I. FIELD OF INVENTION

[001] This application relates to compositions and methods of administering nanoparticle constructs containing an apoptotic or cytotoxic protein or a polynucleotide encoding for the apoptotic or cytotoxic protein, such as Diphtheria toxin A (“DTA”), where the nanoparticle constructs comprise one or more peptides that can bind to reproductive cell receptors and cause cell death, and where the compositions and methods are useful for the sterilization of subjects, including male and female cats and dogs.

II. BACKGROUND OF THE INVENTION

[002] Currently, it is recommended that pet owners and shelters spay or neuter animals, including cats and dogs. Surgical sterilization, e.g., by way of spaying or neutering young animals is considered a responsible way to care for animals. In males, neutering typically involves removal of the testes. In females, it typically involves abdominal surgery to remove one or both ovaries and/or uterus. It is encouraged (and in some countries required for adopted animals) to prevent the births of unwanted litters, which contribute to the overpopulation of unwanted animals in the rescue system. The ASPCA (American Society for the Prevention of Cruelty to Animals®) has indicated the pet homelessness problem results in millions of healthy dogs and cats being euthanized in the United States each year. This is a worldwide problem that is not limited to just the United States. In addition, the ASPCA also indicates there are medical and behavioral benefits to spaying and neutering animals including: preventing certain infections or tumors (e.g., uterine infections and mammary tumors in females and testicular cancer and prostate problems in males); avoiding female pets going into heat; making it less likely for male pets to roam; and possibly leading to better behaved males. Sterilization procedures such as spaying and neutering are common surgeries, but there can be risks, for example with general anesthesia. Additionally, it takes time for the animals to heal after the surgery. Accordingly, bathing must be avoided for, e.g., at least ten days after surgery; the animal must refrain from running or jumping post-surgery; and the incision site must be monitored to avoid infection and proper healing. Accordingly, the present invention provides methods and compositions for sterilizing subjects that can avoid surgery.

[003] Previous attempts to generate non-surgical approaches to animal sterilization have not been successful. This is particularly true of contraceptive vaccines which have been the subject of considerable investment, for instance by governments interested in developing strategies to control feral animal populations. Vaccines, for example, have been used to sterilize females by attempting to permanently remove the primordial follicle population (Aitken et al., 1996). But sterilization via vaccine was somewhat ineffective as infertility takes several months to materialize because the primordial follicle pool is progressively depleted over time. Furthermore, no active immunization approach has ever been found to induce sterility in males.

[004] Accordingly, an aspect of the present invention provides a non-surgical permanent and, in certain embodiments, single dose fertility treatment that is safe and effective in male and female subjects.

[005] Within the male gonads (i.e., testes) are two cell types, Sertoli and Leydig, which are highly differentiated and are necessary for reproductive success, including for example, the maintenance of sperm development and maturation. Without the support function of these two cell types, mature sperm cell pools would not develop, thus resulting in an infertile male. In one aspect, the present invention is directed to permanently disrupting both cell types by delivering a gene, for example, to Sertoli and/or Leydig cells, that will cause cell death. The genetic payload can be delivered intravenously, for example, by a lipid sphere (nanoparticle), which protects the DNA inside until being internalized by the target cell.

[006] Introducing genes into cells to treat human and animal disease and other conditions is coming of age and has commonly involved the use of viral vector technologies. These vectors require the modification of the virus’s genome to incorporate genes of interest to be introduced. The virus is then manufactured with the DNA or other polynucleotide inside. Viral vectors can be limited, however, in their ability to be modified to introduce targeting peptides on their surface.

[007] In contrast to the general practice of using viral vectors to deliver genes, nanoparticle constructs are formulated herein to deliver a gene. Nanoparticles can be formulated with different components and different ratios of components that form the sphere that protects the DNA or other polynucleotide until it enters the cell. Nanoparticles provide versatility of manufacturing. They can be tailored and functionalized to target specific organs by biasing the biodistribution to the site of action of the therapeutic that they deliver. This can also be achieved by grafting or linking ligands to the surface of the nanoparticles, which target a specific receptor expressed by the cells or tissue of interest. Thus, many interactions of the nanoparticle components and targeting ligands bound to the surface of the nanoparticle can be designed for use in the present invention. Nanoparticles themselves are a relatively new drug delivery technology. They are currently used in humans, for example, in the oncology sector for delivering small molecule organic pharmaceuticals (largely chemotherapeutic agents). One exemplary PEGylated liposome is Doxil, indicated for the treatment of AIDS-associated Kaposi’s sarcoma.

[008] For example, to target Sertoli and/or Leydig cells specifically, nanoparticles can be designed to incorporate small peptide sequences on their surface that bind to receptors present on the target cells (e.g., FSH and LH receptors on Sertoli and/or Leydig cells), thereby adding a level of safety preventing the gene drug from being internalized by nonSertoli and/or non-Leydig cells. Herein, the inventors have developed a unique array of peptides that are capable of targeting these cells in vitro and in vivo. These cell-targeting peptides were generated through the use of random peptide phage display technology (Eidne et al, 2000) or by the synthesis of peptides capable of binding to receptors that are restricted to the target cell population (e.g., FSH, LH). The nanoparticles can also be utilized, for example, to target Sertoli and/or Leydig cells, gonocyte cells (precursors of spermatogonia), or to target female cells such as primordial follicle cells, or primordial germ cells in utero.

[009] It is well known in the art that both male and female germ cells are highly vulnerable to ionizing radiation. When tissues are irradiated, highly reactive hydroxyl radicals are generated that induce the rapid onset of lipid peroxidation chain reactions as well as concomitant oxidative damage to proteins and nucleic acids that, together, propel affected cells down an apoptotic pathway leading to cell death (Sakashita et al., 2010). In order to recapitulate the oxidative stress created by ionizing radiation, previous studies included using redox cycling xenobiotics to be carried to target cell types by an appropriate peptide and to induce a local burst of free radical generation (Aitken et al. 2013). This approach was previously successful in disrupting spermatogenesis and generated high levels of oxidative stress in the female germ line, but the levels of oxidative stress achievable with the redox cycling quinones was not sufficient to induce cell ablation with a high level of efficiency, particularly in females. [0010] In certain aspects of the invention, therefore, targeting peptides are incorporated on the surface of nanoparticles that encompass an apoptotic or cytotoxic protein or recombinant viral-based (e.g., recombinant lenti viral-based) DNA construct encoding the apoptotic or cytotoxic protein. Targeting peptides based on, e.g., FSH and LH expressed on the lentiviral coats can be grafted or linked to a nanoparticle to allow specific delivery to, for example, Sertoli and/or Leydig cells in males and primordial follicle cells in females. Species- and cell-specific promoters may also be incorporated to further reduce off-target effects. In certain aspects of the invention, targeting peptides are incorporated on the surface of nanoparticles that encompass a linear DNA construct that encodes an apoptotic or cytotoxic protein.

[0011] As a result of the present invention, sterility may be achieved following a single administration of nanoparticles. Accordingly, sterilization of stray/homeless animals will no longer require a surgical process which can be expensive, time consuming and stressful for the animals. Alongside this, the numbers of animals euthanized as a result of overcrowding in shelters will also be reduced (as a result of fewer litters being produced due to sterilization). Furthermore, this technology could also be used for routine neutering of domestic pets (cats and dogs) as opposed to the surgical processes currently in practice. This technology could both reduce the costs of this procedure and result in a reduced impact on the animals due to lack of anesthetic requirements and surgery.

III. SUMMARY OF THE INVENTION

[0012] In accordance with the present invention, nanoparticle constructs, pharmaceutical compositions comprising the nanoparticle constructs, and methods of sterilizing a subject comprising administering to the subject an effective amount of the nanoparticle construct or pharmaceutical composition are provided.

[0013] The disclosure provides a nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell; and b) an apoptotic or cytotoxic protein or a polynucleotide encoding an apoptotic or cytotoxic protein.

[0014] The disclosure also provides a nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell; and b) a reporter protein or a polynucleotide encoding a reporter protein.

[0015] The disclosure also provides a nanoparticle construct comprising: a) one or more peptides that each bind a receptor of a reproductive cell; b) an apoptotic or cytotoxic protein or a polynucleotide encoding an apoptotic or cytotoxic protein; and c) a reporter protein or a polynucleotide encoding a reporter protein.

[0016] In some embodiments, the nanoparticle construct comprises a polynucleotide encoding the apoptotic or cytotoxic protein.

[0017] In some embodiments, the apoptotic protein is Diphtheria toxin fragment A (DTA).

[0018] In some embodiments, the DTA comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0019] In some embodiments, the reporter protein is selected from red fluorescent protein (RFP), green fluorescent protein (GFP), and enhanced green fluorescent protein (EGFP).

[0020] In some embodiments, the reproductive cell is a Sertoli cell and/or a Leydig cell.

[0021] In some embodiments, the one or more peptides bind a receptor of a Sertoli cell and/or a Leydig cell.

[0022] In some embodiments, the one or more peptides bind a follicle-stimulating hormone receptor of a Sertoli cell and/or the one or more peptides bind a luteinizing hormone receptor of a Leydig cell.

[0023] In some embodiments, the one or more peptides bind a follicle-stimulating hormone receptor of a Sertoli cell.

[0024] In some embodiments, the one or more peptides bind a luteinizing hormone receptor of a Leydig cell.

[0025] In some embodiments, the reproductive cell is a gonocyte, a primordial follicle cell, and/or a primordial germ cell.

[0026] In some embodiments, the one or more peptides bind an anti- Mullerian hormone on a primordial follicle cell.

[0027] In some embodiments, the one or more peptides bind a primordial germ cell.

[0028] In some embodiments, the one or more peptides contain a terminal cysteine residue. [0029] In some embodiments, the polynucleotide encoding the apoptotic or cytotoxic protein is operatively linked to a promoter. In some embodiments, the promoter is a cellspecific promoter. In some embodiments, the promoter is selected from the group consisting of ABP, Rhox5, and HSD17B3. In some embodiments, the promoter is a species-specific promoter.

[0030] In some embodiments, the nanoparticle construct comprises poly(lactide-co- glycolide) (PLGA).

[0031] In some embodiments, the nanoparticle construct comprises polyethylene glycol (PEG). In some embodiments, the nanoparticle construct comprises maleimide- terminated PEG. In some embodiments, the nanoparticle construct comprises DSPE-PEG.

[0032] In some embodiments, the nanoparticle construct comprises cationic lipids, ionizable lipids, and/or helper lipids.

[0033] In some embodiments, the one or more peptides are conjugated to lipids.

[0034] In some embodiments, the ligand density of the nanoparticle construct is optimized.

[0035] In some embodiments, the polynucleotide is condensed with protamine, lysine, or polylysine.

[0036] In some embodiments, the apoptotic or cytotoxic protein or the polynucleotide is encapsulated within the nanoparticle construct.

[0037] In some embodiments, the nanoparticle construct comprises a nanoparticle having a PLGA matrix with maleimide terminated PEG lipids; wherein the one or more peptides are conjugated to the maleimide terminated PEG lipids; and wherein the apoptotic or cytotoxic protein or the polynucleotide is encapsulated in the nanoparticle.

[0038] In some embodiments, the nanoparticle construct comprises a viral vector comprising the polynucleotide encoding the apoptotic or cytotoxic protein. In some embodiments, the viral vector is selected from an adenoviral vector, AAV vector, poxvirus vector, and lentiviral vector. In some embodiments, the viral vector is a lentiviral vector.

[0039] In some embodiments, the nanoparticle construct further comprises a fluorescent label.

[0040] The disclosure also provides a pharmaceutical composition comprising the nanoparticle construct of any of the preceding embodiments.

[0041] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. [0042] In some embodiments, the pharmaceutical composition comprises an aqueous solution.

[0043] In some embodiments, the composition or the nanoparticle construct is freeze- dried.

[0044] The disclosure also provides a method of sterilizing a subject comprising administering to the subject an effective amount of the nanoparticle construct or pharmaceutical composition of any one of the preceding embodiments.

[0045] In some embodiments, the nanoparticle construct or pharmaceutical composition is freeze-dried as a powder and dispersed in an aqueous medium prior to administration.

[0046] In some embodiments, the composition is administered via injection. In some embodiments, the administering comprises a one-time injection.

[0047] In some embodiments, the administering is intravenous, intraperitoneal, or intratesticular administration. In some embodiments, the administering is intravenous injection. In some embodiments, the administering is a one-time intravenous injection into the cephalic vein.

[0048] In some embodiments, the effective amount comprises a dosage capable of inducing sterilization of the subject.

[0049] In some embodiments, sterilization or ablation occurs within 24 hours, 48 hours, or 72 hours after injection.

[0050] In some embodiments, sterilization is permanent.

[0051] In some embodiments, the effective amount comprises a dosage capable of inducing ablation of at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of one or more reproductive cell types of the subject. In some embodiments, the effective amount comprises a dosage capable of inducing ablation of at least about 60% of one or more reproductive cell types of the subject. In some embodiments, the effective amount comprises a dosage capable of inducing ablation of at least about 80% of one or more reproductive cell types of the subject.

[0052] In some embodiments, the one or more reproductive cell types of the subject is Sertoli cells and/or Leydig cells.

[0053] In some embodiments, the one or more reproductive cell types of the subject is gonocytes, primordial follicle cells, and/or primordial germ cells. [0054] In some embodiments, the subject is a cat or dog. In some embodiments, the subject is a cat. In some embodiments, the subject is a dog. In some embodiments, the subject is a male cat or dog. In some embodiments, the subject is a female cat or dog.

[0055] In some embodiments, the subject is pre -pubescent. In some embodiments, the subject has reached pubertal maturation.

[0056] The disclosure also provides a method of determining uptake efficiency of a nanoparticle construct, comprising: a. administering to a subject the nanoparticle construct of a previous embodiment (comprising a) one or more peptides that each bind a receptor of a reproductive cell; and b) a reporter protein or a polynucleotide encoding a reporter protein) or a composition comprising the nanoparticle construct; and b. assessing ligand density on the nanoparticle surface, wherein increased ligand density or transfection efficiency for the nanoparticle as compared to a control or reference value is indicative of optimal uptake efficiency.

[0057] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0058] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0059] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiment(s) and together with the description, serve to explain the principles described herein.

IV. SEQUENCE LISTING

V. DETAILED DESCRIPTION

[0060] The present invention further relates to nanoparticle constructs (e.g., targeting peptides conjugated to the lipids on the surface of the nanoparticle and a nucleotide sequence encapsulated in the nanoparticle) and compositions thereof. The present invention further relates to a method of sterilizing a male or female cat or dog comprising administering to the male or female cat or dog an effective amount of the nanoparticle constructs or compositions thereof, where the nanoparticle constructs comprise one or more peptides that each bind to one or more receptors of one or more reproductive cells; and an apoptotic or cytotoxic protein or polynucleotide encoding the apoptotic or cytotoxic protein.

[0061] As used herein, “nanoparticle” may be interpreted to also mean “nanoparticle construct” with the proper context set forth above.

[0062] In certain aspects, the nanoparticle comprises peptides on its surface that target a receptor on a reproductive cell.

[0063] As used herein, “reproductive cell(s)” refer to any cell(s), ablation (i.e., selective reduction or removal) of which could affect or result in sterilization of a subject. Accordingly, “reproductive cells” as used herein include germ cells, as well as cells whose function effects the development or maturation of reproductive systems (“reproductive supportive cells”) during embryogenesis, including but not limited to, for example, Sertoli cells, Leydig cells, cells of the primordial follicle, or granulosa cells. Other such “reproductive cells” or “reproductive supportive cells” would be known to a person of ordinary skill in the art. [0064] In general, the target peptide can be from any vertebrate source (e.g., any species or breed of dogs or cats).

[0065] In certain aspects, the peptides target FSH and/or LH receptors on Sertoli and/or Leydig cells. In certain aspects, the target peptide is directed to both Sertoli and Leydig cells. It is known in the art that the number of Leydig cell numbers is correlated with Sertoli cell number in adults (mature subjects). It was previously shown in cell ablation studies that ablation (i.e., selective reduction or removal) of Sertoli cells also results in reduction of Leydig cell numbers by 75% (Rebourcet et al (2014); Development 141:2139- 2149; Rebourcet et al., 2017; Endocrinology, 158(9): 2955-2969). A reduction of Leydig cells have been seen when Sertoli cell population is decreased by about 50% or more (Rebourcet et al., 2017; Endocrinology, 158(9): 2955-2969). Ablating Leydig cells along with Sertoli cell ablation, may ablate testosterone production to a degree to confer infertility. Ablating Sertoli cells alone will also impact Leydig cell function. In other aspects, the target peptide is directed to Sertoli cells alone.

[0066] As used herein, Anti-Mullerian hormone (“AMH”) is a hormone playing a role in growth differentiation and folliculogenesis. AMH expression inhibits the development of the female reproductive tract in the male embryo and thus is critical to sex differentiation during fetal development.

[0067] In certain aspects of the invention, the peptide targets the AMH receptor on the primordial follicle.

[0068] As used herein, “gonocytes” relate to precursors of spermatogonia that differentiate from primordial germ cells. As used herein, the “primordial follicles” are the first class of follicles formed in mammalian ovaries.

[0069] In certain aspects of the invention, the peptide targets receptors on reproductive cells selected from primordial germ cells or gonocytes, and primordial follicle cells.

[0070] In certain aspects, the sequences may be modified to include a terminal cysteine, which may be used to attach the peptide to the nanoparticle. In certain aspects, the sequences may be modified, for example to purify protein from cell material for use in in vitro experiments, for example.

[0071] In certain aspects, the invention relates to administering to a male cat or dog an apoptotic or cytotoxic protein or transgene encoding an apoptotic or cytotoxic protein. [0072] As used herein, an “apoptotic protein” is a protein that can cause cell death. In the context of this invention, apoptosis of certain reproductive cells can lead to sterilization.

[0073] In certain aspects, the apoptotic protein is Diphtheria toxin fragment A (DTA). DTA has been used in unrelated indications such as glioblastoma multiforme and prostate cancer. Diphtheria toxin (“DTX”) is a single protein composed of two components; fragment A and fragment B. Fragment B is the component responsible for binding to the HBEGF receptor (the heparin-binding EGF-like growth factor receptor), which leads to its entry into the cell. Fragment B binds to the HBEGF receptor present on the surface of many cell-types, leading to internalization and delivery of fragment A into the cells. Once inside the host cell, fragment A is able to block protein synthesis of the host cell. This induces death of the specific host cell. Without fragment B, fragment A cannot enter any other non-host cell, and is thus inactive or inert without it.

[0074] Accordingly, one benefit of using diphtheria toxin for controlled cell ablation of a target cell is that it has not been shown to have a toxic effect on other cell types or systems in vivo (Rebourcet et al., 2017; Endocrinology, 158(9): 2955-2969). Diphtheria toxin was previously shown to be a means of selectively ablating Sertoli cells from the testis of adult transgenic mice carrying the diphtheria toxin receptor on the surface of these cells. Use of Diphtheria toxin was shown to be an acute means to specifically induce ablation of Sertoli cells with cell death occurring within 24 hours (Rebourcet et al., 2017; Endocrinology, 158(9): 2955-2969). Following induction of Sertoli cell apoptosis by injection of 100 ng DTX (diphtheria toxin), for example, specific and complete ablation of Sertoli cells was observed, which was unchanged 1-day post ablation (Rebourcet et al (2014); Development, 141 :2139-2149). Diphtheria toxin was previously shown to have dose-dependent effects in Sertoli cells in mouse models. As a further example, the Rebourcet 2017 article cited above, which is incorporated by reference herein in its entirety, shows that treatment of neonatal mice with Ing DTX (diphtheria toxin), for example, caused a variable -50% reduction in Sertoli cell numbers and lOng caused complete Sertoli cell ablation. The same article reports treatment of adult mice with lOng and 25ng of DTX caused a clear and variable (mean -50%) reduction in cell numbers and 50ng DTX caused complete Sertoli cell ablation. The article further reports that there was no recovery in Sertoli cell number for up to 90 days after partial ablation of Sertoli cells in adult animals, suggesting the effects of DTX may be permanent. (Rebourcet et al., 2017; Endocrinology, 158(9): 2955-2969). [0075] As used herein, DTA comprises the amino acid sequence of SEQ ID No. 1 or a variant thereof having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In general, the variations can comprise modifications, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present disclosure, a “polypeptide” or “peptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate or may be accidental, such as through mutations of hosts which produce the proteins or errors.

[0076] In certain aspects, the nanoparticle construct further comprises a viral vector (i.e., a recombinant viral-based DNA construct). In certain aspects, the viral vector is selected from an adenoviral vector, AAV vector, poxvirus vector, and lentiviral vector. In certain aspects, the nanoparticles comprise a lentiviral vector. In certain aspects, the lentiviral constructs are self-inactivating and/or cannot be re-activated by serendipitous viral infections to cause insertional mutagenesis and cannot activate surrounding genes due to the mutations in the 3’ viral long terminal repeat. In certain aspects, the tumorigenic WPRE common to many lentiviral vectors have been replaced with a non-tumorigenic OPRE. This can further negate the already minimal risk of tumor development in treated animals. In certain aspects other viral vectors known in the art (defined elsewhere in this application) can be used. In certain aspects, the nanoparticles comprise a lentiviral vector containing the coding sequence for DTA.

[0077] In certain aspects, the nanoparticle construct further comprises a transgene coding a reporter protein (e.g., a fluorescent reporter protein) or a fluorescent label. This labeling is useful, for example, to determine body distribution and cellular uptake in vitro or in vivo). For example, a fluorescent reporter transgene delivered by these nanoparticles allows for analysis of tissues collected post-mortem using fluorescent imaging equipment and detection of the fluorescent protein within fixed tissues. In certain aspects, the fluorescent protein is red fluorescent protein (RFP), green fluorescent protein (GFP), or enhanced green fluorescent protein (EGFP). In certain aspects, the fluorescent label is fluorescent isothiocyanate (FITC)-dextran. FITC-dextran loaded NPs can be used to optimize targeting ligand density and confirm the targeting specificity and uptake efficacy of NP preparations. [0078] In certain aspects, the polynucleotide is operatively linked to a promoter (i.e., a specific component controlling delivered gene expression carried within the nanoparticle). In certain aspects, the promoter can be cell-specific or species-specific (e.g., specific for cats or dogs, or specific for a particular breed of cat or dog). Use of the promoter can provide an additional aspect of cell specificity, to enable the delivered gene to be ‘switched on’ when delivered to the cell types of interest. In certain aspects, the promoter is cell-specific. In certain aspects, the cell-specific promoters are selected from ABP, Rhox5, or HSD17B3.

[0079] In general, the compositions comprise a nanoparticle. Nanoparticles can be lipid-based, polymer-based, or be a polymer/lipid hybrid. Nanoparticles provide many unique advantages over conventional delivery systems. For example, nanoparticles can encapsulate various therapeutics, such as nucleic acids, proteins and small compounds, and enhance both drug stability and bioavailability thus leading to substantial reduction in dosage as well as dose frequency. In certain aspects, the nanoparticles encapsulate an apoptotic or cytotoxic protein or polynucleotide encoding the apoptotic or cytotoxic protein. Targeting ligands, particularly peptides, can also be incorporated onto the nanoparticle surface in order to enable drug delivery to specific cell populations, achieving improvements in efficacy with a corresponding reduction in unwanted effects. Targeting ligands can be introduced on the surface of the nanoparticles with varied densities, which can affect uptake efficiency of the nanoparticles. As non-limiting examples, the nanoparticles can be polymer-based, lipid- based, a polymer/lipid hybrid, or can comprise biocompatible inorganic nanovectors based on layered-double hydroxide strategy. Nanoparticle constructs (e.g., nanoparticle itself together with the targeting peptides conjugated to the lipids of the nanoparticle and nucleotide sequence encapsulated in the nanoparticle) created from biocompatible materials approved for human use have been widely reported in the literature for targeting human diseases. The activity of a nanoparticle construct can depend on, for example, the materials used in the nanoparticle, the type of peptides, peptide density, as well as length of spacer-peptide ligands as well as the testing of cell- specific promoters.

[0080] In certain aspects, the nanoparticle is polymer-based. In certain aspects, the nanoparticle comprises components selected from poly(lactide-co-glycolide) (“PLGA”), PED+linear and dendritic polymers. In certain aspects, the nanoparticle comprises PLGA. PLGA will form the particle matrix to encapsulate payloads, degrading under physiological conditions known by a person of ordinary skill in the art to effect drug release. The degradation time of PLGA nanoparticles can be altered from days to years to achieve a safe and sustained action by varying the polymer molecular weight, the composition of the block copolymer and the structure of the nanoparticles. In certain aspects, the nanoparticle comprises PLGA and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (“DSPE-PEG”). In certain aspects, the nanoparticles further comprise cationic lipids, ionizable lipids, and other helper lipids. Nonlimiting examples of helper lipids include DC- Cholesterol, dilinoleylmethyl-4-dimethylaminobutyrate, DLin-KC2-DMA, DLin-MC3- DMA, and Lipofectamine 3000.

[0081] In certain aspects, the polynucleotide to be delivered by the nanoparticle construct is condensed with protamine, lysine, or polylysine to further enhance transfection efficiency prior to encapsulation by the nanoparticles. As used herein, “transfection efficiency” refers to the percentage of cells successfully transfected by nanoparticle delivery as evidenced, for example, by the polynucleotide payload delivery to the nucleus resulting in transgene expression as related to an entire population. In certain aspects, the polynucleotide is delivered inside a PLGA nanoparticle, wherein the polynucleotide is condensed with protamine, lysine, or polylysine.

[0082] In certain aspects, the nanoparticle comprises PEG (“poly(ethylene glycol)”). PEGylation can minimize non-specific particle uptake and clearance by immune cells. In certain aspects, the nanoparticle comprises a maleimide-terminated PEG, e.g., as a surface coating. Such a coating allows for conjugation of the targeting peptide (synthesized with a terminal cysteine) to the PEG via a covalent thiol-maleimide linkage. Each peptide may be engineered to contain a terminal cysteine for coupling purposes and attachment to the nanoparticle surface may be achieved using maleimide-terminated PEG. The peptide may comprise a terminal cysteine to couple with maleimide-terminated PEG via covalent thiol- maleimide linkage. In certain aspects, the one or more peptides are conjugated to lipids on the surface of the nanoparticle construct.

[0083] As used herein, “ligand density” of targeted nanoparticles refers to the density of targeting peptide/ligand on the nanoparticle surface. Ligand density relates to the ability of the nanoparticles to navigate across biological barriers and promote cell-specific uptake. Generally, there is an optimal ligand density for a nanoparticle construct and targeted receptor that can be determined by a person of ordinary skill in the art. The ligand density of targeted nanoparticles may be indicative of the uptake efficiency of the nanoparticles and may reflect targeted delivery. [0084] As used herein, “N/P ratio” refers to the ratio of positively charged amine (N) groups of the polymer to negatively charged phosphate (P) groups of DNA, which is one characteristic of nanoparticles in affecting gene transfection efficiency and cytotoxicity.

[0085] Various other properties characterized, such as surface chemistry, drug loading, stability in blood serum, and release profile of payloads can be indicative of cell expression or viability. Modern spectroscopic (NMR, MS, UV-Vis, HPLC, IR) and other known techniques in the art (dynamic light scattering, size exclusion chromatography, gel electrophoresis, electron microscopy, X-ray photoelectron spectroscopy) can also be used to characterize a full range of nanopharmaceutical properties including surface chemistry, drug loading, stability in blood serum, and release profile of payloads in biological media etc.

[0086] In certain aspects, the nanoparticle constructs are formulated into compositions comprising a pharmaceutically acceptable carrier. In some aspects, the compositions comprise compendial and widely used excipients. In certain aspects, the composition comprises at least one excipient.

[0087] In certain aspects the compositions comprise a freeze-dried or lyophilized powder, or aqueous solution. In certain aspects, the compositions comprise a freeze-dried powder that can be re-dispersed in aqueous medium prior to administration. This can benefit storage and shelf life.

[0088] In general, a subject is administered the nanoparticle construct or compositions thereof at an effective dosage. In certain aspects, the subject is administered a composition containing the nanoparticles described above at an effective dosage. In certain aspects, the effective dosage comprises an effective amount of the nanoparticles or compositions thereof sufficient to induce ablation of cells.

[0089] In certain aspects, administration of the effective amount of nanoparticle constructs or compositions thereof described above results in partial or complete cell ablation of the reproductive cells. Even partial ablation of the cells results in significant disruption of the blood testes barrier, which has been shown to lead to infertility in males. In certain aspects, the percentage of cells ablated comprise at least about 50%, at least 60%, at least about 70%, at least about 80%, or at least about 90% of cells. In certain aspects, complete cell ablation occurs. In certain aspects, at least about 60% of cells are ablated.

[0090] In certain aspects, administration refers to intravenous, intraperitoneal, or intratesticular administration, although other methods of administration known by persons of ordinary skill in the art in delivering nanoparticle constructs can also be used. In certain aspects, the nanoparticle constructs or compositions thereof are administered by intravenous administration or injection.

[0091] In certain aspects, the nanoparticle constructs or compositions thereof are administered in a single dose or multiple doses. In certain aspects, administration comprises a single dose. In certain aspects, administration comprises a single intravenous injection. In certain aspects, administration comprises a single intravenous injection into the cephalic vein.

[0092] In certain aspects, the subject receiving the administration of the nanoparticle constructs or compositions thereof described above are mature or pre-pubescent. Pubertal maturation in males is defined as the presence of motile sperm in the ejaculate (male dogs), or presence of penile spines (male cats). Pubertal maturation in females is defined as the females undergoing at least one normal estrous cycle (female dogs: presence of serosanginuous vulvar discharge, vulvar swelling and increase of the plasma progesterone concentration > 9nmol/L indicating that ovulation has occurred). In certain aspects, the subjects are mature or have reached pubertal maturation.

[0093] In certain aspects, the subject receiving the administration of the nanoparticle constructs or compositions thereof described above are mature or pre-pubescent cats or dogs. In certain aspects, the subject is selected from a mature male dog, mature male cat, mature female dog, or mature female cat.

[0094] In certain aspects, induction of cell death occurs within 24 hours, 48 hours, or 72 hours of injection or ablation. In certain aspects, induction of cell death leads to sterilization of the subject. In certain aspects, sterilization of the subject is permanent.

VI. ADDITIONAL DEFINITIONS

[0095] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0096] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. [0097] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0098] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages, for example, may mean ±5% of the value being referred to. For example, about 100 means from 95 to 105.

[0099] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[00100] The terms “nucleic acid,” “nucleotide,” and “polynucleotide’ are well known in the art. A “nucleic acid” as used herein will generally refer to a molecule (i.e., strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. The term “polynucleotide” refers to at least one molecule of greater than about 100 nucleobases in length. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g. an adenine "A," a guanine "G." a thymine "T" or a cytosine "C") or RNA (e.g. an A, a G. an uracil "U" or a C). The term “oligonucleotide” refers to a molecule of between about 3 and about 100 nucleobases in length. The term “nucleic acid” also refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

[00101] As used herein, the term “gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. A “gene” refers to coding sequence of a gene product, as well as non-coding regions of the gene product, including 5’UTR and 3’UTR regions, introns and the promoter of the gene product. These definitions generally refer to a single- stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule. Thus, a nucleic acid may encompass a double-stranded molecule or a double-stranded molecule that comprises one or more complementary strand(s) or “complement(s)” of a particular sequence comprising a molecule. The term “gene” refers to the segment of DNA involved in producing a polypeptide chain, it includes regions preceding and following the coding region as well as intervening sequences (introns) between individual coding segments (exons).

[00102] As used herein, the term “binds” (e.g., to a receptor) is a term that is well understood in the art, and methods to determine such binding are also well known in the art. A molecule is said to exhibit “binding” if it reacts, associates with, or has affinity for a particular cell or substance and the reaction, association, or affinity is detectable by one or more methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance devices, or etc.

[00103] As used herein, “nanoparticle” and “nanoparticle construct” refer to materials with overall dimensions in the nanoscale, i.e., under about 100 nm. Nanoparticles can be prepared for a variety of materials, for example, lipids and polymers.

[00104] As used herein, the term “vector” includes any genetic element, including, but not limited to, a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, minichromosome, expression vector, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer nucleic acid molecules to cells. Vectors are well known in the art and include, but are not limited to, cloning and expression vectors, as well as viral vectors. As used herein, “viral vector” includes DNA vectors, RNA vectors, and circular or linear vectors and refers to the recombinant viral-based (e.g., recombinant lentiviral-based DNA) construct. The vectors may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vector, lentivirus vectors, adenovirus vectors, helper-dependent adenoviral vectors (HD Ad), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, pox virus vectors, and retrovirus vectors. In certain aspects, the nanoparticle constructs and compositions thereof comprise a lentivirus. For a lentivirus, the lentivirus may be integrating or non-integrating.

[00105] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Peptides, oligopeptides, dimers, multimers, and the like, are also composed of linearly arranged amino acids linked by peptide bonds, and whether produced biologically, recombinantly, or synthetically and whether composed of naturally occurring or non- naturally occurring amino acids, are included within this definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include co- translational and post-translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases and prohormone convertases (PCs)), and the like. Furthermore, for purposes of the present invention, a “polypeptide” encompasses a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art), to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods. Polypeptides or proteins are composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, has a well-defined conformation. Proteins, as opposed to peptides, generally consist of chains of 50 or more amino acids. For the purposes of the present invention, the term “peptide” as used herein typically refers to a sequence of amino acids of made up of a single chain of D- or L- amino acids or a mixture of D- and L- amino acids joined by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length.

[00106] As used herein, the term “wild-type” refers to a non-mutated version of a polypeptide that occurs in nature, or a fragment thereof. A wild-type polypeptide may be produced recombinantly.

[00107] As used herein, the term “variant” means a biologically active polypeptide having at least about 50% amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N- or C-terminus of the polypeptide.

[00108] A variant can have, for example, at least 1, 2, 3, 4, or 5 amino acids substituted by a different amino acid. In certain aspects, a variant has at least about 50% sequence identity with the reference polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N- or C-terminus of the polypeptide.

[00109] As used herein, the term “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of sequences being compared.

[00110] As used herein, an “amino acid substitution” refers to the replacement of one amino acid in a polypeptide with another amino acid. An amino acid substitution can be a non-conservative substitutions, which will entail exchanging a member of one of these classes with another class. An amino acid substitution can be a conservative substitution.

[00111] Nonlimiting exemplary conservative amino acid substitutions are shown in Table 1. Amino acid substitutions may be introduced into a molecule of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC or enhanced pharmacokinetics.

[00112] Table 1

[00113] Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

[00114] To “reduce” or “inhibit” means to decrease, reduce, or arrest an activity, function, parameter, or amount as compared to a reference or control. In certain aspects, “reduce” or “inhibit” refers to the ability to cause an overall decrease of about 20%, about 50%, about 75%, about 85%, about 90%, or about 95%, or greater. In some embodiments, the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time. A “reference” or “control” as used herein, refers to any sample, standard, or level that is used for comparison purposes.

[00115] The terms “subject,” “individual,” and “patient” are used interchangeably herein, and refer to the subject to whom treatment with the nanoparticles or compositions thereof according to the present invention, is provided. As used herein, a “subject" means a human or animal. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a cat or a dog. A subject can be male or female. Additionally, a subject can be an adult or can be pre -pubescent.

[00116] As used herein, the terms “administering,” and “introducing” are used interchangeably herein and refer to the placement of the nanoparticles or compositions thereof into a subject by a method or route which results in at least partial localization of the nanoparticles at a desired site. The compositions and nanoparticle constructs of the present invention can be administered by the methods described herein and any appropriate route known to one of ordinary skill in the art that results in partial or complete apoptosis of reproductive cells in a subject.

[00117] The term “effective amount” as used herein refers to a sufficient amount of pharmacological composition to provide the desired effect. Thus, it is not possible to specify the exact “effective amount.” However, for any given case as set forth in detail herein, an appropriate “effective amount” can be determined by one of ordinary skill in the art. The efficacy of treatment can be judged by an ordinarily skilled practitioner.

[00118] A “composition” or “pharmaceutical composition” are used interchangeably herein refers to a composition that usually contains an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to cells. Exemplary compositions and pharmaceutical compositions are described in detail herein. The cells may be part of a subject, for example for therapeutic, diagnostic, or prophylactic purposes. The cells may also be cultured, for example cells as part of an assay for screening potential pharmaceutical compositions, and the cells may be part of a transgenic animal for research purposes. The composition can also be a cell culture, in which a polypeptide or polynucleotide encoding a metabolic regulator of the present invention is present in the cells and/or in the culture medium.

[00119] “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. The acceptability of treatment can be judged by an ordinarily skilled practitioner.

[00120] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in maintaining the activity of or carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. As set forth in detail herein, a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In addition to being “pharmaceutically acceptable” as that term is defined herein, each carrier must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. [00121] Definitions of other common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9). Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN- 10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[00122] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, as defined by the claims.

VII. EXAMPLES

Example 1. Design and testing of nanoparticles

[00123] FSH and LH binding ligands were previously identified. The present example compares different nanoparticle constructs (e.g., varying the materials used in the assembly of the nanoparticles, the type of peptides, peptide density, as well as length of spacer-peptide ligands as well as the testing of cell-specific promoters) to determine the optimal compositions to be tested in vitro and in vivo (mouse) with GFP as a reporter, including optimization of promoters to drive transgene expression.

[00124] In general, nanoparticles will comprise poly(lactide-co-glycolide) (PLGA) and PEG polymers. PLGA will form the particle matrix to encapsulate payloads, degrading under physiological conditions in order to effect drug release. DNA can be condensed with protamine or cationic polyethylenimine (PEI, 2kDa) can be incorporated to further enhance transfection efficiency prior to encapsulation. PEG will form the surface coating on the particle matrix to minimize non-specific particle uptake and clearance by immune cells. Maleimide-terminated PEG will be incorporated into the surface coating, allowing subsequent conjugation of the targeting peptide, synthesized with a terminal cysteine, to the particle surface via a covalent thiol-maleimide linkage. Targeting ligands will be introduced onto the surface of PEG-PLGA nanoparticles with varied densities. The resulting targeted nanoparticles will be fluorescently labelled and investigated for body distribution and cellular uptake via flow cytometry and fluorescence microscopy, where fluorescently labelled non-targeted nanoparticles will be used as control. The uptake efficiency of nanoparticles will be correlated with the ligand density on particle surface, which will elucidate the optimum ligand density needed to achieve the most efficient targeted delivery. Transfection efficiency can also be used to assess uptake efficiency of the nanoparticles. The nanoparticles with optimum uptake will then be loaded with genetic payloads (EGFP and DTA gene constructs) to confirm that the latter are expressed in a cell specific manner and, in the case of DTA, precipitate a loss of cell viability. Development of the nanoparticles will include examination of materials used in the assembly of the particles, the type of peptide, peptide density, as well as length of spacer-peptide ligands as well as the testing of cell-specific promoters.

[00125] The nanoparticles are initially designed with an EGFP transgene. In the best performing constructs, the EGFP transgene will be replaced by a DTA transgene. Constructs will utilize well-characterized cell-specific promoters (e.g. ABP, Rhox5, HSD17B3 (male)), initially driving EGFP (control vectors), and later, DTA (induction of cell death). Promoter regions from cat, dog, human, and mouse may also be evaluated. The promoter/gene sequences will be produced as single molecules of DNA using commercial oligonucleotide synthesis, and embedded into lentiviral constructs using standard cloning techniques.

[00126] Completed constructs will undergo DNA sequencing and then functional validation (expression of EGFP, or induction of cell death, respectively) via transient transfection into primary cells (Sertoli cells and Leydig cells). The best performing constructs will be packaged into the nanoparticles.

[00127] For packaging into PLGA nanoparticles, it will be assessed whether the condensation of nucleic acid cargo with either protamine or polylysine will further enhance transfection efficiency. Gene delivery to the target cell type by the nanoparticles will be followed by integration of the genetic payload into the cell genome and expression of the transgene (EGFP or DTA respectively). The delivery of vectors containing the EGFP construct will identify targeted cells and be used both to demonstrate efficacy of the targeting technology and the safety of the system by highlighting any off-target gene delivery. Example 2. In vitro and in vivo testing of nanoparticle comprising DTA gene

[00128] In this example, the DTA gene will be incorporated and again tested in vitro and in vivo to confirm targeting and effectiveness of the nanoparticles construct designed in Example 1. One objective is to test in vitro on cultures expressing the FSH receptor for determination of apoptosis. Another objective is to test lead candidates in vivo with immunohistochemistry and use of an optical dissector to determine the absence of target cells (8 weeks post-injection).

A. In vitro

[00129] In order to configure the nanoparticles with the appropriate composition, ligand density, peptide coupling/spacing arrangement and genetic payload, in vitro models will be used. These include: primary cultures of Sertoli cells and cell lines expressing the FSH or EH receptor developed in-house.

[00130] A single cellular model (FSH peptide-functionalized nanoparticles binding to FSH-R-expressing stable cell line) will initially be used to monitor reporter protein (e.g., GFP) expression to determine relative rates of nanoparticle incorporation and expression. This should rapidly lead to identification of exemplary nanoparticle compositions (in terms of the ligand density on the nanoparticle surface and optionally including linkage spacers). These in vitro studies will help identify exemplary SC-targeting and LC-targeting nanoparticles for confirmation of GFP gene expression in vivo.

[00131] Once the ability of the nanoparticle construct to achieve reporter protein expression in vitro is confirmed, nanoparticles containing a genetic payload encoding DTA under the regulation of cell- and species- specific promoters will be constructed.

[00132] The ability of these nanoparticles to induce cell death in vitro will be tested by conducting dose- and time- dependent studies using the model systems (LH peptide-functionalized nanoparticles targeting TM3 cells and FSH peptide-functionalized nanoparticles targeting FSH-R-expressing cells). Experiments involving the exposure of control cells (e.g., GC1) not expressing receptors for these gonadotrophins will also be conducted. These in vitro data may confirm the nanoparticles are capable of delivering cell death in a highly selective, targeted, efficient manner.

B. In vivo

[00133] The ability of the nanoparticle constructs described above to target Sertoli cells (male) and Leydig cells (male) to induce cell ablation can next be assessed in systematic studies. The structure of the nanoparticles will be based on the in vitro studies described above, which will have led to exemplary nanoparticle compositions in terms of lipid- PEG content, the density and configuration of the cell-specific targeting peptides incorporated into the nanoparticle surface and the structure of the genetic payload inserted into the nanoparticle interior.

[00134] Using a single dose administration regime, two potential nanoparticle formulations will initially be assessed in vivo-. (1) nanoparticles expressing FSH peptides to target Sertoli cells in males; and (2) LH-functionalized nanoparticles to target Leydig cells in males. Each of these formulations will be assessed at 3 different doses.

[00135] The in vivo assessment of the biological efficacy of the nanoparticles will be conducted in juvenile adult Swiss mice (6-8 weeks old). The in vivo assessment will be conducted with functionalized nanoparticles initially administered by intraperitoneal injection.

[00136] The nanoparticles will only be administered on a single occasion and the effects of the treatment will be monitored at autopsy 8 weeks later. Controls animals will receive injections of identical nanoparticles minus the genetic payload. A total of 10 animals will be included in each experimental group (n=10 SC-targeting; n=10 LC-targeting; n=10 controls, x 2 promoters for each cell-type; total mice = 60) in order to provide sufficient data for statistical analyses by ANOVA.

[00137] To assess EGFP expression, target (testes) and off-target (liver, brain, kidney) tissues will be collected, fixed for 24 hr in 4% paraformaldehyde in PBS, sectioned and examined by fluorescence microscopy employing an excitation wavelength of 488 nm.

[00138] To assess DTA expression, two different promoters and three doses will be tested, the identical dose to the GFP-expressing nanovectors plus two others to be determined empirically with a view to identifying the minimal dose to achieve sterility. (n=10 SC-1285 targeting per dose; n=10 LC-targeting per dose; n=10 controls per dose, per dose = 90 mice in total). Cell counts will be conducted using stereological methods as described previously (Rebourcet et al., 2017). These cell counts will focus on the particular cell type targeted for ablation - Sertoli cells and Leydig cells.

[00139] A histological assessment of the quality of spermatogenesis and gonadal architecture respectively will occur in the same tissues.

[00140] Immunohistochemical determination of induced apoptosis will be supported by stereological analyses employing the optical disector technique, to assess the induction of target cell ablation by quantifying the number of Sertoli and Leydig- cells in each testis. The nanoparticles may be capable of inducing cell death in > 80% of the targeted cell population (Sertoli and Leydig cells) in vivo.

[00141] The in vivo studies will involve detailed monitoring of the animals to ensure general health and wellbeing (body weight, posture, vocalization, physical activity) and include, at the time of autopsy, a visual inspection of the major organ systems for signs of systemic damage.

C. Long-term fertility assessment

[00142] Any nanoparticle constructs that are found to possess biological activity in the in vivo assessment above will be submitted for fertility trials in male mice to confirm that a state of sterility has been induced. Fertility assessments will be conducted to ensure that target cell ablation is accompanied by permanent infertility. Fertility trials will be conducted 8 weeks after inoculation and repeated at 12 months to ensure that the infertility induced is permanent.

[00143] These fertility trials will involve housing treated male mice (n=20 SC- targeting; n=20 LC targeting; n=20 controls; + 60 female partners) with mating partners and monitoring the incidence of pregnancy in addition to a full histological examination of the major organ systems, 10 days after mating and 8 weeks after inoculation with the nanoparticles.

Example 3. Xenograft study in mice

[00144] In vivo mouse testing to confirm nanoparticle activity will be performed with the cat and dog gonadal tissue xenograft model in which immunologically compromised nude mice are xenografted with cat and dog ovarian and testicular tissue. This permits testing of efficacy directly in cat and dog gonadal tissue in vivo.

[00145] Mice are naturally resistant to Diphtheria toxin (the murine HBEGF receptor binds the toxin only poorly). A model will be used, in which expression of a transgene for Diphtheria toxin fragment A (DTA) is driven inside cells to induce cell death. This model was previously used to specifically ablate the Sertoli cell population from mouse embryos in vivo, with no off-target effects (Rebourcet, O'Shaughnessy et al. 2014).

A. Xenografting procedure

[00146] Owner-consented cat and dog ovaries and testes will be collected at 4 months of age during routine neutering. [00147] Male CD1 nude mice will be anesthetized by inhalation of isofluorane and castrated/ovarectomised. Removal of gonads ensures that the host mouse gonadotrophins are high in order to drive the maturation of the gonadal tissue; an important and well- characterized interaction between the hypothalamus/pituitary and the xenografted gonad (Honaramooz, Snedaker et al. 2002, Mitchell, Saunders et al. 2010). Subsequently, steroid hormone production by the gonads feeds back to the brain to maintain steady state gonadal function (Honaramooz, Snedaker et al. 2002).

[00148] In males, small pieces (3 mm ³ approx.) of donor testis tissue from a total of six male dogs and six male cats will be inserted subcutaneously under the dorsal skin after aseptic preparation of the skin of the mice using povidone or chlorhexidine scrubbing plus swabbing with ethanol. Between 4-6 testis grafts from a single cat/dog will be inserted on either side of the midline; six mice per cat/dog donor (72 nude mice in total). All surgical procedures will be carried out within a Class II containment cabinet and mice will be housed in individually ventilated cages (IVC) in order to provide a sterile environment and avoid any potential for transmission of pathogens within/between species. Mice will receive analgesia (Rimadyl LA, Pfizer, New York, USA) in the drinking water for 1 day pre- and 5 days postsurgery, as well as antibiotics (Baytril, Bayer, Germany) for 5 days post-surgery, and will be monitored twice daily.

B. Characterization of xenografts

[00149] A genetic pay load containing a DNA sequence encoding only fragment A of the toxin is delivered into cells, through binding of nanoparticles to endogenous cellspecific receptors (e.g. FHSR, AmhR etc). Once inside, the cell expression of the transgene for fragment A of the toxin is induced, which then functions to drive cell death (e.g., within 24 hours). Cell targeting and expression of GFP (controls) or induction of cell ablation (DTA nanoparticles) will then be assessed.

[00150] Host mice will be anesthetized until unconscious and then culled by cervical dislocation and grafts will be retrieved by dissection. Xenografts will be weighed and the percentage survival of xenografts will be recorded. Fragments of pre-graft, xenograft and equivalent age-matched control tissues will be snap frozen for molecular analysis, and the remainder fixed for 2 hours in Bouin’s fixative, transferred to 70% ethanol and then processed into paraffin blocks using standard procedures.

[00151] For testes, the presence of spermatogenesis, recording the most advanced stage of germ cell development observed for each graft, will be determined histologically, and compared to non-grafted age-matched controls, with reference to control data (e.g., previous published studies (Snedaker, Honaramooz et al. 2004, Abrishami, Abbasi et al. 2010)). Immunohistochemical markers for each cell-type (e.g. SOX9 - Sertoli cells; aSMA -Peritubular Myoid cells/vascular smooth muscle; 3BHSD - Leydig cells, Stra8 - Spermatogonia; SOG1- Spermatocytes; Protamine 1 or PGK1/2 - Spermatids; Mac2/CD68 - Macrophages; CD 31 -

Endothelial cells) will be assayed to establish any changes in cellular ratios. Markers reflective of targeting such as GFP, or cleaved caspase 3 (apoptosis marker) will also be examined.

Example 4. Preliminary clinical trials- male and female cats and dogs

[00152] The two most efficacious nanoparticle constructs based on the studies performed above (male and female) will be used in in vivo testing in dogs and cats.

[00153] The pilot study will include 6 male dogs with one construct. Following a successful pilot study, the wider trial in both genders in both cats and dogs will be completed.

[00154] In general, to investigate whether steroid hormone production is only affected in the gonadal cells and not in adrenocortical cells, before and every 2 months after the treatment, or if clinical symptoms suggestive of abnormal adrenal steroidogenesis are present, an ACTH stimulation test (with measurement of cortisol and aldosterone) will be performed. In addition, the circulating ACTH/cortisol ratio and aldosterone/renin ratio will be determined in dogs and cats in which the gene(s) encoding steroidogenic enzymes have been targeted before and every 2 months after the treatment.

A. Male Dogs

[00155] A total of 6 adult male beagle dogs with two scrotal testes will be used in this study. Before treatment, a general physical examination will be followed by an andrological examination involving ultrasonographical measurement of prostate dimensions. In addition, a pre-treatment Gonadotropin-releasing hormone (“GnRH”) stimulation test (De Gier, Buijtels, et al. 2012), will be performed to study the effects of the treatment on the hypothalamic-pituitary-gonadal (HPG) axis and spermatogenesis.

[00156] Circulating LH and testosterone concentrations will be determined before and after the administration of a single intravenous bolus of a GnRH analogue (Buserelin). Furthermore, semen will be collected and evaluated for volume, progressive motility, morphology and sperm count.

[00157] The nanoparticles will be administered by a single intravenous injection into the cephalic vein.

[00158] After the treatment, a general physical examination and andrological examination are performed on a weekly basis. Semen evaluation and a GnRH stimulation test will be performed on a monthly basis after the treatment. A unilateral orchiectomy will be performed under general anaesthesia, 6 months after treatment. The removed testis will be examined for the presence of spermatogenesis and overall histological changes. Three and 6 months after hemicastration, the semen will be evaluated and a GnRH stimulation test will be performed to show persistence of the treatment effect.

B. Male Cats

[00159] Six adult Domestic Shorthaired tomcats will be used. Only cats with two scrotal testes will be included in the study. Treatment will occur similarly to that for the dogs. Before- and on a monthly basis after the treatment, a GnRH stimulation test as described above will be performed, the testes size will be determined and the penis will be examined for the presence of penile spines, which is correlated to the plasma testosterone concentration. Semen will be collected by urethral catheterization (Zambelli, Prati et al., 2008).

[00160] The removed testis will be examined using identical endpoints to those used for dogs (see above). Three and 6 months after hemicastration the semen will be evaluated and GnRH stimulation test will be performed.

C. Female Dogs

[00161] Six adult anestrous female dogs will be used. All dogs will be examined thrice weekly for swelling of the vulva and serosanguineous vaginal discharge, signifying the onset of pro-estrus (Schaefers-Okkens 2010). Plasma progesterone concentration will be measured thrice weekly from the start of pro-estrus until it exceeded 13-16 nmol/1, at which time ovulation is assumed to occur (Okkens, Bevers et al. 1985, Concannon, Hansel et al. 1977, Wildt, Panko et al. 1979). Anestrus is defined as the period from 100 days after ovulation to the onset of pro-estrus, as indicated by vulvar swelling and serosanguineous discharge. Only female dogs who showed regular ovulatory estrous cycles will be included in this study. [00162] A general physical examination will be performed before the start of the study, as well as a GnRH stimulation test (but measuring estradiol instead of testosterone).

[00163] The nanoparticles will be administered by a single intravenous injection into the cephalic vein.

[00164] Additionally, a GnRH stimulation test will be performed on a monthly basis until 6 months after treatment and at 9 and 12 months after treatment, and the plasma progesterone will be determined every 2 weeks to evaluate reproductive (endocrine) function. If the plasma progesterone concentrations remain < 1 ng/ml and when no signs of pro-estrus have been detected for a period longer than the mean interestrus interval + 2 months, it will be attempted to induce estrus by using a slow release GnRH analogue implant (Suprelorin®, Virbac). In healthy, untreated female dogs, (pro)estrus can be expected within 10 days after Suprelorin® administration (Fontaine, Mir et al. 2011, Maenhoudt, Santos et al. 2012). One month after Suprelorin® administration a unilateral ovariectomy will be performed under general anesthesia. The dissected ovary will be examined for targeting success, the presence of steroidogenic enzymes and follicular development. In case of complete disruption of ovarian function, it is expected that (pro) estrus will fail to occur, the plasma progesterone concentration will remain below 1 ng/ml, which indicates infertility.

D. Female Cats

[00165] Six adult Domestic Shorthaired female cats will be used. The female cats will be housed in a group and a vasectomized tomcat will be introduced to enhance estrus detection and induction of ovulation. Before and after the treatment, the female cats will be examined twice weekly for the presence of estrous signs: rolling, head rubbing, treading with hind legs, lordosis and tail deviation. Once a month the plasma progesterone concentration will be determined until the end of the study. A plasma progesterone concentration > 1 ng/ml indicates the presence of a corpus luteum. The fecal oestradiol concentration will be determined before- and on a weekly basis after the treatment.

[00166] The nanoparticles will be administered by a single intravenous injection into the cephalic vein.

[00167] Before and every two months until 6 months after treatment, and at 3 and 6 months after unilateral ovariectomy, a GnRH stimulation test will be performed. If estrous signs failed to occur at 6 months after the treatment, it will be attempted to induce estrus by using Suprelorin®. In anestrous female cats, estrus can be expected within 12 days (Zambelli, Bini et al. 2015). One month after Suprelorin® administration a unilateral ovariectomy will be performed under general anesthesia and six months later, the other ovary will be removed. The ovaries will be histologically examined for the presence of folliculogenesis.

[00168] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[00169] While various specific embodiments have been illustrated and described, it will be appreciated that changes can be made without departing from the spirit and scope of the invention(s).