Compositions and methods for preventing or delaying puberty in prepubertal non-human animals and humans

Abstract

The present invention relates to compositions and methods for reducing fertility and / or blocking puberty in prepubertal non-human subjects (e.g., kittens and puppies) by administering a composition comprising a vector comprising a nucleic acid encoding Mullerian inhibitory factor (MIS) functionally linked to one or more regulatory elements. In some embodiments, administration is by single or multiple injections. Another aspect relates to methods and compositions for delaying puberty in human female subjects by administering recombinant human MIS variant protein. TIFF2024511413000036.tif29170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 164,254, filed March 22, 2021, and U.S. Provisional Application No. 63 / 197,061, filed June 4, 2021, the contents of which are incorporated by reference in their entireties for all purposes.

[0002] Sequence Listing This application is filed in electronic format with a Sequence Listing, which is provided in a file entitled 2022-03-03_01133-0001-00PCT_Seq_List_ST25, created on March 4, 2022, and 62 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] Field The present application relates to compositions and methods of administering MIS proteins and vectors containing nucleic acids encoding MIS proteins to reduce fertility and / or block or delay puberty in prepubertal non-human subjects (e.g., kittens and puppies) and human subjects. [Background technology]

[0004] background Müllerian inhibitory factor (MIS), also known as anti-Müllerian hormone (AMH), is a 140 kDa disulfide-linked homodimeric glycoprotein member of the large transforming growth factor-β (TGFβ) multigene family of glycoproteins. All proteins in this gene family are produced as dimeric precursors and undergo post-translational processing for activation, thus requiring cleavage and dissociation to release the bioactive C-terminal fragment. Similarly, the 140 kilodalton (kDa) disulfide-linked homodimer of MIS is proteolytically cleaved to yield its active C-terminal fragment.

[0005] MIS is a reproductive hormone produced in the fetal testis that inhibits the development of female secondary sexual structures in males. The pre-sexisting fetus is bipotential, and MIS controls, in part, the preference for male Wolffian ducts (i.e., prostate, vas deferens) over female Müllerian ducts (i.e., fallopian tubes, uterus, vagina) during male development.

[0006] The human MIS gene is located on chromosome 19 and its expression is sexually dimorphic. In males, MIS expression begins in the fetal testes at the 9th week of gestation and persists at high levels until puberty, when expression levels fall dramatically. In females, MIS is only produced postnatally in granulosa cells at levels similar to those in adult males from prepuberty until menopause, when expression ceases. In male fetuses, MIS triggers the degeneration of the Müllerian ducts, the precursors of the fallopian tubes, uterus, cervix, and upper third of the vagina.

[0007] Endogenously, MIS is produced by granulosa cells and is a key gatekeeper for the recruitment of primordial follicles to the growing pool. In females, MIS is expressed by granulosa cells of the ovary after sexual differentiation of the Müllerian ducts. There is a correlation between MIS production and the number of growing follicles, and MIS is used clinically to estimate the size of the ovarian reserve (total pool of follicles) because MIS is secreted continuously throughout the ovarian cycle (Kalaiselvi et al., 2012). In males, overexpression of MIS inhibits steroidogenesis in Leydig cells, thereby significantly reducing testosterone levels (Teixeira et al, 1999).

[0008] It was previously discovered that human MIS protein stops follicle formation at an early stage of primordial follicle development. It was previously demonstrated that administration of human MIS protein to sexually mature female mice in a fertile mouse model, for example via gene transfer, can block follicle maturation and oocyte release, thus inhibiting ovulation. It was further demonstrated that administration of human MIS protein to sexually mature female mice significantly reduced reproductive rates (WO2015 / 089321).

[0009] Currently, pet owners and shelters are encouraged to spay or neuter animals, including cats and dogs. Surgically spaying or neutering young animals is considered a responsible way of caring for animals. In females, this typically involves abdominal surgery to remove one or both ovaries and / or the uterus. In males, this typically involves surgical removal of the testes. Spaying and neutering are recommended (and in some countries required for animals to be adopted) to prevent unwanted litters that lead to overpopulation of unwanted animals in rescue facilities. The ASPCA (American Society for the Prevention of Cruelty to Animals®) indicates that the problem of stray pets causes millions of healthy dogs and cats to be euthanized each year in the United States. In addition, the ACPCA also indicates that there are medical and behavioral benefits to spaying and neutering animals, including preventing certain infections or tumors (e.g., uterine infections and breast tumors in females, testicular cancer and prostate problems in males), keeping female pets from going into heat, reducing the likelihood of male pets roaming, and potentially resulting in better-behaved males. Spaying and neutering are common surgical procedures, but they can have risks, for example, involving general anesthesia. Additionally, animals take time to heal after surgery. Thus, bathing should be avoided, for example, for at least 10 days after surgery. After surgery, animals should refrain from running or jumping. Incision sites should be monitored to avoid infection and for proper healing.

[0010] Kittens and puppies are more desirable for adoption than adult cats and dogs. Animal shelters currently surgically spay or neuter kittens and puppies before adoption, often as early as 8 weeks of age. Recommendations suggest that female kittens should be surgically spayed before they reach approximately 5 months of age and female puppies before they reach approximately 6 months of age. Thus, there is a need to reduce fertility and / or prevent puberty in prepubertal animals, including kittens and puppies, and a simple method to achieve long-term sterilization may serve as an alternative to spay or neuter. Summary of the Invention

[0011] In accordance with the present invention, one aspect of the technology disclosed herein relates to a composition comprising a vector comprising a nucleic acid encoding a Mullerian inhibitory factor (MIS) protein, and a method is provided for reducing fertility and / or preventing or delaying puberty in a prepubertal subject (e.g., a kitten or puppy, or a human subject), comprising administering an effective amount of the composition to the subject.

[0012] Thus, one aspect of the present invention provides a method for reducing fertility and / or blocking puberty in prepubertal animals, including kittens and puppies, by administering MIS protein, for example, via gene transfer.If MIS is administered to prepubertal animals, including kittens and puppies, before puberty, a single treatment may prevent prepubertal animals from entering puberty or reduce fertility of prepubertal animals.Long-term sterilization in prepubertal animals, including kittens and puppies, may be an alternative to surgical ovariectomy or castration.Therefore, one aspect of the present invention relates to compositions and methods for administering MIS protein (for example, by a viral vector encoding MIS protein) to reduce fertility and / or block puberty in prepubertal non-human subjects, such as kittens and puppies.

[0013] Another aspect of the technology described herein relates to a method of reducing fertility in a prepubertal non-human subject, comprising administering to the subject an effective amount of a composition comprising a recombinant feline or canine MIS protein disclosed herein, or a vector comprising a nucleic acid encoding the recombinant feline or canine MIS protein, operably linked to one or more regulatory elements.

[0014] Another aspect of the technology described herein relates to a method of blocking puberty in a prepubertal non-human subject, comprising administering to the subject an effective amount of a composition comprising a vector comprising a nucleic acid encoding a MIS protein, operably linked to one or more regulatory elements.

[0015] Another aspect of the technology described herein relates to a method for delaying puberty in a prepubertal human subject, e.g., a female human subject, comprising administering to the subject an effective amount of a composition comprising a recombinant human MIS (rhMIS) protein disclosed herein. In some embodiments, the method reversibly delays puberty in a prepubertal human subject. In some embodiments, the recombinant human MIS protein is a mature protein generated from a preprotein derived from a human MIS protein, comprising at least a change in amino acid 450 of SEQ ID NO:4 from Q to R or a conservative amino acid of R (Q450R). In some embodiments, the conservative amino acid of R is K. Such a method for delaying puberty in a human female subject is useful, for example, when a subject needs to delay puberty, e.g., before starting a gender reassignment procedure and / or surgery, or before a female subject starts a procedure to transition from female to male, to provide the subject with more time. In some embodiments, the method for delaying puberty in human female subjects is useful, for example, when the subject has disease or disorder associated with reduced bone growth, and when puberty causes bone growth to stop.In some embodiments, the method for delaying puberty in human female subjects is useful, for example, when the subject has idiopathic precocious puberty.In some embodiments, the method for delaying puberty in human female subjects is useful, for example, when the subject has sex development difference, and it is useful to delay puberty by administering modified hMIS protein, so that the subject can be administered gender-affirming treatment before or prior to the secondary sexual characteristics that are the change of puberty.

[0016] In some embodiments, the present disclosure also provides a composition comprising a modified feline MIS protein, wherein the modified feline MIS protein is a chimeric feline MIS protein comprising the amino acid sequence of SEQ ID NO:3, or a recombinant feline MIS protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of amino acids 22 to 575 of SEQ ID NO:1.

[0017] In some embodiments, the present disclosure also provides a composition comprising a nucleic acid encoding a chimeric feline MIS protein comprising the amino acid sequence of SEQ ID NO:3, wherein the nucleic acid has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0018] In some embodiments, the disclosure also provides a composition comprising a nucleic acid encoding a modified canine MIS protein (clMIS) comprising the amino acid sequence of SEQ ID NO:15, wherein the nucleic acid has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:16.

[0019] In some embodiments, the prepubertal non-human subject is a kitten or a puppy.

[0020] In some embodiments, the prepubertal non-human subject is female. In some embodiments, the prepubertal non-human subject is male.

[0021] In some embodiments, the MIS protein is a) the wild-type feline MIS protein, the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:18, or an amino acid sequence having at least 80%, at least 85%, at least 90%, 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 or SEQ ID NO:18; b) a wild-type canine MIS protein, an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, 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:2; or c) a wild-type human MIS protein, an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 80%, at least 85%, at least 90%, 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:4; Includes.

[0022] In some embodiments, the prepubertal non-human subject is a kitten that is 12 months or less, 11 months or less, 10 months or less, 9 months or less, 8 months or less, 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, or 2 months or less in age. In some embodiments, the prepubertal non-human subject is a kitten weighing 2 kg or less.

[0023] In some embodiments, the prepubertal non-human subject is a puppy under 24 months of age, under 22 months of age, under 20 months of age, under 18 months of age, under 16 months of age, under 14 months of age, under 12 months of age, under 11 months of age, under 10 months of age, under 9 months of age, under 8 months of age, under 7 months of age, under 6 months of age, under 5 months of age, under 4 months of age, under 3 months of age, or under 2 months of age.

[0024] In some embodiments, the prepubertal non-human subject is weaned. In some embodiments, the prepubertal non-human subject is not weaned.

[0025] In some embodiments, the vector is a viral vector, a plasmid, a cosmid, or a phagemid. In some embodiments, the vector is a viral vector. In some embodiments, the composition further comprises a cell comprising the vector.

[0026] In some embodiments, the composition comprises a sterile injectable solution. In some embodiments, the composition comprises a sterile injectable aqueous solution. In some embodiments, the composition comprises a lipid, a lipid emulsion, a liposome, a nanoparticle, or an exosome.

[0027] In some embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a poxvirus vector, or a lentivirus vector. In some embodiments, the vector is an AAV vector. In some embodiments, the vector is an AAV9 vector.

[0028] In some embodiments, the one or more regulatory elements comprise a promoter element. In some embodiments, the one or more regulatory elements comprise a promoter element and an enhancer element. In some embodiments, the one or more regulatory elements comprise a constitutively active promoter.

[0029] In some embodiments, the composition comprises a pharma- ceutically acceptable carrier.

[0030] In some embodiments, administration is via injection. In some embodiments, administration is via intravenous, subcutaneous, or intramuscular administration. In some embodiments, administration is via intramuscular administration. In some embodiments, administration is via a single injection. In some embodiments, administration is via a one-off single injection. In some embodiments, administration is via a single dose split into multiple injections. In some embodiments, administration is via a single dose split into two injections.

[0031] In some embodiments, the effective amount of the composition administered to a prepubertal non-human subject is 1×10 per kilogram of the subject's body weight. 13 Vector genome: 5×10 12 Vector genome: 1×10 12 Vector genome: 5×10 11 vector genome or less, or 1 × 10 11 Below the vector genome.

[0032] In some embodiments, the concentration of MIS protein in serum of the prepubertal non-human subject 6 or 6 months, 9 or 9 months, 12 or 12 months, 15 or 15 months, or 24 or 24 months after administration of the composition is greater than 250ng / ml, greater than 300ng / ml, greater than 400ng / ml, greater than 500ng / ml, greater than 600ng / ml, greater than 700ng / ml, greater than 800ng / ml, greater than 900ng / ml, greater than 1μg / ml, greater than 1.5μg / ml, greater than 2μg / ml, greater than 3μg / ml, greater than 4μg / ml, greater than 5μg / ml, greater than 6μg / ml, greater than 7μg / ml, greater than 8μg / ml, greater than 9μg / ml, greater than 10μg / ml, or greater than 11μg / ml. In some embodiments, the MIS protein concentration is determined by ELISA.

[0033] In some embodiments, the prepubertal non-human subject is female and, following administration of the composition, (a) does not develop ovarian follicles, (b) does not develop follicles with viable eggs, (c) does not undergo puberty, (d) does not show signs of estrus, and / or (e) is infertile.

[0034] In some embodiments, the prepubertal non-human subject is male and, following administration of the composition, (a) does not undergo puberty, and / or (b) is infertile.

[0035] The present disclosure also provides vectors containing a nucleic acid encoding feline or canine Mullerian inhibitory factor (fcMIS or clMIS). In some embodiments, the vector comprises a nucleic acid encoding a feline Müllerian inhibitory factor (MIS) protein operably linked to one or more regulatory elements, wherein (a) the feline MIS protein comprises a wild-type feline MIS protein having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:18, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22 to 588 of SEQ ID NO:1 or amino acids 22 to 589 of SEQ ID NO:18, or (b) the feline MIS protein comprises a chimeric feline MIS protein having an amino acid sequence of SEQ ID NO:3, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22 to 572 of SEQ ID NO:3. In some embodiments, the nucleic acid of the vector encodes a feline MIS protein comprising a protein having at least 85% sequence identity to amino acids 22-588 of SEQ ID NO:1 or 22-589 of SEQ ID NO:18, wherein the amino acid residue Q at position 478 of SEQ ID NO:1 or 479 of SEQ ID NO:18 is changed from Q to a conservative amino acid, R (arginine). In some embodiments, the nucleic acid of the vector encodes a feline MIS protein comprising a protein having at least 85% sequence identity to amino acids 22-572 of SEQ ID NO:3, wherein the amino acid residue Q at position 465 of SEQ ID NO:3 is changed from Q to a conservative amino acid, R (arginine), e.g. K (lysine). In some embodiments, the conservative amino acid R is K.

[0036] In some embodiments, the vector comprises a nucleic acid encoding a canine Mullerian inhibitory factor (MIS) protein operably linked to one or more regulatory elements, the canine MIS protein comprising a wild-type canine MIS protein having an amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-588 of SEQ ID NO:2. In some embodiments, the vector nucleic acid encodes a canine MIS protein comprising a protein having at least 85% sequence identity to amino acids 23-543 of SEQ ID NO:2, wherein amino acid residue Q at position 462 of SEQ ID NO:2 is changed from Q to R (arginine) or the conservative amino acid of R. In some embodiments, the conservative amino acid of R is K.

[0037] In some embodiments, the modified feline Müllerian inhibitory factor (MIS) protein is (a) a feline MIS protein comprising a non-MIS leader sequence in place of amino acids 1-21 of SEQ ID NO:1 or amino acids 1-21 of SEQ ID NO:18 and comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-588 of SEQ ID NO:1 or amino acids 22-589 of SEQ ID NO:18, or (b) a chimeric feline MIS protein comprising a non-MIS leader sequence in place of amino acids 1-21 of SEQ ID NO:3 and having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-572 of SEQ ID NO:3, or (c) a chimeric feline MIS protein comprising a non-MIS leader sequence in place of amino acids 1-21 of SEQ ID NO:3 and having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-572 of SEQ ID NO:3, or (d) a chimeric feline MIS protein comprising a non-MIS leader sequence in place of amino acids 1-21 of SEQ ID NO:3 and having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-572 of SEQ ID NO:3, or The feline MIS protein is generated from a feline MIS proprotein selected from modified feline MIS proteins (LR-fcMIS) comprising amino acids of SEQ ID NO:14 or SEQ ID NO:20 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-588 of SEQ ID NO:14 or amino acids 22-589 of SEQ ID NO:20. In some embodiments, the feline MIS protein comprises a protein having at least 85% sequence identity to amino acids 22-588 of SEQ ID NO:1 or amino acids 22-589 of SEQ ID NO:18, wherein the amino acid residue Q at position 478 of SEQ ID NO:1 or position 479 of SEQ ID NO:18 is changed from Q to R (arginine) or the conservative amino acid R.In some embodiments, the chimeric feline MIS protein comprises a protein having at least 85% sequence identity to amino acids 22-572 of SEQ ID NO:3, wherein the amino acid residue Q at position 465 of SEQ ID NO:3 is changed from Q to R (arginine) or a conservative amino acid of R, such as K (lysine). In some embodiments, the non-leader sequence of the modified feline MIS protein is selected from any of SEQ ID NOs:9-13.

[0038] In some embodiments, the modified canine Mullerian inhibitor (MIS) protein is generated from a canine MIS proprotein, which comprises a non-MIS leader sequence in place of amino acids 1-21 of SEQ ID NO:2 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-588 of SEQ ID NO:2, or an amino acid sequence of SEQ ID NO:15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:15. In some embodiments, the canine MIS comprises a protein having at least 85% sequence identity to amino acids 23-543 of SEQ ID NO:2, wherein amino acid residue Q at position 462 of SEQ ID NO:2 is altered from Q to R (arginine) or a conservative amino acid of R. In some embodiments, the conservative amino acid of R is K.

[0039] The present disclosure also provides pharmaceutical compositions comprising any of the modified feline MIS proteins disclosed herein or any of the modified canine MIS proteins disclosed herein.

[0040] The disclosure also provides a method of reversibly delaying puberty in a prepubertal human female subject, comprising administering to the subject an effective amount of a composition comprising recombinant human Mullerian inhibitory factor (rhMIS) protein, wherein the recombinant human MIS protein comprises amino acid residues 25-560 of SEQ ID NO:4, or a protein having at least 85% sequence identity to SEQ ID NO:4, where amino acid residue 450 of SEQ ID NO:4 has been changed from Q to the conservative amino acid R or R. In some embodiments, the rhMIS protein is generated from a rhMIS proprotein comprising a non-MIS leader sequence in place of amino acids 1-24 of SEQ ID NO:4 and an amino acid sequence having at least 85% sequence identity to amino acids 25-560 of SEQ ID NO:4, where amino acid residue 450 of SEQ ID NO:4 has been changed from Q to the conservative amino acid R or R. In some embodiments, the conservative amino acid R is K. In some embodiments, the rhMIS protein comprises a protein having at least 19-554 amino acid sequence of SEQ ID NO:7, or a protein having at least 85% sequence identity to SEQ ID NO:7. In some embodiments, the rhMIS protein comprises a protein having at least 19-554 amino acid sequence of SEQ ID NO:7, or a protein having at least 85% sequence identity to SEQ ID NO:7, and a non-MIS leader sequence selected from any of SEQ ID NOs:9-13, or a non-leader sequence having at least 85% sequence identity to any of SEQ ID NOs:9-13. In some embodiments, the recombinant human MIS protein is made or produced by a nucleic acid disclosed in WO2015089321.

[0041] In some embodiments, the prepubertal human female subject is in need of delaying puberty. In some embodiments, the prepubertal human female subject is in need of delaying puberty has one or more conditions selected from gender dysphoria, intersex, atypical genitalia at birth, both male and female genitalia at birth, mosaic genetics, Klinefelter's syndrome, central or peripheral precocious puberty (CPP) or congenital adrenal hyperplasia (CAH).

[0042] The present disclosure also provides a method of detecting anti-fcMIS or anti-clMIS antibodies in a non-human subject administered a viral vector expressing fcMIS or clMIS, comprising the steps of: (a) obtaining a sample from a non-human subject administered a viral vector encoding fcMIS or clMIS, optionally wherein fcMIS or clMIS comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:20; (b) optionally isolating recombinant fcMIS or clMIS, optionally wherein the recombinant fcMIS or clMIS comprises a FLAG tag; (c) adding fcMIS or clMIS to a support, optionally wherein the support is an ELISA plate; (d) adding a test sample to the support; and (e) incubating the support with a detectable antibody, optionally wherein the detectable antibody is goat anti-IgG. Also provided is a method comprising the steps of: (a) carrying out an enzyme substrate reaction, wherein the enzyme substrate is HRP; and (b) carrying out an enzyme substrate reaction, wherein the enzyme substrate is HRP. In some embodiments, the test sample is diluted with blocking buffer before being added to the support. In some embodiments, the method further comprises incubating the support with bovine serum albumin before adding the test sample to the support. In some embodiments, the method further comprises one or more washing steps to remove excess MIS or excess detectable antibody.

[0043] Description of a specific sequence Table 1 provides a list of the specific sequences referenced herein.

[0044] Table 1. Description of specific sequences TIFF2024511413000002.tif84169TIFF2024511413000003.tif245169TIFF2024 511413000004.tif245169TIFF2024511413000005.tif244169TIFF20245114130 00006.tif246169TIFF2024511413000007.tif243169TIFF2024511413000008.t if243169TIFF2024511413000009.tif243169TIFF2024511413000010.tif168169 [Brief description of the drawings]

[0045] [Figure 1A] Figures 1A-1H show pilot studies with the fcMISv1 feline transgene in vitro, in mice, and in cats. Figure 1A shows a sequence alignment of divergent sections of the feline MIS protein from the domestic cat genome release 8.0 (fcMISv1; SEQ ID NO:3 positions 361-467) and the feline MIS protein from version 9.0 (fcMISv2; SEQ ID NO:1, positions 361-480). [Figure 1B] Figures 1A-1H show pilot studies with the fcMISv1 feline transgene in vitro, in mice, and in cats. Figure 1B shows colloidal blue stained protein gel electrophoresis of Flag affinity purified Flag-fcMISv1 and Flag-fcMISv2 proteins (2 μg) with and without in vitro plasmin cleavage. Recombinant human LR-hsMIS and mouse Flag-mmMIS (cleaved and uncleaved) served as controls. [Figure 1C]Figures 1A-1H show pilot studies with the fcMISv1 feline transgene in vitro, in mice, and in cats. Figure 1C shows representative western blots of tissue lysates from mice treated with 5e12vg / kg AAV9-fcMISv1 or 5e12vp / kg AAV9-empty negative control, and recombinant LR-hsMIS (100ng), a positive control. Blots were probed with antibodies against the C-terminus of MIS, or B-actin and GAPDH as loading controls. [Figure 1D] Figures 1A-1H show pilot studies with the fcMISv1 feline transgene in vitro, in mice, and in cats. Figure 1D shows representative fetal rat urogenital ridge sections stained with H&E after incubation with purified Flag-fcMISv1 (uncleaved or cleaved) protein, or CHO cell clone conditioned medium containing fcMISv1, adjusted to 5 μg / mL (non-transfected CHO conditioned medium negative control) CHO feline MIS protein. MIS activity was scored into grades 0-5 representing the degree of Müllerian duct degeneration. (F=Wolf's duct; M=Müllerian duct). [Figure 1E] Figures 1A-1H show pilot studies with the fcMISv1 feline transgene in vitro, in mice, and in cats. Figure 1E shows representative gross morphology of the uterine horns and ovaries of mice treated with AAV9-fcMISv1 (5e12vg / kg) or control AAV9-empty (5e12vp / kg) at 50 days post-treatment. [Figure 1F] Figures 1A-1H show pilot studies with the fcMISv1 feline transgene in vitro, in mice, and in cats. Figures 1F-1H show serum MIS and anti-fcMISv1 antibody titers in three cats after treatment with 5e12vg / kg fcMISv1. [Figure 1G] See legend to Figure 1F. [Figure 1H] See legend to Figure 1F. [Diagram 2]Figure 2 shows histological analysis of the uterus and ovaries of cats treated with fcMISv1. Three years after treatment with AAV9-fcMISv1, three female cats were ovariectomized and uterine and ovarian histology examined. Note that subject 11WBL24 developed rapid and robust immunity to the fcMISv1 transgene, has multiple corpora lutea in the ovaries, few primordial follicles, and cystic endometrial hyperplasia in the uterus. In contrast, subject 11WBL25, who maintained MIS levels in the μg / ml range, has an abundant population of primordial follicles in the ovaries, normal corpora lutea, and normal endometrium. [Figure 3A] Figures 3A-3G show the cloning of feline MIS AAV9 vector and validation in mice. Figure 3A shows the design of the codon-optimized feline MIS transgene. [Figure 3B] Figures 3A-3G show the cloning of feline MIS AAV9 vectors and validation in mice. Figure 3B shows Western blots of conditioned media from stable CHO clones overexpressing human, mouse, and feline transgenes and MIS protein (100 ng) purified by Flag affinity and cleaved or not in vitro by plasmin. Blots were probed with an antibody against the C-terminus of MIS. [Figure 3C] Figures 3A-3G show the cloning and validation in mice of feline MIS AAV9 vector. Figure 3C shows representative fetal rat urogenital ridge sections stained with H&E after incubation with purified protein or conditioned medium adjusted to 5 μg / ml of human MIS protein or feline MIS protein. MIS activity was scored on a grade of 0-5 representing the degree of Müllerian duct degeneration. (F=Wolf's duct, M=Müllerian duct). [Figure 3D]Figures 3A-3G show the cloning of feline MIS AAV9 vector and validation in mice. Figure 3D shows representative western blots of tissue lysates from mice treated with 5e12vg / kg AAV9-fcMISv2 or 5e12vp / kg AAV9-empty negative control, and recombinant LR-hsMIS (100ng) as a positive control. Blots were probed with antibodies against the C-terminus of MIS, or B-actin and GAPDH as loading controls. [Figure 3E] Figures 3A-3G show the cloning of feline MIS AAV9 vector and validation in mice. Figure 3E shows serum concentrations of MIS measured by ELISA in mice after treatment with 5e12vg / kg or 1e13vg / kg AAV9-fcMISv2. [Figure 3F] Figures 3A-3G show the cloning of feline MIS AAV9 vectors and validation in mice. Figure 3F shows representative central sections of ovaries 4 weeks after treatment with 5e12vg / kg or 1e13vg / kg AAV9-fcMISv2 or 5e12vp / kg AAV9-empty negative control. [Figure 3G] Figures 3A-3G show the cloning and validation of feline MIS AAV9 vector in mice. Figure 3G: Total number of individual follicles in the whole ovary (N=5 / group). [Figure 4] FIG. 4 shows the mean serum MIS concentrations (μg / ml) in adult cats from pilot study 1 treated with AAV9-chimeric feline MIS (5e12 vector particles (vp) / kg) and cats from pilot study 2 after injection with low (5e12 vp / kg) or high (1e13 vp / kg) doses of AAV9-wt feline MIS, as described in Example 1.D. [Figure 5A]Figures 5A-5C show individual profiles of serum MIS concentrations (μg / ml) (squares) and circulating anti-MIS antibody concentrations (circles) in each adult cat from pilot study 2 after injection with low dose AAV9-wt feline MIS (5e12 vp / kg, n=3) (Figure 5A), high dose AAV9-wt feline MIS (1e13 vp / kg, n=3) (Figure 5B), or control empty vector particles (5e12 vp / kg, n=3) (Figure 5C), as described in Example 1.D. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6A] Figures 6A-6G show evaluation of AAV9-fcMISv2 in domestic cats. Figure 6A shows sexually mature female domestic cats were treated intramuscularly with 5e12vg / kg (low MIS) or 1e13vg / kg (high MIS) AAV9-fcMISv2 or 5e12vp / kg AAV9-empty vector (control). Fertility was assessed during two mating studies ending at the 1- and 2-year mark after treatment. Serum concentrations of MIS (Figure 6B), luteinizing hormone (LH; Figures 6C and 6E), and inhibin B (Figures 6D and 6E) were measured by ELISA in cats after AAV9-fcMISv2 treatment. Figure 6F shows concentrations of estradiol (E2) and progesterone (P4) in dried fecal pellets collected from cats throughout the pre- and post-treatment periods. FIG. 6G shows the assessment of estrus and luteal phase frequency based on fecal steroid profiles in the pre- and post-treatment periods. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 6G] See legend to Figure 6A. [Figure 7A]Figures 7A-7G show viral vector shedding assessment and individual serum fcMISv2 and anti-drug antibody profiles in domestic cats treated with AAV9-fcMISv2 or empty vector control. Figures 7A-7D show viral genome quantification by qPCR in blood (Figure 7A), stool (Figure 7B), urine (Figure 7C), and oral swab (Figure 7D) samples after treatment. Figures 7E-7G show serum MIS and anti-fcMISv2 antibody titers in individual cats after AAV9-fcMISv2 treatment (Figures 7E-7F) or empty vector control treatment (Figure 7G). [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 7G] See legend to Figure 7A. [Figure 8A-1] Figures 8A-8I show sex steroid and cyclicity assessments in cats treated with AAV9-fcMISv2. Concentrations of E2 and P4 were in dried fecal pellets collected from cats throughout the pre-treatment (left panel) and post-treatment (right panel) periods. Peak steroid concentrations above baseline were used to assess estrus and luteal phase. [Figure 8A-2] See legend to Figure 8A-1. [Figure 8B-1] See legend to Figure 8A-1. [Figure 8B-2] See legend to Figure 8A-1. [Figure 8C-1] See legend to Figure 8A-1. [Figure 8C-2] See legend to Figure 8A-1. [Figure 8D-1] See legend to Figure 8A-1. [Figure 8D-2] See legend to Figure 8A-1. [Figure 8E-1] See legend to Figure 8A-1. [Figure 8E-2] See legend to Figure 8A-1. [Figure 8F-1] See legend to Figure 8A-1. [Figure 8F-2] See legend to Figure 8A-1. [Figure 8G-1] See legend to Figure 8A-1. [Figure 8G-2] See legend to Figure 8A-1. [Figure 8H-1] See legend to Figure 8A-1. [Figure 8H-2] See legend to Figure 8A-1. [Figure 8I-1] See legend to Figure 8A-1. [Figure 8I-2] See legend to Figure 8A-1. [Figure 9A] Figures 9A-9D show reproductive behavior during mating studies and assessment of MIS levels in mating and control kittens. Figures 9A-9B show control and AAV9-fcMISv2 female cats introduced to male breeders. Behavior was recorded by video capture and assessed for success and failure of breeding attempts. [Figure 9B] See legend to Figure 9A. [Figure 9C] Figures 9A-9D show the assessment of reproductive behavior during the mating study and MIS levels in mating and control kittens. Figure 9C shows MIS levels during mating. [Figure 9D] Figures 9A-9D show the assessment of reproductive behavior during the mating study and MIS levels in mated and control kittens. Figure 9D shows MIS levels in kittens born to control females. [Figure 10] FIG. 10 shows a Western blot of transient transfection of feline and canine vectors in CHO cells. [Figure 11] FIG. 11 shows a Western blot of transient transfection of feline and canine vectors in COS7 cells. [Figure 12]FIG. 12 shows qPCR of transient transfection of feline and canine vectors in COS7 cells. [Figure 13] 13A-13B show enrichment media from CHO clones (FIG. 13A) and urogenital ridge regression bioassays (FIG. 13B). [Figure 14] FIG. 14 shows total follicle numbers and vector dose response in mice treated with AAV9-fcMISv2 30 days later. [Figure 15] Figures 15A-15D show the number of primary follicles (Figure 15A), secondary follicles (Figure 15B), antral follicles (Figure 15C), and corpora lutea (Figure 15D) in mice 30 days after treatment with 1e13vg / kg AAV9-empty, AAV9-fcMISv2, or AAV9-LRclMIS. [Figure 16] FIG. 16 shows assessment of circulating mis protein by ELISA (ANSH) 4 weeks after treatment with 1e13 vg / kg. [Figure 17] Figures 17A-17B show qPCR quantification of viral genomes in muscle (Figure 17A) and liver (Figure 17B) of mice 30 days after treatment with 5e12 vg / kg. [Figure 18] FIG. 18 shows assessment of MIS protein cleavage by Western blot in liver lysates after 30 days of treatment with 1e13vg / kg. [Figure 19] FIG. 19 shows assessment of MIS protein expression by ELISA following treatment with 5e12vg / kg AAV.MYO and AAV9-HR vectors delivering fcMISv2 (SEQ ID NO:1). [Figure 20-1] FIG. 20 shows the MIS and inhibin B profiles of kittens. [Figure 20-2] See description of Figure 20-1. [Figure 21] Figures 21A-21B show normalized inhibin B in female kittens (Figure 21A) and male kittens (Figure 21B). [Figure 22]FIG. 22 shows the anti-MIS neutralizing antibody profile in kittens compared to the positive control subject 11WBL24. [Figure 23A] Figures 23A-23C show 23 fecal steroid profiles in subjects M200586, M200667, and M200756. [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 24] Figures 24-24B show uterine horn measurements taken by transabdominal ultrasound. Measurements were taken on cats treated with 5e12vg / kg AAV9-fcMISv2 (low), 1e13vg / kg AAV9-fcMISv2 (high), or 5e12vp / kg AAV9-empty vector (control). Figure 24A shows measurements for all treated cats. In Figure 24B, "Treatment" represents the average data point for cats treated with the "low" and "high" doses. [Diagram 25] Figures 25A-25B show uterine horn measurements taken by transabdominal ultrasound 6-10 months after treatment. Measurements shown were corrected for cat age. Measurements were taken on cats treated with 5e12vg / kg AAV9-fcMISv2 (low), 1e13vg / kg AAV9-fcMISv2 (high), or 5e12vp / kg AAV9-empty vector (control). Figure 25A shows measurements for all treated cats. In Figure 25B, "Treatment" represents the average data point for cats treated with the "low" and "high" doses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Detailed Description The present invention relates to compositions and methods of administering a nucleic acid encoding a Müllerian inhibitory factor (MIS) protein (e.g., administering a viral vector comprising a nucleic acid encoding a MIS protein) to reduce fertility and / or block puberty and / or block reproduction in prepubertal non-human subjects, such as kittens and puppies.

[0047] As described in detail herein, administration of MIS by gene delivery to reduce fertility and / or block puberty is currently in clinical development in prepubertal non-human subjects, including male and female kittens and puppies.

[0048] A. MIS as an Agent for Long-Term Reduction of Fertility and / or Blocking Puberty in Non-Human Subjects, E.g., Kittens and Puppies As discussed herein, one aspect of the present invention relates to administering (i.e., by gene transfer) nucleic acid encoding MIS protein to prepubertal non-human subjects (e.g., kittens or puppies) as a method of long-term reduction of fertility and / or prevention of puberty, for example, as an alternative to surgical ovariectomy or castration.Therefore, a single injection of vector (e.g., viral vector) expressing MIS protein may be a safe and effective alternative to surgical ovariectomy or castration in prepubertal kittens and puppies.The method disclosed herein can be used to reduce fertility in kittens and puppies and / or prevent kittens and puppies from reaching puberty.

[0049] As used herein, "prepubertal" refers to a subject who has not reached puberty and is considered sexually immature.

[0050] In some embodiments, the subject that can be treated includes any non-human prepubertal subject, for example, the prepubertal subject that will undergo surgical ovariectomy or castration.Surgical ovariectomy or castration is common in, for example, kittens, cats, puppies, and dogs.In some embodiments, the prepubertal subject is a kitten or puppy or any animal that has not undergone puberty.In some embodiments, the subject can be administered with the nucleic acid that codes for MIS protein as a single dose (for example, by a viral vector).In some embodiments, the dose can be administered as a single injection or divided into multiple injections.

[0051] In some embodiments, the prepubertal subject is a kitten. In other embodiments, the prepubertal subject is a puppy.

[0052] In some embodiments, the prepubertal kitten is a female kitten. In some embodiments, the prepubertal kitten is a male kitten. In some embodiments, the prepubertal kitten is 12 months or less, 11 months or less, 10 months or less, 9 months or less, 8 months or less, 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, or 2 months or less. In some embodiments, the prepubertal kitten weighs 2 kg or less.

[0053] In some embodiments, the prepubertal puppy is a female puppy. In some embodiments, the prepubertal puppy is a male puppy. In some embodiments, the prepubertal puppy is 24 months or less, 22 months or less, 20 months or less, 18 months or less, 16 months or less, 14 months or less, 12 months or less, 11 months or less, 10 months or less, 9 months or less, 8 months or less, 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, or 2 months or less.

[0054] B. MIS as a drug to increase sperm count and / or sperm concentration One aspect of the present disclosure relates to administering (i.e., by transgenesis) MIS protein or nucleic acid encoding MIS protein to non-human male subjects (e.g., endangered or rare animals) as a method of increasing sperm count and / or sperm concentration. In some scenarios, it may be beneficial to increase sperm count and / or sperm concentration in subjects to aid in the collection and storage of sperm samples for future artificial insemination. Thus, a single injection of a vector (e.g., a viral vector) expressing MIS protein may be a safe and effective method for increasing sperm count and / or sperm concentration in non-human male subjects. The methods disclosed herein can be used to increase fertility in non-human male subjects.

[0055] In some embodiments, the non-human male subject is adult or sexually mature.Methods for determining the adulthood or reproductive maturity of non-human male subjects are known to those skilled in the art.Examples of these methods include: (1) measuring sex hormones (e.g., testosterone, progesterone, and estrogen); and (2) analyzing the morphological signs of reproductive maturity (i.e., puberty), such as penis and testis enlargement, the presence of penile spine, and other morphological changes of genitalia.

[0056] C. MIS Protein as an Agent for Delaying Puberty in Human Subjects In some embodiments, the subject is a prepubertal human subject, for example a female prepubertal human subject, and the method comprises administering to the subject an effective amount of a composition comprising recombinant human MIS protein as disclosed herein.In some embodiments, the subject is a human female subject who needs to delay puberty, for example, to provide the subject with more time before starting gender reassignment treatment and / or surgery, or before the female subject starts gender transition treatment from female to male, or to provide the subject with more time to fully understand the subject's gender identity.For illustrative purposes only, the term "human female subject" refers to a subject who is typically assigned female biological sex at birth (e.g., has XX chromosomes and / or female genitalia appearance, or has female biological sex appearance, or external and / or internal female reproductive anatomy). In some embodiments, the "human female subject" referred to herein may also include a subject who is assigned intersex at birth, or who has atypical genitalia at birth, or who has both male and female genitalia, or who has only internal (but not external) female reproductive anatomy, or who has only external (but not internal) female reproductive anatomy, or who has mosaic genetics (some chromosomes labeled XY and others labeled XX), or Klinefelter syndrome (individual has XXY chromosomes), in which case the subject is referred to or assigned a non-binary gender. Simply by way of explanation, the method includes administering to a human subject an effective amount of a composition comprising a recombinant human MIS protein as disclosed herein to delay puberty, and the delay in puberty is for the period during which the human MIS is administered to the subject. In some embodiments, puberty is delayed by about 6 months, or about 8 months, or about 12 months, or about 18 months, or about 2 years, or about 3 years, or about 4 years, or about 5 years, or more than 5 years.In some embodiments, puberty in a human subject is not arrested, or rather, is reversibly delayed for a period of time, and puberty proceeds in the human subject or resumes at some point once the subject ceases to be administered the recombinant human MIS protein. In some embodiments, if the subject undergoes gender reassignment, puberty resumes in the reassigned gender following treatment with recombinant human MIS protein and cessation of treatment.

[0057] In some embodiments, the human female subject has idiopathic precocious puberty, a condition in which a child's body begins to change into an adult's body too early. In precocious puberty, the development of physical characteristics that would normally occur during puberty (the period of life when rapid physical changes and physiological aging occur, including the development of reproductive capabilities) occurs early. Puberty usually occurs between the ages of 13-15 in boys and between the ages of 9-16 in girls. In some embodiments, the human female subject has precocious puberty, or central precocious puberty (CPP) or peripheral precocious puberty, a condition in which puberty begins at or before the age of 8 in girls. Signs and symptoms of precocious puberty include development of at least one or more of the following before the age of 7 in girls and before the age of 9 in boys: breast growth, first period (onset of menstruation) in girls, maturation of external genitalia, pubic or underarm hair, growth spurt, acne, and adult body odor. In some embodiments, human female subjects have atypical genitalia at birth, and have both male and female genitalia.CPP can be caused by one or more of the following: tumors of the brain or spinal cord (central nervous system), brain defects present at birth, such as excess fluid accumulation (hydrocephalus) or non-cancerous tumors (hamartomas), radiation to the brain or spinal cord, damage to the brain or spinal cord, McKeown-Albright syndrome (a genetic disease that affects bone and skin color and causes hormone problems), congenital adrenal hyperplasia (a group of genetic disorders involving abnormal hormone production by the adrenal gland), or hypothyroidism.Peripheral precocious puberty in girls can be associated with one or more of ovarian cysts or tumors, and tumors of the adrenal gland or pituitary gland that release estrogen or testosterone, McKeown-Albright syndrome, or exposure to external sources of estrogen or testosterone, such as creams or ointments.

[0058] In some embodiments, the human female subject has gender dysphoria. In some embodiments, the human female subject has congenital adrenal hyperplasia (CAH).

[0059] In some embodiments, the human female subject has a disease or disorder associated with decreased bone growth and where bone growth stops due to puberty.

[0060] D. Mullerian inhibitory factor (MIS) protein Without wishing to be bound by any theory, Müllerian inhibitory factor (MIS) is a member of the TGFβ multigene family of glycoproteins. All proteins within this gene family are produced as dimeric precursors that undergo post-translational processing for activation and therefore require cleavage and dissociation to release the bioactive C-terminal fragments. MIS is a 140 kDa dimer of identical 70 kDa disulfide-linked monomers, each of which consists of a 57 kDa N-terminal domain and a 12.5 kDa carboxyl terminus (C-terminus). Thus, MIS contains two identical monomers (hence the term "homodimer"), each of which contains two domains, the N-terminal domain and the C-terminal domain, which are held together non-covalently. The purified C-terminal domain is the biologically active portion, and cleavage is required for activity. The N-terminal domain may aid in protein folding in vivo and facilitate delivery of the C-terminal peptide to its receptors, e.g., MISRI and MISRII. Uncleavable mutants of MIS are biologically inactive.

[0061] The active carboxy-terminal domain shares amino acid homology with other TGFβ family members, e.g., TGF-B1, 2, and 3, inhibins, activins, and bone morphogenetic proteins, as well as with numerous growth and differentiation factors (GDFs). The structure of the MIS carboxy-terminal domain is supported by seven cysteines involved in both intra- and intermolecular disulfide bonds that lead to its structural stability, as revealed by homology to the TGFβ three-dimensional structure using molecular modeling (Lorenzo, Donahoe, et al., unpublished data).

[0062] Like other TGFβ family members, MIS can be cleaved by plasmin to yield its amino- and carboxy-terminal domains. This proteolytic process is required for physiological activity and occurs at a site located similarly to the dibasic cleavage site found in the TGFβ sequence. The resulting products are tightly bound in a non-covalent complex that dissociates at low pH. Thus, technically complex and time-consuming protocols of plasmin treatment and size-exclusion chromatography are required to enhance or complete the separation of the carboxy-terminus from the amino-terminus.

[0063] Although the feline MIS gene was previously cloned, it was discovered that the extent of the MIS sequence in older feline genome versions may have been incomplete. Previous efforts to clone the missing region were only partially successful, likely due to high GC content and secondary structures, which led to structural rearrangement artifacts. By piecing together partial sequences obtained by Sanger sequencing and relying on the high homology of the C-terminus of MIS in Carnivora, we obtained a modern chimeric feline MIS transgene synthesized with synonymous codon usage that reduced the GC content sufficiently to allow the construction of viral packaging. When the latest, more comprehensive ("Version 9") build of the feline genome was released (see GenBank Accession No. GCA_000181335.4), this chimeric feline MIS construct (SEQ ID NO:3) was found to introduce 17 amino acid substitutions and omit two peptide motifs totaling 13 AA for a total of 30 amino acid mismatches (but was still bioactive in the UGR assay). A wild-type feline MIS construct (SEQ ID NO:1) was prepared that corresponds to the feline V9.0 reference sequence and has optimized codon usage to reduce GC content (SEQ ID NOs:1 and 5). An update to the feline V9.0 sequence has since been released (see GenBank Accession No. GCA_000181335.5). This latest feline V9.0 genome sequence information includes the latest feline MIS protein sequence (SEQ ID NO:18). The updated feline v9.0 MIS sequence (SEQ ID NO:18) differs from the original feline v9.0 MIS sequence (SEQ ID NO:1) by the presence of one additional alanine residue within the alanine-rich region at positions 370-376 of SEQ ID NO:18 compared to 370-375 of SEQ ID NO:1. This difference in one alanine residue in the feline MIS region is not expected to affect its processing, activity, and / or antigenicity.

[0064] In some embodiments, the MIS protein comprises a wild-type feline MIS (fMIS) protein. In some embodiments, the MIS protein comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the MIS protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the MIS protein comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the MIS protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO:18. In some embodiments, the MIS protein comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the MIS protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, 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:3.

[0065] In some embodiments, the present disclosure includes compositions comprising chimeric feline MIS proteins. In some embodiments, the chimeric feline MIS proteins comprise the amino acid sequence of SEQ ID NO:3. In some embodiments, the chimeric feline MIS proteins comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the present disclosure includes compositions comprising chimeric feline MIS proteins comprising at least amino acids 22-572 of SEQ ID NO:3 or a protein having at least 85% sequence identity to amino acids 22-572 of SEQ ID NO:3, wherein the endogenous chimeric feline MIS protein leader sequence of residues 1-21 of SEQ ID NO:3 is replaced with a non-MIS leader sequence as disclosed herein.

[0066] In some embodiments, the MIS protein comprises a wild-type canine MIS protein. In some embodiments, the MIS protein comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the MIS protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0067] In some embodiments, the disclosure includes compositions comprising a wild-type canine MIS protein. In some embodiments, the MIS protein comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the MIS protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the disclosure includes compositions comprising a canine MIS protein comprising at least amino acids 23-572 of SEQ ID NO:2, or a protein having at least 85% sequence identity to amino acids 23-573 of SEQ ID NO:2, wherein the endogenous canine MIS leader sequence of residues 1-22 of SEQ ID NO:2 has been replaced with a non-MIS leader sequence disclosed herein.

[0068] "Percent sequence identity" as used herein in the context of two or more polypeptide sequences refers to two or more sequences or subsequences that are identical, or have a certain percentage of identical amino acid residues or conservative substitutions thereof, when compared and aligned for maximum correspondence, as determined using one of the following sequence comparison algorithms or by visual inspection. By way of example, a first amino acid sequence can be considered similar to a second amino acid sequence when it is at least 50%, 60%, 70%, 75%, 80%, 90%, or even 95% identical to the second amino acid sequence, or when it is conservatively substituted, when compared to the same number of amino acids contained in the first sequence, or when compared to an alignment of polypeptides aligned by computer similarity programs known in the art, as discussed below.

[0069] It will be understood by those skilled in the art that the cloned gene can be readily manipulated to alter the amino acid sequence of the MIS protein. The cloned gene for the MIS protein can be manipulated by a variety of well-known techniques for in vitro mutagenesis, particularly to generate variants of the native protein that can be used in accordance with the methods and compositions described herein.

[0070] Variations in the primary structure of MIS protein useful in the present invention may include, for example, deletions, additions, and substitutions. Substitutions may be conservative or non-conservative. Differences between the native protein and variants generally preserve desirable properties, reduce or eliminate undesirable properties, or add desirable or new properties.

[0071] The mature wild-type MIS protein is first produced as a prohormone containing an N-terminal leader sequence corresponding to amino acid residues 1-21 of the wild-type feline MIS protein of SEQ ID NO:1 or SEQ ID NO:18, amino acid residues 1-22 of the wild-type canine MIS protein of SEQ ID NO:2, and amino acid residues 1-24 of the wild-type human MIS protein of SEQ ID NO:4. This leader sequence is cleaved to generate the mature MIS protein. The mature protein is further cleaved at the RAQ / R furin cleavage site (amino acid residues 476-479 of the wild-type feline MIS of SEQ ID NO:1; amino acid residues 477-480 of the wild-type feline MIS of SEQ ID NO:18; or amino acid residues 463-466 of the chimeric feline MIS protein of SEQ ID NO:3) to generate the N-terminal and C-terminal domains. The N-terminal and C-terminal MIS domains homodimerize with another wild-type feline MIS protein containing the N-terminal and C-terminal domains to form a mature protein. In some embodiments, the RAQ / R furin cleavage site of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:3 can be modified. In some embodiments, the Q amino acid residue at position 478 of SEQ ID NO:1, or position 479 of SEQ ID NO:18, or position 465 of SEQ ID NO:3 can be changed from Q to R (arginine) or a conservative amino acid of R, such as K (lysine).

[0072] In all aspects of the invention, the MIS protein or nucleic acid sequence encoding the MIS protein for use in the methods and compositions disclosed herein may have a non-endogenous MIS leader sequence, where the native MIS leader sequence has been replaced with a different leader sequence, such as, for example, the human serum albumin (HSA) leader sequence. In some embodiments, the MIS protein or nucleic acid sequence encoding the MIS protein for use in the methods and compositions disclosed herein is a modified MIS protein in which the primary RAQ / R cleavage site (corresponding to amino acids 476-479 of wild-type feline MIS of SEQ ID NO:1, corresponding to amino acids 477-480 of wild-type feline MIS of SEQ ID NO:18, corresponding to amino acids 460-463 of wild-type canine MIS of SEQ ID NO:2, and corresponding to amino acids 448-451 of wild-type human MIS of SEQ ID NO:4) has been changed to RAR / R, and / or the endogenous MIS leader sequence has been replaced with an albumin leader sequence. The MIS protein useful in the methods disclosed herein may be a wild-type MIS, or a MIS variant, such as LR-MIS, LRF-MIS, etc., as disclosed in WO2015089321, which is incorporated herein in its entirety.

[0073] In some embodiments, the non-endogenous leader sequence for use in the present invention is a functional fragment or variation of the HSA leader sequence disclosed in WO2015089321, US Patent No. 5,759,802, EP2277889, each of which is incorporated herein by reference in its entirety. Other leader sequences are included for use in the MIS protein disclosed herein, for example, to replace the endogenous leader sequence. Such leader sequences are well known in the art, and include the leader sequence comprising an immunoglobulin signal peptide fused with tissue-type plasminogen activator propeptide (IgSP-tPA), as disclosed in US2007 / 0141666, which is incorporated herein by reference in its entirety. Many other signal peptides are used for the production of secreted proteins. One of these is mouse immunoglobulin signal peptide (IgSP, EMBL accession number M13331). IgSP was first identified by Loh et al. (Cell. 33:85-93) in 1983. IgSP is known to provide good expression in mammalian cells. For example, European Patent No. 0382762 discloses a method for producing horseradish peroxidase by constructing a fusion polypeptide between IgSP and horseradish peroxidase. Other leader sequences include, but are not limited to, for example, the MPIF-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession No. AAB51134); stanniocalcin signal sequence; invertase signal sequence; yeast mating factor alpha signal sequence (e.g., the K. lactis killer toxin leader sequence); hybrid signal sequences; HSA / MFα-1 hybrid signal sequence (also known as HSA / kex2); K. lactis killer / MFα-1 fusion leader sequence; immunoglobulin Ig signal sequence; fibulin B precursor signal sequence; clusterin precursor signal sequence; and insulin-like growth factor binding protein 4 signal sequence. Examples of these are disclosed in WO2015089321, which is incorporated herein by reference in its entirety.

[0074] In some embodiments, a non-endogenous leader sequence for use in the present invention is a variant or fragment of SEQ ID NO:9 having at least 85%, or at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO:9. TIFF2024511413000011.tifA functional fragment or variation of the azuroginin (Azuro or "A") leader sequence comprising 4128 amino acids.

[0075] In some embodiments, the non-endogenous leader sequence for use in the present invention is an HSA sequence and is a functional fragment of SEQ ID NO:10, such as, or at least 23, or at least 22, or at least 21, or at least 20, or at least 19, or at least 18, or at least 17, or at least 16, or at least 15, or at least 14, or at least 13, or at least 12, or at least 11, or at least 10, or less than 10 contiguous or non-contiguous amino acids of SEQ ID NO:10. Modified versions of the HSA leader sequence are also encompassed for use in the present invention and are disclosed in U.S. Patent No. 5,759,802, which is incorporated herein by reference in its entirety. In some embodiments, the HSA leader sequence is TIFF2024511413000012.tif12128 or variations thereof, which are disclosed in European Patent EP2277889, which is incorporated herein in its entirety. HSA signal sequence Variants of the prepro region of TIFF2024511413000013.tif5128 include fragments, e.g., the preregion of the HSA signal sequence. TIFF2024511413000014.tif5128 or its variants, e.g. Includes TIFF2024511413000015.tif4128.

[0076] Certain homologues and functional derivatives and functional fragments of the feline, canine, and human MIS proteins are known in the art or can be identified by one of skill in the art by expression of MIS from an expression library.

[0077] E. Delivery of MIS Protein via Gene Transfer Accordingly, in one aspect, the invention relates to a method of reducing fertility and / or blocking puberty in prepubertal non-human subjects, including kittens and puppies and other animals, comprising administering to the subject a composition comprising a vector that includes a nucleic acid encoding a MIS protein (e.g., a wild-type MIS protein or a variant MIS protein).

[0078] In some embodiments, nucleic acids encoding MIS proteins can be effectively used to reduce fertility and / or block puberty in prepubertal non-human subjects via gene transfer. The general principle is that the nucleic acid is introduced into target cells within the subject (in vivo) or outside the subject and the cells are transferred to the subject (ex vivo), where the nucleic acid is transcribed into the MIS protein.

[0079] Thus, in some embodiments, the methods described herein can reduce fertility and / or block puberty in a prepubertal non-human subject following a single injection of a composition comprising a vector comprising a nucleic acid encoding a MIS protein, and the composition administered to the subject can maintain MIS expression at or above a threshold level, which is the minimum MIS level that may be required to reduce fertility and / or block puberty.

[0080] It should be noted that the threshold level may depend on the subject, the species of the subject, and / or the age or maturity of the subject. For example, in veterinary applications, there are various practical situations in which sterilization is desirable.

[0081] Entry into a cell can be facilitated by suitable techniques known in the art, such as providing the nucleic acid in a suitable vector, or encapsulating the nucleic acid in liposomes.

[0082] A desirable mode of gene transfer is one that provides the nucleic acid so that it replicates within the cell, enhancing and prolonging the desired effect. Thus, the nucleic acid is operably linked to an appropriate regulatory element, e.g., a promoter, e.g., the native promoter of the corresponding gene, a heterologous promoter that is endogenously active in liver, nerve, bone, muscle, skin, joint, or cartilage cells, or a heterologous promoter that can be induced by a suitable agent.

[0083] Thus, in some embodiments, a vector (e.g., a viral vector) comprises a nucleic acid encoding a wild-type MIS protein, e.g., comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:18, SEQ ID NO:2, or SEQ ID NO:4. In some embodiments, the vector comprises a nucleic acid encoding a feline MIS protein of SEQ ID NO:1 or SEQ ID NO:18. In some embodiments, the vector comprises a nucleic acid encoding a canine MIS protein of SEQ ID NO:2. In some embodiments, the vector comprises a nucleic acid encoding a human MIS protein of SEQ ID NO:4.

[0084] In some embodiments, the vector comprises a nucleic acid encoding a protein having an amino acid sequence that has 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 a MIS protein naturally produced in a subject. In some embodiments, the vector comprises a nucleic acid encoding a MIS protein, or a functional fragment thereof, that comprises an amino acid sequence that has 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, SEQ ID NO:18, SEQ ID NO:2, or SEQ ID NO:4.

[0085] In some embodiments, the vector comprises a nucleic acid encoding a MIS protein of SEQ ID NO: 3. In some embodiments, the vector comprises a nucleic acid encoding a MIS protein comprising an amino acid sequence having 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:3.

[0086] In some embodiments, the vector comprises a nucleic acid sequence encoding a MIS protein, wherein the nucleic acid comprises a nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0087] In some embodiments, the vector comprises a nucleic acid sequence encoding a MIS protein, wherein the nucleic acid comprises a nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0088] Various vectors can be included that contain the nucleic acid encoding MIS protein for use in the method of the present invention.In some embodiments, said vector is an expression vector.For example, expression vectors compatible with eukaryotic cells, preferably expression vectors compatible with vertebrate cells, can be used to generate recombinant constructs for producing viral vectors with the nucleic acid encoding MIS protein disclosed herein for recombinant expression of such MIS protein.Eukaryotic cell expression vectors are well known in the art and are available from several commercial suppliers.

[0089] Nucleic acid can be introduced into target cells by any suitable method. For example, nucleic acid encoding MIS protein can be introduced into cells by transfection (e.g., calcium phosphate or DEAE-dextran mediated transfection), lipofection, electroporation, microinjection (e.g., by direct injection of naked DNA), biolistics, infection with viral vectors containing nucleic acid, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, nuclear transfer, etc.

[0090] Alternatively, in some embodiments, plasmid expression vectors can be used, including pcDNA3.1, pET vectors (Novagen®), pGEX vectors (GE Life Sciences), and pMAL vectors (New England labs., Inc.) for protein expression in E. coli host cells, such as BL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta(DE3), and Origami(DE3) (Novagen®). Inc.); for expression in mammalian cell lines, e.g., CHO, COS, HEK-293, Jurkat, and MCF-7, the strong CMV promoter-based pcDNA3.1 (Invitrogen™ Inc.) and pCIneo vectors (Promega); for adenovirus-mediated gene transfer and expression in mammalian cells, the replication-incompetent adenovirus vectors pAdenoX, pAd5F35, pLP-Adeno-X-CMV (Clontech®), pAd / CMV / V5-DEST, pAd-DEST vectors (Invitrogen™ Inc.); For use with the Retro-X™ system from Clontech for retroviral-mediated gene transfer and expression in mammalian cells, the retroviral vectors include, but are not limited to, pLNCX2, pLXSN, and pLAPSN; for lentiviral-mediated gene transfer and expression in mammalian cells, the retroviral vectors include, but are not limited to, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (INVITROGEN™ Inc.) for lentiviral-mediated gene transfer and expression in mammalian cells; and for adeno-associated virus-mediated gene transfer and expression in mammalian cells, the adenovirus-associated virus expression vectors include, but are not limited to, pAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vectors (Stratagene®).

[0091] In some embodiments, the nucleic acid (e.g., DNA, modRNA, or RNAa) encoding the MIS protein disclosed herein can be suitably administered as a vector, e.g., a viral vector. In some embodiments, the vector is a viral vector.

[0092] The viral vector system that can be utilized in the present invention includes, but is not limited to, (a) adenovirus vector; (b) retrovirus vector, such as lentivirus vector, mouse Moloney leukemia virus, etc.; (c) adeno-associated virus vector; (d) herpes simplex virus vector; (e) SV40 vector; (f) polyomavirus vector; (g) papillomavirus vector; (h) picornavirus vector; (i) poxvirus vector, such as orthopox, such as vaccinia virus vector or avipox, such as canarypox or fowlpox; and (j) helper-dependent or gutless adenovirus.Replication-defective viruses may also be advantageous.In a preferred embodiment, the vector is an adeno-associated virus vector.

[0093] In some embodiments, viral vectors such as adeno-associated virus (AAV) vectors are used.AAV usually infects mammals, including humans, but is non-pathogenic, and has been developed and used as gene therapy vectors in clinical trials in the United States and Europe (Daya and Berns, Clinical Microbiology Reviews 2008, 21, 583-593).AAV vectors can be prepared by any one of a number of methods available to those skilled in the art. Exemplary AAV vectors are disclosed in Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146, incorporated herein by reference; Gao et al., Gene Therapy 2005, 5, 285-297; Vandenberghe et al., Gene Therapy 2009, 16, 311-319; Gao et al., PNAS 2002, 99, 11854-11859; Gao et al., PNAS 2003, 100, 6081-6086; Gao et al., J. of Virology 2004, 78, 6381-6388.

[0094] It should be noted that the selection of a specific type of AAV vector may depend on the target tissue.In some embodiments, the vector is an adeno-associated virus (AAV) vector.In some embodiments, the AAV vector is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.HR, AAVrh.10, AAVMYO or AAV2.5.In some embodiments, the AAV is AAV9.In some embodiments, the AAV vector for expressing MIS protein is AAV9 as disclosed in the examples herein.

[0095] Adenoviruses are other viral vectors that can be used in gene transfer methods. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, where they cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) provide a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995).

[0096] Retroviral vectors can also be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid encoding the MIS protein is cloned into one or more vectors, which facilitates gene delivery into a subject.

[0097] In another embodiment, the vector is a poxvirus, such as a vaccinia virus, such as an attenuated vaccinia, such as Modified Virus Ankara (MVA) or NYVAC, an avipox, such as fowlpox or canarypox.

[0098] In another embodiment, lentiviral vectors are used, such as the HIV-based vectors described in US Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0099] The vector may or may not integrate into the genome of the cell. The construct may contain viral sequences for transfection, if desired. Alternatively, the construct may be integrated into a vector capable of episomal replication, such as EPV and EBV vectors.

[0100] Generally, to ensure expression of the construct for expressing a nucleic acid, e.g., DNA, modRNA, or RNAa, encoding the MIS protein disclosed herein in a target cell, the construct can be operably linked to regulatory elements, e.g., promoters, enhancers, etc. Other details of the vectors and constructs are described in further detail below.

[0101] As used herein, the term "tissue-specific promoter" refers to a nucleic acid sequence that serves as a promoter, i.e., regulates the expression of a selected nucleic acid sequence operably linked to the promoter, and selectively affects expression of the selected nucleic acid sequence in particular cells of a tissue, e.g., in cells derived from the ovary.

[0102] The term "constitutively active promoter" refers to a promoter of a gene that is constantly expressed in a particular cell. Exemplary promoters for use in mammalian cells include the cytomegalovirus (CMV), CMV early enhancer / chicken beta actin (CBA) promoter, and the like.

[0103] The term "inducible promoter" refers to a promoter of a gene that can be expressed in response to a particular signal, for example, in response to the addition or reduction of an agent. Non-limiting examples of inducible promoters are "tet-on" and "tet-off" promoters, or promoters that are regulated in particular tissue types.

[0104] In some embodiments, the regulatory element comprises a constitutively active promoter. In some embodiments, the regulatory element comprises a CMV early enhancer / chicken beta actin (CBA) promoter.

[0105] In some embodiments, the composition being administered comprises inducible vector.The use of inducible vector for regulating gene expression or protein synthesis is known in the art.See, for example, WO1993022431, US20110301228, US6500647, WO2005053750 or US6784340, which are incorporated herein by reference.

[0106] In some embodiments, the MIS protein is expressed by an inducible vector, which may include one or more regulatory elements, e.g., promoters, enhancers, etc., operably linked to a polynucleotide encoding the MIS protein, and the regulatory elements are capable of controlling the expression level of MIS.

[0107] Typical regulatory elements include, but are not limited to, transcriptional promoters, inducible promoters and transcriptional elements, any functional sequences for controlling transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences for controlling the termination of transcription and / or translation. The term "regulatory element" includes nucleic acid sequences such as initiation signals, enhancers, and promoters that induce or control the transcription of an operably linked protein coding sequence. In some cases, the transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the expression of the recombinant gene in the cell type in which it is intended to be expressed. It is also understood that the recombinant gene may be under the control of the same transcriptional regulatory sequence or a transcriptional regulatory sequence that is different from the sequence that controls the transcription of the native protein. In some cases, the promoter sequence is recognized by the synthetic machinery of the cell, or the synthetic machinery introduced, necessary to mediate the transcription of a particular gene.

[0108] The regulatory sequence may be one regulatory sequence or multiple regulatory sequences, or may be modified regulatory sequence or fragments thereof.Modified regulatory sequence is a regulatory sequence whose nucleic acid sequence is changed or modified by some means, such as, but not limited to, mutation, methylation, etc.The regulatory sequence useful in the method disclosed herein is a promoter element (e.g., enhancer or repressor) that is sufficient to control promoter-dependent gene expression for cell type-specific, tissue-specific, or inducible expression by external signals or drugs.Such elements may be located in the 5' or 3' region of the natural gene, or may be located inside an intron.

[0109] In some embodiments, when the MIS protein encoded by the viral vector is endogenously expressed in the subject, the expression level of the MIS protein disclosed herein may be constant for a desired period of time, for example, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 5 years, or throughout the life of the subject.In some embodiments, the expression of the MIS protein disclosed herein may be maintained at or above a therapeutically effective dosage level for a desired period of time.

[0110] In some embodiments, other expression vectors can be used.For example, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, and such vectors can be integrated into host genome or replicate autonomously in a particular cell.Other types of expression vectors known to those skilled in the art that perform equivalent functions can also be used.

[0111] Another gene transfer approach is ex vivo gene transfer, which involves introducing nucleic acid into cells in tissue culture and delivering the transduced cells to a subject. Nucleic acid may be introduced into cultured cells by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Usually, the transfer method includes introducing a selection marker into cells. The cells are then placed under selection to isolate cells that have taken up and are expressing the introduced nucleic acid. These cells are then delivered to a subject.

[0112] In certain embodiments, nucleic acids encoding MIS proteins can be introduced into cells by electroporation (see, e.g., Wong and Neumann, Biochem. Biophys. Res. Commun. 107:584-87 (1982)) and biolistics (e.g., gene guns; Johnston and Tang, Methods Cell Biol. 43 Pt A:353-65 (1994); Fynan et al., Proc. Natl. Acad. Sci. USA 90:11478-82 (1993)).

[0113] In certain embodiments, the nucleic acid sequence encoding the MIS protein can be introduced into the target cell by transfection or lipofection. Suitable agents for transfection or lipofection include, for example, calcium phosphate, DEAE dextran, lipofectin, lipfectamine, DIMRIEC, Superfect, and Effectin (Qiagen), unifectin, maxifectin, DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyldioctadecylammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide), HDEAB (Nn-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene, poly(ethyleneimine) (PEI), and the like (see, for example, Banerjee et al., J. Immunol. 1999, 144:1311-1323, 1999). al., Med. Chem. 42:4292-99 (1999); Godbey et al., Gene Ther. 6:1380-88 (1999); Kichler et al., Gene Ther. 5:855-60 (1998); Birchaa et al., J. Pharm. 183:195-207 (1999).

[0114] A variety of delivery systems are known and can be used to directly administer therapeutic agents, for example, encapsulating vectors containing nucleic acids encoding MIS proteins in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, and receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). U.S. Patent No. 5,676,954 (incorporated herein by reference) reports the injection of genetic material complexed with cationic liposome carriers into mice. U.S. Patent No. 4,897,355, U.S. Patent No. 4,946,787, U.S. Patent No. 5,049,386, U.S. Patent No. 5,459,127, U.S. Patent No. 5,589,466, U.S. Patent No. 5,693,622, U.S. Patent No. 5,580,859, U.S. Patent No. 5,703,055 and International Publication No. WO94 / 9469 (which are incorporated herein by reference) provide cationic lipids for use in transfecting DNA into cells and mammals.U.S. Patent No. 5,589,466, U.S. Patent No. 5,693,622, U.S. Patent No. 5,580,859, U.S. Patent No. 5,703,055 and International Publication No. WO94 / 9469 (which are incorporated herein by reference) provide methods for delivering DNA-cationic lipid complexes to mammals. Such cationic lipid complexes or nanoparticles can be used to administer a therapeutic agent, e.g., a vector containing a nucleic acid encoding the MIS protein, and can also be used to deliver a protein, e.g., the MIS protein.

[0115] F. Detection of antibodies against MIS One aspect of the disclosure relates to detecting antibodies against feline MIS (fcMIS, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20) or canine MIS (cMIS, e.g., SEQ ID NO:3 or SEQ ID NO:15). In some embodiments, it may be beneficial to ascertain whether a subject has an immunological response to an administered vector composition, e.g., an AAV vector, a poxvirus vector, or a lentivirus vector expressing fcMIS or clMIS. In some embodiments, ascertaining the immunological response comprises detecting IgG antibodies against fcMIS or clMIS in the subject. A test sample may be taken from the subject for use in detecting antibodies against fcMISv1 or clMIS. In some embodiments, the test sample comprises blood or serum.

[0116] In some embodiments, a method for detecting antibodies to fcMIS or clMIS in serum includes the steps of: (a) optionally isolating recombinant fcMIS or clMIS, e.g., FLAG-tagged fcMIS or FLAG-tagged clMIS; (b) adding the isolated fcMIS or clMIS to a support, where optionally the support is an ELISA plate; (c) adding a test sample to the support; (d) incubating the support with a detectable antibody, where optionally the detectable antibody is goat anti-cat IgG (H+L) HRP; and (e) performing an enzyme substrate reaction, where optionally the enzyme substrate is HRP.

[0117] In some embodiments, the isolated fcMIS or clMIS can be isolated according to materials and methods known to those skilled in the art. In some embodiments, the isolated fcMIS is any exemplary fcMIS protein disclosed herein, such as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:18, or SEQ ID NO:20. In some embodiments, the isolated clMIS is any clMIS protein disclosed herein, such as SEQ ID NO:2 or SEQ ID NO:15. In some embodiments, the recombinant fcMIS or clMIS has a purification tag, such as a His tag or a FLAG tag. In some embodiments, the recombinant fcMIS is a FLAG-tagged fcMISv1 or a FLAG-tagged fcMISv2 or a recombinant FLAG-tagged clMIS. In some embodiments, the isolated fcMIS or clMIS is purified from cell culture conditioned medium.

[0118] In some embodiments, the method for detecting antibodies against fcMIS or clMIS includes attaching isolated fcMIS or clMIS to the surface of a support, such as an ELISA plate. In some embodiments, an excess of isolated fcMIS or clMIS is added to the support to completely cover the surface of the support with fcMIS or clMIS. In some embodiments, the isolated MIS and the support are incubated for about 30 minutes, 12 hours, 24 hours, or overnight. In some embodiments, the isolated fcMIS or clMIS and the support are incubated at about room temperature, 4°C, 20°C, 25°C, 30°C, or 37°C. After this incubation step, in some embodiments, excess fcMIS or clMIS that is not attached to the support is washed away with an appropriate buffer or washing solution.

[0119] In some embodiments, after the support is coated with isolated fcMIS or clMIS, a blocking step is performed. This blocking step is performed before test sample is added to the support. In some embodiments, the blocking step comprises incubating the support coated with fcMIS or clMIS with a blocking solution. In some embodiments, the blocking solution comprises phosphate buffered saline (PBST) containing bovine serum albumin, goat serum, and / or low concentration of Tween 20 detergent.

[0120] A test sample from a subject (e.g., a subject that is administered with a vector, e.g., a viral vector expressing fcMIS or clMIS) can be added to the support coated with fcMIS or clMIS. In some embodiments, the test sample is diluted with a suitable buffer before being added to the support. In some embodiments, the test sample is diluted with the above-mentioned blocking buffer. In some embodiments, the test sample is diluted 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500, or 1 / 1000. In some embodiments, blocking buffer or suitable coating buffer is used to be blank.

[0121] In some embodiments, anti-fcMIS antibody or anti-clMIS antibody is used as positive control.In some embodiments, anti-fcMIS antibody or clMIS antibody is diluted with appropriate buffer and then added to support.In some embodiments, anti-fcMIS antibody or anti-clMIS antibody is diluted with above blocking buffer.In some embodiments, anti-fcMIS antibody or anti-clMIS antibody is diluted 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500, or 1 / 1000.

[0122] In some embodiments, whole molecule IgG antibody, such as cat or dog IgG antibody, is used as antibody standard for detection by ELISA. In some embodiments, whole molecule IgG antibody is diluted with appropriate buffer and then added to support. In some embodiments, whole molecule IgG antibody is diluted with blocking buffer as above. In some embodiments, whole molecule IgG antibody is diluted 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500, or 1 / 1000.

[0123] After the test sample, anti-fcMIS antibody or anti-clMIS antibody, whole molecule IgG antibody standard, and blank are added to the fcMIS-coated or clMIS-coated support, the support is incubated at an appropriate temperature for a certain amount of time. In some embodiments, the support is incubated for about 30 minutes, 1 hour, 12 hours, 24 hours, or overnight. In some embodiments, the support is incubated at about room temperature, 4°C, 20°C, 25°C, 30°C, or 37°C. After the incubation step, the support is washed by 3, 5, or more washing steps with an appropriate buffer or washing solution.

[0124] In some embodiments, after the washing step, the support is incubated with a detectable antibody, such as goat anti-IgG horseradish peroxidase (HRP). Goat anti-IgG HRP is used according to the manufacturer's recommendations. In some embodiments, the support is incubated for about 30 minutes, 1 hour, 12 hours, 24 hours, or overnight. In some embodiments, the support is incubated at about room temperature, 4°C, 20°C, 25°C, 30°C, or 37°C. In some embodiments, the support is incubated in the dark. To detect anti-MIS antibodies in the test sample, an enzyme substrate reaction, such as an HRP enzyme substrate reaction, is performed according to the manufacturer's recommendations.

[0125] G. Administration of the Composition In some embodiments, the amount of the composition administered is "effective" when it is sufficient to reduce fertility and / or block puberty in a non-human animal or sufficient to delay puberty in a human subject. In some embodiments, the concentration of MIS protein in the serum of a prepubertal non-human subject (e.g., a kitten or puppy) 6 or 9 months, 12 or 15 months, or 24 or 24 months after administration of the composition is greater than 250 ng / ml, greater than 300 ng / ml, greater than 400 ng / ml, greater than 500 ng / ml, greater than 600 ng / ml, greater than 700 ng / ml, greater than 800 ng / ml, greater than 900 ng / ml, greater than 1000 ng / ml, greater than 1500 ng / ml, greater than 2000 ng / ml, greater than 3000 ng / ml, greater than 400 ng / ml, greater than 500 ng / ml, greater than 600 ng / ml, greater than 700 ng / ml, greater than 800 ng / ml, greater than 1000 ng / ml, greater than 15 ... The amount of a composition comprising a vector (e.g., a viral vector) comprising a nucleic acid encoding a MIS protein is administered to a subject (e.g., as a one-off administration) such that the MIS concentration is greater than 1, greater than 900 ng / ml, greater than 1 μg / ml, greater than 1.5 μg / ml, greater than 2 μg / ml, greater than 3 μg / ml, greater than 4 μg / ml, greater than 5 μg / ml, greater than 6 μg / ml, greater than 7 μg / ml, greater than 8 μg / ml, greater than 9 μg / ml, greater than 10 μg / ml, or greater than 11 μg / ml.

[0126] MIS protein concentration can be determined by any number of protein quantification methods understood in the art, including antibody-based assays.For example, enzyme-linked immunosorbent assay (ELISA) methods can be used to quantify MIS protein concentration, such as commercial kits including AMH Gen II ELISA (Beckman Coulter, Catalog No. A73818).Anti-human MIS / AMH IgG antibodies can cross-react with and bind to feline MIS protein and canine MIS protein.In some embodiments, MIS protein concentration is determined by ELISA.

[0127] In some embodiments, a composition comprising a vector (e.g., a viral vector) comprising a nucleic acid encoding a MIS protein can be administered at one time. In some embodiments, the composition can be divided into multiple subdoses, e.g., 2-4 subdoses, and administered over a period of time, e.g., at appropriate intervals throughout the day, or on other suitable schedules. In some embodiments, administration can be chronic, e.g., one or more doses and / or treatments per day for weeks or months. Dosage should not be so large as to cause adverse side effects. In some embodiments, administration comprises a single dose. In some embodiments, administration comprises dividing a single dose into two or more doses.

[0128] In some embodiments, the composition comprises a MIS protein that is a native (i.e., wild-type) feline MIS protein corresponding to SEQ ID NO:1 or SEQ ID NO:18. In some embodiments, the MIS protein is a native (i.e., wild-type) canine MIS protein corresponding to SEQ ID NO:2.

[0129] In some embodiments, the composition comprises a modified feline MIS protein corresponding to SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:3, or a protein having at least 85% sequence identity to amino acids 22-588 of SEQ ID NO:1, at least 85% sequence identity to amino acids 22-589 of SEQ ID NO:18, or at least 85% sequence identity to 22-572 of SEQ ID NO:3, wherein the RAQ / R furin cleavage site of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:3 is modified. In some embodiments, residue 478 of SEQ ID NO:1 or residue 479 of SEQ ID NO:18 is changed from Q to R (Q478R) or K (Q478K). In some embodiments, residue 465 of SEQ ID NO:3 is changed from Q to R (Q465R) or K (Q465K), and optionally, the endogenous leader sequence of 1-21 of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:3 is replaced with a non-MIS leader sequence disclosed herein.

[0130] In some embodiments, the MIS protein is a native (i.e., wild-type) canine MIS protein corresponding to SEQ ID NO:2. In some embodiments, the composition comprises a modified canine MIS protein corresponding to SEQ ID NO:2, or a protein having at least 85% sequence identity to at least amino acids 23-573 of SEQ ID NO:2, wherein the RAQ / R furin cleavage site of SEQ ID NO:2 has been modified. In some embodiments, residue 462 of SEQ ID NO:2 has been changed from Q to R (Q462R) or K (Q462K), and optionally, the endogenous leader sequence at 1-22 of SEQ ID NO:2 has been replaced with a non-MIS leader sequence disclosed herein.

[0131] In some embodiments, the MIS protein is a native (i.e., wild-type) human MIS protein corresponding to SEQ ID NO:4. In some embodiments, the recombinant hMIS protein comprises a combination of a non-MIS leader sequence, or functional fragment thereof, in place of the MIS leader sequence at amino acids 1-25 of SEQ ID NO:4, and at least one amino acid modification between residues 448-452 of SEQ ID NO:4 to increase cleavage compared to the absence of the modification, and the recombinant MIS protein has increased cleavage and is produced in greater amounts in vitro when compared to the wild-type MIS protein corresponding to the amino acid residues of SEQ ID NO:4. In some embodiments, the recombinant hMIS protein for use in the methods and compositions herein lacks a leader sequence (i.e., the leader sequence is truncated) but is produced from processing of a hMIS preprotein that includes a non-MIS leader sequence in place of the endogenous MIS leader sequence at amino acid residues 1-25 of SEQ ID NO:4. That is, in some embodiments, recombinant hMIS protein for use in the methods disclosed herein can be produced from a preproprotein that contains a non-MIS leader sequence or a functional fragment thereof in place of the MIS leader sequence of amino acids 1-25 of SEQ ID NO:4, and the leader sequence is cleaved during production. In some embodiments, the MIS protein is a recombinant modified human MIS protein corresponding to SEQ ID NO:7. In some embodiments, the composition comprises a modified human MIS protein having at least 85% sequence identity to at least amino acids 25-560 of SEQ ID NO:4, and the RAQ / R furin cleavage site of SEQ ID NO:4 is modified. In some embodiments, residue 450 of SEQ ID NO:4 is changed from Q to R (Q450R) or K (Q450K), and optionally, the endogenous leader sequence of 1-24 of SEQ ID NO:4 is replaced with a non-MIS leader sequence disclosed herein, such as, but not limited to, a leader sequence selected from any of SEQ IDs: 9-13.In some embodiments, the recombinant MIS protein, such as the human MIS protein, does not contain a FLAG tag or other tags.

[0132] In some embodiments, the human recombinant MIS protein disclosed herein is administered to a human subject to delay puberty in a human female subject, e.g., to reversibly delay puberty. In some embodiments, the MIS protein (e.g., a protein comprising amino acids having at least 85% sequence identity to 25-560 of SEQ ID NO:4, where residue 450 of SEQ ID NO:4 is changed from Q to R (Q450R) or K (Q450K), and optionally, the endogenous leader sequence of 1-24 of SEQ ID NO:4 is replaced with a non-MIS leader sequence) is administered to a female subject simultaneously with, before, or after a treatment for central precocious puberty, such as, but not limited to, any one or more of the following treatments selected from GnRH (gonadotropin releasing hormone), GnRH agonist, GnRH analog, metformin, progestin, or superagonist treatment. GnRH analogs include, but are not limited to, leuprolide acetate (leupron depot), or triptorelin (TRELSTAR™, Triptodur Kit). Other treatments for delaying puberty or for central precocious puberty or precocious puberty include SUPPRELIN LA™ (histrelin), LUPRON DEPOT-PED™, TAMOXIFEN™, LEUPROLIDE™, SOLTAMOX™, VANTAS™, HISTRELIN™, SYNAREL™, TRIPTORELIN™, TRIPTODUR™, NAFARELIN™, FENSOLVI™. In some embodiments, administration of the rhMIS variant proteins disclosed herein to a female subject to delay puberty is administered to the subject for a period of 1-3 months, 3-6 months, 6-12 months, 12-18 months, 18-24 months, or 2-3 years, 3-4 years, or 4-5 years, or 5-6 years, or greater than 6 years. Treatment may be daily or weekly, for example, using a pump, implant, or transdermal skin patch.In some embodiments, administration of the rhMIS variant proteins disclosed herein to female subjects to delay puberty is administered to female subjects with central precocious puberty or peripheral precocious puberty as young as 5 years of age, and may be in the age range of 6-16 years if the subject has gender dysphoria and puberty needs to be delayed to give the subject more time to fully understand their gender identity or before gender reassignment procedures and / or surgery, etc. Methods of administering rhMIS proteins are disclosed in U.S. application US20200071376 or US20160039898, which are incorporated herein by reference in their entireties.

[0133] In some embodiments, the composition comprises a chimeric feline MIS protein corresponding to SEQ ID NO: 3. In some embodiments, the MIS protein is a recombinant protein or a functional fragment or derivative or variant thereof.

[0134] In some embodiments, the administration of the composition comprising the vector (e.g., viral vector) comprising the nucleic acid encoding the MIS protein disclosed herein may be a one-time administration. In some embodiments, the administration of the composition disclosed herein is by pulsed administration. In some embodiments, an effective dose of the composition can be administered as two, three, four, five, six or more subdoses, which are administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In some embodiments, administration is repeated to maintain a desired level of the active ingredient in the body.

[0135] In some embodiments, an effective amount of a composition comprising a viral vector encoding a MIS protein is administered at a dose of 1×10 per kilogram of body weight of a prepubertal subject (e.g., a kitten or puppy). 13 The vector genome dose was 1 x 10 13 At a dose of 5 × 10 vector genome or less 12 Vector genome dose: 5 × 10 12At a dose of 1 × 10 vector genome or less 12 At a dose of 5 × 10 vector genome or less 11 at a dose of 1 x 10 vector genome or less 11 The vector genome can be provided in a dose of 1×10 per kilogram of body weight of a prepubertal subject (e.g., a kitten or puppy). In some embodiments, an effective amount of a composition comprising a viral vector encoding a MIS protein is provided in a dose of 1×10 per kilogram of body weight of a prepubertal subject (e.g., a kitten or puppy). 11 Vector genome ~ 1 × 10 13 In some embodiments, an effective amount of a composition comprising a viral vector encoding a MIS protein can be provided in a dose of 1×10 per kilogram of body weight of a prepubertal subject (e.g., a kitten or puppy). 11 Vector genome ~ 1 × 10 12 The vector may be provided in a dose of the genome.

[0136] Vector genome (vg) and vector particle (vp) concentrations may be determined by art-recognized methods of genome copy quantification, such as by PCR (e.g., droplet digital PCR (ddPCR) technology) and comparison to standards. Viral particle (vp) concentrations may be determined by art-recognized methods of quantification, such as by SDS-PAGE followed by Coomassie or silver staining and comparison to standards.

[0137] When determining the effective amount of the composition containing the vector (e.g., viral vector) containing the nucleic acid encoding MIS protein to be administered, the circulating plasma level of MIS protein, the formulation toxicity, and the state of fertility are evaluated.Such effective dose, including the lowest effective dose to produce a therapeutic effect, generally depends on the above factors.The dosage level selected also depends on various factors, including the activity of the specific compound of the present invention used, the route of administration, the administration time, the excretion rate of the specific compound used, the duration of treatment, the other drugs, compounds, and / or materials used in combination with the specific compound used, the age, sex, weight, condition, overall health, and previous medical history of the subject being treated, and similar factors well known in the medical field.

[0138] For example, an effective amount can be estimated in an animal model that achieves a desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in other subjects. In general, a therapeutically effective amount depends on the desired therapeutic effect.

[0139] In some embodiments, a composition comprising a vector (e.g., a viral vector) comprising a nucleic acid encoding the MIS protein disclosed herein can be administered to a prepubertal subject by any suitable and / or conventional injection administration route (e.g., intramuscular, intravenous, subcutaneous, or intraarterial).

[0140] When the composition containing the vector (e.g., viral vector) containing the nucleic acid encoding the MIS protein disclosed herein is administered, it is generally formulated into an injectable unit dosage form (e.g., solution, suspension, or emulsion).The pharmaceutical preparation suitable for injection includes sterile aqueous solution or dispersion, and sterile powder for reconstitution into injectable sterile solution or dispersion.The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), suitable mixture thereof, and / or vegetable oil.

[0141] The term "unit dosage" form as used herein refers to physically discrete units suitable as unitary dosage forms for the mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are determined by and directly depend on (a) the unique characteristics of the vector (e.g., viral vector) as disclosed herein and the particular biological effect to be achieved, and / or (b) the route of administration.

[0142] H. Pharmaceutical Compositions In some embodiments, a vector (e.g., a viral vector) comprising a nucleic acid encoding a MIS protein disclosed herein can be formulated by any suitable means as a pharmaceutical composition, e.g., a sterile injectable solution, which can be prepared by incorporating the composition in the required amount of an appropriate solvent, along with various other ingredients, as desired.

[0143] In certain embodiments, the composition comprising the vector (e.g., viral vector) comprising the nucleic acid encoding the MIS protein disclosed herein can be administered to a subject as a pharmaceutical composition together with a pharma- ceutically acceptable carrier.In certain embodiments, these pharmaceutical compositions optionally further comprise one or more additional therapeutic agents.

[0144] Pharmaceutically acceptable carriers are well known to those skilled in the art.For compositions formulated as liquid solution, acceptable carriers include saline and sterilized water, and may optionally include antioxidants, buffers, bacteriostatic agents and other common additives.Those skilled in the art can further formulate the compound of the present invention in an appropriate manner and according to generally accepted methods, for example, the methods disclosed in Reming'on's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1990.

[0145] Pharmacological formulations of compositions comprising vectors (e.g., viral vectors) containing nucleic acids encoding the MIS proteins disclosed herein can be administered to a subject in any pharma- ceutically acceptable carrier, such as injectable formulations containing various vehicles, adjuvants, additives, and diluents.

[0146] Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters such as isopropyl myristate can also be used as the solvent system of the compound composition. In addition, various additives that enhance the stability, sterility, and isotonicity of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. In many cases, it is desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents that delay absorption, for example, aluminum monostearate and gelatin. However, according to the present invention, any vehicle, diluent, or additive used must be compatible with the compound.

[0147] In another embodiment, the composition comprising the vector (e.g., viral vector) comprising the nucleic acid encoding the MIS protein disclosed herein may comprise lipid-based formulation. In carrying out the present invention, any known lipid-based drug delivery system can be used. For example, multivesicular liposome, multilamellar liposome and unilamellar liposome can all be used, as long as they can establish a sustained release rate of the encapsulated active compound. The method of making sustained release multivesicular liposome drug delivery system is described in PCT Application Publication Nos. WO9703652, WO9513796 and WO9423697, the contents of which are incorporated herein by reference.

[0148] The composition of synthetic membrane vesicles is usually a combination of phospholipids, usually combined with steroids, especially cholesterol.Other phospholipids or other lipids may also be used.Examples of lipids useful in the production of synthetic membrane vesicles include phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides, and preferred embodiments include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylephorin, dioleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.

[0149] When preparing lipid-based vesicles containing nucleic acids encoding MIS proteins, variables such as encapsulation efficiency, lability of the vector and / or nucleic acid, uniformity and size of the resulting vesicle population, compound to lipid ratio, permeability, instability of the preparation, and pharmaceutical acceptability of the formulation should be taken into consideration.

[0150] The compounds utilized in the present invention, such as the nucleic acids encoding the MIS proteins disclosed herein, can be administered parenterally to a subject in the form of slow-release subcutaneous implants or targeted delivery systems, such as polymer matrices, liposomes, and microspheres. Other such implants, delivery systems, and modules are well known to those skilled in the art.

[0151] Prior to introduction, compositions containing vectors (e.g., viral vectors) that contain nucleic acids encoding the same as those disclosed herein can be sterilized by any of the numerous techniques available in the art, including filtration.

[0152] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and / or pharmaceutical compositions of the present invention, are formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0153] definition Unless otherwise specified or implied from the context, the following terms and phrases include the meanings set forth below. Unless otherwise expressly stated or apparent from the context, the following terms and phrases do not exclude the meaning that the terms and phrases have acquired in the art to which they belong. The definitions are provided to aid in the description of certain embodiments and are not intended to limit the invention, the scope of which is limited only by the claims. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0154] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are useful for an embodiment, but embrace the inclusion of elements not expressly stated, whether useful or not.

[0155] The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0156] Except as provided in the operating examples or unless otherwise specified, all numbers expressing amounts of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When the term "about" is used in connection with percentages, it can mean ±5% of the stated value. For example, about 100 means 95 to 105.

[0157] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprises" means "includes". The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example".

[0158] The term "polypeptide" as used herein refers to a polymer of amino acids and their equivalents, and does not refer to a specific length of the product.Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.Derivatives are polypeptides that have conservative amino acid substitutions when compared to another sequence.Derivatives also include other modifications of proteins, including, for example, modifications such as glycosylation, acetylation, phosphorylation, etc.

[0159] The terms "Mullerian inhibitor" and "MIS" are used interchangeably herein and also refer to anti-Mullerian hormone or AMH, and compounds and materials structurally similar to MIS or fragments thereof. "MIS" or "Mullerian inhibitor" refers to a polypeptide and fragments thereof having an amino acid sequence that is at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid residues of SEQ ID NO:1, SEQ ID NO:18, SEQ ID NO:2, or SEQ ID NO:4. The present invention is intended to include variant forms of MIS that have substantially the same or greater biological activity as wild-type MIS. Examples of such variant MIS molecules include deletions, insertions, or changes in the amino acid sequence of wild-type MIS. Furthermore, MIS protein may be obtained using recombinant DNA technology or from chemical synthesis of MIS protein.

[0160] The term "wild-type" refers to a naturally occurring polynucleotide sequence encoding a protein or a portion thereof, or a protein sequence or a portion thereof, as normally occurring in vivo.

[0161] Thus, as disclosed herein, the wild-type amino acid sequence of the feline MIS preproprotein corresponds to SEQ ID NO:1 or SEQ ID NO:18 (amino acid residues 1-21 correspond to the leader sequence). The wild-type amino acid sequence of the canine MIS preproprotein corresponds to SEQ ID NO:2 (amino acid residues 1-22 correspond to the leader sequence). The wild-type amino acid sequence of the human MIS preproprotein corresponds to SEQ ID NO:4 (amino acid residues 1-24 correspond to the leader sequence). The wild-type amino acid sequence of the feline MIS proprotein comprises amino acid residues 22-588 of SEQ ID NO:1 or amino acid residues 22-589 of SEQ ID NO:18. The wild-type amino acid sequence of the canine MIS proprotein comprises amino acid residues 23-572 of SEQ ID NO:2. The wild-type amino acid sequence of the human MIS proprotein comprises amino acid residues 25-560 of SEQ ID NO:4. The proprotein is then post-translationally processed by cleavage as discussed herein to form the biologically active MIS homodimer.

[0162] By "nucleic acid encoding MIS" is meant a nucleic acid that comprises a nucleic acid encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:18, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or that encodes a polypeptide comprising an amino acid sequence having at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:18, SEQ ID NO:2, SEQ ID NO:4, amino acid residues 22-588 of SEQ ID NO:1, amino acid residues 22-589 of SEQ ID NO:18, amino acid residues 23-572 of SEQ ID NO:2, or amino acid residues 26-560 of SEQ ID NO:4.

[0163] The term "variant" refers to any change in the genetic material of an organism, particularly any change (i.e., deletion, substitution, addition, or alteration) in a wild-type polynucleotide sequence or any change in a wild-type protein sequence. The term "mutant" is used synonymously with "variant". Although it is often assumed that changes in genetic material alter the function of a protein, the terms "variant" and "mutant" refer to changes in the sequence of a wild-type protein, regardless of whether the change alters (e.g., increases, decreases, confers a new function) the function of the protein, or whether the change does not affect (e.g., the mutation or variation is silent) the function of the protein. The term mutation is used synonymously herein with polymorphism in this application. A variant may be a natural, synthetic, recombinant, or chemically modified polynucleotide or polypeptide, isolated or produced using methods well known in the art. A variant may include conservative or non-conservative amino acid changes, as described below. A polynucleotide change may result in substitution, addition, deletion, fusion, and truncation of amino acids in a polypeptide encoded by a reference sequence. Variants may also include amino acid insertions, deletions, or substitutions, including insertions and substitutions of amino acids and other molecules that do not normally occur in the peptide sequence on which the variant is based, such as, for example, but not limited to, the insertion of ornithine, which does not normally occur in human proteins.

[0164] The term "nucleic acid" is well known in the art. As used herein, "nucleic acid" generally refers to a molecule (i.e., a strand) of DNA, RNA, or derivatives or analogs thereof, that includes nucleobases. Nucleobases include, for example, the natural purine or pyrimidine bases found in DNA (e.g., adenine "A", guanine "G", thymine "T", or cytosine "C"), or the natural purine or pyrimidine bases found in RNA (e.g., A, G, uracil "U", or C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide", each of which is a subgenus of the term "nucleic acid". The term "oligonucleotide" refers to a molecule between 3 and 100 nucleobases in length. The term "polynucleotide" refers to at least one molecule that is greater than 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 synonyms, analogs of either RNA or DNA made from nucleotide analogs, and, where applicable to the embodiment being described, single-stranded (sense or antisense) and double-stranded polynucleotides. The terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

[0165] The term "gene" as used herein refers to a nucleic acid that includes an open reading frame that encodes a polypeptide, including both exon and (optionally) intron sequences. "Gene" refers to the coding sequence of a gene product, as well as the non-coding regions of the gene product, including the 5'UTR and 3'UTR regions of the gene product, introns, and promoters. These definitions generally refer to single-stranded molecules, but in certain embodiments also encompass additional strands that are partially, substantially, or completely complementary to the single-stranded molecule. Thus, a nucleic acid may encompass a double-stranded molecule, or a double-stranded molecule that includes one or more complementary strands or "complements" of a particular sequence that constitutes the molecule. As used herein, a single-stranded nucleic acid may be designated with the prefix "ss", a double-stranded nucleic acid may be designated with the prefix "ds", and a triple-stranded nucleic acid may be designated with "ts". The term "gene" refers to a DNA segment that is involved in the production of a polypeptide chain. A "gene" includes regions preceding and following the coding region, as well as intervening sequences (introns) between individual coding segments (exons).

[0166] The term "regulatory sequence" is used interchangeably herein with "regulatory element" to refer to a segment of nucleic acid, typically but not limited to DNA or RNA or analogs thereof, that regulates the transcription of a nucleic acid sequence to which it is operably linked, thus acting as a transcriptional modulator. Regulatory sequences regulate the expression of genes and / or nucleic acid sequences operably linked to the regulatory sequence. Regulatory sequences often include "regulatory elements", which are transcription binding domains and nucleic acid sequences recognized by transcription proteins and / or nucleic acid binding domains of transcription factors, repressors, or enhancers, etc. Exemplary regulatory sequences include, but are not limited to, transcription promoters, inducible promoters and transcription elements, any functional sequence that controls transcription, sequences encoding appropriate mRNA ribosomal binding sites, and sequences that control the termination of transcription and / or translation. Regulatory sequences may be a single regulatory sequence or multiple regulatory sequences, or may be modified regulatory sequences or fragments thereof. Modified regulatory sequences are regulatory sequences in which the nucleic acid sequence has been altered or modified by some means, such as, but not limited to, mutation, methylation, etc.

[0167] The term "operably linked" as used herein refers to the functional relationship of a nucleic acid sequence to a regulatory sequence of nucleotides, such as a promoter, an enhancer, a transcriptional and translational stop site, and other signal sequences. For example, functional linkage of a nucleic acid sequence, typically DNA, to a regulatory sequence or promoter region refers to a physical and functional relationship between the DNA and the regulatory sequence or promoter, such that transcription of such DNA is initiated from the regulatory sequence or promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA. To optimize expression and / or in vitro transcription, it may be necessary to modify the regulatory sequence to express the nucleic acid or DNA in the cell type in which the nucleic acid or DNA is expressed. The desirability, i.e., the necessity, of such modifications may be ascertained empirically. An enhancer need not be located in close proximity to the coding sequence whose transcription is enhanced by the enhancer. Furthermore, a gene transcribed from a promoter that is transregulated by a factor transcribed by a second promoter may be said to be functionally linked to the second promoter. In such cases, transcription of the first gene is said to be operably linked to the first promoter, and also operably linked to the second promoter.

[0168] A "promoter" is a region of a nucleic acid sequence at which the initiation and rate of transcription are controlled. A "promoter" may contain elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind to initiate specific transcription of a nucleic acid sequence.

[0169] The term "enhancer" refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Enhancers can function in either orientation and can be located upstream or downstream of the promoter.

[0170] The term "functional," when used in conjunction with "variant" or "fragment," refers to a polypeptide that has a biological activity (either functional or structural) that is substantially similar to the biological activity of the polypeptide, which is a functional derivative, variant, or functional fragment thereof. The term functional derivative is intended to include fragments, analogs, or chemical derivatives of a molecule.

[0171] In this context, the term "substantially similar" means that the biological activity, for example, the activation of the receptor MISRII, is 25% or at least 35%, or at least 50%, preferably at least 60%, 70%, 80%, 90%, 95%, 100%, or even more (i.e., the variant or derivative has greater activity than the wild type) of the reference polypeptide, for example, the activity of the corresponding wild type MIS polypeptide, for example, 110%, 120%, or more. In other words, in this context, a "substantially similar" functional fragment of a MIS protein means that at least 25%, at least 35%, at least 50% of the relevant or desired biological activity of the corresponding reference MIS protein (e.g., wild type MIS protein) is retained. In the case of a functional fragment or peptide of a MIS protein (e.g., SEQ ID NO: 1, 18, 2, or 4) disclosed herein, the functional fragment will be a protein or peptide that contains a portion that retains the activity to activate MISRII.

[0172] The terms "biologically active variant" or "biologically active fragment" are used interchangeably and refer to a compound having a biological activity (either functional or structural) that is substantially similar to the biological activity of the entity or molecule, which is a functional derivative (e.g., a wild-type MIS protein).

[0173] The term "conservative substitution", when describing a polypeptide, refers to a change in the amino acid composition of a polypeptide that does not substantially change the activity of the polypeptide. For example, a conservative substitution refers to the replacement of an amino acid residue with a different amino acid residue that has similar chemical properties. Conservative amino acid substitutions include the replacement of leucine with isoleucine or valine, aspartic acid with glutamic acid, or threonine with serine. A "conservative amino acid substitution" results from the replacement of one amino acid with another amino acid that has similar structural and / or chemical properties, such as the replacement of leucine with isoleucine or valine, aspartic acid with glutamic acid, or threonine with serine. Thus, a "conservative substitution" of a particular amino acid sequence refers to the replacement of an amino acid that is not important for the polypeptide activity, or the replacement of an important amino acid with another amino acid that has similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the activity of the peptide is not reduced when the important amino acid is replaced.

[0174] Conservative substitution tables showing functionally similar amino acids are well known in the art. For example, the following six groups: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W), each contain amino acids that are conservative substitutions for one another (see also Creighton, Proteins, WH Freeman and Company (1984)).

[0175] As used herein, the term "non-conservative substitution" refers to changing an amino acid residue to a different amino acid residue with different chemical properties.Non-conservative substitutions include, but are not limited to, aspartic acid (D) and glycine (G); asparagine (N) and lysine (K); or alanine (A) and arginine (R).

[0176] In some embodiments, individual substitutions, deletions, or additions that change, add, or delete one amino acid or a small percentage of amino acids can also be considered "conservative substitutions" if the change does not reduce the activity of the MIS protein. Insertions or deletions typically range from 1 to 5 amino acids. The choice of conservative amino acid may be based on the location of the amino acid to be substituted within the peptide, for example, whether the amino acid is on the exterior of the peptide and exposed to the solvent, or on the interior and not exposed to the solvent.

[0177] The terms "homology", "identity" and "similarity" refer to the degree of sequence similarity between two peptides or between two optimally aligned nucleic acid molecules. Homology and identity can be determined by comparing positions in each sequence that can be aligned for comparison. For example, this is based on using standard homology software such as BLAST at default positions. When corresponding positions in the compared sequences are occupied by the same base or amino acid, the molecules are identical at that position. When corresponding sites are occupied by similar amino acid residues (e.g., similar in terms of steric and / or electronic properties, e.g., conservative amino acid substitutions), the molecules can be called homologous (similar) at that position. Expression as a percentage of homology / similarity or identity refers to a function of the number of similar or identical amino acids, respectively, at the position shared by the compared sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity with the sequences disclosed herein, but preferably less than 25% identity.

[0178] The term "sequence identity" as used herein means that two polynucleotide or amino acid sequences are identical (i.e., nucleotide-by-nucleotide or residue-by-residue) over a comparison window. The term "percent sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, G, U, or I) or residue occurs, calculating the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percent sequence identity.

[0179] The term "substantial identity" as used herein refers to a feature of a polynucleotide or amino acid sequence comprising a sequence in which the polynucleotide or amino acid has at least 85% sequence identity, preferably at least 90%-95% sequence identity, more usually at least 99% sequence identity, when compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, and frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, where the percent sequence identity is calculated by comparing the reference sequence to a sequence that may contain deletions or additions that total no more than 20 percent of the reference sequence over the comparison window. The reference sequence may also be a subset of a larger sequence. The term "similarity," when describing a polypeptide, is determined by comparing the amino acid sequence and conserved amino acid substitutions of one polypeptide to the sequence of a second polypeptide.

[0180] As used herein, the terms "homologous" or "homolog" are used interchangeably and when used to describe a polynucleotide or polypeptide, indicate that two polynucleotides or polypeptides or their designated sequences are identical in at least 70% of the nucleotides, usually 75%-99% of the nucleotides, more preferably at least 98-99%, with appropriate nucleotide or amino acid insertions or deletions, when optimally aligned and compared, e.g., with BLAST, using default parameters for alignment. As used herein, the terms "homolog" or "homologous" also indicate homology with respect to structure and / or function. With respect to sequence homology, sequences are homologous if they are at least 50%, at least 60, at least 70%, at least 80%, at least 90%, at least 95% identical, at least 97% identical, or at least 99% identical. Determination of homologs of genes or peptides of the present invention can be easily ascertained by one skilled in the art.

[0181] The term "substantially homologous" refers to a sequence that is at least 90%, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical. Homologous sequences may be the same functional gene in different species. The determination of the homolog of the gene or peptide of the present invention can be easily ascertained by one skilled in the art.

[0182] A molecule is said to be "substantially similar" to another molecule if they have substantially similar structures or if they have similar biological activity, for example, if two MIS molecules can activate MISRII or inhibit follicular maturation. Thus, if two molecules have similar activity, even if the structure of one molecule is not found in the other or the sequence of amino acid residues is not identical, they are considered variants and are included for use as disclosed herein. Thus, if two molecules have similar biological activity, even if the structure of one molecule is not found in the other or the sequence of amino acid residues is not identical, they are considered variants as this term is used herein. Thus, nucleic acid sequences and amino acid sequences that have a smaller degree of similarity to MIS protein but have the same biological activity are considered equivalent. When determining polynucleotide sequences, all present polynucleotide sequences that can code for substantially similar amino acid sequences are considered to be substantially similar to the reference polynucleotide sequence regardless of differences in codon sequence.

[0183] By "enhanced proteolytic stability" is meant that the rate or extent of proteolytic degradation of a peptide sequence is reduced by at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% compared to a control sequence under the same conditions (e.g., in vivo or in an in vitro system such as a cell or cell lysate). A peptide with enhanced proteolytic stability may contain any modification, e.g., insertion, deletion, or point mutation, that reduces proteolytic cleavage at a particular site or eliminates a site susceptible to proteolytic cleavage. Proteolytic cleavage sites can be identified based on known target sequences or using computer software (e.g., software described in Gasteiger et al., Protein Identification and Analysis Tools on the ExPASy Server. In John M. Walker, ed. The Proteomics Protocols Handbook, Humana Press (2005)). Alternatively, proteolytic sites can be determined experimentally, for example, by Western blotting of proteins after expression or incubation in cell lines or cell lysates, followed by sequencing of identified fragments to determine the cleavage site.

[0184] The term "recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide that is genomic, cDNA, viral, semisynthetic, and / or synthetic in origin or by manipulation and is not associated with all or a portion of the polynucleotide with which it is naturally associated. The term recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. The term recombinant as used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced. Recombinant is also used herein with respect to a material (e.g., a cell, a nucleic acid, a protein, or a vector) to refer to the material being modified by the introduction of heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).

[0185] The term "subject" may refer to a non-human subject or a human subject. A "non-human subject" refers to an animal (e.g., a kitten or puppy) to which a composition according to the present invention is provided. In certain embodiments, the animal is a vertebrate, including, but not limited to, a mammal, a cat, a dog, a primate, a rodent, a domestic animal, or a game animal. In certain embodiments of the aspects described herein, the subject is a cat or a dog. The subject may be male or female. Furthermore, the subject may be an adult or a prepubescent (e.g., a kitten or puppy).

[0186] As used herein, the terms "administering" and "introducing" are used interchangeably herein and refer to placing the composition comprising the vector comprising the nucleic acid encoding the MIS protein disclosed herein in a subject by a method or route that allows the composition to be at least partially localized at a desired site.The compound of the present invention can be administered by any suitable route that reduces fertility and / or blocks puberty or delays puberty in a subject.

[0187] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein to refer to an amount of a pharmacological composition sufficient to produce a desired effect. As detailed herein, the appropriate "effective amount" in any given case can be determined and judged by one of ordinary skill in the art.

[0188] As used herein, the terms "prevent", "preventing" and "prevention" refer to the avoidance or delay of the onset of a biological state, symptom or marker, such as puberty, estrus or fertility. The terms "prevent", "preventing" and "prevention" include the avoidance or delay of the onset of a biological state, symptom or marker (e.g., puberty, estrus or fertility) compared to the state, symptom or marker of a control, untreated subject or treated subject at a reference point. The terms "prevent", "preventing" and "prevention" not only include avoidance or delay, but also include the reduction in the severity or extent of any biological state, symptom or marker.

[0189] The term "delay" or "delaying" as used herein with respect to delaying puberty refers to the postponement or cessation or interruption of puberty in a subject for a specified period of time, with puberty resuming after treatment has ceased.

[0190] As used herein, the terms "reduce", "reducing" and "reduction" refer to a reduction in the severity or extent of a biological state, symptom or marker. The terms "reduce", "reducing" and "reduction" include a reduction in the severity or extent of a biological state, symptom or marker (e.g., puberty, estrus or fertility) compared to a control, untreated subject, or treated subject at a baseline.

[0191] For example, "reduce," "reducing," and "reduction" refer to a statistically significant amount of decrease, and can include a decrease in the severity or extent of a condition, symptom, or measurable marker by at least 10%, e.g., at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 90%-95%, 90%-99%, 10%-95%, 10%-99%, or even 100% (i.e., no symptoms or measurable markers) as compared to a control or reference.

[0192] "Composition" or "pharmaceutical composition" are used interchangeably herein to refer to a composition that typically contains excipients, e.g., pharma- ceutically acceptable carriers, that are conventional in the art and suitable for administration to a cell. Exemplary compositions and pharmaceutical compositions are detailed herein.

[0193] "Pharmaceutically" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to a mammal, as appropriate.

[0194] 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, that is involved in maintaining the activity of the agent or carrying or transporting the agent from one organ or part of the body to another. As described in detail herein, a pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary. In addition to being "pharmaceutically acceptable" as this term is defined herein, each carrier must also be "acceptable" in the sense of being compatible with other ingredients of the formulation.

[0195] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A plasmid is one type of genus encompassed by "vectors." A viral vector is one type of genus encompassed by "vectors."

[0196] The term "viral vector" refers to the use of a virus or virus-associated vector as a carrier of a nucleic acid construct into a cell. The construct can be integrated and packaged into a non-replicating, defective viral genome, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), including retrovirus and lentivirus vectors, for infection or transduction into a cell. The vector may or may not be integrated into the cell genome. The construct may contain viral sequences for transfection, if desired. Alternatively, the construct may be integrated into a vector capable of episomal replication, such as EPV and EBV vectors.

[0197] The term "inducible vector" refers to a vector that can control gene expression. For example, the level of gene expression can be increased, decreased, or reduced to zero. In some embodiments, an inducible vector can include a switch that controls gene expression.

[0198] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2 SD) or greater.

[0199] Definitions of common terms in cell and molecular biology are provided in The Merck Manual of Diagnosis and Clinical Trials, published by Merck Research Laboratories. Th"The Encyclopedia of Molecular Biology," 19th Edition, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, 1994 (ISBN 0-632-02182-9), published by Blackwell Science Ltd. VCH Publishers, Inc. Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, 2009 (ISBN-10: 0763766321), published by Jones & Bartlett Publishing; Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, 1995 (ISBN 1-56081-569-8), published by VCH Publishers, Inc.; and Current Protocols in Protein Sciences 2009, Wiley Intersciences, edited by Coligan et al.

[0200] It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc. described herein, 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 disclosure, which is defined only by the claims. EXAMPLES

[0201] Example 1 - Pilot study of AAV9-MIS gene transfer as long-term non-surgical contraception in adult female cats A. In vitro, mouse, and adult cat pilot studies using the first generation chimeric feline MIS (fcMISv1) transgene Gene delivery of an AAV9 viral vector containing a human MIS transgene into sexually mature female mice has been shown to induce lifelong contraception without detectable side effects (WO2015 / 089321).

[0202] material and method A first-generation chimeric feline (felis catus) MIS transgene, fcMISv1 (SEQ ID NO:3), was synthesized based on the partial wild-type MIS sequence of the feline genome version 8.0, which was completed using the consensus Carnivora sequence. This partial MIS sequence corresponds to a GC-rich region encoding amino acids 361-466 at the C-terminus of the MIS prodomain, which is not involved in receptor binding, and was found to contain 30 amino acid differences when compared to the feline genome version 9.0 (Figure 1A). In this study, a viral vector containing the chimeric feline MIS transgene (AAV9-fcMISv1) was designed.

[0203] Recombinant fcMISv1 (SEQ ID NO:3) protein and Flag-tagged variants were produced in CHO cells and subsequently purified. CHO-K1 (ATCC; #CCL-61) cells were kept in culture in DMEM culture medium containing 5% FBS and 1% PenStrep. CHO-K1 cells were plated in 6-well plates for transfection. When 80% confluent, CHO-K1 cells were transfected with cat MIS plasmid (Genscript) expressing fcMISv1 or fcMISv2. In total, 5.1 μg of plasmid was transfected in each well with 15.3 μl of Fugene6 (Promega, #E2691) (1:6 mass / volume ratio). Transfection efficiency was confirmed to be over 80% by transfecting the same plate with a GFP-expressing plasmid (PCDNA3-eGFP-N1). Culture conditioned medium was harvested after 72 hours and used for urogenital ridge regression assays, ELISA, or Western blots.

[0204] Experiments in mice were performed using 6-week-old Nu / Nu nude mice (Gnotobiotic Mouse Cox7 Core, Boston) approved by the National Institutes of Health and the Harvard Medical School Institutional Animal Care and Use Committee, following experimental protocol 2014N000275 approved by Massachusetts General Hospital. Mice were housed under 12-h light / 12-h dark conditions and provided with food and water ad libitum. Each mouse received a single intraperitoneal (ip) injection of 5e12vg / kg, 1e13vg / kg, or 5e12vg / kg AAV9-fcMISv1 or empty vector. Blood was collected from the mouse cheek before vector injection and weekly thereafter. One month after vector delivery, mice were euthanized and mouse ovaries were collected and fixed overnight in formalin before preparation in formalin.

[0205] For Western blots, freshly dissected mouse tissues or transfected cells were homogenized in 250 μL of RIPA Lysis Buffer System containing protease inhibitor cocktail and PMSF (Santa Cruz Biotechnology, Santa Cruz, CA), sonicated twice for 15 s at 11% amplitude, and centrifuged at 20,800 g for 15 min at 4°C. Supernatants were harvested and protein content was measured using Pierce BCA Protein Assay (ThermoFisher Scientific, Rockford, IL). Samples were prepared with 50 or 100 μg of protein extract, 4X sample buffer, and RIPA buffer to a final volume of 25 μL and electrophoresed in Nupage 4-12% Bis Tris 1.5 mm gels (ThermoFisher Scientific, Rockford, IL). Proteins were transferred to Nu-PAGE PVDF membranes and blocked with 5% milk for 1 h. The primary goat anti-MIS C-terminus antibody, MIS C-20 (Santa Cruz, Santa Cruz, CA) 1:200 in 5% milk was incubated overnight, followed by incubation in donkey anti-goat IgG HRP 1:2,000 for 1.5-2 hours. ProSignal Dura ECL reagent was applied for 1-2 minutes and the membrane was exposed for 4 minutes. The membrane was then stripped, blocked in milk for 30 minutes, incubated in goat anti-β-actin (Santa Cruz, Santa Cruz, CA) for 2 hours, donkey anti-goat 1:2,000 for 1.5-2 hours, and imaged as before. The membrane was again stripped and incubated with anti-GAPDH antibody.

[0206] Female cats were treated with the AAV9-fcMISv1 vector. On day 0 of the study, cats were anesthetized using a ketamine / dexmedetomidine combination with partial reversal using atipamezole. A single injection (0.51–1.30 ml total volume) was administered into the right caudal thigh muscle of each individual. Cats were single-housed under biosafety level 2 (BSL-2) containment for 7 days before being returned to group housing.

[0207] Three adult female cats were injected intramuscularly with 5e12 vector particles (vp) / kg of AAV9-chimeric feline MIS (AAV9-fcMISv1). Table 2 shows the weight and age of the subjects at the time of injection. Viral shedding in feces, urine, rectal swabs, and oral swabs was assessed by viral genome qPCR.

[0208] (Table 2) Target AAV9-chimeric cat MIS TIFF2024511413000016.tif32166

[0209] In some cases, in situ hybridization was performed using the RNAscope 2.5 HD Reagent Kit (RED, ACD Bio, # 322350) as previously described (Saatcioglu et al., 2019). Ovarian tissue sections from cats were hybridized with probes designed to identify AMH and AMHR2, following brief xylene deparaffinization and heat-induced epitope retrieval according to the manufacturer's instructions. Tissues were then hybridized for 2 h and processed for standard signal amplification steps and chromogen development. Finally, slides were counterstained with hematoxylin, air-dried, and coverslipped with EcoMount.

[0210] result Both the chimeric feline MIS protein (fcMISv1; SEQ ID NO:3) and the flag-tagged version (flag-fcMISv1) were biologically active. Flag-fcMISv1 was expressed and purified from CHO cells (Figure 1B) and incubated with fetal rat urogenital ridge sections. As shown in Figure 1D, flag-fcMISv1 induced Müllerian duct regression in fetal rat urogenital ridge in vitro. In nude mice, the protein was delivered using AAV9 vectors as follows: (1) 5e12vg / kg AAV9-fcMISv1 or (2) 5e12vp / kg AAV9-empty negative control. Expression of fcMISv1 (containing both cleaved and uncleaved versions; MISc and proMIS, respectively, in Fig. 1C) from the AAV9-fcMISv1 vector was confirmed in the quadriceps, body wall, kidney, spleen, pancreas, and liver of treated mice (Fig. 1C). As shown in Fig. 1E, fcMISv1 was also biologically active in vivo, as it demonstrated induction of ovarian dysgenesis by day 50 (Fig. 1E).

[0211] However, in female cats treated with 5e12vg / kg AAV9-fcMISv1, chimeric feline MIS protein was rapidly excreted within 7 days. Accordingly, levels of the inflammatory marker C-reactive protein peaked on day 7 but returned to baseline by day 14. All three cats showed initial high circulating chimeric feline MIS protein in the blood (day 7: 6.44μg / ml+ / -0.03). However, expression was gradually lost in two of the three cats over the course of two years. These two cats did not maintain serum MIS levels in the μg / ml range. Furthermore, high titers of anti-MIS antibodies could be detected within 14 days of treatment (Figures 1F-1H). This is likely due to the immunogenicity of the introduced sequence mismatch. Meanwhile, there was evidence of ovarian suppression in cats that continued to produce MIS (Figure 2).

[0212] B. In vitro activity of wild-type feline MIS protein (fcMISv2) expressed from a codon-optimized transgene. material and method AAV9 vectors expressing wild-type feline MIS (wt-fcMIS or fcMISv2; SEQ ID NO:1) were designed based on the domestic cat genome version 9.0 (Figure 3A). A codon-optimized transgene (SEQ ID NO:5) was designed for feline translation and low GC content to enable efficient viral packaging. A Flag-tagged variant was also designed (Flag-fcMISv2) for production and purification in CHO cells. Flag-fcMISv2 inhibited the endogenous activating cleavage of proMIS but not MIS. N+C It could be processed in vitro by plasmin to generate dimers (Fig. 3B).

[0213] Materials and methods relating to CHO cells and Western blots were as described in Example 1.A. above.

[0214] Urogenital ridge regression assays were performed as previously described (Pepin 2013). Briefly, E14.5 female rat embryo urogenital ridges were dissected, cultured at the medium / air interface of agar-coated steel grids, and treated with conditioned medium containing 5 μg / ml human MIS or feline MIS or mock as a negative control for 72 h at 37 °C in humidified 5% CO2. After incubation, samples were fixed in Zamboni buffer, dehydrated overnight in a tissue processor in several steps, and embedded in paraffin. Ridge sections (8 μm) were stained with hematoxylin and eosin. They were then scored by two independent individuals from 0 (no regression) to 5 (complete regression of the Müllerian duct).

[0215] result Both purified Flag-fcMISv2 and enzymatically cleaved Flag-fcMISv2 induced Müllerian duct regression to grade 4 (out of 5) in the fetal rat urogenital ridge bioassay when adjusted to 5 μg / ml (Figure 3C). Concentrated conditioned medium from CHO cells with stable fcMISv2 overexpression also induced Müllerian duct regression in the fetal rat urogenital ridge (Figure 3C).

[0216] C. MIS levels and activity in mice following AAV9-fcMISv2 treatment To avoid possible transgene immunogenicity, the AAV9-fcMISv2 vector was also evaluated by intraperitoneal injection in nude mice.

[0217] material and method To enhance transcription, the fcMISv2 vector construct was further optimized by using a CMV enhancer, the ubiquitous chicken β-actin promoter, a synthetic intron, and a rabbit β-globin polyadenylation signal to terminate the 3'UTR (Gao et al., 2002), and packaged into an AAV9 viral vector (AAV9-fcMISv2). Given the efficient transduction of muscle tissue by AAV9 serotypes and the relatively low abundance of AAV9 pre-existing antibodies in cats (Adachi et al., 2020; Li et al., 2019), this vector was used to deliver fcMISv2 (SEQ ID NO:1) to this species.

[0218] Each mouse was given a single intraperitoneal (ip) injection of 5e12vg / kg or 1e13vg / kg AAV9-fcMISv2 or 5e12vg / kg empty vector. Blood was collected from the mouse cheek before vector injection and weekly thereafter. One month after vector delivery, mice were euthanized and mouse ovaries were collected, fixed in formalin overnight, and then mounted in formalin.

[0219] For follicle count analysis in mice, formalin-fixed paraffin-embedded mouse ovaries were serially sectioned at 5 microns and one section out of every five sections was saved for follicle quantification as previously described. After hematoxylin / eosin staining, slides were photographed one by one and follicles with nucleated oocytes were quantified. Follicles with one layer of squamous granulosa cells were considered as primordial follicles, follicles with one layer of cuboidal granulosa cells were considered as primary follicles, follicles with several layers of cuboidal granulosa cells were considered as secondary follicles, and finally, those showing follicular cavity were considered as tertiary or antral follicles. Factor Five was applied to obtain the final number of follicles. One ovary from 4–5 mice per group was quantified.

[0220] result One month after treatment, fcMISv2 protein (SEQ ID NO:1) was expressed in several tissues, including quadriceps and body wall muscles and peritoneal organs such as kidney, spleen, pancreas, and liver, and was efficiently cleaved by endogenous proteases (Figure 3D). After treatment with 5e12vg / kg or 1e13vg / kg AAV9-fcMISv2, the transduced tissues secreted MIS protein into the circulation, and in turn maintained MIS levels above 0.5μg / ml, which is above the 0.25μg / ml target level required to ensure contraception in mice (Kano, 2017) (Figure 3E). One month after treatment with 5e12vg / kg or 1e13vg / kg AAV9-fcMISv2, the ovaries of nude mice already showed visible hypoplasia (Figure 3F) and a significant reduction in follicle numbers from all stages of follicular maturation (Figure 3G).

[0221] D. A Pilot Study of Adult Female Cats Treated with AAV9-fcMISv2 Pilot study and summary of results The effect of the AAV9-fcMISv2 vector on feline reproduction was studied in domestic cats (Felis silvestris catus).Adult female cats were injected with AAV9-wt feline MIS (AAV9-fcMISv2) or an empty vector control as follows: (1) high dose AAV9-fcMISv2 (1e13vg / kg; n=3); (2) low dose AAV9-fcMISv2 (5e12vg / kg; n=3); and (3) control empty vector (5e12vp / kg; n=3).

[0222] Estradiol (E2) and progesterone (P4) metabolites were quantified from fecal samples collected 3X / week beginning 6 months prior to treatment. Cats were also monitored for behavioral signs of estrus. One cat in the high dose group exhibited transient injection site edema. No other injection site reactions were observed. Physical examinations, blood tests, and well-being assessments performed throughout the study were otherwise unremarkable. Serum MIS concentrations and anti-transgene antibody titers were monitored by ELISA during the 8-month observational study following injection. After the initial 8-month observation period, cats were monitored over a 4-month breeding trial and underwent ongoing observation to ascertain whether pregnancy was blocked, as well as to monitor continued steroid production and ovarian protein levels.

[0223] All cats treated with AAV9-fcMISv2 displayed circulating MIS levels throughout the study, including the mating period (Figure 4). Overall, MIS levels appeared to decline initially and reach stable MIS expression levels over the years of observation for both treatment groups, reflecting the characteristic stabilization of expression from persistent viral genomes following AAV gene transfer. Serum concentrations of MIS remained stable over the study period, averaging 2.88 μg / ml + / - 2.32 in the low group and 11.78 μg / ml + / - 2.51 in the high group at 6 months, versus basal levels in controls (5 ng / ml + / - 1). No anti-MIS antibodies were detected in any of the AAV9-fcMISv2-treated cats during this period. In contrast to these results, the two cats in the first pilot study that had lost MIS gene expression had antibody titers of 58 μg / ml ± 25 and MIS levels of 0.07 μg / ml ± 0.07 at 6 months.

[0224] Five of six treated cats maintained circulating MIS concentrations above 1 μg / ml over the first year of observation (Figures 5A and 5B). At 1 year, in the low dose (5e12vp / kg) group, subject 17LPY6 had a circulating MIS concentration of 3.34 μg / ml, subject 17LR06 had a circulating MIS concentration of 2.38 μg / ml, and subject 17LRE4 had a circulating MIS concentration of 0.82 μg / ml (1.65-0.82 μg / ml during mating). In the high dose (1e13vp / kg) group, all cats had high circulating MIS concentrations at 1 year, subject 17LRI5 had a circulating MIS concentration of 5.78 μg / ml, subject 17LRJ1 had a circulating MIS concentration of 9.28 μg / ml, and subject 17ERG2 had a circulating MIS concentration of 5.36 μg / ml.

[0225] Figures 5A, 5B, and 5C show individual profiles of serum MIS concentrations (μg / ml) (squares) and circulating anti-MIS antibody concentrations (circles) over time in adult cats following injection with low dose AAV9-wt feline MIS (5e12 vp / kg, n=3) (Figure 5A), high dose AAV9-wt feline MIS (1e13 vp / kg, n=3) (Figure 5B), or control empty vector particles (5e12 vp / kg, n=3) (Figure 5C).

[0226] In contrast to the first pilot study with AAV9-chimeric feline MIS (AAV9-fcMISv1), AAV9-wild-type feline MIS (AAV9-fcMISv2) did not elicit detectable antibodies to fcMISv2 in any of the treated cats when tested by ELISA.

[0227] Evidence of ovarian function suppression was observed, particularly in the high dose cohort as evidenced by lower E2 / P4 levels and peaks. Values ​​from the low and high dose groups were pooled to compare the mean fecal E2 metabolite concentrations between the two time periods: 6 months pre-injection vs. 6 months post-injection. Analysis revealed that E2 was significantly lower in treated cats (145.4ng / gm dry feces) compared to controls (198.4ng / gm) during the post-injection period (p=0.048). Mean E2 did not differ between pre-injection groups (P=0.262). The number of monthly estrus (defined as 2+ consecutive fecal samples with E2>1.5xE2 baseline) did not differ between groups either pre- or post-injection (P=0.802, P=0.201, respectively). However, treated cats tended to be less in estrus after injection compared with before (pre: 0.89; post: 0.53; P = 0.081).

[0228] Based on these results, a 4-month breeding trial was initiated with a certified breeder male group-housed (8 hours / day, 5 days / week) with 9 females and continuously monitored to record all breeding interactions, see Example 1.E. below.

[0229] material and method Cats were housed in a research colony at the Center for Conservation and Research of Endangered Wildlife (CREW) at the Cincinnati Zoo and Botanical Garden. All procedures were approved by the Institutional Animal Care and Use Committee (identification number 18-132) and the Cincinnati Children's Hospital Medical Center Institutional Biosafety Committee (IBC 2018-0066). All cats treated in this study (n=9) were sexually intact females from different dams, providing genetic diversity among the study population. Upon arrival at CREW, cats were cared for for several months to acclimate to the environment and caregivers, to collect blood, fecal, and urine samples to ascertain baseline levels of hormones of interest, and to ensure that the cats were sexually mature at the time of injections. Cats were group-housed, fed a commercial dry cat food and had free access to fresh water. Two certified breeder male cats (2 and 13 years old) used during the breeding trial were housed singly in separate rooms but were otherwise kept under identical conditions. Physical examination and blood tests (complete blood count (CBC) and serum biochemistry panel) were performed prior to study enrollment.

[0230] Adult female cats were randomly assigned to one of these three treatment groups using the random number generator function in Microsoft Excel. Figure 6A shows a schematic of the study design, and Table 3 shows the age and weight of the subjects at the time of injection.

[0231] Table 3. Target AAV9-wt feline MIS TIFF2024511413000017.tif60166

[0232] On day 0 of the study, cats were anesthetized using a ketamine / dexmedetomidine combination with partial reversal using atipamezole. A single injection (0.51–1.30 ml total volume) was administered to each cat into the right caudal thigh muscle. Cats were single-housed under biosafety level 2 (BSL-2) containment for 7 days before being returned to group housing.

[0233] Female cats were assessed daily for general well-being during the first 2 weeks. No adverse events were observed. Physical and blood tests were performed 2 weeks prior to treatment, on day 0 (pre-MIS treatment), and repeated every 3 months until the first year of the study, then every 6 months thereafter. All results were unremarkable. Injection sites were examined daily for 14 days, weekly for the next 2 weeks, and then monthly thereafter. One cat in the group receiving 1e13vg / kg AAV9-fcMISv2 (i.e., the high dose group) showed minor edema at the injection site on days 3 and 4. No evidence of pain-stimulated behavior, temperature changes, or tissue mass formation was observed, and the edema had resolved by day 5. No other injection site reactions were observed.

[0234] Viral shedding in feces and body fluids was measured by quantitative PCR performed at the University of Florida Powell Gene Therapy Center Toxicology Core. Fecal and urine samples were collected daily from each cat in Biolevel Safety-2 housing during the 7-day post-treatment period. Oral swabs were obtained on days 0 (pre-treatment), 2, 7, and 14. Whole blood (jugular, cephalic, or lateral saphenous) was collected on days 0 (pre-treatment), 2, 21, 28, and thereafter up to 6 months into microtainer EDTA tubes. Fecal samples were placed in plastic bags and sealed. Urine, oral swabs, and blood samples were transferred to 1.8 ml cryovials. All samples were stored at -20°C until analysis.

[0235] For feline MIS protein analysis, venous blood samples were collected on days 0 (pretreatment), 2, 7, 14, 21, 28, and monthly thereafter until year 2. Blood was collected into serum separator tubes, allowed to clot for approximately 15 minutes, and centrifuged at 1534 g for 10 minutes. Collected serum was transferred to 1.8 ml cryovials and stored at -80°C until analysis. MIS levels were measured using AMH Gen II ELISA Ruo (Beckman Coulter, Miami, FL). Briefly, feline serum was diluted in diluent as follows: 1:10 for all controls, day 0 low dose, and day 0 high dose cats; 1:1000 to 1:500 for subsequent low dose cats, 1:100 for subject 17LRE4 and exceptions; and 1:2000 to 1:1000 for high dose cats. The remainder of the ELISA was followed according to the manufacturer's instructions.

[0236] An ELISA assay was developed to measure anti-fcMISv1 / v2 IgG in feline serum without using an antibody sandwich. In this ELISA, recombinant FLAG-tagged fcMISv1 (FLAG-fcMISv1) or FLAG-tagged fcMISv2 (FLAG-fcMISv2) was purified from conditioned medium. FLAG-fcMISv1 or v2 protein (i.e., capture protein) was added directly to an ELISA plate (Immulon HB2 ELISA plate, Thermo Fisher Scientific, Rochester NY cat. 3455) at 5 μg / ml. The ELISA plate was directly coated with FLAG-tagged wt feline MIS protein rather than immobilized using rabbit anti-FLAG antibody. The rabbit anti-FLAG antibody may be a source of high background due to cross-reactivity with the developing antibody. Standard wells were coated with whole molecule feline IgG (Rockland Antibodies and Assays, Limerick, PA cat. 002-0102-0002) diluted 8 times 3-fold in coating buffer (CB) starting at 900ng / ml. Control and blank wells received coating buffer only (alternating capture protein rows with control rows). Plates were then incubated for 30 minutes at RT and overnight at 4°C. Two rinses were performed and plates were blocked for 2.5 hours with 200μL / well of 1% bovine serum albumin (Jackson Laboratories cat. 001-000-162) + 7.5% normal goat serum (Abcam cat. Ab7481) dissolved in PBST. Samples were diluted 1:100 in blocking buffer, added and plates were incubated for 1 hour at RT. After five more washes, the plates were incubated with goat anti-cat IgG (H+L) HRP (Novus Biologicals cat. NBP73347) 1:10,000 in PBST for 1 h at 4°C in the dark. Plates were rinsed five times and the enzyme substrate reaction was performed. For hormone metabolite analysis, fecal samples were collected three days a week without continuation, starting 6 months before MIS treatment and continuing until 2 years after treatment.To facilitate identification of fecal samples from group-housed females, a unique combination of commercially available food dyes (Wilton Industries, Woodridge, IL, USA) and / or glazes (Dixon Ticonderoga Company, Appleton, WI, USA) was given to each cat in a small amount of canned food each day prior to sample collection. The collected fecal samples could then be used to identify each individual cat by the color of the dye or the presence of the glaze. Samples were sealed in plastic bags, labeled with the name and collection date, and stored at -20°C until processing.

[0237] For fecal E2 and P4 analysis, fecal samples were freeze-dried in a plastic bag by a freeze dryer (Labconoco Corp., Kansas City, MO, USA), crushed into a fine powder, and then weighed (250 ± 5 mg) into a labeled 15 ml polypropylene conical tube. Each sample was then extracted overnight (≥ 12 h) on a mechanical rocker by adding 2.5 ml of 90% ethanol (or 1:10 w:v). Extracted samples were then centrifuged at 1000 g for 15 min, the supernatant was pipetted out, and samples were stored at −20 °C in 2.0 ml cryovials until analysis. All enzyme immunoassay (EIA) procedures were modified from previously published methods. To ascertain estrogen (E2), a polyclonal antibody (R0008) raised against 17β-estradiol was used together with horseradish peroxidase (HRP) ligand. In contrast, to quantify progestogens (P4), the Arbor Assays P4 mini-kit (ISWE003, Arbor Assays, Ann Arbor, MI, USA) was used, which contained both antibodies and HRP. Both assays have been used previously in this laboratory and have been validated for use in domestic cats. Samples and standards were analyzed in duplicate for both assays.

[0238] For statistical analysis of fecal hormone metabolites, hormone baseline values ​​were calculated for each female based on the sampling period of 6 months before treatment. Data were analyzed using the R statistical package hormLong. Baseline E2 and P4 values ​​for each individual animal were calculated using an iterative process excluding all points that exceeded the mean + 1.5 standard deviations. Estrous phases were defined as 2 or more consecutive fecal samples with E2 values ​​> 1.5 times the baseline. Luteal phases were defined as 6 or more consecutive fecal samples with P4 values ​​> 1.5 times the baseline. Means of number of estrous phases, number of luteal phases, and fecal metabolite concentrations were compared between two periods: 6 months before treatment vs. 22 months after treatment following a 2-month post-treatment transition period. Periods of pregnancy and lactation were excluded from the analysis. To account for the different lengths of time during each sampling period, estrus was reported as the number of estrus in a one-month period (total number of estrus / days in sampling period x 30 days) and luteal phase was reported as the number of luteal phases in a six-month period (total number of luteal phases / days in sampling period x 180 days). Data were analyzed using ANOVA as a randomized complete block design, where period and treatment group (and their interactions) were fixed effects and individual animals were included as random effects (blocks). Tukey's multiple mean comparison test was used for pairwise comparisons. Analyses were performed using SAS Studio software (Release: 3.8, Enterprise Edition, SAS Institute Inc., Cary, NC, USA).

[0239] For serum LH analysis, diluted serum samples (1:5) were analyzed utilizing a double antibody EIA modified from Graham et al., Zoo Biol, 20:227-236 (2001). NIH-bovine LH standards, controls, and samples were added to plate wells in duplicate. After overnight incubation, biotinylated NIH-ovine LH was added to all wells and allowed to compete for 4 hours at room temperature. Plates were then incubated with streptavidin-peroxidase after competition. The EIA for feline serum was validated by demonstrating similarity between dilutions of pooled serum and the standard curve. Additionally, bovine LH (used for standards) was added to feline serum samples to generate dose-response curves. Intra- and interassay coefficients of variation were 3.5% and 8%.

[0240] For statistical analysis of serum LH, serum LH samples were divided into three groups: pre-treatment / transition (transition period immediately before treatment to 2 months; pre-transition), early post-treatment (months 3-8, early post-treatment), and late post-treatment (months 15-20, late post-treatment). These groups were chosen to avoid sampling periods during breeding trials or subsequent pregnancy and lactation. Analyses were performed using SAS® Studio software, Release: 3.8. Serum LH changes between time (samples) in treated versus untreated cats were assessed using generalized linear mixed-effects models in the MIXED procedure with treatment, time (three levels: pre-treatment / transition, early post-treatment, and late post-treatment) as fixed effects, and treatment x time interaction and cat (individual) as a random block, with Satterthwaite adjustment for degrees of freedom. Time levels within each treatment group were compared using Tukey's multiple mean comparison test. Significant differences were declared at P<0.05.

[0241] For blood inhibin B analysis, inhibin B was measured using the Cat INHB-B ELISA Kit (Mybiosource.com, San Diego, Calif.). Undiluted feline serum samples were assayed using the manufacturer's protocol.

[0242] result Viral genomes were detected in blood on day 2 and remained elevated for 2 months before rapidly declining over the 3-4 month period, likely reflecting infected cell turnover (e.g., liver cells) releasing cell-free DNA (Figure 7A). Viral shedding was detected in urine on day 1 and steadily declined over 7 days, whereas measurements in oral swabs and stool samples showed variability between individuals and groups (Figures 7B-7D).

[0243] Circulating MIS levels were initially strong but gradually decreased over the course of the first year, eventually reaching a relative plateau in the second year and remaining above 0.5 μg / ml (Figures 7E-7G). One treated cat, subject 17LRE4, in the group treated with 5e12vg / kg AAV9-fcMISv2 (i.e., the low-dose group), had the lowest circulating MIS levels, averaging 0.93 μg / ml during the first mating trial and 0.61 μg / ml during the second mating trial (Figure 7E). Interestingly, subject 17LRE4 also had 100-fold less viral genome by day 2 compared to other cats in the subject 17LRE4 group, suggesting misinjection or pre-existing vector immunity (Figure 7E).

[0244] Importantly, unlike the strong anti-transgene antibody responses observed with fcMISv1 (Figures 1F-1H), anti-fcMISv2 antibodies were not detected above background using direct capture ELISA in any cat given AAV9-fcMISv2 (Figures 7E-G).

[0245] As previously observed, there is a delay of approximately one month between AAV9-MIS treatment and complete ovarian function suppression in mice, which likely corresponds to the time required for a cohort of growing pre-existing follicles to complete maturation and be cleared from the ovaries (Kano et al., 2019, 2017). Feline reproductive hormones were measured and compared during the first 3 months of treatment and the remainder of the post-injection period. Hormones measured were luteinizing hormone (LH), a pituitary feedback hormone (Figure 6C), and inhibin B, a marker of growing follicles (Figure 6D).

[0246] Serum LH means were calculated over three time periods: (1) the transition period immediately prior to treatment through 2 months (Pre / Transition), (2) the early post-treatment period (3-8 months; Post-Early), and the late post-treatment period (15-20 months; Post-Late). Analysis revealed that in control cats, LH was not different at any time period. However, in AAV9-MIS-treated cats, LH was elevated in both post-treatment periods compared to Pre / Transition (P = 0.0008, Post-Early; P = 0.0036 Post-Late) (Table 4; Figure 6E). There was no difference between the early and late post-treatment periods in treated cats.

[0247] (Table 4) Serum LH TIFF2024511413000018.tif21136 Data shown as mean (SEM).

[0248] Conversely, inhibin B was significantly decreased throughout the entire period in the high-dose group (Figures 6D-6E). These profiles suggest hypergonadotropic hypogonadism, especially at the high dose.

[0249] Fecal estradiol (E2) and progesterone (P4) were measured in fecal samples three times a week from 6 months before injection until the end of the study (2 years) (Figures 8A-8I and Table 5). Reproductive cyclicity was confirmed in all females before treatment. There is a delay of approximately 1 month between AAV9-fcMISv2 treatment and complete ovarian function suppression in mice, which likely corresponds to the time required for already developing follicles to fully mature and be removed from the ovaries (Kano et al., 2019, 2017; Meinsohn et al., 2021). The timing of ovarian function suppression by MIS in cats is uncertain, but a 2-month delay between MIS treatment and effect is estimated. Therefore, the mean fecal E2 and P4 metabolite concentrations were compared 6 months before treatment and 22 months after treatment after a 2-month transitional phase (Figure 6F). A significant increase in E2 was observed in the controls during the post-treatment observation and breeding periods (P=0.0008). No differences were observed between the periods in the low- or high-dose AAV9-fcMISv2 groups. After treatment, the mean P4 metabolite concentration decreased in the high-dose group (P=0.0029). The low-dose group trended downward (P=0.0986), but the controls did not differ between time points.

[0250] Table 5. Fecal E2 and P4 profiles TIFF2024511413000019.tif45164 Data shown as mean (SEM). ** indicates P<0.01.

[0251] Fecal E2 and P4 levels were used to infer the timing of estrus and luteal phase (Figures 6F, 9A and 9B, Table 6). The frequency of estrus per month and the frequency of luteal phases per 6 months were compared between 6 months before treatment and 24 months after treatment (excluding the 2-month transition phase). No difference in estrus frequency was observed for any of the groups (Figure 6G). Luteal phase frequency was reduced in the high-dose AAV9-fcMISv2 group (P=0.0263), but not in the low-dose group or in controls (Figure 6G).

[0252] Serum LH means were calculated in two periods: samples taken before MIS treatment and during the 2-month transition period (pre / transition) and samples taken after treatment (excluding breeding trial, pregnancy, and lactation periods; post-Tx). Data from the two treatment groups were pooled. Analysis revealed no difference in LH in control cats (Figure 6E). However, post-Tx LH was significantly greater than pre / transition LH in MIS-treated cats (P=0.0001).

[0253] Table 6. Estrous and luteal phase evaluation TIFF2024511413000020.tif26164 Data shown as mean (SEM). * indicates p>0.05.

[0254] E. Repeat breeding extension material and method In the repeat breeding study, a certified breeder male cat was transferred to a group-housed room with nine AAV-MIS research females (three control, six AAV-MIS-treated). To facilitate incorporation, males were pre-exposed to the females in a controlled manner (housed in a cat carrier) in the room. Males were housed with the females for 8 h / day, 5 days / week for 4 consecutive months. Reproductive activity was documented by a combination of direct observation and remote audio / video monitoring. Each female was assessed weekly by abdominal palpation and ultrasound examination to confirm pregnancy status (i.e., fetal presence and viability). All females were pre-operant conditioned to spontaneously accept these procedures with minimal restraint or disturbance. Pregnant females were re-evaluated by ultrasound examination every 3 weeks to monitor fetal development and viability. Females remained group-housed until approximately day 50 of gestation and then transferred to a birthing room with individual caging for later natural birth (typically approximately day 63–65 after breeding). Pregnant females were monitored daily by caretakers in person and remotely via an internet-accessible video camera linkage until the expected time of parturition.

[0255] At 8 and 20 months after vector treatment (Figure 6A), two 4-month breeding trials were initiated using a different certified breeder male for each trial. This male was group-housed with nine females for 8 h / day, 5 days / week and continuously video-monitored to record all breeding interactions. Weekly transabdominal ultrasound examinations were performed to assess pregnancy status. Interactions were assessed by video review and scored as breeding success (defined as intromission and an appropriate response from the female) or breeding attempt (defined as the male attempting to mount the female or successfully mounting without intromission). The identity of each female was confirmed for interactions by a member of the animal care staff.

[0256] In this example, a "breeding bout" is defined as a period of consecutive, repetitive breeding behavior. A "breeding bout" consists of a queen successfully breeding with a male (on one or several consecutive days). A "breeding bout" is typically a period of estrus during which a female accepts a male, but in rare cases, there may be a prolonged estrus (i.e., 8 days or more) or multiple overlapping estrus periods resulting in a truly long breeding bout. For example, subject 17LRJ1 was observed to have an atypical breeding bout consisting of approximately 125 confirmed breeding events over a 33-day period.

[0257] For statistical analysis of the breeding trials, the number of females bred, the total number of breeding bouts, the number of luteal phases after breeding, the number of pregnant females, and the total number of kittens born were compared between treatments within each breeding trial using chi-squared tests of homogeneity, where the null hypothesis was uniform distribution among treatments. Analyses were performed using SAS Studio software (Release: 3.8, Enterprise Edition, SAS Institute Inc., Cary, NC, USA).

[0258] result For both trials, all control females became pregnant after the first breeding bout (Tables 7 and 9). Controls gave birth to 2-4 healthy kittens in each litter. In contrast, AAV9-MIS-treated females did not give birth during either trial, and no gestational sacs or fetuses were observed during weekly ultrasound examinations.

[0259] Table 7. Reproductive activity, ovulation, and pregnancy occurrence in AAV9-fcMIS-treated cats versus controls. TIFF2024511413000021.tif50165 * There were differences between the control and treatments within each breeding trial (P<0.05).

[0260] Two of the AAV9-MIS treated cats exhibited reproductive activity during the trials (Tables 7 and 10). Subject 17LRJ1 (high dose AAV9-fcMISv2) allowed one breeding bout during each trial. Subject 17LRE4 (low dose AAV9-fcMISv2) allowed six breeding bouts during the first trial and one breeding bout during the second trial. No luteal phase was detected in fecal hormone analysis after any bout (Table 7). No reproductive behavior was observed from other AAV9-MIS treated females.

[0261] Using chi-squared tests for homogeneity, no differences were found between the number of females allowed to breed or the total number of breeding bouts. There were significant differences in the number of luteal phases following breeding bouts (P = 0.0498), the number of females who became pregnant (P = 0.0498), and the total number of kittens born (P < 0.0001; Table 7).

[0262] To determine whether the introduction of males affected E2 levels during estrus, the E2 peak in fecal pellets during the mating period was compared with the E2 peak in fecal pellets during the pretreatment period when females were not cohabiting with males. A significant increase in the E2 peak was only found in control cats after the introduction of males (Table 8).

[0263] Table 8. Estradiol peaks during mating studies TIFF2024511413000022.tif26128 data expressed as (SEM)

[0264] Maternal-fetal transmission of MIS was not assessed as no kittens were born to treated females, however the reference value in male kittens born to control females was confirmed to be 254 ng / ml ± 72 (Figure 9D).

[0265] Table 9. First breeding trial TIFF2024511413000023.tif253144TIFF2024511413000024.tif25336S: Breeding success, A: Breeding attempt

[0266] Table 10. Second breeding trial TIFF2024511413000025.tif255105S: Breeding success A: Breeding attempt * Reproductive success was also seen on 01 / 04 / 21(1), 01 / 13 / 21(1), and 01 / 14 / 21(2). An estrus peak was detected on 01 / 09 / 21 (but was excluded from the peak analysis because it occurred during pregnancy).

[0267] F. Discussion Intraperitoneal administration of AAV9 in mice primarily transduces skeletal muscle and liver cells, which in turn act as in vivo bioreactors secreting MIS for systemic delivery to the ovaries (Kano et al., 2017). In cats, MIS levels during the early period of sampling followed a rapid evolution of decreasing concentrations over time until a relative plateau was reached. This rapid decline could be due to the turnover of short-lived transduced cells (e.g., liver cells), which could also be the source of viral genomes observed in the blood for as long as the first 3 months, and was offset by a balanced increase in secretion from long-lived muscle fibers that maintained long-term expression of the fcMISv2 transgene. We therefore hypothesized that muscle-tropic viral vectors could be beneficial for lifelong production of transgenes at contraceptive levels.

[0268] In AAV9-fcMISv2-treated cats, we observed a significant reduction in P4 levels and luteal phase, strongly suggesting the absence of ovulation. However, we observed a relatively modest decline in inhibin B, whereas there was no difference in E2 levels. These data are consistent with the previously demonstrated inhibition of preantral follicle maturation in AAV9-MIS-treated mice, which significantly reduced circulating inhibin B and inhibited cycling, but maintained E2 levels at nearly half of baseline (Kano et al., 2017). Later studies demonstrated a stall in preantral follicle maturation due to inhibition of granulosa cell differentiation by MIS (Meinsohn et al., 2021). Thus, this suggests that a pool of immature follicles unable to ovulate is maintained in treated animals and continues to produce E2. This has important implications for the use of recombinant MIS or MISR2 agonists as contraceptives in humans, as it means that estrogen levels may not be suppressed by these therapies, and therefore hormonal health may be maintained in the absence of cycles.

[0269] While the timing of onset of contraceptive efficacy was not directly tested in this study, contraception was observed 8 months after treatment. Hormonal profiles of AAV9-fcMISv2-treated cats indicate that most changes in reproductive hormones stabilized by 3 months, suggesting a possible upper limit for the contraceptive efficacy of MIS. Similarly in mice, adult females treated with AAV9-LRMIS and mated with certified male breeders were fertile for the first 30 days but remained infertile thereafter (Kano et al., 2019, 2017).

[0270] Interestingly, control females showed a significant E2 rise during the post-treatment period (Figure 6E). This period included 8 months of cohabitation with an intact male. In sheep, the ram effect is well described (Martin et al., 1986), where socio-sexual stimulation introducing an intact male to a seasonally non-estrus female can induce an LH surge and ovulation. More recently, it has been shown that the LH surge is due to increased E2 secretion (Fabre-Nys et al., 2015). The Vandenbergh effect, where the presence of a male leads to a surge in serum E2 in females and accelerates puberty, is best described in mice but also occurs in several other rodents as well as other species including pigs and cattle (reviewed by (deCatanzaro, 2015)). A similar phenomenon has been documented in cats, where the non-mating presence of males can increase the rate of spontaneous ovulation (Gudermuth et al., 1997). Therefore, the E2 increase observed in control females may be due to the effect of the presence of males in close proximity. Both treatment groups showed a small but non-significant E2 increase (Figure 6E), suggesting that AAV9-fcMISv2 treatment may have reduced this response.

[0271] Previously ovariectomized female cats experienced increased LH due to lack of negative feedback from ovarian-derived E2 (Bateman et al., 2017; Concannon et al., 1989; Johnson and Gay, 1981; Rohlertz et al., 2012). Although E2 did not appear to be suppressed based on fecal hormone data, the LH elevation in MIS-treated cats (Figure 6C) suggests some degree of ovarian dysfunction. When estrus frequency was ascertained by assessing successive E2 elevations in fecal samples, no differences were observed in MIS-treated cats compared to controls (Figure 6F). However, when estrus was defined behaviorally by females allowing mounting and copulation, the effect of treatment can be clearly observed. All three control females mated repeatedly with both males, whereas four of the six MIS-treated females rejected every mating attempt by the breeder male during both breeding trials (Tables 7 and 10). Although mate choice among females may have influenced reproductive activity, the use of two different breeder males (of widely different phenotypes and ages) and the consistent receptivity of randomly assigned control females to both males argue against this being a major factor.

[0272] As AAV9-fcMISv2 vector-mediated contraception has been proposed as an alternative to ovariohysterectomy, the long-term health of the retained reproductive tract is an important consideration. Cystic endometrial hyperplasia-pyometra complex is a clinically significant and potentially life-threatening disease in intact female cats. Ovarian hormones contribute to the pathogenesis, with P4 playing a major role. The disease is characterized by endometrial hyperplasia, cystic dilatation of endometrial glands, uterine inflammation, and purulent secretions (Agudelo, 2005). The overall incidence is unknown. However, a study evaluating the histopathology of 106 reproductive tracts from clinically healthy cats submitting for elective ovariohysterectomy detected cystic endometrial hyperplasia in 21 (~20%) and pyometra in 2 (~2%) (Binder et al., 2019).

[0273] The six treated females in this study received AAV9-fcMISv2 injections approximately 3 years ago (March 2019). Physical examination and transabdominal ultrasound performed every 3 months and blood tests evaluated every 6 months revealed no evidence of cystic endometrial hyperplasia-pyometra or abnormalities in systemic blood parameters of any female. As these females are still intact, there is no uterine histopathology data available for evaluation. However, three females treated with AAV9-fcMISv1 during the pilot study were ovariectomized and histologically examined 40 months after treatment (age 9–10 years). One female (subject 11WBL25) produced minimal antibodies against the fcMISv1 protein (SEQ ID NO:3) and maintained serum MIS protein levels above the target level (0.50 μg / ml) (Figures 1F–1H). The female's reproductive system showed normal endometrium and quiescent ovaries containing only primordial follicles (Figure 2). In contrast, histological analysis of the female with the highest anti-fcMISv1 antibody level and the lowest serum MIS level (subject 11WBL24) showed multiple corpora lutea (indicating natural ovulation as this female was never housed with males) and cystic endometrial hyperplasia (Figures 1F-1H and 2). These data suggest that high MIS may have a protective effect on uterine health by preventing natural ovulation in intact females.

[0274] One important aspect of this MIS-induced protection is likely the reduction in long-term P4 exposure as it occurs during the non-pregnant luteal phase. In the high-dose group, both mean P4 levels and spontaneous ovulation were significantly reduced after treatment (Figures 6E-6F). Spontaneous ovulation can occur at a high rate (>80%) in group-housed female cats not in direct contact with males (Gudermuth et al., 1997) and may contribute to the cystic endometrial hyperplasia-pyometra complex by long-term P4 effects on the endometrium. In one case study, 45% of cats evaluated for inflammatory uterine disease or infertility had active corpora lutea resulting from spontaneous ovulation at the time of the study (Lawler et al., 1991).

[0275] Similarly, high MIS levels appear to inhibit the occurrence of the luteal phase induced by natural breeding, as demonstrated in two breeding trials (Tables 9-10).

[0276] Although serum MIS levels mirrored the AAV9-fcMISv2 dose-response relationship (approximately 2-fold greater in high-dose than in low-dose cats) (Figure 9C), inhibition of breeding-induced ovulation was observed in both dose groups. One female in the low group that was allowed to breed (subject 17LRE4) had the lowest MIS levels during each trial (0.92 μg / ml for trial 1 and 0.61 μg / ml for trial 2). However, both MIS values ​​remained above 0.50 μg / ml, twice the 0.25 μg / ml threshold required for complete contraception in mice (Kano et al., 2017). In contrast, one female in the high treatment group that was allowed to breed (subject 17LRJ1) had consistently high serum MIS (Figure 9C). During the second breeding trial, this cat's mean MIS level of 4.0 μg / ml coincided with an atypical 33-day breeding bout consisting of 125 recorded intercourses (Table 10). For comparison, three control females had a mean (SE) reproductive success of 24.7 (9.9) in a bout lasting 4.3 (0.7) days during the same trial.

[0277] These findings demonstrate an overall decrease in fecal P4 concentrations, a decrease in spontaneous ovulation, and a complete inhibition of coitus-induced ovulation after AAV9-fcMISv2 treatment. It remains unclear whether these reproductive changes are caused by the inability of follicles to complete maturation and ovulate in response to the LH surge or impaired LH surge per se. Ovariohysterectomized cats (with high basal LH) are able to generate an LH surge even after gonadotropin-releasing hormone (GnRH) treatment, suggesting that the pituitary gland still responds to breeding-inducing hormonal stimulation. However, other studies have shown that breeding early in estrus (day 1) is unlikely to induce an LH surge (approximately 43% ovulation rate), as can occur with high MIS, primarily due to low concentrations of E2. Although the timing of behavioral estrus may not be consistent relative to the final stages of follicular development, the E2 threshold required for behavioral estrus appears to be lower than that produced by fully mature follicles (Shille et al., 1979). Thus, sustained E2 elevation may be an important prerequisite for stimulating the hypothalamus and / or pituitary to respond to intercourse with the ovulatory reflex (Banks and Stabenfeldt, 1982) and may be impaired by high MIS concentrations.

[0278] Taken together, these data support the use of AAV9-fcMISv2 as a long-lasting contraceptive in adult cats. Although continued long-term transgene expression needs to be further investigated, it remains above the contraceptive range at 2 years in the low dose (5e12vg / kg) and high dose (1e13vg / kg) groups. Furthermore, no toxicity was observed in adult cats treated with MIS (Example 1.D. above). The MIS concentrations demonstrated in gene therapy for contraception in adult cats (Figures 7E-7F) and in newborn male kittens (0.5 μg / ml; Figure 9D) are within the same order of magnitude. Endogenous MIS concentrations in this range have also been observed in other mammalian species, including baby boys (Lee et al., 1997).

[0279] Interestingly, it has also been shown that MIS may modulate pituitary gonadotropins to increase the LH / FSH ratio (Cimino et al., 2016; Garrel et al., 2016; Tata et al., 2018). AAV9-fcMISv2-treated cats showed an early LH decline followed by high baseline levels (Figures 6C and 6E), consistent with hypergonadotropic hypogonadism. However, given that ultra-substance MIS primarily inhibits follicles at gonadotropin-independent primordial and early preantral stages in mice, it is unclear how gonadotropin modulation may contribute to contraception. Overall, the observed reduction in luteal phase, P4 levels, mating behavior, and lack of conception strongly support the hypothesis that MIS inhibits ovulation by blocking follicular maturation to the ovulatory stage, but maintains a pool of follicles capable of producing ovarian hormones. Furthermore, the hormonal data suggest that complete ovarian suppression with MIS is not necessary for contraception, given that suppression was mild (with E2 and inhibin B) and animals in the low-dose group in which estrus and mating events were recorded were also infertile.

[0280] G. References TIFF2024511413000026.tif180164TIFF2024511413000027.tif245164TIFF2024511413 000028.tif238164TIFF2024511413000029.tif245165TIFF2024511413000030.tif12142

[0281] Example 2 - Vector design for MIS delivery This example describes vector design efforts for optimal expression of MIS in cats and dogs. The fcMISv2 protein (SEQ ID NO:1) and clMIS protein (SEQ ID NO:2) were modified as follows: (1) the leader sequence was replaced with a human albumin leader sequence, and (2) the glutamine (Q) in the MIS cleavage site was replaced with an arginine (R). Such leader and cleavage site ("LR") modified fcMISv2 and LR modified clMIS transgenes were engineered and cloned into both pcDNA3.1 and pAAV vectors. The protein sequence of the LR-fcMISv2 transgene is SEQ ID NO:14, and the protein sequence of the LR-clMIS transgene is SEQ ID NO:15.

[0282] A. Effect of LR vector modification in CHO and COS7 cells material and method Materials and methods relating to CHO cells were as described above in Example 1.A.

[0283] COS7 cells (African green monkey kidney cell line; American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin in a humidified 5% CO2 incubator at 37°C. COS7 cells were plated in 6-well culture dishes (Corning Life Sciences) containing DMEM with 1% fetal bovine serum (FFBS) (to reduce bovine MIS) and transfected with pcDNA3.1 expression plasmids containing fcMISv2, LR-fcMISv2, clMIS, or LR-clMIS using Fugene6 (Promega) (1:6 mass / volume ratio) according to the manufacturer's instructions. Transfection efficiency was confirmed to be >80% by transfecting the same plates with a GFP expression plasmid (PCDNA3-eGFP-N1). Conditioned medium was collected after 72 hours and used for Western blot analysis.

[0284] CHO or COS7 cells were transfected with pcDNA3.1 or pAAV vectors containing fcMISv2 or clMIS transgenes. Transient and stable CHO clones were used to compare transcript levels, protein levels, and cleavage ratios of fcMISv2 (SEQ ID NO:1), LR-fcMISv2 (SEQ ID NO:14), clMIS (SEQ ID NO:2), and LR-clMIS (SEQ ID NO:15). Materials and methods related to cells, transfection, and Western blot were as described above in Example 1.A. Materials and methods related to ELISA were as described above in Example 1.D.

[0285] result In transiently transfected CHO cells, CHO medium conditioned with LR-fcMISv2 for 48 h had much higher levels of cleavage with a strongly cleaved mature MIS band (FIG. 10; LR-Fc-MIS 48h) compared to medium from CHO clones producing fcMISv2 (FIG. 10; Fc-MIS 3dcsf medium). This cleavage ratio can be compared between controls present on a Western blot, including purified human recombinant protein (FIG. 10; LR-MIS 25ng), FLAG-tagged mouse MIS or F-mmMIS (FIG. 10; MF-MIS 25ng cleavage), LR-mmMIS conditioned medium (FIG. 10; LR-MM-MIS), and serum-free medium of fcMISv2-producing CHO clones prior to purification (FIG. 10; Fc-MIS 3dcsf medium), as well as CHO cell medium conditioned 48 hours after transient transfection with pCDNA3.1 plasmids encoding pCDNA-LR-fcMISv2 (FIG. 10; LR-Fc-MIS 48h), pCDNA-clMIS (FIG. 10; CL-MIS 48h), and pCDNA-LR-clMIS (FIG. 10; LR-CL-MIS 48h). This reproduces the effects observed in the human and mouse transgenes shown here as controls. Similarly, clMIS was largely intact, whereas LR-clMIS was almost completely cleaved. These data support the hypotheses that (1) the LR modification facilitates proteolytic cleavage and cross-species activation, and (2) the unmodified transgene, especially canine clMIS, is poorly cleaved by CHO cells.

[0286] To ensure that the variable cleavage ratios were not an artifact of CHO cells or vectors (pcDNA3.1 vs. pAAV-CB6-PI; Figure 10), the experiment was repeated with COS7 cells (Figure 13). Strong MIS expression from pcDNA-based vectors was also observed in COS7 cells (Figure 13). Again, cleavage of MIS from LR-based constructs was compared to non-LR variants, and enhanced cleavage was observed in the LR-based constructs (Figure 11). These data strongly support the use of LR modifications to enhance activation cleavage. The observed cleavage effect appears to be entirely post-translational, as the various constructs were transcribed to relatively similar levels when compared by RT-qPCR (Figure 12).

[0287] Finally, the recombinant proteins were evaluated in a rat urogenital ridge bioassay to compare biological activity. Using stably transfected CHO clones, clones were screened by ELISA for expression of MIS protein. Clones that showed high protein expression were used in production cultures. Media from production cultures were harvested and concentrated to a total protein concentration of 5 μg / ml. Concentrations were adjusted based on ELISA values. ELISA may in some cases underestimate the total amount of protein in non-LR variants due to possible detection bias (discussed in Example 2.E below), so the observed increased potency achieved by LR modification and increased cleavage may also be underestimated. The data demonstrate that the stable high-expressing CHO clones recapitulated the enhanced cleavage of fcMISv2 and clMIS LR variants (Figure 13A). This translated into increased activity in a rat urogenital ridge assay that scores Müllerian duct regression from 0 to 5 (Figure 13B). Overall, these data demonstrate that all of the transgenes discussed in this example were biologically active.

[0288] B. Capsid Serotype and Transgene Modification in Mice A benchmark for vector activity was established using AAV9-fcMISv2 as a reference vector, allowing one of skill in the art to make comparisons between numerous vectors and to extrapolate results to those observed in cats using the same virus batch.

[0289] material and method Materials and methods related to the CHO cells and mouse experiments were as described above in Example 1.A. Materials and methods related to follicle counts in mice were as described above in Example 1.C. In this example, vectors were administered to mice by injection, and mouse experiments were performed generally as described in Example 1.A. Mouse ovaries were harvested 30 days after administration. Complete sectioning and follicle counts were performed on ovarian sections by at least two independent, blinded observers.

[0290] result The first two AAV9-fcMISv2 concentrations administered to mice were 5e12vg / kg and 1e13vg / kg, the same concentrations used in cats, which inhibited follicle formation in a dose-independent manner, likely due to a saturation effect at the high doses of MIS (Figure 3G).

[0291] To compare inter-capsid and inter-transgene activity, non-saturating doses of vectors were used in the next experiment. A dose-response experiment was performed with AAV9-fcMISv2 to be able to quantify the improvement on the inhibitory effect of the vector. AAV9-fcMISv2 vectors were administered at 1E10, 1E11, 5e11, and 1e12vg / kg. A dose-response effect was observed in the inhibition of growing follicles (FIG. 14), with 1e11vg / kg being a potential dose with partial but strong inhibition. This 1e11vg / kg dose may be used in future virus studies to lead to high MIS concentrations in the blood.

[0292] C. AAV9-LR-clMIS activity in mice In anticipation of treating dogs, AAV9-clMIS or AAV9-LR-clMIS were tested for activity in mice. Materials and methods for this example were as described in Examples 2.A. and 2.B above. Both vectors were able to suppress ovarian function at high doses (1e13vg / kg) in mice. Despite low expression detected in the blood (Figure 16; Example 2.D. below), LR-clMIS showed a strong suppressive effect at this dose comparable to AAV9-fcMISv2 (Figures 15A-15D). 1e13vg / kg can be used as a high vector dose in dogs, potentially resulting in a high enough level of MIS to saturate the suppressive effect.

[0293] D. Feline and canine MIS levels in mice material and method Materials and methods related to mouse experiments were as described above in Example 2.B. ELISAs were performed as described above in Example 1.D. except that the Ansh Labs AMH ELISA kit (Ansh Labs Catalog No. AL-105) was used instead of the Beckman AMH Gen II ELISA kit as follows:

[0294] result The 5e12vg / kg AAV9-clMIS and AAV9-LR-clMIS vectors produced lower than expected MIS concentrations, i.e., in the 100-200ng / ml range. This experiment was repeated with a higher vector concentration of 1e13vg / kg. Again, MIS levels in mice were lower than expected for AAV9-clMIS (Figure 16). However, MIS concentrations were nearly twice as high for AAV9-LR-clMIS as for the unmodified AAV9-clMIS. Based on this data, translational and post-translational processing of the canine transgene appears to be less efficient than that of the feline transgene. However, MIS processing improved with the introduction of the LR modification, so that circulating MIS is within the expected contraceptive range in cats and dogs.

[0295] Furthermore, because the Ansh Labs ELISA was capable of detecting both feline and canine MIS and results were generally comparable to the Beckman ELISA (described above in Example 1.D.), the Ansh Labs ELISA was used in subsequent analysis of blood MIS levels.

[0296] E. Viral infectivity across tissue types material and method Materials and methods relating to cells, mice, transfection, and Western blots were as described above in Example 2.A.

[0297] result To ensure that the variable serum MIS detection among the various vectors was not due to differences in virus batch quality (which may affect virus infectivity), the presence of vector DNA in different tissues was compared. Tissues from mouse quadriceps ("Muscle" in FIG. 17A) and liver were analyzed 30 days after treatment with vectors expressing fcMISv2 (SEQ ID NO:1), LR-fcMISv2 (SEQ ID NO:14), clMIS (SEQ ID NO:2), and LR-clMIS (SEQ ID NO:15). In general, equal amounts of vector DNA were present in tissues for all vectors. Although an excess of AAV9-fcMISv2 vector DNA was found in the liver compared to the other vectors (FIGS. 17A-17B), the amount of DNA was orders of magnitude too low to explain the observed differences in circulating MIS as measured by ELISA. It was therefore recognized that the quality of the vector and / or virus preparations may affect MIS levels in animals.

[0298] Secretion and cleavage of MIS in target tissues (liver and muscle tissues) from mice treated with AAV9-fcMISv2, AAV9-LR-fcMISv2, AAV9-clMIS, or AAV9-LR-clMIS were evaluated. The two vector concentrations used were 5e12vg / kg and 1e13vg / kg. Several antibodies were evaluated for compatibility with mouse lysates in Western blots. One antibody, LSBio rabbit anti-AMH, was able to reliably detect MIS in Western blots, but only in tissues where AAV9-fcMISv2 or AAV9-LR-fcMISv2 were present at high doses, i.e., conditions with high vector concentrations in the liver. This Western blot is consistent with both fcMISv2 (SEQ ID NO:1) and LR-fcMISv2 (SEQ ID NO:14) being produced at higher levels in the liver than clMIS and LR-clMIS. Furthermore, cleavage of the LR-fcMISv2 variant (SEQ ID NO:14) exceeded that of fcMISv2 (SEQ ID NO:1) in the liver (Figure 18).

[0299] The cleavage ratio of feline MIS to canine MIS can be quantified in blood by ELISA. Analysis of the cleavage ratio can provide information on the benefits of LR modification in vivo and the ability of muscle cells to efficiently cleave the LR modified transgene, which may be particularly important for the muscle-tropic vectors described herein.

[0300] F. Evaluation of muscle-tropic vectors This example describes studies to determine whether infecting cell types with slow cell turnover rates would promote long-term production of the MIS transgene. For example, muscle cells generally have a slower turnover rate than liver cells.

[0301] material and method AAV.MYO and AAV9.HR muscle-tropic vectors were used to deliver fcMISv2. 5e12vg / kg of vector was administered by ip injection to nude mice. Materials and methods related to mouse experiments were as described in Example 1.A.

[0302] result Both the AAV.MYO-fcMISv2 and AAV9.HR-fcMISv2 muscle-tropic vectors produced lower serum MIS levels than AAV9-fcMISv2 (Figure 19).

[0303] Example 3 - Efficacy of AAV-wt feline MIS treatment to prevent kittens from entering puberty and to provide long-term sterility G. Preventing puberty in cats (kittens) Many animal shelters surgically spay or neuter kittens as early as 8 weeks of age prior to adoption. Recommendations suggest that kittens should be surgically spayed or neutered before reaching approximately 5 months of age. However, the long-term developmental effects of increasing MIS levels in prepubertal kittens (and puppies) are unknown. Nevertheless, the ability of MIS treatment via gene delivery in cats (kittens) to block puberty as well as any other associated developmental effects was evaluated. Additionally, parameters for using AAV-wt feline MIS when given as a single injection for long-term reduction in fertility in kittens and / or to block kittens from entering puberty were evaluated. As an example, this study also serves as a non-human animal model to evaluate MIS treatment via gene delivery or MIS protein delivery to delay puberty in human subjects.

[0304] material and method The kittens and virus preparations used in this example were generated in breeding studies as described above in Examples 1.D. and 1.E.

[0305] The timing of kitten injection with AAV-wt feline MIS (AAV9-fcMISv2) depended on the timing of parturition and weaning. Healthy kittens were weaned by 8 weeks of age. Healthy kittens (e.g., 9 females, 3 males) approximately 3 months of age (e.g., 10-12 weeks of age) were randomized into control and treatment groups. Kittens were intramuscularly injected with AAV9-fcMISv2 or empty vector control as follows: (1) high dose AAV9-fcMISv2 (1e13vg / kg, e.g., 3 females and 1 male); (2) low dose AAV9-fcMISv2 (5e12vg / kg, e.g., 4 females and 1 male); and (3) control empty vector (5e12vp / kg, e.g., 2 females and 1 male). Table 11 shows the kitten sampling and monitoring schedule.

[0306] Table 11. Kitten monitoring schedule TIFF2024511413000031.tif23796

[0307] Kittens were housed in cages for 5 days (2-3 kittens from the same treatment group / cage) and then transferred to single group enclosures for further monitoring. During this period, pooled fecal and urine samples and individual oral swabs were collected daily from group-housed kittens to assess viral shedding. qPCR of viral genomes in individual blood, mixed urine, mixed feces, and individual oral swabs was monitored to analyze viral shedding. Blood was sampled 7 days after injection, weekly for the remainder of the month, and monthly after the first month. Urine, feces, and oral swabs were collected daily for the first week.

[0308] Food dyes were mixed with cat food and given to each individual kitten separately (three times a week) starting two weeks prior to injection and continuing for nine months (e.g., approximately one year of age) after injection. Kittens from the same treatment group were housed together so fecal samples were evaluated for the presence of estrogen and progesterone metabolites (females) or testosterone metabolites (males) to confirm the onset of puberty and reproductive maturity. As described above in Example 1.D., dyes / cat-specific food dyes and / or glitter were mixed with cat food and given to each individual cat separately to facilitate identification of cat-specific feces. Kittens were weighed weekly starting two weeks prior to injection until one year of age.

[0309] Whole blood samples (1 ml, minimum) were collected from each kitten (1X / month) beginning immediately prior to injection to assess serum MIS, inhibin B, anti-MIS antibodies, and LH concentrations. Additional blood samples were collected on days 7, 14, 21, and 28 post-injection for both treated and control kittens.

[0310] Safety monitoring included daily assessments of general health and regular evaluations of the injection sites (daily for 14 days, weekly until the second month, then monthly thereafter). Physical examinations and CBC / biochemistry evaluations were performed immediately prior to injection and then every 3 months until 1 year of age.

[0311] The postnatal health of all kittens was monitored throughout their development up to puberty. Kittens were assessed for signs of behavioral estrus using video and direct observation. Because untreated kittens can enter puberty between 4 and 6 months of age, the study continued until 10 months of age. If there were no signs of estrous cycling cats by 10 months of age, a follow-up breeding study was considered.

[0312] Sex steroids E2 and P4 were measured in fecal samples. Fecal samples were freeze-dried and processed for E2 and P4 extraction. Fecal hormone analysis in females showed evidence of puberty by demonstrating ovarian cyclicity based on a gradual increase in basal estrogen over time and the occurrence of estrogen spikes that coincide with estrus. Young females do not typically ovulate naturally, and therefore fecal progesterone may not be informative, although female kittens may appear to acquire the ability to ovulate naturally as they age, and therefore may regain the luteal phase as they approach the end of their yearly period.

[0313] In females, transabdominal ultrasound was performed monthly and uterine horn measurements were recorded. Measurements were taken for each horn, as close to the bifurcation as possible. The widest and narrowest diameters were measured and divided by 2 to obtain the long and short axes. The area was calculated as an ellipse (π x long axis x short axis) and the mean area of ​​a given horn (mm 2 ) was reported.

[0314] In males, a rise in fecal testosterone was indicative of puberty, demonstrated by the presence of penile spines and sperm production. Every 3 months after treatment, male kittens were anesthetized with a ketamine-dexmedetomidine combination for attempted semen collection using a standardized electroejaculation protocol. Recovered samples were evaluated for the presence of sperm, fluid volume, sperm concentration, motility, and morphology. Testicular dimensions and penile morphology (i.e., presence of spines) were also evaluated.

[0315] Since male and female kittens were group-housed until 1 year of age, some reproductive activity may have occurred in the later months, especially among the control cats. Beginning at 6 months of age, females were evaluated weekly by abdominal ultrasound to confirm pregnancy status. Any observed reproductive activity between cats was recorded.

[0316] result The prepared virus used in this study (previously prepared as described in Example 1.E.) was confirmed to be effective. The mean circulating MIS concentration in kittens was measured at 4 months. In the low dose group, i.e., 5e12vg / kg, the circulating MIS concentration was 12.93μg / ml. In the high dose group, i.e., 1e13vg / kg, the circulating MIS concentration was 18.49μg / ml. In the control group, the circulating MIS concentration was 8.36ng / ml. These values ​​were higher than those measured at the same time point in adult cats, which averaged 3.48μg / ml in the low dose and 15.17μg / ml in the high dose. This strong and stable transgene expression may be due to the continued and proportionally increasing contribution of muscle cells during the development of the kittens (Figure 20). In the controls, as expected, endogenous MIS was high in young kittens, especially males, and slowly declined to low ng / ml levels as males reached sexual maturity. Inhibin B levels in young kittens were expected to reflect this pattern of high MIS expression followed by a decline. However, a significant increase in inhibin B was observed in contrast to MIS. This increase was associated with puberty, and inhibin B levels were maintained in adults. Initial analysis suggested that a slight decline in inhibin B could be observed during the first month after treatment (approximately 7 months of age), especially in control animals, followed by a slight increase until 4 months after treatment (approximately 10 months of age), when kittens are expected to approach puberty (Figures 21A-21B). Ongoing monitoring during puberty would provide information on MIS effects on hormones, especially inhibin B, in female high-dose groups that appeared to have a premature relative induction compared to controls or low doses (Figure 21A).

[0317] Furthermore, no significant production of anti-MIS antibodies was observed in blood samples from kittens given vectors expressing fcMISv2 (Figure 22). In contrast, subject 11WBL24, who received AAV9-fcMISv1 during the first pilot study, showed strong anti-MIS antibody induction over the same time frame (Figure 22).

[0318] The effect of fcMISv2 on female kittens was studied. Fecal P4 levels from female kittens increased gradually over time (Figure 23A-C). This P4 increase may provide information about reproductive maturation. All three kittens in Figure 23A-C, subjects M200586, M200667, and M200756, were littermates and were expected to mature relatively synchronously. The P4 values ​​in prepubertal cats were significantly lower than those observed with ovulation, and the duration was significantly longer than the normal luteal phase. Previous studies have not documented P4 changes in cats going through puberty. This P4 rise may indicate the onset of puberty. Further characterization of the fecal estrogen peak (indicative of estrus) in these same three cats (subjects M200586, M200667, and M200756) may inform whether at least some of these cats are or are not exhibiting reproductive cyclicity. These results and the profile of the remainder of the female cohort are pending.

[0319] Uterine horn measurements were performed in treated female cats after treatment with 5e12vg / kg AAV9-fcMISv2 (low), 1e13vg / kg AAV9-fcMISv2 (high), or 5e12vp / kg AAV9-empty vector (control). A decrease in uterine horn area was observed in cats treated with high or low doses of AAV9-fcMISv2 (Figures 24A-25B), which may indicate contraception in the cats over the time frame of the study. There was no difference in uterine horn area between control and treated cats 6 months after treatment (i.e., when the cats were approximately 9 months old). However, over time, uterine area appeared to increase slightly in control cats, whereas treated cats showed a slight decrease. These results may indicate that control females were actively cycling and possibly ovulating, which led to proliferation and involution of the uterine glands, whereas treated cats did not show these cyclic changes (suggesting ovarian function suppression). The large SEM of the control means indicates that uterine area in controls changed considerably over time, whereas treated females showed little variation in area between individuals. Fecal hormone assays (when completed) should provide more insight into the relationship between uterine area change and ovarian hormone profiles.

[0320] The effect of fcMISv2 on male kittens was also studied. Both low and high doses of AAV9-fcMISv2 (i.e., 5e12vg / kg and 1e13vg / kg, respectively) did not significantly reduce sperm counts in male kittens, subjects 21LRS71 and 21LRS72, by 8 months of age, and these parameters continued to improve as sexual maturation was completed (Table 12). Furthermore, sperm from male kittens were fully functional when evaluated in an IVM / IVF trial with domestic cat oocytes (Table 13). In contrast, the control male cat (subject 21LRS73) appeared to mature correctly by 5 months of age, with the largest testes of the three males, a fully formed preputial cavity, and characteristic penile spine (indicative of testosterone production), but did not produce meaningful numbers of sperm. Insufficient motile sperm were collected from subject 21LRS73 to assess fertility in vitro. The cause of infertility in subject 21LRS73 was unknown but was not assumed to be a result of AAV9 treatment.

[0321] Sperm count was not adversely affected by AAV9-fcMISv2 treatment in subjects 21LRS71 and 21LRS72. In fact, sperm count and sperm concentration were higher than those observed in control subject 21LRS73 (Table 13). These increases in AAV9-fcMIMSv2-treated cats were unexpected. The data disclosed in this example support the use of AAV9-fcMISv2 in scenarios where increased sperm count and / or sperm concentration may be desirable, such as sperm collection methods for artificial insemination of endangered or rare animals.

[0322] Table 12. Reproductive maturation of male kittens following prepubertal treatment with AAV9-fcMISv2 gene therapy TIFF2024511413000032.tif197159TIFF2024511413000033.tif220158

[0323] Table 13. In vitro oocyte maturation / fertilization to assess sperm function in male kittens prepubertally treated with AAV9-fcMISv2 TIFF2024511413000034.tif90158

[0324] H. Follow-up breeding studies material and method One or more follow-up breeding studies are contemplated. For example, if there are no signs of cycling cats by 10 months of age and / or if females do not breed and / or become pregnant during the time periods set forth above in this example, subsequent breeding trials may be warranted using certified breeder males.

[0325] In general, materials and methods for this breeding study were as described in Example 1.E. above, with the following modifications. In this breeding study, a certified breeder male cat, subject 18IDG51, was moved into a group room with six AAV9-fcMISv2 research females (one control, five AAV-MIS treated). The cats were obtained from Marshall BioResources. A second certified breeder male, subject 17CCW45, was moved into a second room with three AAV9-fcMISv2 females (one control, two treated) born in CREW (sirened by subject 18IDG51). In the week leading up to the start of the breeding trial, both males were exposed to the females in a controlled manner (housed in cat carriers) in their respective rooms to facilitate integration. Each male was then housed with each female for 8 hours per day (8am-4pm), five days per week (Monday-Friday) for four consecutive months.

[0326] After the cats were first introduced on the first day, the cat handler remained in the room for 10 minutes to monitor the cat interactions and ensure that no excessive aggression occurred between the male and female. The handler performed additional monitoring (approximately 5 minutes) through the window of the cat room after leaving the room, and then periodically (every 30-60 minutes) throughout the first day and the remainder of the week. Some aggression was expected between the cats during the initial direct exposure, including minor physical trauma (e.g., scratches) toward either the male or female. Safe refuge spaces (e.g., cat carriers, cat condos, and shelves without doors) were available within the room to allow the cats to escape from any aggressors. Significant and / or continued fighting responses required intervention by the handler and physical isolation of the cat. The handler assessed the cats daily for any evidence of trauma, and veterinary staff provided medical treatment as needed. The cat handler used a remote baby monitor to listen for vocalizations (related to mating activity or aggression) from the cat room throughout the day while working elsewhere in the cat colony. Two video cameras in each cat room recorded animal interactions continuously throughout the day (8am-4pm). All video footage was reviewed by CREW volunteers to identify any potential reproductive activity. These observations served to document occurrences of reproductive activity that did not result in ovulation and / or conception, as well as to verify expected birth data for any pregnant cats. Any reproductive activity observed by cat caretakers was recorded in a diary, noting the identity of the female and the time for later video review.

[0327] Each female was evaluated weekly by abdominal palpation and ultrasound to confirm pregnancy status (i.e., the presence and viability of the fetus). All females were pre-operant conditioned to spontaneously undergo these procedures with minimal restraint or disturbance.

[0328] All pregnant females were re-evaluated by ultrasound every 3 weeks to monitor fetal development and survival. Females remained group-housed until approximately day 50 of gestation and were then transferred to a birthing room with individual caging for later natural parturition (typically approximately days 63-65 after breeding). Pregnant females were monitored daily in person and remotely via an internet-accessible video camera linkage until the expected time of parturition.

[0329] Pregnant females were monitored daily by caretakers in person (i.e., 8 am–4 pm) and then continuously by CREW volunteer observers until the expected time of parturition (i.e., 4 pm–8 pm) via an internet-accessible video camera linkage. Caretakers and veterinary staff were notified when any female went into labour. If difficult labour occurred, kittens were delivered by Caesarean section at the discretion of the designated veterinarian. Kittens were given an initial physical examination within 24 hours of birth, weighed daily (for the first month after parturition, then weekly) to monitor growth, and provided supportive care as needed.

[0330] Any stillborn kittens or dead neonates underwent a complete necropsy, including sex assessment and reproductive anatomy. The entire reproductive system was harvested and fixed for later histological evaluation to assess possible MIS effects on intrauterine reproductive system development.

[0331] Viable kittens were preferentially raised by the queen until weaning (typically at about 8 weeks of age). Kittens were hand-reared as necessary in certain cases, e.g., abandonment, aggression, or overgrooming by the dam, or were raised by other queens.

[0332] After weaning, kittens may be adopted as companion animals or may remain in the CREW colony for future studies. If viable female kittens were born to AAV9-fcMISv2-treated females, these kittens were evaluated for fertility until puberty, i.e., 7-8 months of age. Blood samples were collected within 12 hours of birth from kittens born to treated females to assess serum MIS levels. If AAV9-fcMISV2K-treated females did not become pregnant from natural breeding, fertility status was evaluated to determine whether these treated females were effectively spayed.

[0333] During the breeding trial, excreted fecal samples were collected three times a week from each female for hormone monitoring. As described above in Example 1.D. and Example 3.A., dyes / cat-specific food colors and / or glitter were mixed with cat food and fed separately to each cat to facilitate identification of cat-specific feces. Similarly, blood samples (minimum volume of 1 ml each) were collected monthly from each female to assess serum MIS concentrations. To minimize handling discomfort, blood samples (≧1 ml) were collected by medial saphenous or cephalic vein puncture after sedation with low-dose combinations of ketamine, dexmedetomidine, and / or butorphanol combinations and partial reversal with atipamezole. For collection of small blood volumes (<1 ml) and / or collection from pregnant females, blood samples were collected by venipuncture using only manual restraint (i.e., cats were immobilized in nylon holding bags). The procedure was stopped if the cat appeared to be stressed by it.

[0334] Example 4 - Efficacy of AAV-wt canine MIS treatment to prevent pups from entering puberty and to provide long-term sterility I. Preventing puberty in dogs (puppies) The ability of MIS treatment by gene delivery to block puberty in dogs (puppies) and any other related developmental effects are evaluated.Furthermore, the parameters for using canine MIS when given as a single injection (e.g., by gene delivery) to reduce fertility in puppies long term and / or to block puppies from entering puberty are evaluated.For example, this can also serve as a non-human animal model for evaluating MIS treatment via gene delivery or MIS protein delivery to delay puberty in human subjects.

[0335] The timing of puppy injections with AAV-wt canine MIS will depend on the timing of parturition and weaning. Healthy puppies will be weaned by 8 weeks of age. Healthy puppies approximately 3 months of age (e.g., 10-12 weeks of age) will be randomized into control and treatment groups. Puppies will be injected im into the caudal thigh muscle with AAV-wt canine MIS or empty vector control as follows: (1) high dose AAV-wt canine MIS (1e13vg / kg); (2) low dose AAV-wt canine MIS (5e12vg / kg); and (3) empty vector control (5e12vp / kg). Table 14 shows the sampling and monitoring schedule for puppies.

[0336] Table 14: Puppy monitoring schedule TIFF2024511413000035.tif23487

[0337] Puppies are housed in cages for 5 days (2-3 puppies from the same treatment group / cage) and then transferred to single-group enclosures for further monitoring. During this period, pooled fecal and urine samples and individual oral swabs are collected daily from group-housed puppies to assess viral shedding. qPCR of viral genomes in individual blood, mixed urine, mixed feces, and individual oral swabs are monitored to analyze viral shedding. Blood is sampled 7 days after injection, weekly for the remainder of the month, and monthly after the first month. Urine, feces, and oral swabs are collected daily for the first week.

[0338] Food dye is mixed with dog food and fed separately (three times a week) to each individual pup starting two weeks prior to injection and continuing for, for example, nine months (e.g., approximately one year of age) after injection. Since puppies from the same treatment group may be housed together, fecal samples are evaluated for the presence of estrogen and progesterone metabolites (females) or testosterone metabolites (males) to ascertain the onset of puberty and reproductive maturation. The weights of the puppies are measured weekly starting two weeks prior to injection until the end of the study period.

[0339] Whole blood samples (1 ml, minimum) will be collected from each puppy (1X / month) beginning immediately prior to injection to assess serum MIS, inhibin B, anti-MIS antibodies, and LH concentrations. Additional blood samples will be collected on days 7, 14, 21, and 28 post-injection for both treated and control puppies.

[0340] Safety monitoring will include daily assessments of general health and regular evaluations of the injection sites (daily for 14 days, weekly for the second month, then monthly thereafter). Physical examinations and CBC / biochemistry evaluations will be performed immediately prior to injection and then every 3 months until the end of the study period.

[0341] Puppies should be evaluated for signs of behavioral estrus using video and direct observation. If there are no signs of cyclic dogs, a follow-up breeding study may be considered.

[0342] Fecal hormone analysis in females provides evidence of puberty by showing ovarian cyclicity based on a gradual increase in basal estrogen over time and the occurrence of estrogen spikes that coincide with estrus. Young females typically do not ovulate naturally, and therefore fecal progesterone may not be informative, although female pups may appear to acquire the ability to ovulate naturally as they age, and therefore may regain the luteal phase toward the end of the study period.

[0343] In males, a rise in fecal testosterone is an indicator of puberty, demonstrated by the presence of penile spines and sperm production. Every 3 months after treatment, male pups are anesthetized with a ketamine-dexmedetomidine combination for attempted semen collection using a standardized electroejaculation protocol. Retrieved samples are evaluated for the presence of sperm, fluid volume, sperm concentration, motility, and morphology. Testicular dimensions and penile morphology (i.e., presence of spines) are also evaluated.

[0344] Since male and female pups will be group-housed until the end of the study period, some reproductive activity may occur during the later months, especially among the control dogs. During the later months, females will be evaluated weekly by abdominal ultrasound to confirm pregnancy status. Any observed reproductive activity between dogs will be recorded.

[0345] J. Follow-up Reproduction Studies One or more follow-up breeding studies may be considered. For example, if there is no sign of cycling dogs and / or if females do not breed and / or become pregnant during the study period set forth above in this example, subsequent breeding trials may be warranted using certified breeder males.

[0346] In these follow-up breeding studies, certified breeder male dogs are transferred to a group-housing room with AAV-wt canine MIS research females. To facilitate integration, males are pre-exposed to the females (housed in dog carriers) in a controlled manner in the room. Males are housed with the females for 8 h / day, 5 days / week for 4 consecutive months. Reproductive activity is documented by a combination of direct observation and remote audio / video monitoring. Each female is evaluated weekly by abdominal palpation and ultrasound to confirm pregnancy status (i.e., presence and viability of fetuses). All females were pre-operant conditioned to willingly accept these procedures with minimal restraint or disturbance.

[0347] Any pregnant females will be re-evaluated by ultrasound every 3 weeks to monitor fetal development and survival. Females will remain group-housed until suitable for transfer to a birthing room with individual caging for later natural birth. Pregnant females will be monitored daily in person and remotely via an internet-accessible video camera linkage until the expected time of birth.

[0348] Example 5 - Efficacy of recombinant hMIS protein treatment to delay the onset of puberty in human subjects To demonstrate that MIS treatment can delay puberty, by way of example, Examples 2 and 3 evaluate puberty delay in cats (kittens) and dogs (kittens) as exemplary non-human animals.

[0349] Example 4 evaluates the ability of recombinant human MIS protein produced from the proprotein of SEQ ID NO:7 to reversibly delay puberty in human female subjects. That is, administration of LR-MIS protein in delaying puberty in female subjects was evaluated and found to be reversible. hMIS protein can be administered to prepubertal human female subjects at an age when puberty has not yet begun, or between about 8 and 16 years of age.

[0350] Human rhMIS protein, e.g., LR-MIS of SEQ ID NO:7, was produced according to the methods disclosed in US Application US20200071376, which is incorporated herein in its entirety. The availability of biologically active rhMIS protein that can be produced and purified to high yields using CHO cells allows for high dose and long-term in vivo dosing that was previously not feasible with the insufficiently truncated wild-type protein, nor with the commercially available C-terminal recombinant MIS protein, which was found to be inactive. For example, we have previously demonstrated that incubation of fetal (E14.5) female rat urogenital ridges in ex vivo culture with 5 μg / ml rhMIS for 72 hours resulted in near complete degeneration of the Müllerian duct, whereas the R&D Systems (Minneapolis, Minn.) C-terminal MIS has no observable activity during the Müllerian duct bioassay, which is the gold standard for testing hormone potency and specificity.

[0351] Treatment of mice with rhMIS protein results in reversible ovarian quiescence.

[0352] rhMIS protein can be administered subcutaneously (sc), intravenously (iv), or intraperitoneally (ip), each resulting in a half-life of approximately 4 hours, reaching peak concentrations (Cmax) at 4 hours, 30 minutes, and 2 hours, respectively. The preferred delivery route for rhMIS protein was subcutaneous, as subcutaneous absorption kinetics were most favorable. However, when using osmotic pumps, intraperitoneal implantation was found to be optimal, producing consistent delivery for up to one week (see, e.g., Figure 1F of US2020 / 0071376). rhMIS activity was remarkably stable, with material recovered from pumps implanted in mice for one week retaining full biological activity in a rat urogenital ridge bioassay (data not shown).

[0353] Administration of rhMIS to the female subject may be via any of the means disclosed in US Application US20200071376, which is incorporated herein in its entirety. In some cases, administration may be via a pump (e.g., an osmotic pump) or a transdermal patch, or the like.

[0354] To confirm that rhMIS protein can inhibit primordial follicles with the goal of elucidating the kinetics of ovarian reawakening, the effects of transient rhMIS protein treatment are measured in prepubertal female mice, a representative animal model of prepubertal female humans. rhMIS protein is administered sc twice daily (every 12 hours) at 1.5 mg / kg, which in silico pharmacokinetic modeling predicted would maintain circulating rhMIS levels above the 0.25 μg / ml target level. Actual circulating rhMIS levels can be measured by ELISA 12 hours after injection, which is the trough level, and were maintained above the 0.25 μg / ml target threshold, although concentrations gradually decreased over the course of 35 days of treatment. Ovaries from treated mice can be evaluated, and significant size reduction is assessed by analysis of representative midsections after 35 days of treatment. Inhibition of follicular development is evidenced by the presence of fewer primary follicles and no secondary or antral follicles in the ovaries. To ensure that the ovaries have been released from quiescence and folliculogenesis has resumed, the ovaries are evaluated after rhMIS protein treatment is discontinued. Ovarian volume in mice can be evaluated over a period of 15 days. From the increase in size, it can be seen that primary follicles gradually increase in size from day 3 to day 10, secondary follicles begin to appear on day 5 and increase to levels similar to controls by day 15, at which time some antral follicles can be observed.

[0355] To test the efficacy of rhMIS protein to reversibly delay puberty, we chose to use osmotic pumps implanted ip in C57BL / 6N female mice. This allows for very precise delivery of rhMIS (see US20200071376, Figure 1F). In this model, osmotic pumps filled with 100 μl of 1200 μg / ml rhMIS solution diluted in saline, or control pumps filled with saline, were implanted. Pumps were replaced every 7 or 5 days (see, for example, US20200071376, Figure 5E). After 2 weeks, mice were sacrificed, ovaries were harvested, serially sectioned, and follicle numbers were counted. A significantly higher ovarian reserve was observed in mice implanted with rhMIS-eluting pumps compared to controls with saline pumps. Treatment with hrMIS did not significantly affect primordial or growing follicles within this short period of time. However, there was a trend towards fewer growing follicles compared with saline-only controls.

[0356] One might envisage the use of low-dose rhMIS to slow down but not completely stop the activation of primordial follicles, thus reversibly delaying the onset of puberty.

[0357] In conclusion, it has been demonstrated herein that the efficacy of high doses of AAV-wt feline MIS or AAV-wt canine MIS can irreversibly block feline and canine puberty, respectively, in Examples 1-3, and that administration of rhMIS can reversibly delay the onset of puberty in a mouse animal model in Example 4. This demonstrates that rhMIS is a potent inhibitor of primordial follicle activation.

Claims

1. A method for reducing fertility in a prepubertal non-human subject, comprising administering to the subject an effective amount of a composition comprising a vector comprising a nucleic acid encoding a Mullerian inhibitory factor (MIS) protein, operably linked to one or more regulatory elements.

2. The method of claim 1, wherein the prepubertal non-human subject is a kitten or a puppy.

3. 2. The method of any one of the preceding claims, wherein the prepubertal non-human subject is female.

4. The MIS protein a) the wild-type feline MIS protein, the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:18, or an amino acid sequence having at least 80%, at least 85%, at least 90%, 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 or SEQ ID NO:18; b) a wild-type canine MIS protein, an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, 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:2; c) a feline MIS protein comprising a non-MIS leader sequence instead of amino acids 1 to 21 of SEQ ID NO:1 or amino acids 1 to 21 of SEQ ID NO:18 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22 to 588 of SEQ ID NO:1 or amino acids 22 to 589 of SEQ ID NO:18, optionally wherein the non-MIS leader sequence comprises amino acids 1 to 18 of SEQ ID NO:14, or d) a modified feline MIS protein (LR-fcMIS) comprising the amino acids of SEQ ID NO:14 or SEQ ID NO:20 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 22-588 of SEQ ID NO:14 or amino acids 22-589 of SEQ ID NO:20; 2. The method of any one of the preceding claims, comprising:

5. 2. The method of any one of the preceding claims, wherein the prepubertal non-human subject is a kitten under 12 months of age, under 11 months of age, under 10 months of age, under 9 months of age, under 8 months of age, under 7 months of age, under 6 months of age, under 5 months of age, under 4 months of age, under 3 months of age, or under 2 months of age.

6. 2. The method of any one of the preceding claims, wherein the prepubertal non-human subject is a kitten weighing 2 kg or less.

7. 20. The method of any one of the preceding claims, wherein the prepubertal non-human subject is a puppy up to 24 months old, up to 22 months old, up to 20 months old, up to 18 months old, up to 16 months old, up to 14 months old, up to 12 months old, up to 11 months old, up to 10 months old, up to 9 months old, up to 8 months old, up to 7 months old, up to 6 months old, up to 5 months old, up to 4 months old, up to 3 months old, or up to 2 months old.

8. 20. The method of any one of the preceding claims, wherein the prepubertal non-human subject is weaned.

9. The method of any one of claims 1 to 7, wherein the prepubertal non-human subject has not been weaned.

10. The method of any one of the preceding claims, wherein the vector is a viral vector, a plasmid, a cosmid, or a phagemid.

11. The method of any one of the preceding claims, wherein the vector is a viral vector.

12. The method of any one of the preceding claims, wherein the composition further comprises a cell comprising the vector.

13. The method of any one of the preceding claims, wherein the composition comprises a sterile injectable solution.

14. The method of any one of the preceding claims, wherein the composition comprises a sterile injectable aqueous solution.

15. 2. The method of any one of the preceding claims, wherein the composition comprises a lipid, a lipid emulsion, a liposome, a nanoparticle, or an exosome.

16. 2. The method of any one of the preceding claims, wherein the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a poxviral vector, or a lentiviral vector.

17. The method of any one of the preceding claims, wherein the vector is an AAV vector.

18. The method of any one of the preceding claims, wherein the vector is an AAV9 vector.

19. 3. The method of any one of the preceding claims, wherein the one or more regulatory elements comprises a promoter element.

20. 3. The method of any one of the preceding claims, wherein the one or more regulatory elements comprise a promoter element and an enhancer element.

21. 3. The method of any one of the preceding claims, wherein the one or more regulatory elements comprises a constitutively active promoter.

22. The method of any one of the preceding claims, wherein the composition comprises a pharma- ceutically acceptable carrier.

23. 20. The method of any one of the preceding claims, wherein the administration is via injection.

24. 20. The method of any one of the preceding claims, wherein the administration is via intravenous, subcutaneous, or intramuscular administration.

25. 20. The method of any one of the preceding claims, wherein the administration is via intramuscular administration.

26. 20. The method of any one of the preceding claims, wherein the administration is via a single injection.

27. 20. The method of any one of the preceding claims, wherein said administration is via a one-time single injection.

28. 2. The method of any one of the preceding claims, wherein the administration is via a single dose divided into multiple injections.

29. 20. The method of any one of the preceding claims, wherein the administration is via a single dose divided into two injections.

30. The effective amount of the composition administered to a prepubertal non-human subject is about 1×10 per kilogram of the subject's body weight. 13 Vector genome: 5×10 12 Vector genome: 1×10 12 Vector genome or less, 5 × 10 11 vector genome or less, or 1 × 10 11 2. The method of claim 1 , wherein the vector genome is:

31. 2. The method of any one of the preceding claims, wherein the concentration of MIS protein in serum of the prepubertal non-human subject 6 or 6 months, 9 or 9 months, 12 or 12 months, 15 or 15 months, or 24 or 24 months after administration of the composition is greater than 250 ng / ml, greater than 300 ng / ml, greater than 400 ng / ml, greater than 500 ng / ml, greater than 600 ng / ml, greater than 700 ng / ml, greater than 800 ng / ml, greater than 900 ng / ml, greater than 1 μg / ml, greater than 1.5 μg / ml, greater than 2 μg / ml, greater than 3 μg / ml, greater than 4 μg / ml, greater than 5 μg / ml, greater than 6 μg / ml, greater than 7 μg / ml, greater than 8 μg / ml, greater than 9 μg / ml, greater than 10 μg / ml, or greater than 11 μg / ml.

32. 32. The method of claim 31, wherein the MIS protein concentration is measured by ELISA.

33. and wherein the prepubertal non-human subject is female, and following administration of the composition, a) It prevents follicles from developing, b) failure to develop follicles containing viable eggs; and / or c) being infertile; 10. The method according to any one of the preceding claims.

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