosure

[0128] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding orfollowing individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:1. A method for augmenting the health of an oocyte, the method comprising introducing into the oocyte one or more of:i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6;ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4;iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9; iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises aAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1;v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) orthe protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis orthe protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5.2. The method of aspect 1, wherein the method comprises introducing into the oocyte at least one, at least two, or at least three of the nucleic acids comprising a coding sequence of a gene listed in Table 6 or the encoded proteins.3. The method of aspect 1 or 2, wherein the method comprises introducing into the oocyte one, two, or three of the nucleic acids comprising a coding sequence of a gene listed in Table 6 orthe encoded proteins, wherein the genes are ribosomal protein SA (Rpsa), nucleophosmin 1 (Npml), or steroid 5 alpha-reductase 3 (Srd5a3).4. The method of any one of aspects 1-3, wherein the method comprises introducing into the oocyte one, two, or three of mRNAs encoding the proteins ribosomal protein SA (Rpsa), nucleophosmin 1 (Npml), and steroid 5 alpha-reductase 3 (Srd5a3).5. The method of any one of aspects 1-4, wherein the method comprises introducing into the oocyte at least one, at least two, or at least three of the nucleic acids comprising a coding sequence of a gene listed in Table 4 or the encoded proteins.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 6. The method of any one of aspects 1-5, wherein the method comprises introducing into the oocyte an mRNA encoding the protein zona pellucida glycoprotein 2 (Zp2).7. The method of aspect 1, wherein the method comprises introducing into the oocyte at least one, at least two, at least three, at least four, at least five, or all of the nucleic acids comprising a coding sequence of a gene listed in Table 9 or the encoded proteins.8. The method of aspect 7, wherein the method comprises introducing into the oocyte at least one, at least two, at least three, or at least four of a mRNA encoding Zp2, a mRNA encoding Rpsa, a mRNA encoding Npml and a mRNA encoding Srd5a3.9. The method of any one of aspects 1-8, wherein the oocyte is: an isolated ex vivo oocyte, an ex vivo oocyte surrounded by granulosa cells, present in an ex vivo follicle, present in an ex vivo ovarian tissue, or an ex vivo oocyte present in an ovarian organoid, wherein the oocyte is from a human subject or differentiated in vitro from a human pluripotent stem cell and / or wherein the granulosa cells are from the human subject or a granulosa cell line.10. The method of aspect 9, wherein follicle is a pre-antral stage follicle.11. The method of aspect 9, wherein the oocyte is in vivo in a human subject.12. The method of aspect 9 or 10, wherein the human subject is a female older than 30 years or 35 years.13. The method of any one of aspects 1-12, wherein the nucleic acid is RNA, optionally, wherein the RNA is an mRNA, further optionally wherein the mRNA comprises a 5'UTR-coding sequence-3'UTR-polyA tail.14. The method of any one of aspects 1-12, wherein the nucleic acid is DNA.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 15. The method of any one of aspects 1-14, wherein the nucleic acid is in an expression vector.16. The method of aspect 15, wherein the expression vector is viral expression vector.17. The method of aspect 16, wherein the viral expression vector is a lentiviral or adenoviral vector.18. The method of any one of aspects 1-17, wherein the nucleic acid or protein is in a vesicle or a lipid nanoparticle (LNP).19. The method of aspect 18, wherein the method comprises contacting the oocyte with the vesicle or LNP.20. The method of any one of aspects 1-17, wherein introducing the nucleic acid or protein comprises injecting the nucleic acid or protein into the oocyte or wherein introducing the nucleic acid or protein into the oocyte comprises introducing the nucleic acid or protein into granulosa cells or granulosa cell line, wherein the . granulosa cells or cells of the granulosa cell line surround the oocyte.21. The method of any one of aspects 1-20, wherein the nucleic acid or protein comprises a modification.22. The method of aspect 21, wherein the nucleic acid comprises a modification to increase stability, resistance to nucleases, and / or serum half-life.23. The method of aspect 21, wherein the protein comprises a modification to increase stability, resistance to nucleases, and / or serum half-life.24. The method of any one of aspects 1-23, wherein augmenting the health of the oocyte comprises improved oocyte maturation as compared to maturation of a control oocyte in which the nucleic acid or protein has not been introduced.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-19025. The method of any one of aspects 1-23, wherein augmenting the health of the oocyte comprises improved embryo development upon fertilization of the oocyte as compared to embryo development upon fertilization of a control oocyte in which the nucleic acid or protein has not been introduced.26. The method of any one of aspects 1-25, wherein the oocyte is a cryopreserved oocyte, a freshly isolated oocyte, an ex vivo cryopreserved oocyte or wherein the oocyte is ex vivo and the method further comprises cryopreserving the oocyte.27. The method of any one of aspects 1-26, wherein the method further comprises contacting the oocyte with a growth factor prior to the introducing.28. A composition for augmenting an oocyte's health comprising:i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6;ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4;iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9; iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1;Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) orthe protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis orthe protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5,wherein the nucleic acid is present in a lipid nanoparticle (LN P), a vesicle, or a viral expression vector, the protein is present in a LNP or a vesicle or is conjugated to a heterologous amino acid sequence, optionally the composition further comprises an oocyte, an ex vivo follicle comprising an oocyte, ovarian tissue comprising an oocyte, or an ovarian organoid comprising an oocyte.29. The composition of aspect 28, wherein the viral expression vector is lentivirus or adenovirus.30. The composition of aspect 28 or 29, wherein the oocyte is a human oocyte.31. The composition of any one of aspects 28-30, wherein the composition further comprises one or more RNA-binding proteins (RBPs).32. The composition of any one of aspects 28-31, wherein the composition further comprises a GTPase.33. The composition of aspect 32, wherein the GTPase is Dynamin 2.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 34. A granulosa cell line genetically modified to overexpress mRNA or protein encoded by the mRNA as compared to expression level of the mRNA or protein in a non-modified parental granulosa cell line, wherein the granulosa cell line is genetically modified to overexpress one or more of:i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6;ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4;iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9; iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1;v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) orthe protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis orthe protein, wherein the nucleic acid comprises a coding sequence of aAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5.35. The genetically modified granulosa cell line of aspect 34, wherein the cell line is produced from an embryonic stem cell or an induced pluripotent stem cell.36. The genetically modified granulosa cell line of aspect 34 or 35, wherein the cell line overexpresses one or more of a mRNA encoding Zp2, a mRNA encoding Rpsa, a mRNA encoding Npml and a mRNA encoding Srd5a3.37. The genetically modified granulosa cell line of any one of aspects 34-36, wherein the cell line overexpresses one, two, three, four, or five of mRNAs comprising coding sequence of a gene listed in Table 9.EXAMPLES

[0129] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0130] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0131] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in theAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.

[0132] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.1. Introduction

[0133] The oocyte expands 300x in volume during its most rapid phase of growth, and it was hypothesized that building blocks from surrounding granulosa cells support the growth and augment health of the oocyte during its maturation. This process is highly supported by surrounding granulosa cells, which directly transport transcripts and organelles to the oocyte to ensure healthy development. The inventors found evidence that transcripts and organelles are transported to the oocyte during its period of inflationary growth. It is showed using high resolution microscopy that mRNAs and mitochondria localize to protrusions known as transzonal projections (TZPs) that originate from encapsulating granulosa cells and contact the oocyte. Labeled mRNAs loaded into granulosa cells and mixed with the secondary stage oocyte ended up in the oocyte. To identify the specific transcripts that are conveyed into the oocyte, follicles are reconstituted in vitro from two distantly-related strains of mice, C57BI6 and Castaneous, with an average of 1 SNP per kb across their genomes. Isolated granulosa cells from Castaneous were aggregated around secondary oocytes from C57BI6 and cultured for one week. Oocytes from the reconstituted follicles were re-isolated, subjected to RNA-seq, and sequence polymorphisms were analyzed.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0134] In three trials, it is found that more than 1000 transcripts in the oocyte were from Castaneous and had therefore originated from the granulosa cells. Many of these transcripts also decrease with age, and there is published evidence that the density of TZPs decreases with age, which the inventors hypothesize may decrease the efficiency of transport to the oocyte. The list of transported transcripts and their protein products are therapeutic targets for augmenting the health of oocytes during in vitro maturation (IVM) or with systemically taken drugs.2. Benefits

[0135] The number and proportion of healthy eggs decrease dramatically during the 4th decade, and 1 / 6 people experience infertility according to a recent WHO study. A therapy to augment the health of eggs during in vitro maturation would increase the success of IVF clinics during fertility treatment. Systemic therapies for sustained use would have application for preserving and augmenting fertility as well as delaying menopause.3. Methods3.1 Mouse handling

[0136] All mouse work was performed under the University of California, San Francisco (UCSF) Institutional Animal Care and Use Committee guidelines in an approved facility of the Association for Assessment and Accreditation of Laboratory Animal Care International.3.2 Follicle enzymatic digestion and reaggregation

[0137] P12-P15 mice were euthanized via cervical dislocation, ovaries were removed and placed in pre-warmed L15 media on a 37 °C heated microscope stage mounted on a dissecting microscope in a biosafety cabinet with HEPA filter. 2-3 ovaries were placed in ImL L15 with 25 pg / mL Liberase and 200 pg / mL DNase I and ripped apart with dissection forceps. After 20 minutes a plOOO pipette pre-coated with 30% BSA and then "rinsed" 5x in L15 was used to triturate samples for 30 seconds. After another 10 minutes (total of 30) 100 pl of 30% BSA was added and the samples were triturated again. Samples were then added to a 200 pm filter stacked on a 40 pm filter) and then rinsed. The 200 pm filter removes un-dissociated oocyte tissue and the 40 pm filter allows primordial and small primary follicles to pass through. The 200 pm filter was removed and the 40 pmAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 filter was rinsed 3x with L15. The 40 pm filter was then inverted and follicles were eluted off with 1 mL 0.05% Trypsin. After 10 minutes 100 pl BSA was added and the sample was triturated until all oocytes were bare (about 20 times). The sample was then passed through a pre-rinsed 20 pm filter to collect granulosa cells. Granulosa cell suspensions were visually inspected to confirm that they contained no oocytes. The 20 pm was then rinsed 3x with L15 and then inverted and oocytes were eluted off with L15. Oocytes and granulosa cell suspensions were transferred to separate tubes and were then spun at 750 ref for 5 minutes. As much supernatant was removed as possible without disrupting the cells and replaced with adKSOM supplemented with 10 nm Estradiol 100 ng / mL GDF9 and 10 lU / mL FSH.

[0138] Oocyte and granulosa cell suspensions were then combined according to experimental setup, added to Ultra Low attachment wells and spun at 750 ref for 30 seconds. Plates were placed in incubators and removed each of the first 3 days to be spun at 750 ref for 30 seconds. Half of the supplemented adKSOM was exchanged with fresh supplemented adKSOM on the third 3 day and then left undisturbed in the incubator for 3 more days. Reaggregates were then either fixed with 2% PFA directly in the culture wells for 10 minutes for staining or oocyte were re-isolated.

[0139] Oocytes were re-isolated by transferring reaggregates to warm 0.05% Trypsin, with BSA quenching after 5 minutes. Oocytes then were transferred through 3 L15 washes and visualized on a brightfield microscope. Only bare oocytes where granulosa cells were completely removed were selected for RNA isolation.3.3 Intact follicle isolation and staining

[0140] Follicles were mechanically isolated in warmed L15 media (11415064 Gibco) on a heated stage by ripping ovaries with forceps. Enzymatic isolation of follicles with collagenase or Liberase causes TZPs to collapse. Follicles were transferred via EZ-Grip pipette to fresh / warm L15 media and then fixed in 2% PFA (cat 043368.9M Thermo Scientific) for 10 minutes with the PFA being directly added to the media. All wash / incubation sets were performed in glass-well dishes on an orbital shaker. Follicles were then washed 3 times for 5 minutes in 0.05% PBS-Tween (cat P7949-500ML Sigma-Aldrich) and then permeabilized in 0.01% PBS-Triton (cat X100-1L Sigma-Aldrich) for 20 minutes or one hour specifically for the anti-mCherry / tdTomato staining. Blocking was performed in 0.05% PBS-Tween with 5% donkey serum (S30-100mL Sigma-Aldrich) and 1%Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 BSA (cat A30075 RPI Research Products) and then incubated overnight with primary antibodies diluted in Toyobo Can Get Signal Solution A (cat NKB-501 Toyobo). Follicles were then washed 3 times for 5 minutes in 0.05% PBS-Tween and then incubated for 2 hours in secondary antibody diluted in Toyobo Can Get Signal Solution B (cat NKB-601 Toyobo). Some samples were then incubated with WGA for 1 hour, Phalloidin for 30 minutes, DAPI for 15 minutes or DRAQ5 for 15 minutes. When staining was finished, follicles were mounted in a 50 / 50 mixture of Aqua Polymount (18606-20 Polysciences) and RapiClear 1.52 (RC1520001 SUNJin labs) on slides with coverslips for imaging.

[0141] List of stains and primary / secondary antibodies used:• Phalloidin 405 (A30104 Invitrogen) or 488 (A12379 Invitrogen) • DAPI (EN62248 Invitrogen), DRAQ5 (62251 Thermo Scientific)• WGA 680 (W32465 Invitrogen)• Goat anti-FMRP (Knockdown validated, PA5-18742 Invitrogen)• Rabbit anti-TDP43 (Knockout validated, AB109535 abeam)• Rabbit anti-mCherry (Cross-reacts with tdTomato, AB167453 abeam) • Donkey anti-goat 488 (A11055 Invitrogen), 594 (A11058 Invitrogen) • Donkey anti-rabbit 488 (A21206 Invitrogen), 555 (A31572 Invitrogen), 594 (A21207 Invitrogen).3.4 Microscopy

[0142] For these experiments, a confocal microscope, such as Nikon Spatial Array Confocal (NSPARC) detector, was used.3.5 RNA extraction

[0143] Following dissociation from follicle reaggregations, 25-35 oocytes were transferred to phase lock gel heavy tubes. 100 pl of Trizol and then 50 pl chloroform were added. Tubes were inverted for 15 seconds and then incubated at room temperature for 3 minutes. Tubes were then centrifuged at 12,000 ref at 4C for 30 minutes. The aqueous phase was then transferred to a new RNase-free tube and 50 pl ice-cold isopropanol, 10 pl 3M sodium acetate, and 2.5 pl glycogen were added. The samples were mixed gently but thoroughly and then placed at -20 °C for 60 minutes to encourage precipitation. Tubes were then centrifuged at 12,000 ref at 4 °C for 30 minutes. Supernatants were discarded and pellets were rinsed in 150 pl 70% ice-cold ethanol (diluted with RNase-free water) andAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 then centrifuged at 7,500 ref for 5 minutes at room temperature. This step was repeated. As much ethanol as possible was then removed without disturbing the pellet and tubes were left open under a sterile flame to dry for 5 minutes. Pellets were then resuspended in 10 pl nuclease-free water at 65 °C for 5 minutes. These samples were then either immediately shipped for sequencing or stored at -80 °C.3.6 EU incorporation and processing

[0144] P12-P15 mice were injected with 1 mg 5-ethynyl-uridine dissolved in sterile saline. These mice were sacrificed 16-18 hours later and follicles were either mechanically isolated and processed for imaging or enzymatically isolated for reaggregations.Enzymatically isolated E U-fol I icles were processed as described above to isolate EU-labelled granulosa cells and re-aggregated with oocytes from a separate litter. Fixed whole follicles were fixed and washed as described above and processed according to Click-iT kit instructions.3.7 Bioinformatics

[0145] Following C57BL / 6J (B6) oocytes aggregate with CAST / EiJ (CAST) granulosa cells and subsequent removal of the granulosa cells, total RNA was isolated from three replicates of B6 oocytes (details to follow here). Total RNA samples were sent on dry ice to Novogene, and sequencing libraries were constructed using the low input SMART Seq V4 kit (Takara Bio) according to manufacturer's protocols. Libraries were sequenced 2 x 150bp paired-end on the NovaSeq platform (Illumina). Approximately 100 million reads were generated in the first sample, and ~65 million reads were generated from the subsequent two replicates. FastQC (Andrews, 2010) was used to assess read quality, and TrimGalore (Krueger F. 2015) was used to remove Illumina adapter sequences and trim the 2 x 150bp reads down to the highest quality 75bp. The forward read of each trimmed pair was used fortranscript quantification. Trimmed reads were first aligned to the B6 transcriptome (genome version: GRCM39. Gene annotation version: vl05) using Bowtie aligner (Langmead et al., 2009), allowing for zero mismatches, with parameters "- best -strata -a -v 0 -un". Any reads failing to map perfectly to the reference B6 transcriptome were aligned a second time to a "CAST-specific" transcriptome containing all CAST SNPs and indels that differ from the reference. Reads that did not map (or mapped with >= 1 mismatch) to the B6 reference transcriptome but mapped perfectly (0 mismatches) to the CAST transcriptome were considered likely to be of CAST strain origin. Many additional reads ofAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 CAST origin will likely be missed by this conservative read trimming and alignment strategy, which was selected to minimize false positive CAST read assignments due to sequencing errors or overly permissive alignment parameters.

[0146] To estimate transcript abundance levels from reads of CAST origin, we applied an Expectation-Maximization algorithm as implemented in RSEM (RNA-Seq by Expectation-Maximization) (Li & Dewey, 2011). Gene ontology enrichment analysis was then performed using the enrichGO function implemented in the clusterProfiler Bioconductor R package (Wu et al., 2021) to determine the biological relevance and specific pathways involved in the overlap genes.4. Results4.1 Granulosa cells re-establish contact with pre-antral oocytes following enzymatic separation and reaggregation

[0147] Most studies performed on granulosa cell-oocyte interactions in the transzonal region are performed on cumulus oocyte complexes removed from antral follicles. However, the rate of oocyte growth plateaus at antral stages (FIG. 5C) (Eppig 2001, Griffin et al. 2006), suggesting that biosynthetic requirements peak earlier in growth during primary and secondary stages. Therefore, ex vivo protocols was developed for investigating the dynamics of granulosa cell-oocyte interactions during pre-antral stages. Previous work demonstrated that mechanically separated granulosa cells re-grow TZPs and re-attach to oocytes isolated from antral follicles (El-Hayek et al. 2018). Based on our experience, which is consistent with other publications, pre-antral oocytes cannot be mechanically denuded without first disrupting the integrity of the granulosa epithelium, such as via alginate culture (Wang et al. 2024).

[0148] Therefore, granulosa cells and oocytes were isolated from primary and secondary stage follicles of juvenile mouse ovaries by enzymatic digestion followed by filtering and then preantral follicles were reconstructed in vitro by reaggregating granulosa cells with oocytes (FIG. 5D). In our culture system some oocytes become fully reencapsulated by granulosa cells and some only partially. From the observations it is believed that this is due to separation of oocytes and granulosa cells based on cytoplasmic densities when the ultra-low attachment dishes were centrifuged to encourage reaggregation (FIG. 5E). Imaging with phalloidin confirmed that granulosa cells regrowAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 oocyte directed TZPs in these preantral follicle reaggregates (FIG. 5F). It is concluded that secondary stage oocytes and granulosa cells follicles can be reaggregated and cultured ex vivo to recapitulate connectivity via TZPs in preantral follicles.4.2 Labelled RNA transits from granulosa cells to oocytes

[0149] To investigate whether RNA can transit from granulosa cells to the oocyte, RNA is labeled in vivo and then analyzed intact and re-aggregated preantral follicles.Neonatal mice were injected with the uridine analog 5-ethylnyl-uridine ( EU ), which incorporates into nascent RNA, isolated pre-antral follicles 16 hours later and detected EU by click chemistry. In whole-mounted follicles variable labelling of granulosa cells by EU was observed, likely due to their proliferation during this stage. Labeled RNA was not detected in the oocyte nucleus; however, EU labeled RNA puncta present at the oocyte cortex were observed (FIG. 1C). It was hypothesized that this RNA was newly imported from granulosa cells. To exclude the possibility that EU-RNA was synthesized in oocytes, granulosa cells isolated from EU-injected mice were re-aggregated with oocytes from an unlabeled mouse. After growth of this reaggregated follicle for six days, EU-la bel led RNA was detected in the oocytes (Figure ID, E). This result indicates that RNA transcribed in granulosa cells can transit to mouse oocytes.4.3 Chimeric follicle reaggregates identify transferred transcripts that are involved in oocyte maturation and preimplantation development.

[0150] Next an approach to identify specific RNAs that move from the granulosa cells to the oocyte in pre-antral follicles was implemented. Reaggregation was carried out with granulosa cells and oocytes from two distantly related strains of inbred mice.Compared to the C57BL6 / J (B6) reference genome NCBIM37, the transcriptome of CAST / Eij (Castaneus) contains single nucleotide polymorphisms (SNPs) at approximately 1 in every 217 bases and indels at approximately 1 in every 1650 bases (Munger at al. 2014). This degree of polymorphism provides wide transcriptome coverage when comparing RNAs from B6 and CAST origin. Reaggregations of B6 oocytes with CAST granulosa cells were cultured for 6 days before dissociation and bulk RNA-sequencing on 15-30 B6 oocytes in three replicates (Fig 2A).

[0151] Following bioinformatic analysis, approximately 1,400 transcripts present in all three B6 oocyte replicates that originated from CAST were identified. Of the 500Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 transcripts with highest read counts, 273 were present in all three replicates, giving us high confidence in the replicability of our findings (Fig. 2C).

[0152] This will highlight that the mostly highly significant categories in the granulosa-derived transcripts are oocyte maturation (prevention of polyspermy, spindle localization), the subcortical maternal complex (SMC), and functional categories (fertility, maternal effect, embryo turning).

[0153] Geneset analysis of the 1,330 protein-coding transcripts from CAST present in all three B6 oocyte replicates was performed. The most significant pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was pyruvate metabolism. Oocytes are known to lack the enzymes to metabolize glucose into pyruvate, which has been shown to move from granulosa cells to oocytes through gap junctions in TZPs. Among the strongest enrichments for mammalian phenotype was maternal effect genes; mutations in these genes do not affect the health of the mother but result in infertility or subfertility by compromising function of the oocyte or resulting embryo (Li et al. 2010). Maternal effect genes can influence the levels of pyruvate metabolites within an embryo by regulating the expression of genes involved in pyruvate metabolism, which is crucial for early embryonic development, potentially impacting the embryo's energy production and developmental fate; essentially, the mother's genetic makeup can affect the availability of pyruvate for the developing embryo through the expression of specific genes during oogenesis.

[0154] Components of the Subcortical Maternal Complex (SCMC), which is required for storage of maternally-deposited proteins in oocytes and is involved in proper meiotic spindle positioning, organelle patterning, cytoskeletal organization, epigenetic programming, and mRNA translation (Bebbere et al. 2021, Jentoft et al. 2023) were enriched among the CAST-derived RNAs captured in B6 oocytes. Specifically, seven of the eight genes encoding components of the Subcortical Maternal Complex (SCMC), were present in all three of our replicates and the eighth was present in two replicates (Fig 2C).

[0155] Other maternal effect genes that may be associated with the SCMC, but also may have other functions, were also present in all three of our replicates.

[0156] Additionally, transcripts for the maternal-effect gene Zarl were present in all three replicates. Zarl promotes coalescence of oocyte structures known as mitochondrial-associated membraneless compartments (MARDO), which store maternal mRNAs that are necessary for oocyte maturation (Cheng et al. 2022). Mutations in Zarl are also associatedAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 with primary ovarian insufficiency (POI). POI is a polygenic condition and individuals suffer from a premature exhaustion of the oocyte pool. Seven other transcripts for genes associated with POI (Ke et al 2023, Neyroud et al. 2023) were also present in all three of our replicates (Fig 2C). Together these analyses reveal that granulosa cell-derived transcripts in the oocyte overwhelmingly function in oocyte maturation and early embryo development.

[0157] Since many of the granulosa cell-transcribed RNAs detected in oocytes have been historically viewed as "oocyte genes", first it was checked whether they were present at detectable levels in granulosa cells. It was also ensured that there was little to no contamination in our sequencing samples. Filter sizes were selected to exclude primordial and small primary follicles during initial sorting. Furthermore, all cell suspensions were visually inspected to ensure contaminating cell types were excluded, such as ensuring there were no oocytes in granulosa cell suspensions prior to re-aggregation and that re-isolated oocytes were fully denuded prior to sequencing. Transcripts that are used as markers of granulosa cells, such as foxl2, were detected in only one replicate with a negligible read count, and transcripts of amhr2 were not detected at all, providing that there was little to no contamination in oocytes from Cast granulosa cells. The complimentary analysis for oocyte specific genes to ensure no Cast oocyte contamination is difficult given that we are challenging the notion of "oocyte genes."

[0158] However, transcripts of the pluripotency gene oct4 (pou5fl), which is downregulated in primordial germ cells during late embryogenesis and then re-expressed in growing oocytes (Pesce at al. 1998), were not present in any replicates.4.4 Trafficking-associated RNA-binding proteins co-localize with TZPs

[0159] The enrichment of oocyte maturation, maternal effect genes and embryo development processes in Geneset analysis of granulosa-derived mRNAs in oocytes suggests that the transfer process is selective. Based on the number of polymorphisms between Cast and B6 genomes and the stringency conditions of our analysis pipeline, the theoretical maximum number of transcripts that could have identified is 8,120. Across 3 replicates, 1,453 transcripts, or 18% of the maximum are detected. Therefore, this likely indicates that a mechanism based on specific properties traffics this subset of RNAs, as opposed to a non-selective transfer of all transcripts present in the granulosa cell cytoplasm to the oocyte.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0160] When RNAs are trafficked, they associate with RNA-binding proteins (RBPs) in phase-separated structures called ribonucleoprotein particles (RNPs). RNPs, though small, can be resolved with light microscopy. RNA-binding proteins (RBPs) stabilize RNAs in addition to regulating their translation and trafficking. To test the possibility that granulosa-derived RNAs in the oocyte are bound by RBPs, an analysis resource that ranks RBPs based on the number of motif instances present in a set of RNAs (Bouvrette et al. 2020) is used. Among the top 50 RBPs with the most frequent motif instances in the collection of RNAs (Table 8), both TARDBP and FMRP stood out for their well-characterized roles in RNA-trafficking in neurons (Thelen and Kye 2020, Prashad and Gopal 2021). Additionally, FMRP is well-known to be associated with Premature Ovarian Insufficiency and FMRP-GFP transfected into bovine granulosa cells has been observed to transit to oocytes (Allen et al.2021, Macaulay et al 2014, Nenonene et al. 2023).

[0161] Table 8. Top 50 RBPs with the most frequent motif instances in the collection of granulosa cell-derived RNAs we identified in the oocyteAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0162] The zona pellucida is a matrix composed of glycoproteins separating the oocyte from granulosa cells that grows to ~6 pm thick in mice. Given this distance and density, it was reasoned that RNP trafficking would most likely occur on the granulosa cell membrane structures that span the zona. Whole-mount immunofluorescence in intact secondary follicles with confocal microscopy revealed puncta of TARDBP and FMRP within the zona pellucida that colocalized with TZPs in (Fig.3. 3A, B). By super-resolution microscopy, TZP-associated FMRP puncta at various positions between the granulosa layer as well as proximal to the oocyte membrane was observed (Fig 3A). These results are consistent with the hypothesis that TARDBP and FMRP are involved in transferring RNAs via TZPs. It was also once again performed E U-la be I ling of nascent RNA in follicles and saw coAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 localization of FMRP with RNA puncta and TZPs. Together, the predicted binding of granulosa-derived RNAs to RBPs TARDBP and FMRP and the localization of both along actin perforations in the zona pellucida support the hypothesis that RNPs traffic from granulosa cells into the oocyte through TZPs or similar structures.5. Effect of granulosa cell extracellular vesicles on oocyte quality

[0163] It is well known that oocyte quality decreases with maternal age. The findings reported here that granulosa cell-derived mRNAs in the oocyte disproportionately include those required for oocyte maturation and embryo development (i.e. maternal effect genes) suggests that mRNA transfer promotes the health of the oocyte and future embryo. Furthermore, the reduction in the density of transzonal projections (TZPs) from the granulosa cell to the oocyte in aged follicles (Converse et al. 2023) may suggest that diminished transfer of mRNAs during aging contributes to the decline in oocyte quality. The capacity of mRNAs derived from granulosa cells to improve various metrics of oocyte quality in aged C57BI6 / J mice is analyzed.

[0164] The effect of granulosa cell vesicles (or vesicle contents) on mouse oocyte quality using multiple metrics is tested. Isolated extracellular vesicles will be delivered directly to granulosa cells or microinjected into oocytes in an ex vivo culture model of secondary or later follicles from aged C57BI6 / J mice to maturation. Multiple metrics are measured:

[0165] 1) Proliferation and apoptosis in granulosa cells of 2°-3° follicle (compare frequency of cells by immunofluorescence for Ki67, cCasp3, gH2AX ) (Wang et al. 2024) (FIG. 1)

[0166] 2) Follicle diameter and growth rate during culture from 2° or later to maturation (Wang et al. 2024)

[0167] 3) Frequency of chromosome spindle abnormality (Wang et al. 2024).

[0168] 4) Rate of fertilization and development to 2-cell, blastocyst stage following In vitro maturation (IVM) and IVF.

[0169] 5) N umber of live births following transfer of IVF embryos to pseudopregnant female mouse.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 7. Additional Uses

[0170] 1) Egg Freezing: Improved success rates of cryopreserved oocytes by delivering an mRNA encoding a sequence of a gene listed in Tables 1-5. Studies in human and mouse show a compromised rate of fertilization and embryo development from oocytes after cryopreservation (Oktay, Cil, and Bang 2006; Walker, Lanes, and Ginsburg 2022).

[0171] 2) Facilitating RNA transfer: Transfer of mRNAs (or vesicles) between granulosa cells and oocyte may be augmented with strategies to increase the density of TZPs. Such strategies include use of growth factors such as EGF and small molecules.8. Selection of Transcripts for introduction into oocytes

[0172] We began with the set of transcripts bearing CAST SNPs that were detected in bulk RNAseq of the C57BI6 oocyte following a coculture experiment of granulosa cells and oocytes from the two different mouse strains. To broadly identify mRNAs putatively transcribed in granulosa cells and transported to the oocyte, we relaxed the requirement for CAST transcripts to be detected in oocytes in a minimum of 2 replicates at >10 TPM. This yields 1,069 additional genes for a total of 2,336; we refer to this set as "Doherty genes". We further prioritized these genes through the following analyses (FIG. 6).

[0173] Comparison to age-related changes in granulosa cells. Based on our hypothesis that oocyte transcripts are imported from granulosa cells, it follows that such genes must be transcribed in granulosa cells. To test this, we interrogated mouse granulosa cell bulk RNAseq datasets, as they contain the deepest reads. A previous study by Liu and colleagues (Liu et al. 2023) profiled mouse C57BI6 granulosa cells by bulk RNAseq from cumulus-oocyte complexes that were collected after superovulation and visually staged as growing oocytes (GO) that have not yet resumed meiosis or slightly more mature fully grown oocytes (FGO). These stages are relevant since the transzonal projections that link granulosa cells to the oocyte have not yet broken down. Cross-referencing to the transcriptomic data from granulosa cells surrounding GO or FGO, we detected expression above 10 TPM for 2,161 and 2,087, respectively of the 2,336 Doherty genes; this near complete overlap confirms that 2,168 of the 2,336 Doherty genes in oocytes could indeed have been transcribed in granulosa cells during the relevant phase of growth before final maturation.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190

[0174] We hypothesize that a treatment of aged or unhealthy oocytes by delivering transcripts to granulosa cells will be most effective for transcripts that decline with age in granulosa cells. To identify relevant age-dependent transcripts in granulosa cel Is, we compared genes decreased in granulosa cells from aged (10 month old) relative to young (6 week old) C57BI6 mice from Liu et al. datasets. We found that 1,271 of the Doherty genes also decline at least 1.2-fold in aged granulosa cells surrounding GO oocytes. Similarly, 1,800 of the Doherty genes decline at least 1.2-fold in aged granulosa cells from FGOs, and 966 decline at both stages. This analysis supports our hypothesis and suggests that 2,105 of the 2,336 Doherty genes decrease by at least 1.2-fold with age in granulosa cells and therefore their restoration could lead to increased transcript levels received by the oocyte.

[0175] Oocyte aging and transplant analysis. We hypothesize that transport of granulosa cell mRNAs to the growing and immature oocyte declines with age. To test this, we took advantage of oocyte RNA-seq data from a recent study in mice using follicles in similar stages of growth. Wang and colleagues (Wang et al. 2024) isolated secondary stage follicles from young (2-3 month) and aged (14-15 month) mice of the ICR strain, cultured in hydrogels for 2 days and then transplanted the denuded oocyte to a different follicle. This Recombined Cultured Follicle (RCF) was further cultured for 4-5 days to reach the antral stage of growth. At this point, oocytes were recovered and subjected to single-cell RNA-seq. We cross-referenced the genes that were more abundant in young oocytes of RCFs compared to aged oocytes and found that 362 Doherty genes matched the "young oocyte genes" of Wang et al with a cutoff of 1.2 fold-change and p<0.05, and 127 Doherty genes matched the cutoff of 1.5 fold-change and p<0.05.

[0176] Using their RCF system to study aging of the oocyte versus granulosa cells, Wang et al. found that aged oocytes transplanted to a young follicle environment were improved in their rate of in vitro maturation; upon fertilization, the rate of progression through stages of embryonic development to the 2-cell, morula and blastocyst stage, and live birth upon uterine transfer were all improved compared to control aged oocytes transplanted to an aged RCF. We hypothesize that this functional improvement relies upon mRNAs transported from the young granulosa cells to the aged oocyte (AY) in the RCF. To test this hypothesis, we compared the genes that Wang et al. found by RNAseq to be increased in aged oocytes that grew in a young follicle (AY RCF) compared to those transplanted to an isochronic aged follicle (AA RCF); we termed these "age-reversal genes".Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 We found that 270 Doherty genes were also 1.2-fold-changed and p<0.05, and 157 Doherty genes were 1.5-fold changed and p<0.05 in Wang's age-reversal gene set. Finally, we cross-referenced the set of genes that increase in Wang's aged oocytes in young chimeric (AY) RCFs compared to young oocytes in young (YY) RCFs; we termed these "rejuvenation-only genes". We found that 143 Doherty genes were 1.2-fold increased and p<0.05 in the rejuvenation-only geneset and 47 were 1.5 fold increased and p<0.05. The intersection of "age-reversal" and "rejuvenation-only" genes was 28 of the Doherty genes, while 385 matched either criterion and 663 matched young oocyte, age-reversal or rejuvenation-only genes. The high degree of concordance of Doherty genes with the Wang et al. RNAseq datasets aligns with our hypothesis that granulosa-derived transcripts conveyed into the growing oocyte contribute to its competence for maturation, meiotic resumption, meiotic progression, and embryonic development; furthermore this concordance indicates that rescuing the declining levels of these transcripts in aging or poor quality oocytes could increase success rates of maturation, fertilization and preimplantation development in vitro or in vivo.

[0177] Functional analysis. We hypothesized that granulosa-mediated supplementation of specific transcripts to the oocyte promotes its maturation, resumption and progression through meiosis, and transition to the embryo. Hence, we reasoned that the most critical and therapeutically-relevant transcripts should be essential to the oocyte and fertility. To prioritize these transcripts, we intersected the Doherty genes with genes identified from Mouse Genome Informatics Phenome Database (MGI phenotypes). We searched for mouse alleles of all types (excluding quantitative trait loci) indexed in MGI with the following four terms: "female infertility", "reduced female fertility", "oocyte meiotic defect" and "embryonic lethality before implantation". In cross-comparing, we obtained 97 and 65 Doherty genes for "female infertility" and "reduced female fertility", respectively. The term "embryonic lethality before implantation" matched 61 Doherty genes, including 6 overlapping with fertility. Finally, 77 Doherty genes corresponded to alleles with phenotypes classified as "oocyte meiotic defect", and 49 of these overlapped with fertility and meiosis phenotypes. In addition, we manually curated phenotypes from literature searches in PubMed to match 9 more Doherty genes. We further cross-referenced to human genes reported in a recent publication (Chen et al. 2025) as previously known causative infertility genes; of the 37 reported, 15 human homologsAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 matched Doherty genes and all but 1 had been identified with the MGI Phenome Database. Of the 123 novel infertility genes identified by human exome sequencing by Chen and colleagues, 22 matched Doherty genes of which 3 were identified through the MGI Phenome Database. As a final criteria for functional gene prioritization, we reasoned that critical and therapeutically-relevant genes are dosage-sensitive, such that their augmentation would promote oocyte or embryo health. We identified dosage-sensitive genes by searching MGI Phenome Database with the term "haploinsufficient"; this matched 2 Doherty genes. As an alternative approach, we performed a literature search in PubMed with a subset of Doherty genes and identified 15 additional matches that are dosage sensitive, 13 of which were found by other functional criteria described above. Together, these functional analyses prioritized 226 Doherty genes, with 133 of these in 3 of the CAST / B6 experimental replicates.

[0178] Multifactoral analysis of genes. To prioritize the most therapeutically relevant Doherty genes, we considered the functional or phenotypic criteria together with the analyses of datasets on oocyte age, follicle transplant, granulosa cell expression and age-related changes. Of the 226 Doherty genes that we validated with causing at least one relevant phenotype— female infertility, subfertility, oocyte meiotic defect, preimplantation embryo lethality, and dosage sensitivity in mice, and human exome sequencing— we found that 223 also passed our threshold in at least one other cross-referenced dataset. While these analyses depend upon the current state of knowledge, we present this strategy as a framework for prioritizing the most potent transcripts. With this approach, we show an example of a cocktail of mRNAs that could be delivered to granulosa cells in order to augment the health and competence of an oocyte for growth, maturation, meiotic resumption, and embryo development in Table 9.

[0179] Table 9. Twenty-one selected transcripts are shown with gene name, mRNA length, relevant phenotype, oocyte stages in which each declines in granulosa cell data from (Liu et al. 2023), conditions in which each gene was found to change with age or transplant in chimeric RCFs by (Wang et al. 2024), and considerations such as counts per million (CPM). ** indicates threshold of 1.5 fold-change and p<0.05, * indicates 1.2 foldchange and p<0.05.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 > > >Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190Table 10. Gene name, Sequence Identifiers (SEQ ID NO) for nucleic acid sequence (DNA or RNA) encoding the corresponding protein, Sequence Identifiers (SEQ ID NO) for mRNAAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 sequence encoding the corresponding protein with the native 5'UTR (untranslated region) and native 3'UTR for each RNA:

[0180] Modeling an in vitro therapeutic. Based on the intersections between Doherty genes and differentially expressed genes in both growing oocytes and granulosa cells in young and reproductively-aged mice, we reasoned that augmenting theseAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 transcripts in immature germinal vesicle-stage (GV) oocytes surrounded by cumulus cells could improve the efficiency of in vitro maturation (IVM). To introduce mRNAs into intact mouse cumulus-oocyte complexes (COCs), we used a low-invasive method for electroporation previously shown to have no measurable impact on the rate of maturation and or development to the blastocyst stage of embryogenesis (Satouh et al. 2024). We observed that electroporated Egfp mRNA was conducive to survival of COCs obtained after superovulation. Furthermore, GFP fluorescence could be detected in cumulus granulosa cells after 2-3 hours and was not observed in oocytes until the following day (FIG. 7). This may reflect either transport of the Egfp mRNA from granulosa cell to oocyte, or a delayed but direct electroporation of the mRNA into the oocyte.

[0181] FIG. 7. Dynamics of GFP expression after mRNA delivery to COCs. Following electroporation of eGfp mRNA into COCs, GFP fluorescence is shown at various time intervals with differential interference contrast (DIC) above and excitation for GFP below. GFP signal can be detected in the cumulus cells at 2-3 hours and in some oocytes at 18 hours. Scale bar, 100 um.

[0182] To test whether augmenting specific granulosa-to-oocyte transported transcripts could improve oocyte maturation and early embryo development, we delivered a cocktail of mRNAs to immature GV-stage COCs mechanically retrieved from reproductively-aged mice followed by IVM and in vitro fertilization (IVF). We used C57BI6 / J mice at 9 months of age, a timepoint shown by our group and others to exhibit reproductive aging phenotypes resembling those in women in their 30s and early 40s. These phenotypes include decreased counts of growing follicles, reduction in the number of oocytes retrieved after stimulation with gonadotropins, and significantly diminished probability of oocytes developing to the 2-cell as well as the blastocyst stage of development after IVF (Gaylord et al. 2025).

[0183] We compared the selected synthesized mRNAs against two controls: eGFP mRNA and total poly adenine (polyA) enriched mRNA isolated from pooled young mouse granulosa cells collected either immediately after isolation or following short-term in vitro culture. Among the example list of 21 therapeutically-relevant genes in FIG. 6 that passed all of our criteria, Zp2, Rpsa, Npml and Srd5a3 were chosen as a representative list in consideration of their differing functions and divergent levels of expression in oocytes and granulosa cells. Considering that length of untranslated regions (UTRs) is important inAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 regulating the transport as well as stability of mRNAs and oocytes maintain mRNAs for up to many decades (Mayr 2017; Navarro et al. 2021), we reasoned that therapeutic mRNAs that include UTRs. Constructs were designed for each of the four genes that included their native 5' UTR, coding region, 3' UTR and a polyA tail of 120 bases. To enhance stability of the mRNA and minimize toxicity, in vitro transcription was performed with the uridine analog Nl-methypseudouridine.

[0184] The performance of the four synthesized mRNAs was measured by the rate of development to 2-cell and blastocyst stage of development following IVM and IVF. For each condition a cohort of four 9-month- females was hormone-primed with HyperOva, and 40-50 GV-stage COCs obtained were electroporated with synthesized mRNAs, GFP mRNA or PolyA-enriched granulosa cell mRNA. After 18 hours in IVM media, the COCs were fertilized with sperm from proven males (who had previously fathered) and their progress scored visually. At 24 hours post fertilization, we observed that 88.2% of COCs that received the 4 synthesized mRNAs advanced to the 2-cell stage, compared to 66.7% and 71.7% from GFP and PolyA-enriched conditions, respectively; the synthesized mRNAs delivered before IVM+electroporation+IVF produced a similar result to IVF alone (Gaylord et al. 2025) from mature oocytes collected from the oviduct after superovulation of C67BI6 / J mice at the same age (85.3% p=0.7816 ; FIG. 8). The transition to the 2-cell stage is among the earliest and most age-sensitive outcomes of maternal aging (Cimadomo et al. 2018); since zygotic transcription has not yet begun, this stage of development relies entirely on maternal mRNAs and is thus an important readout of oocyte health. By 96 hours we observed that 90% of the 2-cell embryos advanced to blastocyst stage after receiving 4 synthesized mRNAs, compared to 46% in the control GFP mRNA group (p=0.0005; FIG. 9). Using Kaplan-Meier statistics, we compared the probability of survival each group of GV-stage oocytes from fertilization and found that the 4 synthesized mRNA cocktail group was improved at the blastocyst stage (p=0.0002; FIG. 10). This result supports our hypothesis that mRNAs transcribed in granulosa cells for delivery to the oocyte can be harnessed to augment the quality of oocytes therapeutically. Critically, this test-case demonstrates that delivering specific mRNAs to immature GV-staged oocytes can improve their rate of IVM and early development, bringing them to an equivalent competence as mature oocytes used for traditional IVF. Given that 15-30% of oocytes retrieved during an average cycle areAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 immature (GV-stage), this finding has the potential to increase the efficiency and lower the cost of IVF.

[0185] FIG. 8. Effect of mRNA delivery to COCs on oocyte competence to the 2-cell stage. The frequency of oocytes retrieved from cohorts of 9-month-old C57BI / 6J females that reached the 2-cell stage of development. For the IVF-only control, COCs were collected from superovulated females treated with HyperOva and hCG (n = 8) and subjected directly to IVF [from our prior publication (Gaylord et al. 2025)]. For the GFP, PolyA-enriched, and Synthesized mRNA groups, immature COCs were collected from HyperOva-primed females (n = 4 per group), electroporated (EP) with Gfp mRNA, PolyA-enriched mRNA from young mouse granulosa cells, or 4 synthesized mRNAs (Zp2, Rpsa, Npml and Srd5a3), and subjected to IVM and IVF. Statistical significance between groups was assessed using Fisher's exact test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0186] FIG. 9. Effect of mRNA delivery to COCs on oocyte competence to the blastocyst stage. From cohorts of 9-month-old C57BI / 6J females, the frequency of 2-cell embryos that reached the blastocyst stage of development. For the IVF-only control, COCs were collected from superovulated females treated with HyperOva and hCG (n = 8) and subjected directly to IVF [from our prior publication (Gaylord et al. 2025)]. For the GFP, PolyA-enriched, and Synthesized mRNA groups, immature COCs were collected from HyperOva-primed females (n = 4 per group), electroporated (EP) with Gfp mRNA, PolyA-enriched mRNA from young mouse granulosa cells, or 4 synthesized mRNAs (Zp2, Rpsa, Npml and Srd5a3), and subjected to IVM and IVF. Statistical significance between groups was assessed using Fisher's exact test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0187] FIG. 10. The probability of survival of zygotes from COCs from HyperOva-primed 9-month-old C57BI / 6J mice was compared between groups receiving 4 synthsized mRNAs (Zp2, Rpsa, Npml and Srd5a3), polyA mRNA enriched, and Egfp controls using Kaplan-Meier statistics; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.MethodsMice

[0188] All mouse work was performed under the University of California, San Francisco (UCSF) Institutional Animal Care and Use Committee guidelines in an approved facility of the Association for Assessment and Accreditation of Laboratory Animal CareAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 International. All mice used in this study were on a C57BL / 6J background (The Jackson Laboratory, stock 000664).Collection of GV COCs

[0189] C57BL / 6J female mice (9-months of age) were intraperitoneally injected with 100 pL of HyperOva (Cosmo Bio Co., Ltd., KYD-010-06-EX). Females were euthanized 46 hours after hormone priming by cervical dislocation, and ovaries were immediately dissected. Following removal of surrounding adipose tissue, the ovaries were transferred to a 35 mm petri dish containing ~2 mL of Leibovitz's L-15 medium (Gibco, 11415064) maintained at 37°C on a heated stage. Germinal vesicle (GV)-stage cumulus oocyte complexes (COCs) were released by repeated puncturing of the ovaries using two 25-gauge stainless steel needles. COCs were collected using an EZ-Grip pipette fitted with a 200-pm stripper tip (CooperSurgical) and transferred to a fresh dish containing ~2 mL Opti-Mem medium (ThermoFisher Scientific) for electroporation.Gfp, Zp2, Srd5a3, Rpsa, and Npml mRNA synthesis

[0190] Unmodified GFP in vitro-transcribed (IVT) mRNA was designed and synthesized by OZBiosciences (MRNA15-100). Four therapeutic IVT mRNAs encoding Zp2, Srd5a3, Rpsa, and Npml were synthesized by Gene Universal, Inc., with uridine residues substituted by Nl-Methylpseudouridine. All mRNAs were initially reconstituted in nuclease-free water at a concentration of 1 pg / pL.Primary murine ovarian follicle isolation

[0191] Postnatal day (P) 12 to P15 C57BL / 6J mice were euthanized via cervical dislocation, ovaries were removed and placed in pre-warmed L15 media (Gibco, 11415064) on a 37°C heated microscope stage mounted on a dissecting microscope in a biosafety cabinet with HEPA filter. 2-3 ovaries were placed in 1 mL L15 with 25 pg / mL Liberase (Sigma-Aldrich, 5401119001) and 200 pg / mL DNase I (Qiagen, 79254) and ripped apart with dissection forceps (Fine Science Tools, 11251-20). After 20 minutes a plOOO pipette precoated with 30 % BSA (RPI Corp, A30075-100.0) and then "rinsed" 5x in L15 was used to triturate samples for 30 seconds. After another 10 minutes (total of 30) 100 pl of 30% BSA was added and the samples were triturated again. Samples were then added to a 200 pm filter (PluriSelect, 43-10200-40) stacked on a 40 pm filter (PluriSelect, 43-10040-40) and then rinsed. The 200 pm filter removes un-dissociated oocyte tissue and the 40 pm filter allows primordial and small primary follicles to pass through. The 200 pm filter was removed and the 40 pm filter was rinsed 3x with L15. The 40 pm filter was then invertedAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 and follicles were eluted off with 1 mL 0.05% Trypsin-EDTA (Gibco, 25300-054). After 10 minutes 100 pil BSA was added and the sample was triturated until all oocytes were bare (about 20 times). The sample was then passed through a pre-rinsed 20 pm filter (PluriSelect, 43-10020-40) to collect granulosa cells. Granulosa cell suspensions were visually inspected to confirm that they contained no oocytes. The 20 pm filter was then rinsed 3x with L15 and then inverted and oocytes were eluted off with L15. The granulosa cell suspension was transferred to a tube and was spun at 750 ref for 5 minutes. As much supernatant was removed as possible without disrupting the cells and replaced with EmbryoMax Advanced KSOM Embryo Medium (Sigma-Aldrich, MR-101-D) supplemented with 10 nm -Estradiol (Sigma-Aldrich E8875), 100 ng / mL GDF9 (R&D Systems, 739G9010) and 10 lU / mL FSH (Sigma-Aldrich, F4021). Granulosa cell suspensions were either processed immediately for RNA extraction or plated in 2-D for expansion.2-D expansion of isolated primary murine granulosa cells

[0192] Granulosa cell growth medium was prepared using DMEM / F12 (Gibco, 21331) supplemented with 5% fetal bovine serum (FBS; Cytiva, SH30071.03), 1% GlutaMAX (Gibco, 35050), 1% penicillin-streptomycin (Gibco, 15140), and Primocin (InvivoGen, ant-pm-1) at a final concentration of 100 pg / mL. Medium was filter-sterilized, and basic fibroblast growth factor (bFGF; 10 pg / mL stock) was added fresh at a 1:2000 dilution to achieve a final concentration of 5 ng / mL immediately prior to use.

[0193] For plate coating, Matrigel (Corning, 354277) was thawed overnight at 4 °C and diluted 1:1000 in cold DMEM / F12 using chilled tubes and pipette tips. Six-well plates were pre-chilled and coated with 1 mL diluted Matrigel per well, then incubated at room temperature for 1 hour. Excess Matrigel was removed prior to cell seeding. Cells were counted and plated at a density of 3 x io5cells per well in Matrigel-coated six-well plates. Cultures were maintained with medium changes every other day. Cells were passaged by adding 1 mL of TrypLE Express Enzyme (Gibco, 12605010) for 5 minutes, blocked 1:1 with media and then spun for 5 min at 200g. The supernatant was removed, cells were resuspended in 1 mL fresh media and plated on freshly coated matrigel plates at 0.22e6 cell per well of a 6 well plate.

[0194] At harvest, wells were washed once with PBS, and cells were extracted for RNA isolation by adding 1 mL TRIzol per well followed by mechanical scraping with a cell scraper.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 Isolation of mRNA from primary murine granulosa cells

[0195] Total RNA from granulosa cell suspensions or plated 2-D expansion cultures was extracted with the Direct-zol RNA MiniPrep with TriReagent kit (Zymo Research, R2051-A) followed by the RNA Clean & Concentrator kit (Zymo Research, R1017) per kit recommendations. Total RNA concentration was measured using both the Nanodrop (ThermoScientific) and Qubit Fluorometer (ThermoScientific). PolyA tail enrichment to isolate mRNA was performed using the NEBNext High Input Poly(A) mRNA isolation module (E3370S) per manufacturer instructions.Electroporation of GV COCs

[0196] Electroporation was performed using a NEPA21 electroporator (NEPA GENE, Chiba, Japan) as previously described (Kaneko & Mashimo 2015; Satouh et al. 2024).Electroporation was carried out in a total volume of 40 pl_ Opti-MEM containing mRNA (300 ng). Three mRNA conditions were tested:• GFP mRNA alone at 300 ng per 40 ul electroporation media (7.5 microgram / mL).• PolyA-enriched mRNA pooled from non-cultured and cultured postnatal granulosa cells, contributing ~100 ng and ~200 ng, respectively (300 ng total).• Synthesized therapeutic mRNAs (Zp2, Srd5a3, Rpsa, and Npml), pooled at 75 ng each (300 ng total).

[0197] COCs were isolated from n = 4 animals, with approximately 40-50 COCs pooled per condition, and transferred into the electroporation chamber. Opti-MEM volume was adjusted as needed to achieve a target impedance of 530 - 555 O prior to pulse delivery. Electroporation was performed using a poring pulse (225 V; 2.0 ms pulse width; 50 ms interval; 4 pulses; 10 % decay; positive polarity), followed by transfer pulses (20 V; 50 ms pulse width; 50 ms interval; 5 pulses; 40 % decay; alternating polarity).

[0198] Control (GFP mRNA) or experimental (PolyA-enriched or synthesized therapeutic mRNA) conditions were performed in separate electroporation rounds, with control samples processed first. Three washes with Opti-MEM were performed between rounds to minimize cross-contamination. Following electroporation, COCs were immediately subjected to in vitro maturation (IVM).Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 In vitro maturation of GV COCs

[0199] COCs were moved to a droplet containing IVM Medium (MEM Alpha medium: Gibco, 12571-063; EGF: 4 ng / mL, R&D Systems, 2028-EG-200; hCG: 1.2 I.U. / mL, Ilex Life Sciences, A225005; FSH: 0.1 I.U. / mL, Sigma-Aldrich, F4021; FBS: 5 %, Gibco, 10438026) under mineral oil (EMD Millipore, ES-005-C) in a 6-well glass bottom plate (Cellvis, P06-20-1-N) and allowed to mature for 18 hours at 37 °C and 5 % CO2.In vitro fertilization

[0200] The epididymis was removed from proven C57BL / 6J breeding males and sperm were isolated via 25 G stainless injection needle in a pre-warmed drop of CARD FERTIUP (Cosmo Bio Co., Ltd., KYD-002-05-EX) under mineral oil (EMD Millipore, ES-005-C) for capacitation (~1 hour, 37 °C and 5 % CO2). Sperm from the same male was used for females from different experimental conditions on the day of the experiment.

[0201] In parallel, COCs were allowed to capacitate for 40-50 minutes (37°C and 5 % CO2) in a pre-warmed drop of CARD Medium (Cosmo Bio Co., Ltd., KYD-003-EX) before fertilization with 3 pL of sperm isolated from the outer perimeter of the sperm drop. After 3 hours of fertilization time (37°C and 5 % CO2), oocytes were washed four times in CARD mHTF (Cosmo Bio CO., Ltd., KYD-008-02-EX) to remove excess sperm and cumulus cells on a heated stage (37°C). The oocytes viable after fertilization were then incubated (37°C and 5 % CO2) and, 24 hours after fertilization, the embryos that successfully reached the 2-cell stage were moved to a fresh drop of pre-warmed, equilibrated EmbryoMax Advanced KSOM Embryo Medium (Sigma-Aldrich, MR-101-D) under mineral oil for washing and counting, all performed on a heated stage (37°C). The 2-cell embryos were then incubated (37°C and 5% CO2) and, 96 hours after fertilization, the number of embryos that successfully reached the blastocyst stage were scored.Statistical analysis

[0202] Data were plotted in a Kaplan-Meier-style plot, where the curves represent proportions of oocytes that survived to each developmental stage, conditional on successful transfer.Microscopy and image analysis of COCs

[0203] All images were acquired on the Leica THUNDER Imager 3D Cell Culture microscope at a magnification of 20X, z step size 15 pm. All images were processed using Fiji (ImageJ) or Imaris (Bitplane, version 10.2).Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 Bibliography1. Chen B, Wang W, Shi J, Sun X, Guan Y, et al. 2025. Genetic landscape of human oocyte / embryo defects. Cell Genomics. 101012.2. Cimadomo D, Fabozzi G, Vaiarelli A, Ubaldi N, Ubaldi FM, Rienzi L. 2018. Impact of maternal age on oocyte and embryo competence. Front Endocrinol (Lausanne). 9:327.3. Gaylord EA, Foecke MH, Samuel RM, Soygur B, Detweiler AM, et al. 2025.Comparative analysis of human and mouse ovaries across age. Science.390(6778):eadx0659.4. Kaneko T, Mashimo T. 2015. Simple genome editing of rodent intact embryos by electroporation. PLoS ONE. 10(ll):e0142755.5. Liu C, Zuo W, Yan G, Wang S, Sun S, et al. 2023. Granulosa cell mevalonate pathway abnormalities contribute to oocyte meiotic defects and aneuploidy. Nat. Aging 6. Mayr C. 2017. Regulation by 3'-Untranslated Regions. Annu. Rev. Genet. 51:171-94.7. Navarro E, Mallen A, Hueso M. 2021. Dynamic Variations of 3'UTR Length Reprogram the mRNA Regulatory Landscape. Biomedicines. 9(11).8. Satouh Y, Suzuki E, Sasaki K, Sato K. 2024. Improved low-invasive mRNA electroporation method into immature mouse oocytes visualizes protein dynamics during developmentt. Biol. Reprod. 111(4):931-41.9. Wang H, Huang Z, Shen X, Lee Y, Song X, et al. 2024. Rejuvenation of aged oocyte through exposure to young follicular microenvironment. Nat. Aging. 4(9): 1194— 1210.

Claims

Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190CLAIMSWhat is claimed is:

1. A method for augmenting the health of an oocyte, the method comprising introducing into the oocyte one or more of:i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6; ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4;iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9;iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1;v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2;vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / orAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 vii) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5.

2. The method of claim 1, wherein the method comprises introducing into the oocyte at least one, at least two, or at least three of the nucleic acids comprising a coding sequence of a gene listed in Table 6 or the encoded proteins.

3. The method of claim 1 or 2, wherein the method comprises introducing into the oocyte one, two, or three of the nucleic acids comprising a coding sequence of a gene listed in Table 6 or the encoded proteins, wherein the genes are ribosomal protein SA (Rpsa), nucleophosmin 1 (Npml), or steroid 5 alpha-reductase 3 (Srd5a3).

4. The method of any one of claims 1-3, wherein the method comprises introducing into the oocyte one, two, or three of mRNAs encoding the proteins ribosomal protein SA (Rpsa), nucleophosmin 1 (Npml), and steroid 5 alpha-reductase 3 (Srd5a3).

5. The method of any one of claims 1-4, wherein the method comprises introducing into the oocyte at least one, at least two, or at least three of the nucleic acids comprising a coding sequence of a gene listed in Table 4 or the encoded proteins.

6. The method of any one of claims 1-5, wherein the method comprises introducing into the oocyte an mRNA encoding the protein zona pellucida glycoprotein 2 (Zp2).

7. The method of claim 1, wherein the method comprises introducing into the oocyte at least one, at least two, at least three, at least four, at least five, or all of theAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 nucleic acids comprising a coding sequence of a gene listed in Table 9 or the encoded proteins.

8. The method of claim 7, wherein the method comprises introducing into the oocyte at least one, at least two, at least three, or at least four of a mRNA encoding Zp2, a mRNA encoding Rpsa, a mRNA encoding Npml and a mRNA encoding Srd5a3.

9. The method of any one of claims 1-8, wherein the oocyte is: an isolated ex vivo oocyte, an ex vivo oocyte surrounded by granulosa cells, present in an ex vivo follicle, present in an ex vivo ovarian tissue, or an ex vivo oocyte present in an ovarian organoid, wherein the oocyte is from a human subject or differentiated in vitro from a human pluripotent stem cell and / or wherein the granulosa cells are from the human subject or a granulosa cell line.

10. The method of claim 9, wherein follicle is a pre-antral stage follicle.

11. The method of claim 9, wherein the oocyte is in vivo in a human subject.

12. The method of claim 9 or 10, wherein the human subject is a female older than 30 years or 35 years.

13. The method of any one of claims 1-12, wherein the nucleic acid is RNA, optionally, wherein the RNA is an mRNA, further optionally wherein the mRNA comprises a 5'UTR-coding sequence-3'UTR-polyA tail.

14. The method of any one of claims 1-12, wherein the nucleic acid is DNA.

15. The method of any one of claims 1-14, wherein the nucleic acid is in an expression vector.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-19016. The method of claim 15, wherein the expression vector is viral expression vector.

17. The method of claim 16, wherein the viral expression vector is a lentiviral or adenoviral vector.

18. The method of any one of claims 1-17, wherein the nucleic acid or protein is in a vesicle or a lipid nanoparticle (LNP).

19. The method of claim 18, wherein the method comprises contacting the oocyte with the vesicle or LNP.

20. The method of any one of claims 1-17, wherein introducing the nucleic acid or protein comprises injecting the nucleic acid or protein into the oocyte or wherein introducing the nucleic acid or protein into the oocyte comprises introducing the nucleic acid or protein into granulosa cells or granulosa cell line, wherein the . granulosa cells or cells of the granulosa cell line surround the oocyte.

21. The method of any one of claims 1-20, wherein the nucleic acid or protein comprises a modification.

22. The method of claim 21, wherein the nucleic acid comprises a modification to increase stability, resistance to nucleases, and / or serum half-life.

23. The method of claim 21, wherein the protein comprises a modification to increase stability, resistance to nucleases, and / or serum half-life.Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 24. The method of any one of claims 1-23, wherein augmenting the health of the oocyte comprises improved oocyte maturation as compared to maturation of a control oocyte in which the nucleic acid or protein has not been introduced.

25. The method of any one of claims 1-23, wherein augmenting the health of the oocyte comprises improved embryo development upon fertilization of the oocyte as compared to embryo development upon fertilization of a control oocyte in which the nucleic acid or protein has not been introduced.

26. The method of any one of claims 1-25, wherein the oocyte is a cryopreserved oocyte, a freshly isolated oocyte, an ex vivo cryopreserved oocyte or wherein the oocyte is ex vivo and the method further comprises cryopreserving the oocyte.

27. The method of any one of claims 1-26, wherein the method further comprises contacting the oocyte with a growth factor prior to the introducing.

28. A composition for augmenting an oocyte's health comprising:i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6; ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4;iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9;Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1;v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5,wherein the nucleic acid(s) is present in a lipid nanoparticle (LNP), a vesicle, or a viral expression vector, the protein is present in a LNP or a vesicle or is conjugated to a heterologous amino acid sequence, optionally the composition further comprises an oocyte, an ex vivo follicle comprising an oocyte, ovarian tissue comprising an oocyte, or an ovarian organoid comprising an oocyte.

29. The composition of claim 28, wherein the viral expression vector is lentivirus or adenovirus.

30. The composition of claim 28 or 29, wherein the oocyte is a human oocyte.

31. The composition of any one of claims 28-30, wherein the composition further comprises one or more RNA-binding proteins (RBPs).Atty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-19032. The composition of any one of claims 28-31, wherein the composition further comprises a GTPase.

33. The composition of claim 32, wherein the GTPase is Dynamin 2.

34. A granulosa cell line genetically modified to overexpress mRNA or protein encoded by the mRNA as compared to expression level of the mRNA or protein in a non-modified parental granulosa cell line, wherein the granulosa cell line is genetically modified to overexpress one or more of:i) a nucleic acid encoding a protein associated with embryonic lethality before or after implantation or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 6 and wherein the protein comprises a sequence encoded by a gene listed in Table 6; ii) a nucleic acid encoding a protein associated with subfertility or infertility or primary ovarian insufficiency (POI) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 4 and wherein the protein comprises a sequence encoded by a gene listed in Table 4;iii) a nucleic acid identified as being transferred from granulosa cells to an oocyte or the protein encoded by the nucleic acid, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 9;iv) a nucleic acid encoding a protein that supports pyruvate metabolism in the oocyte or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 1 and wherein the protein comprises a sequence encoded by a gene listed in Table 1;v) a nucleic acid encoding a protein found in a subcortical maternal complex (SCMCs) or the protein, wherein the nucleic acid comprises aAtty. Dkt: UCSF-839WO UCSF Ref. No.: SF24-190 coding sequence of a gene listed in Table 2 and wherein the protein comprises a sequence encoded by a gene listed in Table 2; vi) a nucleic acid encoding a protein encoded by a maternal effect gene (MEG) or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 3 and wherein the protein comprises a sequence encoded by a gene listed in Table 3; and / or vii) a nucleic acid encoding a protein associated with meiosis or the protein, wherein the nucleic acid comprises a coding sequence of a gene listed in Table 5 and wherein the protein comprises a sequence encoded by a gene listed in Table 5.

35. The genetically modified granulosa cell line of claim 34, wherein the cell line is produced from an embryonic stem cell or an induced pluripotent stem cell.

36. The genetically modified granulosa cell line of claim 34 or 35, wherein the cell line overexpresses one or more of a mRNA encoding Zp2, a mRNA encoding Rpsa, a mRNA encoding Npml and a mRNA encoding Srd5a3.

37. The genetically modified granulosa cell line of any one of claims 34-36, wherein the cell line overexpresses one, two, three, four, or five of mRNAs comprising coding sequence of a gene listed in Table 9.

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