WO2011038469A1

WO2011038469A1 - Suppressive agents

Info

Publication number: WO2011038469A1
Application number: PCT/AU2010/001299
Authority: WO
Inventors: Jock Findlay; Jeffrey Kerr; Karla Hutt; Clare Scott; Andreas Strasser; Ewa Michalak
Original assignee: Prince Henry's Institute Of Medical Research; The Walter And Eliza Hall Institute Of Medical Research; Monash University
Priority date: 2009-10-01
Filing date: 2010-10-01
Publication date: 2011-04-07

Abstract

Methods of conserving fertility in a female subject are provided, comprising administering to a female or to oocytes ex vivo an agent which (i) suppresses PUMA expression or PUMA polypeptide activity; (ii) suppress PUMA and NOXA expression or PUMA and NOXA polypeptide activity; (iii) NOXA expression or NOXA polypeptide activity; or (iv) suppresses TP63 expression or TP63 activity. In some embodiments, the female is or will be under cancer treatment; is diagnosis as perimenopausal or menopausal or at risk of developing same within a predetermined time span; or is undergoing IVF. In some embodiments, the agent binds to PUMA, NOXA, or TP63 nucleic acid and suppress PUMA, NOXA or TP63 expression. In other embodiments the agents bind to PUMA, NOXA or TP63 and suppress PUMA, NOXA or TP63 polypeptide activity.

Description

SUPPRESSIVE AGENTS

FIELD The present invention relates generally to the field of therapeutic agents. More particularly, the specification relates to apoptosis induced by agents that promote cellular DNA damage. In some embodiments, the present invention provides therapeutic or prophylactic agents and methods for conserving fertility in a female subject undergoing cancer treatment. BACKGROUND

Bibliographic details of the publications referred to in this specification are also collected at the end of the description. Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country.

The development of small therapeutic agents is a major goal in the pharmaceutical industry. Such agents are potentially relatively inexpensive to manufacture and are less likely to induce adverse immunological responses. One of the difficulties, however, in small therapeutic molecule development is target selection. Many potential targets lack suitability due to their pleiotropic nature and/or due to the level of redundancy in a particular pathway.

Primordial follicles are structures which house immature oocytes, containing the female germ line, arrested in meiotic prophase I. In mammals, the population of primordial follicles is established during foetal and/or early neonatal development, and it is from this original pool of germ cells that all eggs for ovulation are eventually drawn. In the case of female primates, a primordial follicle may exist in stasis for a period of 40 years or more, before being selected to resume meiosis and enter the developmental pathway that ultimately culminates in the ovulation of a mature egg. Extreme longevity, combined with the unusual diplotene state of their nucleus, may make primordial follicle oocytes especially vulnerable to DNA damage. Therefore, to ensure that mutations are not, introduced into the germ line, oocyte genomic integrity is under constant surveillance with detection and repair of DNA damage or elimination, through apoptosis, of those primordial follicles with compromised genomic fidelity. p63 is a member of the p53 transcription factor family that shares significant homology with p53 and has been described as the "Guardian of the Germ-line". p63 mediates a special surveillance system in primordial follicles which rapidly detects and eliminates oocytes by apoptosis in response to DNA damage. Phosphorylation of the p63 isoform, TAp63 (also referred to here as Trp63) is essential for death of oocytes following DNA damage (Suh et al, Nature, 444{Ί\ \9): 624-628, 2006; Livera et al, Reproduction, 735(1): 3-12, 2008). The p63 gene encodes two major isoform subsets, TAp63 (ΤΡ63)-α, β and γ, which have transactivating activity and are pro-apoptotic, and deltaNp63-ct, β and γ (N-terminal truncated forms) which act as dominant negative transcriptional repressors, negatively regulating apoptosis, for example, by competing for TAp63 target genes.

Two distinct mammalian apoptotic pathways exist - "death receptor" ("extrinsic") and "Bcl-2-regulated ("mitochondrial" or "intrinsic") apoptosis signaling, each activating different initiator caspases but converging at the level of effector caspases (reviewed in Adams and Cory, Oncogene, 26: 1324-1337, 2007). The "Bcl-2-regulated" apoptotic pathway is regulated by three sub-groups of proteins from the Bcl-2 family: (i) pro-survival members, such as Bcl-2, that are essential for cell survival; (ii) pro-apoptotic Bax/Bak proteins that are critical for activation of the downstream events in cell demolition; and (iii) pro-apoptotic BH3-only proteins that are required for initiation of apoptosis signalling (reviewed in Happo et al., on-line Encyclopedia of Life Sciences. In Press. Accepted March 18 2009). Certain BH3-only proteins have been found to preferentially bind to particular pro-survival Bcl-2-like proteins, with implications for requirement of BH3-only proteins required to "neutralise" the pro-survival members, thus regulating the decision of life versus death. Bim and Puma are the most "potent" BH3-onIy genes, capable of binding to and sequestering all pro-survival members present. p63, a transcription factor related to p53 the "Guardian of the Genome", is essential for craniofacial, skin and limb development (Yang et al, Nature, 398: 714, 1999). The p63 gene encodes two major isoform subsets: ΤΑρ63-α, β, γ, which have trans-activating activity, and deltaNp63-a, β, γ (N-terminal truncated), which can exert dominant-negative effects as transcriptional repressors (Yang et al, Mol. Cell, 2: 305, 1998). p53 exerts its tumor suppressor function in part through activation of apoptosis requiring transcriptional induction of the BH3-only Bcl-2 family members Puma and Noxa (Villunger et al, Science, 302(5647): 1036-1038, 2003; Jeffers et al, Cancer Cell, 4: 321, 2003). Although p63 over-expression triggers apoptosis in immortalized cell lines (Patel et al., Nucleic Acids Res., 5(5(16): 5139-5151, 2008, Bergamaschi et al, Mol Cell Biol., 24(3): 1341-1350, 2004, Khokhar et al, Cell Res., 18(10): 1061-1073, 2008), how p63 induces apoptosis under physiological conditions remains unclear. No mouse deletion models have as yet been reported to confirm downstream involvement of specific putative p63 targets, in part due to the complexity of the p53 family member interplay in response to DNA damage. Thus, it is not known whether or not Puma or Noxa have critical roles in apoptosis in oocytes as a result of p63 activation.

The treatments for cancer have improved dramatically in the last twenty years and it is estimated, for example, that more than 80% of children diagnosed with cancer are now surviving into adulthood. Further, it is anticipated that by 2010 one in 250 adults will be a survivor of a childhood cancer. Cancer treatments such as chemotherapy, radiation and surgery are all associated with male or female infertility or the early onset of menopause. There are currently no treatments or prophylactic protocols for sterility and the only option for subjects is to bank or store reproductive cells. It has been reported that the greatest concern of many cancer survivors is their ability to have children, i.e. their fertility. There is a need in the art to identify therapeutic or prophylactic protocols for conserving reproductive cells and enhancing fertility in subjects in need thereof. SUMMARY

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an agent" includes one agent, as well as two or more agents; and so forth.

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO: 1), <400>2 (SEQ ID NO: 2), etc. A summary of sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.

In some embodiments, the present invention provides a method of conserving fertility in a female subject, the method comprising administering to the female a composition comprising an agent which suppresses PUMA expression or PUMA polypeptide activity.

As shown in the Examples, mice lacking puma are resistant to the effects of radiation on enhancing apoptosis of ovarian follicles. In some embodiments, the female subject is undergoing or will undergo cancer treatment. Accordingly, the present methods may be used prophylactically or therapeutically.

Administration to the female includes administering to cells or tissues of the female, including oocytes of the female or oocytes or tissues comprising same to be implanted into the female. It is contemplated that in some embodiments the agent binds to PUMA nucleic acid and suppresses PUMA expression. Genes and other genetic material (e.g. isolated and in situ DNAs and RNAs, constructs etc) are represented in italics and their proteinaceous products are represented in non-italicized form. Thus, PUMA polypeptide is the product of the PUMA gene. The term "PUMA" or "PUMA" is used to encompass all homologs, isoforms and variants in any species and in some embodiments, mammalian, including human forms. These definitions are used mutatis mutandis for NOXA and TP63. As used in the description, reference to genes and proteins in upper case indicates that the products are derived from any species.

Accordingly, in some embodiments, the agent comprises or encodes an antisense, siRNA, shR A, miRNA, ribozyme, DNAzyme or other nucleic acid based moiety that suppresses PUMA gene expression. Alternatively, it is contemplated that the agent binds to PUMA polypeptide or a PUMA-binding agent and suppresses PUMA activity.

In an illustrative embodiment, the agent comprises a small inhibitory molecule. Thus, the present invention provides a PUMA antagonist for use in conserving fertility in a female subject in need thereof.

As further shown in the Examples, conservation was enhanced in mice lacking puma and noxa. Accordingly, in another aspect the present invention provides a method of conserving fertility in a female subject, the method comprising administering to the female one or more compositions comprising one or more agents which either alone or together suppress PUMA and NOXA expression or PUMA and NOXA polypeptide activity.

Although the present methods may be used to conserve fertility in any female subject, in a non-essential preferred embodiment, the female is undergoing or will undergo cancer treatment. In some embodiments, the female is diagnosed as perimenopausal or menopausal.

In some embodiments, the female has been diagnosed as at risk of developing perimenopause or menopause within a pre-determined time span or shortly.

In some embodiments, the female is undergoing in vitro fertilisation (IVF) treatment.

In some embodiments, administration to the female is administration ex vivo to oocytes of the female.

In some embodiments, oocytes are contacted ex vivo with a suppressive agent as described herein.

In other embodiments, the agent or agents bind to PUMA and NOXA nucleic acid and suppresses PUMA and NOXA expression.

Conveniently, the agent or agents comprise or encode an antisense, siRNA, shRNA, miRNA, ribozyme, DNAzyme or other nucleic acid based moiety. Alternatively, the agent or agents bind to PUMA and NOXA or a NOXA-binding agent or a PUMA-binding agent and suppress PUMA and NOXA activity.

In some embodiments, the compositions comprise a small inhibitory molecule. In yet another embodiment, it is proposed that in certain subjects, for example those with a low level or activity of PUMA, fertility may be conserved by administration of an agent that suppresses NOXA expression or NOXA activity. Accordingly, the present invention provides a method of conserving fertility in a female subject, the method comprising administering to the female an agent which suppresses NOXA expression or NOXA polypeptide activity. In some embodiments, the female is undergoing or will undergo cancer treatment. In some particular embodiments, the agent binds to NOXA nucleic acid and suppresses NOXA expression. Conveniently, such agents comprise or encode a nucleic acid based moiety such as an antisense, siRNA, shR A, miRNA, ribozyme, DNAzyme moiety.

Alternatively the agent, such as a small inhibitory molecule, binds to NOXA polypeptide or a NOXA-binding agent and suppresses NOXA activity.

PUMA is the transcriptional target of TP63 and, accordingly, in a particular embodiment, the agent reduces the level or antagonises the activity of TP63. In a preferred, non-essential embodiment, isoforms of TP63 which are predominantly expressed in reproductive tissue such as oocytes are preferred targets.

NOXA and PUMA are the transcriptional targets of TP63 and, accordingly, in a particular embodiment, the agent reduces the level or antagonises the activity of TP63. In a preferred, non-essential embodiment, isoforms of TP63 which are predominantly expressed in reproductive tissue such as oocytes are preferred targets.

PUMA and NOXA are the transcriptional target of TP63 and, accordingly, in a particular embodiment, the agent reduces the level or antagonises the activity of TP63. In a preferred, non-essential embodiment, isoforms of TP63 which are predominantly expressed in reproductive tissue such as oocytes are preferred targets.

The prior art suggests that p63 functions to induce apoptosis in oocytes exposed to ionizing radiation and furthermore that this action is part of an essential surveillance system to ensure destruction of damaged oocytes and thus protect the integrity of the female germ- line. However, as shown herein, when oocyte apoptosis prompted by exposure to DNA damaging agents is prevented, DNA repair is sufficient to conserve fertility, allowing production of healthy offspring. Accordingly, in another related aspect, the present invention provides a method of conserving fertility in a female subject, the method comprising administering to the female an agent which suppresses TP63 expression or 10 001299

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TP63 activity. In some embodiments, the female subject undergoes cancer treatment.

In some particular embodiments, the agent binds to TP63 nucleic acid and directly suppresses TP63 expression.

Suitable agents comprises or encode an antisense, siRNA, shR A, miRNA, ribozyme, DNAzyme or other nucleic acid based moiety.

Alternatively, the polypeptide is targeted and the agent binds to TP63 polypeptide or a TP63 -binding agent and suppresses TP63 activity. In some embodiments, the agent comprises a small inhibitory molecule.

To facilitate selective effects, in some embodiments, the agent binds to a complex comprising the TP63 protein bound to regulatory sequences within the PUMA gene and suppresses the activity of the complex.

To facilitate selective effects, in some embodiments, the agent binds to a complex comprising the TP63 protein bound to regulatory sequences within the NOXA gene and suppresses the activity of the complex.

To facilitate selective effects, in some embodiments, the agent binds to a complex comprising the TP63 protein bound to regulatory sequences within the PUMA gene or the TP63 protein bound to regulatory sequences within the NOXA gene and suppresses the activity of these complexes.

In some further embodiments, the TP63 isoform is preferentially expressed in oocytes in the subject. Reference to TP63 includes variant forms of TP63 such as α, β or γ-forms of TP63.

In some embodiments, the TP63 is the a isoform of TP63 or an isoform expressed in oocytes in the subject.

In some embodiments, the agent is an antagonist of PUMA expression, such as a truncated form of TP63 or a variant thereof that binds to PUMA and suppresses PUMA expression. Illustrative PUMA binding agents may be derived from the Bcl-2 family as known in the art.

In accordance with some aspects of the invention, suppression of the level or activity of TP63 or PUMA or NOXA or PUMA and NOXA is proposed to conserve or enhance fertility during or after their exposure to an agent or stimulus that is sufficient to induce apoptosis in ovarian follicles. In accordance with some exemplified aspects of the invention, inhibition or reduction of the level or activity of one or more of these targets is proposed to conserve or enhance female fertility by conserving oocytes in the presence or context of an apoptotic stimulus to oocytes. In some embodiments, partial suppression of apoptosis is preferred in order to maximise the survival of follicles requiring the least DNA or other repair. In some embodiments, this is achieved by targeting only one pro-apoptotic target such as PUMA alone. Thus, in some embodiments, PUMA expression or PUMA activity alone is suppressed. Reference herein to "fertility" includes the ability of a subject to have healthy offspring. Prior to the present invention, it was not known that TAp63 or PUMA , or PUMA and NOXA, can be depleted in subjects to conserve fertility in the face of sterilizing doses of radiation and that subjects would have viable/normal offspring. The term "fertility" includes the ability to conceive and or to reproduce, the ability of a female to carry a baby to term, or the ability of a female subject to have normal or healthy offspring. In some embodiments, enhancing or conserving female fertility includes delaying menopause. In some embodiments, enhancing or conserving female fertility further includes modulating folliculogenesis, oocyte maturation or ovulation.

In some embodiments, the present invention provides a TP63 or PUMA or NOXA suppressive agent for use in conserving fertility or the reproductive ability of a subject. In some embodiments, the subject is undergoing or scheduled for a cancer treatment associated with loss of fertility in some patients.

In a preferred but non-essential embodiment, the targeted TP63 isoform is expressed predominantly in reproductive cells or reproductive tissue. In this way, suppression of TP63 facilitates oocyte survival while reducing any unwanted survival effects on cancerous cells or particular cells that are not reproductive cells.

The suppressive agents and medicaments contemplated for use in the present invention include small chemical molecules, membrane-penetrating polypeptides or peptides, nucleic acid molecules and conjugates or chimeric molecules comprising one or more of these molecules.

Compositions such as pharmaceutical compositions and kits comprising same useful in conserving fertility are also contemplated herein.

The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Some figures contain colour representations or entities. Coloured versions of the figures are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

Figure 1 is a photographic representation of the expression of Puma mR A and PUMA protein in primordial follicle oocytes following γ-irradiation induced DNA damage. Ovaries were harvested from PN5 wt and Puma^'1' (negative control) mice at 0 (untreated control) and 6 h post whole-body γ-irradiation (0.45 Gy). In situ hybridization (top panel) was performed using an anti-sense probe for Puma. Puma mRNA was detected in wt primordial follicle oocytes at 6 h post γ-irradiation. Arrows indicate positively stained primordial follicle oocytes (dark purple/brown staining). Anti-PUMA antibody immunofluorescent staining (bottom panel; green) in wt and Puma^'1' (negative control) primordial follicle oocytes at 6 h post γ-irradiation. Scale bar: 20 m.

Figure 2 is a photographic representation of the expression of Noxa mRNA in primordial follicle oocytes following γ-irradiation induced DNA damage. Ovaries were harvested from PN5 wt and Noxa^'1' (negative control) mice, at 0 (untreated) and 3 h post whole-body γ-irradiation (0.45 Gy). ISH staining was performed using anti-sense probes for Noxa. Arrows indicate positively stained primordial follicle oocytes. Scale bar: 20 m.

Figure 3 is a graphical (A) and photographic (B) representation illustrating loss of PUMA rescues primordial follicle oocytes from DNA damage induced apoptosis. (A) Quantification (means ± SEM) of follicles in wt, Noxa^''^', Puma^''^', Puma^''^"Noxa^''^' and Trp53^~'^' mice that were left untreated or exposed to whole body γ-irradiation (0.45 or 4.5 Gy) at PN5 and analyzed at PN10. Percent primordial follicle survival (compared to untreated) shown above each bar. n=3-8 animals/genotype. For comparison with wt: * p<0.05, ** pO.001. (B) Anti-MSY2 immuno-histochemical staining of ovaries of PN10 wt, Noxa^''^', Puma^''^', Puma^''^" Noxa^''^' and Trp53^''^' mice five days after exposure to 4.5 Gy γ-irradiation or no treatment (control). Arrows indicate intrinsically DNA damage resistant growing (post-primordial) follicles. Black arrow heads indicate primordial follicles. Scale bar: 200 m.

Figure 4 is a photographical (A) and graphical (B and C) representation showing detection of DNA damage in primordial follicle oocytes following γ-irradiation. (A) DNA double- strand breaks were detected in oocytes from untreated wt primordial follicles and 3 h after exposure to whole-body γ-irradiation at the lower (0.45 Gy) and higher (4.5 Gy) doses by immunofluorescent staining with anti- H2AX antibodies (red). Ovaries from Puma '^', Noxa^"'^" and Puma^'1' Noxa^''^' were also analyzed 3 h after low dose (0.45 Gy) γ-irradiation. (B) γ-irradiation-induced DNA damage was quantitated by counting the number of γ-Η2ΑΧ foci within the nucleus (n=50-100 primordial follicles) of wt, Puma^'1', Noxa^'1' and Puma^''^' Noxa^'1' primordial follicles 3 h after exposure to whole-body γ-irradiation (0.45 Gy). No significant difference was observed in the number of foci among genotypes (p>0.05). (C) γ-irradiation-induced DNA damage was quantified by counting the number of γ-Η2ΑΧ foci within the nucleus of wt primordial follicles (n=50-100) 3 h after exposure to whole-body γ-irradiation at the lower (0.45 Gy) and higher (4.5 Gy) dose. ***p<0.001.

Figure 5 is a graphical representation showing fertility of untreated and γ-irradiated wt and Puma^'1' mice. Female mice (PN5) of the indicated genotypes were either left untreated or exposed to whole-body γ-irradiation (0.45 Gy), as described above and after at least 7 weeks of age (i.e. 45 days post γ-irradiation) breeding trials were commenced. Data shown: 1st and 2nd litters from matings to non-irradiated proven males (wt C57BL/6); litters were inspected at 0800 on the day of birth and fostered {Puma^'1' litters only) then inspected twice weekly and at weaning (1st litters only from wt mice). Untreated wt: n=6 litters (6 mothers, 26 healthy pups at weaning); untreated Puma^'1": 6 litters (4 mothers, 26 pups); untreated Puma^'1' Noxa^'1': 12 litters (6 mothers, 31 pups); γ-irradiated wt: 0 (5 mothers, 0 pups); -irradiated Puma^"'^': 13 litters, (8 mothers, 65 pups); γ-irradiated Puma^"'^" Noxa^"'^": 11 litters (7 mothers, 38 pups). ** p value <0.001 for Puma^"'^" γ-irradiated vs wt γ-irradiated or Puma^"1" Noxa^'1" -irradiated vs wt γ-irradiated, for all categories. All other comparisons NS. 2nd litters from Puma^'1' females were included to show protection was not limited to 1st litter. Analysis of first litters only was very similar (p=0.9). Figure 6 is a photographical representation illustrating detection of DNA damage in primordial follicle oocytes following γ-irradiation. Expression of Puma mRNA in primordial follicle oocytes following γ-irradiation induced DNA damage. Ovaries were harvested from PN5 wt and Puma^'1' (negative control) mice at 0 (untreated control), 3 and 6 h post whole-body γ-irradiation. (A) In situ hybridization (anti-sense probe: dark purple/brown staining; sense probe: negative control) indicates Puma mRNA in wt primordial follicle oocytes at 3 and 6 h post γ-irradiation (0.45 and 4.5 Gy). Arrows indicate positively stained primordial follicle oocytes. (B) Negative control for ISH mediated detection of Puma mRNA using Puma^'1' primordial follicles at 3 and 6 h post γ-irradiation (0.45 Gy). Scale bar: 20 μιη.

Figure 7 is a photographical representation illustrating expression of noxa mRNA in primordial follicle oocytes following γ-irradiation induced DNA damage. Expression of Noxa mRNA in primordial follicle oocytes following γ-irradiation-induced DNA damage.

(A) Ovaries were harvested from PN5 wt and Puma^'1" mice, at 0 (untreated) and 3 h post whole-body γ-irradiation (0.45 and 4.5 Gy) and 6 h post whole-body γ-irradiation (4.5 Gy). ISH staining was performed using sense (negative control) and anti-sense probes for Noxa. Arrows indicate positively stained primordial follicle oocytes. (B) Negative controls were performed using Noxa^'1' ovaries (untreated and 3 h post whole-body γ-irradiation (0.45 Gy). Scale bar: 20 μηι.

Figure 8 is a photographical representation illustrating expression of Puma (A) and Noxa

(B) mRNA in Trp53^"'^' primordial follicle oocytes following γ-irradiation-induced DNA damage. Expression of Puma (A) and Noxa (B) mRNA in Trp53^' primordial follicle oocytes following γ-irradiation induced DNA damage. Ovaries were harvested from PN10 Trp53^' mice (untreated negative control) and from PN5 Trp53^'f" mice 3 h post whole-body γ-irradiation (0.45 Gy) and in situ hybridisation using sense (negative control) and anti-sense probes was performed. In situ hybridization indicates Puma (A) and Noxa (B) mRNA in Trp53^' primordial follicle oocytes at 3 h post γ-irradiation. Arrows indicate positively stained primordial follicle oocytes. Scale bar: 20μ^ηι. Figure 9 is a photographical representation illustrating loss of PUMA rescues primordial follicle oocytes from DNA damage induced apoptosis. Loss of Puma rescues primordial follicle oocytes from DNA damage induced apoptosis. Anti MSY2 (A) and anti-GCNA (B) immuno-histochemical staining of ovaries of PN10 wt, Trp53^" , Nox , Pumd and Pumd Noxd mice five days after exposure to 0.45 Gy or 4.5 Gy γ-irradiation or no treatment (control). Arrows indicate intrinsically resistant growing follicles. Black arrow heads indicate primordial follicles. White arrow heads indicate primordial follicle remnants. Scale bars: (A), 200μιη; (B), 20μη .

Figure 10 is a photographical representation showing TUNEL staining in primordial follicle oocytes following γ-irradiation induced DNA damage. TUNEL staining in primordial follicle oocytes following γ-irradiation induced DNA damage. Ovaries were harvested from PN5 wt mice at 0 (untreated control) and 6 h post whole-body γ-irradiation (4.5 Gy). Arrows indicate positively stained primordial follicle oocytes. Scale bar: 20 μηι.

Figure 11 is a graphical representation showing detection of DNA damage in primordial follicle oocytes following γ-irradiation. Detection of DNA damage in primordial follicle oocytes following γ-irradiation. (A) DNA damage was quantitated by counting the number of γ-Η2ΑΧ foci within the nucleus (n=50-100 primordial follicles) of wt and Pumd Noxd primordial follicles 3 h after exposure to the higher dose of whole-body γ-irradiation (4.5 Gy). No significant difference was observed in the number of foci between genotypes (p>0.05). (B) DNA damage was quantified by counting the number of γ-Η2ΑΧ foci within the nucleus of Puma ^~ Noxa ^~ primordial follicles (n=50-100) 3 h after exposure to whole-body γ-irradiation at the lower (0.45 Gy) and higher (4.5 Gy) dose. ***p<0.001.

Figure 12 is a graphical representation illustrating no evidence for protection of primordial oocytes from DNA damage induced death by inhibition of an Imatinib-sensitive kinase. (a) Ovaries were harvested from PN5 wt mice, maintained in whole organ culture and treated with Imatinib (10 μΜ) or vehicle for 2 h, then subjected to γ-irradiation (0.45 Gy) in vitro or left non-irradiated. Quantification (means ± SEM) of follicles and pyknotic bodies analysed at 48 h. (b) Quantification of follicles and pyknotic bodies in wt mice that were treated at PN5 with Imatinib (7.5 mg/kg i.p.) or vehicle for 2 h and then exposed to whole body γ-irradiation (0.45 Gy) or left non-irradiated and analysed at PN10. (c) Ovaries were harvested from wt PN5 mice, maintained in whole organ culture, pre-treated with Imatinib (10 μΜ) or vehicle for 2 h and then treated with cisplatin (20 μΜ) or vehicle. Quantification of follicles and pyknotic bodies was performed after 24 and 48 h. (a-c) For comparison with untreated controls: *p<0.05, **p<0.01, ***p<0.001. n=3-9 ovaries per treatment group.

Figure 13 is a photographic representation of data showing the appearance of ovaries and TUNEL analysis following in vitro treatment of oocytes with Imatinib and cisplatin. 24 (a) and 48 h (b). Ovaries were harvested from wt PN5 mice, maintained in whole organ culture and treated with Imatinib (10 μΜ) or vehicle for two hours, then 2 h later with cisplatin (20 μΜ) or vehicle. MSY2 immuno-staining was used to visualize oocytes and GCNA immuno-staining was used to confirm the identity of primordial follicles. Primordial follicles were not detected and a large amount of pyknosis was observed in ovaries treated with cisplatin alone, or a combination of cisplatin and Imatinib after 48 h in culture. TUNEL staining revealed increased TUNEL-positive (apoptotic) primordial oocytes 24 h following treatment with cisplatin, with or without Imatinib pre-treatment. Limited TUNEL staining was observed at 48 h because most oocytes had earlier degraded. Scale bars indicate: (a), 200 μπι; (b), 20 μπι.

BRIEF DESCRIPTION OF THE TABLES Table 1 provides a description of the SEQ ID NOs provided herein. Table 2 provides a list of non-natural amino acids contemplated in the present invention. Table 3 provides an amino acid sub-classification. Table 4 provides exemplary amino acid substitutions.

Table 5 provides data showing loss of Puma or both Puma and Noxa protects fertility following γ-irradiation. Litters produced by mothers, which were either untreated or γ-irradiated at PN5 (offspring from 4th litters; Puma ^/" or Puma ^/~Noxa^/~); or were untreated Puma '^' daughters of γ-irradiated Puma '^' mothers (produced from matings of γ-irradiated Puma '^' females to Puma '^' males); or which had been γ-irradiated as adults (PN48) (wt or Puma ).

DET AILED DESCRIPTION

The present invention is predicated in part upon the unexpected finding that suppression of PUMA activity is effective in conserving fertility and the ability to produce normal offspring in female mice subject to sterilizing dosages of radiation.

The subject invention is not limited to particular screening procedures for agents, specific formulations of agents and various medical methodologies, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Each embodiment described in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise. As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a medicament" includes a single medicament, as well as two or more medicaments; reference to "an agent" includes a single agent as well as two or more agents; reference to "the invention" includes a single aspect or multiple aspects of an invention; and so on.

The term "complementary" refers to the capability of a single stranded form of an oligonucleotide, polynucleotide or nucleotide sequence to bind to (hybridise with) another oligonucleotide or polynucleotide or nucleotide sequence through specific pairing of bases to form a double stranded stretch of nucleic acid. Oligonucleotides or oligonucleotide subsequence can be described as "complementary" to a target sequence within a polynucleotide, and furthermore, the contact surface characteristics are complementary to each other. The term "complementary" includes base complementarity such as the binding interaction of cytosine and guanine bases or adenine arid thymine or uracil bases within double stranded DNA or RNA molecules, polynucleotides or oligonucleotides. The term complementary also includes the predicted complementarity of nucleic acid molecules, oligonucleotides and polynucleotides, where those predictions are derived from the known sequences of those molecules. Base analogs, such as inosine, may also be complementary to natural base residues, and hence, base analogues may be part of a complementary sequence or oligonucleotide. Degenerate base positions within a degenerate oligonucleotide may complement several different natural bases at the same site in a target sequence. Specifically, within the mixture of oligonucleotides that constitute the degenerate oligonucleotide, at one homologous position in the sequence, the different oligonucleotides have different bases. This invention also encompasses situations in which there is non-traditional base-pairing such as Hoogsteen base pairing which has been identified in certain transfer RNA molecules and postulated to exist in a triple helix. In the context of the definition of the term "complementary", the terms "match" and "mismatch" as used herein refer to the hybridisation potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridise efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that hybridise less effectively or not at all and these may be incorporated into oligonucleotides or nucleic acid molecules in order to modulate binding efficiencies.

The term "subject" as used herein refers to an animal, in particular a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the medical protocol of the present invention. A subject regardless of whether a human or non-human animal or embryo may be referred to as an individual, subject, animal, patient, host or recipient. The present invention has both human and veterinary applications. For convenience, an "animal" specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys and laboratory test animals. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. In a particular embodiment the invention relates to human female fertility. In some embodiments, oocytes of the subject are contacted ex vivo (or in vitro) with a suppressive agent as herein described such as an agent which suppresses PUMA expression or PUMA polypeptide activity, or NOXA expression or NOXA polypeptide activity, PUMA and NOXA expression or PUMA and NOXA polypeptide activity, or TP 63 expression or TP63 activity.

The terms "modulate", "inhibit" or "down regulate", "suppress" and the like include antagonizing, decreasing, reducing and partially inhibiting formation, expression, level or activity of one or more of the herein disclosed targets in relation to conserving or enhancing fertility in a subject.

Using studies of gene deletion in mice, PUMA is proposed as the first essential pro- apoptotic target gene of TP63 following DNA-damage in vivo. A subset of oocytes lacking puma were resistant to lethal doses of γ-irradiation in vivo, and astoundingly were capable of producing phenotypically normal offspring. Thus, it is proposed that therapeutic manipulation of PUMA or PUMA favors survival, accurate repair of DNA defects with rescue of fertility without compromise of oocyte quality. In addition, suppressing NOXA or NOXA activity provides additional conservation effects.

Accordingly, the present invention provides a method of conserving fertility in a female subject in need, the method comprising administering to the female an agent which suppresses PUMA or NOXA expression, or PUMA or NOXA activity. In some embodiments, the female is undergoing or will undergo cancer treatment.

The term "gene" is used in its broadest sense and includes cDNA corresponding to the exons of a gene. Reference herein to a "gene" is also taken to include:-a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or an mRNA or cDNA corresponding to the coding regions (i.e. exons), pre-mRNA and 5'- and 3'- untranslated sequences of the gene.

Reference to "expression" is a broad reference to gene expression and includes any stage in the process of producing protein or RNA from a gene or nucleic acid molecule, from pre- transcription, through transcription and translation to post-translation. Generally, transcription or translation are suppressed. In some embodiments, translation is suppressed by reducing translational efficiency or reducing message stability or a combination of these effects. In some embodiments, splicing of the unprocessed RNA is targeted leading to the production of non-functional or less active protein. In some embodiments, expression is suppressed by agents that alter the reading frame to introduce premature stop codons leading to non-sense mediate decay.

PUMA is the transcriptional target of TP63 in ovaries and, accordingly, in one embodiment, the agent reduces the level or antagonizes the activity of TP63 polypeptides. In a preferred, non-essential embodiment, isoforms of TP63 which are predominantly expressed in reproductive tissue such as oocytes are preferred targets.

As shown in the Examples, mice lacking puma and noxa are better protected than mice lacking puma alone and thus suppressors of the activity of both pro-apoptotic molecules are proposed to have a synergistic effect in subjects.

Accordingly, another embodiment of the present invention provides a method for the treatment or prophylaxis of a subject with reduced fertility or who is at risk of developing same, the method comprising administering to the subject an amount of an agent effective to reduce formation, expression or activity of TP63 or PUMA or NOXA or an agent or agents effective to reduce formation, expression or activity of PUMA and NOXA for a time and under conditions sufficient to conserve oocytes and fertility.

In accordance with one aspect of the present invention, inhibition or down regulation of the level or activity of PUMA or NOXA or PUMA and NOXA is used to protect the fertility of subjects from the effects of agents acknowledged to reduce fertility. Non-limiting examples include chemotherapeutic, radiotherapeutic or radio-chemotherapeutic agents.

Further components of the transcriptional complex comprising TP63 and the PUMA gene or TP63 and the NOXA gene can be identified using methods well known in the art, such as chromatin immunoprecipitation assays (ChIP), transient transfection and luciferase reporter assays, and GST fusion protein association assays, yeast-two-hybrid screening and proteomics techniques. Co-activators of TP63 bind to the N-terminal domain of transactivating TP63 isoforms and may modulate TP63 effects in reproductive cells. Examples of co-activators involved with p53 family members include TATA box protein associated factors (TAFs), p300/CBP and high mobility group (HMG) proteins such as SSRP1.

Reference herein to TP63 includes forms which are pro-apoptotic and have transactivating activity. Reference to antagonists of PUMA includes isoforms of TP63 and in particular, truncated forms of TP63 and active fragments or variants thereof that bind to PUMA and repress transcription. Reference to TP63 includes α, β and γ forms. TP63a is a particular target as it is predominantly expressed in reproductive tissue.

The specification contemplates to cancer treatment, apoptosis-inducing agents and DNA damaging agents as examples of environmental infections or therapeutic agents or events that induce DNA or cellular damage sufficient to trigger reproductive cell apoptosis in a subject. In an illustrative embodiment, the apoptotic stimulus is nucleic acid damage, typically DNA damage. Illustrative DNA damaging agents include: radiation, such as by ultraviolet light, X-rays or gamma rays; various toxins, industrial or environmental chemicals, chemotherapy, radiotherapy or DNA damage associated with advancing age.

In an illustrative embodiment, the agent is a small molecule inhibitor, a nucleic acid molecule or a protein or peptide, such as a stapled peptide or foldamer, or antibody fragment.

Agents which have the potential to act as suppressors include small chemical molecules which can penetrate a cell membrane or via an ion channel or other pore and antigen binding agents which have the capacity for intracellular transmission, such as cartilage fish-derived antibodies (e.g. shark antibodies; see for example, Liu et ah, BMC Biotechnol. 7: 78, 2007). An antigen binding agent, or a functionally active fragment thereof, which has the capacity for intracellular transmission also includes antibodies such as camelids and llama antibodies, scFv antibodies, intrabodies or nanobodies, e.g. scFv intrabodies and V_HH intrabodies. Such antigen binding agents can be made as described by Harmsen & De Haard in Appl. Microbiol. Biotechnol. Nov; 77(1): 13-22, 2007; Tibary et al, Soc. Reprod. Fertil. Suppl. 64: 297-313, 2007; Muyldermans, J. Biotechnol. 74: 277- 302, 2001; and references cited therein. In one embodiment, scFv intrabodies which are able to interfere with a protein-protein interaction are used in the methods of the invention; see for example, Visintin et al, J. Biotechnol, 755:1-15, 2008 and Visintin et al, J. Immunol. Methods, 290(1-2): 135-53, 2008 for methods for their production. For use in the methods of the invention, agents may comprise a cell-penetrating peptide sequence or nuclear-localizing peptide sequence such as those disclosed in Constantini et al, Cancer Biotherm. Radiopharm., 23(1): 3-24, 2008 or International Publication No. WO 2005/086800. Also useful for in vivo delivery are Vectocell or Diato peptide vectors such as those disclosed in De Coupade et al, Biochem J. 390($\2): 407-418, 2005 and Meyer- Losic et al, J Med Chem. 49(23): 6908-6916, 2006. Thus, the invention provides the therapeutic use of fusion proteins of the agents (or functionally active fragments thereof), for example, but without limitation, where the antibody or fragment thereof is fused via a covalent bond (e.g. a peptide bond), at optionally the N-terminus or the C-terminus, to a cell-penetrating peptide or nuclear-localizing peptide sequence. Alternatively, peptides may be linked chemically.

Some suitable peptide or other agents capable of suppressing PUMA or NOXA or PUMA and NOXA or TP63 activity are described.

Examples of antibodies which bind and reduce PUMA activity are described in International Publication No. WO 00/26228. Anti-Puma antibodies can also be obtained from commercial sources such as Cell Signaling Technology (Danvers, MA, USA). Anti- Noxa antibodies include Alexis 114C307 (also known as AB13654).

Natural products, combinatorial synthetic organic or inorganic compounds, peptide/polypeptide/protein, nucleic acid molecules and libraries or phage or other display technology comprising these are all available to screen or test for suitable agents. Natural products include those from coral, soil, plant, or the ocean or Antarctic environments. Libraries of small organic molecules can be generated and screened using high-throughput technologies known to those of skill in this art. See for example United States Patent No. 5,763,623 and United States Application No. 20060167237. Combinatorial synthesis provides a very useful approach wherein a great many related compounds are synthesized having different substitutions of a common or subset of parent structures. Such compounds are usually non-oligomeric and may be similar in terms of their basic structure and function, for example, varying in chain length, ring size or number or substitutions. Virtual libraries are also contemplated and these may be constructed and compounds tested in silico (see for example, US Publication No. 20060040322) or by in vitro or in vivo assays known in the art. Libraries of small molecules suitable for testing are already available in the art (see for example, Amezcua et al, Structure (London), 10: 1349-1361, 2002). Yeast SPLINT antibody libraries are available for testing for intrabodies which are able to disrupt protein-protein interactions (see Visintin et al, supra). Examples of suitable methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. Sci. USA 90: 6909, 1993; Erb et al, Proc. Natl. Acad. Sci. USA 91: 11422, 1994; Zuckermann et al, J. Med. Chem. 37: 2678, 1994; Cho et al, Science, 261: 1303, 1993; Carrell et al, Angew. Chem. Int. Ed. Engl. 33: 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33: 2061, 1994; and Gallop et al, J. Med. Chem. 37: 1233, 1994.

Thus, agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is suited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997; United States Patent No. 5,738,996; and United States Patent No. 5,807,683). Libraries of compounds may be presented, for example, in solution (e.g. Houghten, Bio/Techniques 13: 412-421, 1992), or on beads (Lam, Nature 354: 82-84, 1991), chips (Fodor, Nature 364: 555-556, 1993), bacteria (United States Patent No. 5,223,409), spores (United States Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et ah, Proc. Natl. Acad. Sci. USA, 89: 1865-1869, 1992) or phage (Scott and Smith, Science 249: 386-390, 1990; Devlin, Science 249: 404-406, 1990; Cwirla et al, Proc. Natl. Acad. Sci. USA 87: 6378-6382, 1990; and Felici, J. Mol. Biol. 222: 301-310, 1991).

Reference to "expression" is a broad reference to gene expression and includes any stage in the process of producing protein or RNA from a gene or nucleic acid molecule, from pre- transcription, through transcription and translation to post-translation. Generally, transcription or translation are suppressed. In some embodiments, translation is suppressed by reducing translating efficiency or reducing message stability or a combination of these effectors. In some embodiments, splicing of the unprocessed RNA is targeted leading to the production of non-functional or less active protein. In some embodiments, expression is suppressed by agents that alter the reading frame to introduce premature stop codons leading to non-sense mediate decay.

Nucleic acid molecules including oligonucleotides and vectors such as viruses encoding same are used to suppress gene expression of a gene encoding PUMA or NOXA or TP63. Nucleic acids (including oligonucleotides, including double or single stranded nucleic acid molecules) include DNA (gDNA, cDNA), RNA (sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (SiRNAs), double-stranded RNAs (dsRNA), short hairpin RNAs (shRNAs), piwi-interacting RNAs (PiRNA), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs), small nuclear (SnRNAs) ribozymes, aptamers, DNAzymes or other ribonuclease-type complexes are conveniently employed. Methods of producing chimeric constructs capable of inducing RNA interference in eukaryotic cells are described in the art.

The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,

61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

In some embodiments, the nucleic acid or oligonucleotide comprises a contiguous nucleic acid sequence complementary to a nucleic acid of about 8 to 50 nucleotides selected from those nucleic acid sequences set out in the sequence listing and as described in Table 1. In some embodiments, the suppressive nucleic acid or oligonucleotide is substantially complementary to a region flanking an intron-exon boundary and thereof flanks a junction between two sequences set out in SEQ ID NOs: 43-45, SEQ ID NOs: 46-50, SEQ ID NOs: 51-57, SEQ ID NOs: 58-64, SEQ ID NOs: 65-71, SEQ ID NOs: 72-78, SEQ ID NOs: 79- 81, SEQ ID NOs: 82-86 or SEQ ID NOs: 87-91.

In other embodiments, oligonucleotides bind to target sites within the leader and sequences surrounding the start site.

In some embodiments, a suppressor antisense oligonucleotide or nucleic acid is substantially complementary to an exon sequence set out in SEQ ID NOs: 1-8, SEQ ID NOs: 17-23, SEQ ID NOs: 31-36, SEQ ID NOs: 43, 45, 46, 48, 50, 51, 53, 55, 57, 58, 60,

62, 64, 65, 67, 69, 71, 72, 74, 76, 78, 79, 81, 82, 84, 86, 87, 89 or 91.

In some embodiments, a suppressor antisense oligonucleotide or nucleic acid is substantially complementary to an intron sequence set out in SEQ ID NOs: 44, 47, 49, 52, 54, 56, 59, 61, 63, 66, 68, 70, 73, 75, 77, 80, 83, 85, 88 or 90.

For the avoidance of doubt, the terms "oligonucleotide" and "nucleic acid" include non- naturally occurring modified forms as well as naturally occurring forms. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides. Sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 20 contiguous positions, usually about 20 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA; CLUSTAL described by Jeanmougin et al, Trends Biochem. Sci. 23: 403-405, 1998) or by inspection, or using dot diagrams, and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al, Nucl. Acids Res. 25: 3389, 1997. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel, et al, eds. Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1995.

Sequences that are substantially complementary to a recited sequence have a high level of sequence identity with the recited sequence. In some embodiments, sequence identity is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or more. The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base {e.g., A, T, C, G, I) or the identical amino acid residue {e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison {i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

The sequence of the oligonucleotide or nucleic acid is designed to exhibit suitable energy related characteristics important for duplex formation, specificity, function, transport and nuclease resistance. As known in the art, sequences ideally exhibit minimal self annealing properties, unless required. The computer program, OLIGO, may be used to estimate the behaviour of preferred antisense sequences.

RNA interference (RNAi) includes the process of gene silencing involving double stranded (sense and antisense) RNA which leads to sequence specific reduction in gene expression via target mRNA degradation. RNAi is typically mediated by short double stranded siR As or single stranded microRNAs (miRNA). Broadly, RNAi is initiated when a strand of RNA from either or these molecules forms a complex referred to as an RNA-induced silencing complex (RISC) which targets complementary RNA and suppresses translation. The process has been exploited for research purposes and for therapeutic application (see for example, Izquierdo et al, Cancer Gene Therapy, 72(3): 217-27, 2005) Other oligonucleotides having RNA-like properties have also been described and many more different types of RNAi may be developed. Both RNAi and antisense strategies have been used to induce stop codon suppression via inhibition of eRFl expression (Carnes et al, RNA, 9: 648-653, 2003). Antisense oligonucleotides have been used to alter exon usage and to modulate pre-RNA splicing, see for example Madocsai et al, Molecular Therapy, 12: 1013-1022, 2005 and Aartsma-Rus et al, BMC Med Genet, 8: 43, 2007. Antisense and iRNA compounds may be double stranded or single stranded oligonucleotides which are RNA or RNA-like or DNA or DNA-like molecules that hybridize specifically to DNA or RNA of TP63 or PUMA or NOXA encoding sequences. iRNA compounds are typically approximately 8 to 80 nucleobases in length and specifically hybridize to a nucleic acid region encoding TP63 or PUMA or NOXA as further described herein or as known in the art. In some embodiments, antisense compounds are designed to inhibit production of transactivating (pro-apoptotic) forms of TP63 and in particular a forms or other forms expressed in reproductive cells of the subject. Examples of RNAi molecules that can be used to suppress PUMA activity are described in US Patent Publication No. 20080025958 and Hemann et al, Proc Natl Acad Sci U S A., 101(25): 9333-9338, 2004. Examples of RNAi molecules that can be used to suppress NOXA activity are described in Fernandez et al, Cancer Research, 65(14): 6294-6304, 2005. siRNA may have a first strand and a second strand each strand being approximately 20 to 25 nucleobases in length with the strands being complementary over at least about 19 nucleobases and having on each 3' termini of each strand a deoxy thymidine dimer (dTdT) which in the double-stranded compound acts as a 3' overhang. Alternatively, the double- stranded antisense compounds are blunt-ended siRNAs. Alternatively, single-stranded RNAi (ssRNAi) compounds that act via the RNAi antisense mechanism are contemplated. Further modifications can be made to the double-stranded compounds and may include conjugate groups attached to one of the termini, selected nucleobase positions, sugar positions or to one of the intemucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double-stranded. When formed from two strands, or a single strand that takes the form of a self- complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.

Antisense polynucleotide sequences are an example of a suitable therapeutic suppressor. Polynucleotide vectors, for example, containing all or a portion of PUMA or NOXA or TP63 gene sequences or gene flanking sequences may be placed under the control of a promoter in an antisense orientation and introduced into a cell. Expression of such an · antisense construct within a cell will interfere with gene transcription and translation. Furthermore, co-suppression and mechanisms to induce RNAi (i.e. siRNA or miRNA) may also be employed. Alternatively, antisense or sense molecules may be directly administered. In this latter embodiment, the antisense or sense molecules may be formulated in a composition and then administered by any number of means to target cells.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Once an oligonucleotide is selected, modified forms are made and tested, as known in the art, in order to optimize activity. Nuclease insensitive antisensitive oligonucleotide are preferred as these have a substantially reduced rate of degradation by nucleases, such as RNAses and/or DNAses. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. Illustrative modified oligonucleotides include oligonucleotides such as those comprising morpholine ring (C40N aromatic rings) in place of the natural ribose sugar moiety. Further favourable modified oligonucleotides include 2-O-methyl, PNA, LNA, morpholino or combinations of these in natural (non-modified) variants or analogs. In a preferred non-essential embodiment, antisense oligonucleotides comprise morpholine rings or are morpholino oligonucleotides. In some embodiments, morpholino oligonucleotides comprise a morpholine ring in place of a ribose sugar and a neutral charge backbone.

In some embodiments, oligonucleotides also include 2'-substituted ribonucleotides. The term "2'-substituted ribonucleoside" refers to where the hydroxyl group at the 2' position of the pentose moiety is substituted to produce a 2'-0-substituted ribonucleoside. For example, such substitution is with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an aryl or allyl group having 2-6 carbon atoms or 6-10 carbon atoms, wherein such alkyl, aryl, or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups. The term "2'-substituted ribonucleoside" also includes ribonucleosides in which the 2' hydroxyl group is replaced with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an amino or halo group.

Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphoro-thioates, phosphoro-dithioates, phosphotri-esters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 -5' linkages, 2 -5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. Many of the preferred features described above are appropriate for sense nucleic acid molecules. Thus, in some embodiments, oligonucleotides or nucleic acids are modified to be insensitive to nucleases via various modifications to the natural oligonucleotide or nucleic acid.

In other embodiments, the oligonucleotide or nucleic acid suppresses translation initiation, splicing at a splice donor site or splice acceptor site. In some embodiments, modification of pre-mRNA splicing reduces the activity of PUMA, NOXA or TP63 polypeptides so produced. In other embodiments, modification of splicing alters the reading frame and initiates nonsense mediated degradation of the transcript. Chimeric oligomeric compounds may be formed as composite structures of two or more oligonucleotides, oligonucleotide analogs, oligonucleosides or oligonucleotide mimetics. Routinely used chimeric compounds include but are not limited to hybrids, hemimers, gapmers, extended gapmers, inverted gapmers and blockmers wherein the various point modifications and or regions are selected from native or modified DNA and RNA type units and or mimetic type subunits such as for example locked nucleic acids (LNA), peptide nucleic acids (PNA), morpholinos, and others. The preparation of such hybrid structures is described for example in US Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.

One preferred form of modified oligonucleotide is a morpholino (MO), which are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages (for example, Summerton and Weller, Antisense and Nucleic Acid Drug Development, 7:187-195, 1997). Morpholino nucleic acids typically comprise heterocyclic bases attached to the morpholino ring. A number of linking groups may link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups have been selected to give a non-ionic oligomeric compound. The non-ionic morpholino- based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based oligomeric compounds are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins. (See Genesis, volume 30, issue 3, 2001 and Heasman, J, Dev. Biol, 243, 209-214, 2002). Morpholino-based oligomeric compounds are disclosed in U.S. Patent No. 5,034,506; WO 00024885 and WO 00045167 and are reviewed in Ekker and Landon, Genesis, 30:89-93, 2001. Thus, the terms oligonucleotide and nucleic acid include other families of compounds as well, including but not limited to oligonucleotide analogs, chimeric, hybrid and mimetic forms.

PNA oligonucleotides have favorable hybridization properties, high biological stability and are electrostatically neutral molecules. In PNA oligomeric compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are bound directly or indirectly to an aza nitrogen atoms of the amide portion of the backbone. The preparation of PNA oligomeric compounds is disclosed for example in US Patent Nos: 5,539,082; 5,714,331 ; and 5,719,262. Variants including peptide nucleic acids with phosphate group (PHONA) or locked nucleic acid (LNA) or morpholino backbones or backbones with allyl linkers or amino linkers are also encompassed. Various modified oligonucleotide structures contemplated herein are described in US Patent Publication No. 2002/0125287 and US Patent No. 6,017,786 referred to herein in their entirety.

Aptamers are also contemplated. RNA and DNA aptamers can substitute for monoclonal antibodies in various applications (Jayasena, Clin. Chem., 45(9): 1628-1650, 1999; Morris et /., Proc. Natl. Acad. Sci., USA, 95(6): 2902-2907, 1998). Aptamers are nucleic acid molecules having specific binding affinity to non-nucleic acid or nucleic acid molecules through interactions other than classic Watson-Crick base pairing. Aptamers are described, for example, in United States Patent Nos. 5,475,096; 5,270,163; 5,589,332; 5,589,332; and 5,741,679. An increasing number of DNA and RNA aptamers that recognize their non- nucleic acid targets have been developed by SELEX and have been characterized (Gold et ah, Annu. Rev. Biochaem., 64: 763-797.1995; Bacher et al., Drug Discovery Today, 3(6): 265-273, 1998). Agents that down modulate the formation, interaction, expression or activity of TP63 or PUMA or NOXA may be derived from TP63 or PUMA or NOXA or their encoding sequences or are isoforms or variants, fragments or analogs thereof. Mismatches may be used to modulate binding efficiency of antisense nucleic acids.

Agents may be hydrocarbon-stapled peptides or minature proteins which are alpha-helical and cell-penetrating, and are able to disrupt protein-protein interactions (see for example, Wilder et al, ChemMedChem. 2(8): 1149-1151, 2007; & for a review, Henchey et a!., Curr Opin Chem Biol., 72(6):692-697, 2008).

Reference to "PUMA" or "TP63" or "NOXA" herein includes isoforms, mutants, variants, and homologs or orthologs from other species, including without limitation murine and human forms. Illustrative embodiments are described in SEQ ID NOs: 1 to 42 as described in Table 1. In some embodiments, the agents are derived from nucleic acid molecules such as the nucleotide sequences of TP63 or PUMA or NOXA as described herein or corrected version thereof or are variants thereof. Variants include nucleic acid molecules sufficiently similar to naturally occurring forms of these molecules or their complementary forms over all or part thereof such that selective hybridisation may be achieved under conditions of medium or high stringency, or which have about 60% to 90% or 90 to 98%> sequence identity to the nucleotide sequences defining naturally occurring TP 63 or PUMA or NOXA DNA or RNA sequences as described herein (see Table 1) and over a comparison window comprising at least about 15 nucleotides. Preferably the hybridisation region is about 12 to about 18 nucleobases or greater in length. Preferably, the percent identity between a particular nucleotide sequence and the reference sequence is at least about 80%, or 85%, or more preferably about 90% similar or greater, such as about 95%, 96%, 97%, 98%, 99% or greater. Percent identities between 80% and 100% are encompassed. The length of the nucleotide sequence is dependent upon its proposed function. For example, short interfering RNAs are generally about 20 to 24 nucleotides in length, whereas molecules designed to provide dominant negative functions may require full length or substantially full length molecules. The term "homolog" or "homologs" refers broadly to functionally and structurally related molecules including those from other species. Isoforms, mutants, homologs and orthologs are examples of variants. One isoform of TP63 is an N-terminally truncated transcriptional repressor. In some embodiments, the present invention contemplates the use of full length TP63 or PUMA or NOXA polypeptides or biologically active components or portions (fragments) or stapled peptides of one or more of these molecules as antagonists. Biologically active portions or peptides comprise one or more binding domains. A biologically active portion or stapled peptide of a full length polypeptide can be a polypeptide which is, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 350, or 400 or more amino acid residues in length. The TP63 or PUMA or NOXA polypeptides contemplated herein include all biologically active or naturally occurring forms of as well as biologically active portions and variants thereof. "Variant" polypeptides or peptides include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess at least one biological activity of the native protein. Antagonist variants are selected on the basis that they inhibit or antagonise the biological activity or formation of TP63 or PUMA or NOXA, their encoding genes, a complex comprising TF63/puma or ΎΡ63/ηοχα or a component thereof. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native TP63 or PUMA polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98%) or more sequence similarity with the amino acid sequence for the native protein as determined by contemporary sequence alignment programs using default parameters. A biologically active variant of a TP63 or PUMA or NOXA polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

Active peptides are conveniently between 9 and 32 amino acid residues in length, more preferably between 9 and 31 amino acids in length, even more preferably between 9 and 30 amino acids in length, even more preferably between 9 and 29 amino acids in length, even more preferably between 9 and 28 amino acids in length, even more preferably between 9 and 27 amino acids in length, even more preferably between 9 and 26 amino acids in length, even more preferably between 9 and 25 amino acids in length, even more preferably between 9 and 24 amino acids in length, even more preferably between 9 and 23 amino acids in length, even more preferably between 9 and 22 amino acids in length, even more preferably between 9 and 21 amino acid residues in length, even more preferably between 9 and 20 amino acids in length, even more preferably between 9 and 19 amino acids in length, even more preferably between 9 and 18 amino acids in length, even more preferably between 9 and 17 amino acids in length, even more preferably 9 and 16 amino acid residues in length, even more preferably between 9 and 15 amino acids in length, even more preferably between 9 and 14 amino acids in length, and still even more preferably between 9 and 13 amino acids in length. An especially preferred amino acid sequence is between 9 and 12 amino acid residues in length.

An inhibitory TP63 or PUMA or NOXA polypeptide or peptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by introducing mutations in the encoding DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA, 82: 488-492, 1985, Kunkel et al, Methods in Enzymol., 154: 367-382, 1987, United States Patent No. 4,873,192, Watson et al, "Molecular Biology of the Gene", Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987 and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al, (Natl. Biomed. Res. Found, 5: 345-358, 1978). Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of polypeptides. Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify useful polypeptide variants (Arkin et ai, Proc. Natl. Acad. Sci. USA, 89: 7811-7815, 1992; Delgrave et al, Protein Engineering, 6: 327-331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.

Variant polypeptides or peptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to reference amino acid sequences. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues that may be substituted generally have similar side chains have been defined in the art, which can be generally sub-classified as follows:

Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine. Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine). Suitable negatively charged amino acids include L-aspartic acid, L-glutamic acid, D-aspartic acid, D-glutamic acid, L-P-homoaspartic acid, L-p-homoglutamic acid, L- a-methylaspartic acid, L-a-methylglutamic acid, D-a-methylaspartic acid and D-a- methylglutamic acid.

Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan. Preferably, hydrophobic side chains are selected from L- phenylalanine, L-isoleucine, L-leucine, L-valine, L-methionine, L-tyrosine, D- phenylalanine, D-isoleucine, D-leucine, D-valine, D-methionine, D-tyrosine, L-β- homophenylalanine, L-P-homoisoleucine, L-P-homoleucine, L- -homovaline, L-β- homomethionine, L-P-homotyrosine, aminonorbornylcarboxylate, cyclohexylalanine, L- norleucine, L-norvaline, L-a-methylisoleucine, L-a-methylleucine, L-a-methylmethionine, L-a-methylnorvaline, L-a-methylphenylalanine, L-a-methylvaline, L-a-methyltyrosine, L- a-methylhomophenylalanine, D-a-methylleucine, D-a-methylmethionine, D-a- methylnorvaline, D-a-methylphenylalanine, D-a-methylvaline, D-a-methyltyrosine, D-a- methy lhomopheny 1 alanine residues L-tryptophan, L-3'4'-dichlorophenylalanine, L- 1 'naphthylalanine and L-2'naphthylalanine.

Neutral/polar. The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

This description also characterizes certain amino acids as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the a- amino group, as well as the a-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et ah, 1978 {supra); and by Gonnet et ah, Science, 255(5062): 1443-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid. Suitable small amino acids include glycine, L-alanine, L-serine, L-cysteine, D-alanine, D-serine, D- cysteine, L-P-homoserine, L-p-homoalanine, γ-aminobutyric acid, aminoisobutyric acid, L- a-methylserine, L-a-methylalanine L-a-methylcysteine, D-a-methylserine, D-a- methylalanine and D-a-methylcystine residues. Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 3.

Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional TP63 or PUMA or NOXA polypeptide can readily be determined by assaying its activity. Activities that can readily be assessed are known to those of skill and include assays to determine DNA or protein binding detected by, for example, nuclear magnetic resonance spectroscopy (NMR) where heteronuclear single quantum coherence (HSQC) spectra are observed, Biacore, kinetic, affinity and pulldown analyses. Conservative substitutions are shown in Table 4 below under the heading of exemplary substitutions. More preferred substitutions are shown under the heading of preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, Biochemistry, third edition, Wm.C. Brown Publishers, 1993.

Thus, a predicted non-essential amino acid residue in a TP63 or PUMA or NOXA polypeptide or peptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly or produced synthetically and the activity of the peptide can be determined.

Accordingly, the present invention also contemplates variants of the naturally-occurring TP63 or PUMA or NOXA polypeptide or peptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identity to a reference TP63 or PUMA or NOXA polypeptide sequence as described herein (see Table 1). Moreover, sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more amino acids but which retain certain properties of the reference TP63 or PUMA or NOXA polypeptide are contemplated. The present variant polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to TP63 or PUMA or NOXA polynucleotide sequences, or the non-coding strand thereof.

In some embodiments, variant polypeptides differ from a TP63 or PUMA or NOXA polypeptide sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In another, variant polypeptides differ from the corresponding sequence in any referenced sequence by at least 1% but less than 20%, 15%, 10% or 5% of the residues. If this comparison requires alignment the sequences should be aligned for maximum similarity. ("Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In one embodiment, the differences are differences or changes at a non-essential residue or a conservative substitution. A sequence alignment for TP63 or PUMA or NOXA proteins from a range of mammalian species is used to demonstrate conserved residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of a polypeptide agent of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present.

In other embodiments, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of a TP63 or PUMA or NOXA polypeptide as described herein, and has at least one activity of that TP63 or PUMA or NOXA polypeptide.

In some embodiments, analogs of peptide suppressors of PUMA or TP63 or NOXA have enhanced stability and activity or reduced unfavorable pharmacological properties. They may also be designed in order to have an enhanced ability to cross biological membranes or to interact with only specific substrates. For example, proteins or peptides comprising membrane translocating motifs are conveniently employed as described in International Publication No. WO 2005/086800. The composition may be made recombinantly or using protein chemistry techniques.

Thus, analogs may retain some functional attributes of the parent molecule but may posses a modified specificity or be able to perform new functions useful in the present context i.e., for administration to a subject. Analogs of peptide or polypeptide agents contemplated herein include but are not limited to modification to side chains, incorporating of unnatural, non-proteogenic or non-naturally occurring amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaB¾; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid contemplated herein is shown in Table 2.

Suitable β-amino acids include, but are not limited to, L-p-homoalanine, L- β-homoarginine, L-P-homoasparagine, L- -homoaspartic acid, L-p-homoglutamic acid, L- β-homoglutamine, L-P-homoisoleucine, L-P-homoleucine, L-p-homolysine, L-β- homomethionine, L-β-homo henylalanine, L^-homoproline, L-β-homoserine, L-β- homothreonine, L-β-homotryptophan, L^-homotyrosine, L-β-homovaline, 3-amino- phenylpropionic acid, 3-amino-chlorophenylbutyric acid, 3-amino-fluorophenylbutyric acid, 3-amino-bromopheynyl butyric acid, 3-amino-nitrophenylbutyric acid, 3-amino- methylphenylbutyric acid, 3-amino-pentanoic acid, 2-amino-tetrahydroisoquinoline acetic acid, 3-amino-naphthyl-butyric acid, 3-amino-pentafluorophenyl-butyric acid, 3-amino- benzothienyl-butyric acid, 3-amino-dichlorophenyl-butyric acid, 3-amino-difluorophenyl- butyric acid, 3-amino-iodophenyl-butyric acid, 3-amino-trifluoromethylphenyl-butyric acid, 3-amino-cyanophenyl-butyric acid, 3-amino-thienyl-butyric acid, 3-amino-5- hexanoic acid, 3-amino-furyl-butyric acid, 3-amino-diphenyl-butyric acid, 3-amino-6- phenyl-5-hexanoic acid and 3-amino-hexynoic acid.

Sugar amino acids are sugar moieties containing at least one amino group as well as at least one carboxyl group. Sugar amino acids may be based on pyranose sugars or furanose sugars. Suitable sugar amino acids may have the amino and carboxylic acid groups attached to the same carbon atom, a-sugar amino acids, or attached to adjacent carbon atoms, β-sugar amino acids. Suitable sugar amino acids include but are not limited to:

Sugar amino acids may be synthesized starting from commercially available monosaccharides, for example, glucose, glucosamine and galactose. The amino group may be introduced as an azide, cyanide or nitromethane group with subsequent reduction. The carboxylic acid group may be introduced directly as C0₂, by Wittig reaction with subsequent oxidation or by selective oxidation of a primary alcohol. 2010/001299

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Crosslinkers can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_a and N_a-methylamino acids and the introduction of double bonds between C_a and Cp atoms of amino acids.

Non-limiting examples of chemotherapeutic agents associated with cancer therapy include alkylating agents such as procarbazine, nitrogen mustard, chlorambucil, and platinum compounds such as cisplatin and oxaliplatin, also compounds such as melphalan, mustagen, carmustine and lomustine.

The present invention identifies new targets for the identification of agents useful in conserving or enhancing fertility. The targets include PUMA and TP63 and NOXA either as polypeptides or nucleic acid molecules (R A or DNA as genes in situ, isolated nucleic acid molecules or cellular transcribed RNAs). Targets further include moieties that interact with PUMA or TP63 or NOXA or PUMA or TP63 or NOXA and modulate their activity and also include the complex comprising PUMA and TP63. Typically, agents interact with binding (interacting) or activity sites within a given target molecule. Thus, for the purposes of screening and design the skilled person may use fragments or variants of target molecules to facilitate screening and design.

Screening procedures may conveniently include an assay for the presence of binding between a putative agent and a target as well as screening for a change in function of a complex or the ability to form a complex. One suitable assay includes an amplified luminescent proximity homogenous assay. Biocore assays may also be usefully employed. Such methods known in the art and are described for example in Best et al., PNAS., 101; 10 001299

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17622-17627, 2004, incorporated herein by reference). Cell-based screening procedures are contemplated including the use of puma and or noxa or puma and noxa deficient cells.

Candidate agent may be identified by methods comprising: i) contacting the candidate agent with a system comprising TP63 and or PUMA and or NOXA in nucleic acid or proteinaceous form or a fragment or variant thereof (collectively referred to as the target) and ii) determining the presence of a complex between the agent and the target, a change in activity of the complex or a component thereof, or a change in the level of an indicator of the activity of the complex or a component thereof. One form of assay involves competitive binding assays.

In some embodiments, agents that interact with (e.g. bind to) the present targets or a may be identified in a cell-based assay where a population of cells expressing one or more targets is contacted with a candidate agent and the ability of the candidate agent to interact with the target/s is determined. Preferably, the ability of a candidate agent to interact with the target or component thereof is compared to a reference range or control. In some embodiments, the ability of the candidate agent to interfere with TP63 or PUMA or NOXA activity is determined in vitro. If desired, this type of assay may be used to screen a plurality (e.g. a library) of candidate agents using a plurality of cell populations expressing target/s. If desired, this assay may be used to screen a plurality (e.g. a library) of candidate agents. The cell, for example, can be of prokaryotic origin (e.g. E. coli) or eukaryotic origin (e.g. yeast or mammalian). Further, the cells can express the target/s endogenously or be genetically engineered to express the target/s. In some embodiments, the ability of a candidate agent to modulate a TP63 isoform dependent PUMA gene transcription is assayed in cells transfected with a reporter construct using techniques known in the art.

In some embodiments, drug candidates are tested for their ability to disrupt TP63 binding. In order to modulate apoptosis and/or the level and/or activity of TP63 and or PUMA and NOXA, the agent will be combined with a cell in vitro or in vivo. Combining the agent and the cell may be achieved by any method known in the art. In some embodiments the cell has been isolated from the organism and combining occurs in vitro. In other embodiments the cell has not been isolated from the organism and combining the molecule and the cell occurs in vivo. The molecule may be combined with the cell directly, i.e. applied directly to the cell. Alternatively the molecule may be combined with the cell indirectly, eg by injecting the molecule into the bloodstream of an organism, which then carries the molecule to the cell or via application to the skin or for example, vaginally. The inhibitors, agents and medicaments contemplated for use in the present invention include small chemical molecules, membrane penetrating or membrane penetrating immune-like molecules, cell penetrating peptides, nucleic acid molecules and conjugates comprising one or more of these molecules. Illustrative conjugates include iRNA , molecules conjugated to cell or membrane penetrating peptides capable of delivery to reproductive cells as described in Abes et al,, Journal of Controlled Release, 77(5:304-313, 2006.

Agents are tested inter alia for their ability to modify apoptosis in a cell. Many different methods have been devised to detect apoptosis such as uptake of vital cellular dyes (eosin red, trypan blue, alamar blue), TU EL (TdT-mediated dUTP Nick-End Labeling) analysis, ISEL (in situ end labeling), and DNA laddering analysis for the detection of fragmentation of DNA in populations of cells or in individual cells, Annexin-V staining that measures alterations in plasma membranes, detection of apoptosis related proteins such as caspases (including caspase activity or activation), Bcl-2 family proteins and p53. These are techniques known to the skilled person.

Similarly, methods are known to the skilled person for directly or indirectly detecting the level and/or activity of PUMA or TP63 or NOXA. Protein forms may be monitored immunohistochemically or by immuno-assay. Nucleic acids may be monitored, for example, by any method known in the art such as by quantitative PGR, RT-PCT, dot blots, Northerns, or other forms of marker detection. The protein can be purified from the cell, such as by chromatographic techniques, and compared to the protein purified from a cell which has not been subjected to the method of the invention. PUMA instigates apoptosis by engaging through its alpha helical BH3-domain apoptosis inducing molecules such as Bad and Bax which undergo conformational changes, aggregation and damage mitochondrial membranes leading to cell death. In some embodiments, the ability of U2010/001299

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PUMA and NOXA to promote apoptosis is limited by binding of other Bcl-2 family molecules such as Bcl-2, Mcl-l, Bcl-xL and Al. Puma activity may be measured for example by measuring binding or modified activity between PUMA and its interacting Bcl-2 family partners.

The three-dimensional structures of Tp63 and Puma and Noxa have been determined and this facilitates the design of binding agents that modulate their activity. Three-dimensional representations of the structure of one or more binding sites are used to identify interacting molecules that, as a result of their shape, reactivity, charge potential etc. favorably interacts or associate. In a preferred aspect, the skilled person can screen three-dimensional structure databases of compounds to identify those compounds having functional groups that will fit into one or more of the binding sites. Combinational chemical libraries can be generated around such structures to identify those with high affinity binding to target binding sites. Agents identified from screening compound databases or libraries are then fitted to three-dimensional representations of TP63 or Puma binding sites in fitting operations using, for example docking software programs.

A potential modulator may be evaluated "in silico" for its ability to bind to a TP63 or NOXA or PUMA active site prior to its actual synthesis and testing. The quality of the fit of such entities to binding sites may be assessed by, for example, shape complementarity by estimating the energy of the interaction (Meng et al, J. Comp. Chem., 13: 505-524, 1992).

Once a binding compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e. the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should of course be understood that components known in the art to alter conformation should be avoided. 10 001299

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Putative binding agents may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the one or more binding sites. Selected fragments or chemical entities may then be positioned in a variety of orientations, or "docked," to target binding sites. Docking may be accomplished using software, such as QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM or AMBER. Specialized computer programs may be of use for selecting interesting fragments or chemical entities. These programs include, e.g., GRID (Oxford University, Oxford, UK), 5 MCSS (Molecular Simulations, USA), AUTODOCK (Scripps Research Institute, USA), DOCK (University of California, USA), XSITE (University College of London, UK) and CATALYST (Accelrys).

Useful programs to aid the skilled addressee in connecting chemical entities or fragments include CAVEAT (University of California, USA), 3D database systems and HOOK (Molecular Simulations, USA) De-novo ligand design methods include those described in LUDI (Molecular Simulations, USA), LEGEND (Molecular Simulations, USA), LeapFrog (Tripos Inc.,) SPROUT (University of Leeds, UK) and the like.

Structure based ligand design is well known in the art and various strategies are available which can build on structural information to determine ligands which effectively modulate the activity of PUMA. Molecular modelling techniques include those described by Cohen et ah, J. Med. Chem., 33:883-894, 1990, and Navia et ah, Current Opinions in Structural Biology, 2: 202-210, 1992. Standard homology modelling techniques may be employed in order to determine the unknown three-dimensional structure or molecular complex. Homology modelling involves constructing a model of an unknown structure using structural coordinates of one or more related protein molecules, molecular complexes or parts thereof. Homology modelling may be conducted by fitting common or homologous portions of the protein whose three-dimensional structure is to be solved to the three- dimensional structure of homologous structural elements in the known molecule. Homology may be determined using amino acid sequence identity, homologous secondary structure elements and/ or homologous tertiary folds. Homology modelling can include rebuilding part or all of a three-dimensional structure with replacement of amino acid residues (or other components) by those of the related structure to be solved. Using such a three-dimensional structure, researchers identify putative binding sites and then identify or design agents to interact with these binding sites. These agents are then screened for a modulatory effect upon the target molecule.

In some embodiments, binding agents are designed with a deformation energy of binding of not greater than about 10 kcal/mole, more preferably not greater than 7kcal/mole. Computer software is available to evaluate compound deformation energy and electrostatic interactions. For example, Gaussian 98, AMBER, QUANTA, CHARMM, INSIGHT II, DISCOVER, AMSOL and DelPhi.

Libraries of small organic molecules can be generated and screened using high-throughput technologies known to those of skill in this area. See for example US Patent No. 5,763,263 and US Application No. 20060167237. Combinatorial synthesis provides a very useful approach wherein a great many related compounds are synthesized having different substitutions of a common or subset of parent structures. Such compounds are usually non-oligomeric and may be similar in terms of their basic structure and function, varying in for example chain length, ring size or number or pattern of substitutions. Virtual libraries may also, as mentioned above, be constructed and compounds tested in silico (see for example, US Application No. 20060040322) or in vitro or in vivo assays known in the art.

A TP63 or PUMA or NOXA molecule including homologs from species other than human, may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labelled probe having the sequence of a desired nucleic acid and isolating full-length cDNA and genomic clones containing said nucleic acid sequence. Such hybridization techniques are well known in the art. One example of stringent hybridization conditions is where attempted hybridization is carried out at a temperature of from about 35° C to about 65° C using a salt solution of about 0.9M. However, the skilled person will be able to vary such conditions as appropriate in order to take into account variables such as probe length, base composition, type of ions present, etc. For a high degree of selectivity, relatively stringent conditions such as low salt or high temperature conditions, are used to form the duplexes. Highly stringent conditions include hybridization to filter-bound DNA in 0.5M NaHP04, 7% sodium dodecyl sulphate (SDS), ImM EDTA at 65° C, and washing in O.lxSSC/0.1% SDS at 68° C (Ausubel F.M. et al, eds., Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3, 1989). For some applications, less stringent conditions for duplex formation are required. Moderately stringent conditions include washing in 0.2xSSC/0.1% SDS at 42° C (Ausubel et al., 1989, {supra)). Hybridization conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilize the hybrid duplex. Thus, particular hybridization conditions can be readily manipulated, and will generally be chosen as appropriate. In general, convenient hybridization temperatures in the presence of 50% formamide are: 42° C for a probe which is 95-100% identical to the fragment of a gene encoding a polypeptide as defined herein, 37° C for 90-95% identity and 32° C for 70-90% identity.

One skilled in the art will understand that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5" end of the cDNA. Methods to obtain full length cDNAs or to extend short cDNAs are well known in the art, for example RACE (Rapid amplification of cDNA ends; e.g. Frohman et al, Proc. Natl. Acad. Sci USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon® technology (Clontech Laboratories Inc.) have significantly simplified the search for longer cDNAs. This technology uses cDNAs prepared from mRNA extracted from a chosen tissue followed by the ligation of an adaptor sequence onto each end. PCR is then carried out to amplify the missing 5'-end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using nested primers which have been designed to anneal with the amplified product, typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence. The products of this reaction can then be analyzed by DNA sequencing and a full length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full length PCR using the new sequence information for the design of the 5' primer. Recombinant PUMA or TP63 or NOXA polypeptides or fragments or variants thereof may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. The sequences of human genes and proteins can be found under appropriate database such as Genbank Accession Nos. These sequences and their accession numbers may be updated from time to time and all such modifications are contemplated and encompassed herein. The appropriate nucleic acid sequence may be inserted into an expression system by any variety of well known and routine techniques, such as those set forth in Sambrook et ah, (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbour laboratory Press, Cold Spring Harbour, NY, 1989). In some embodiments, PUMA and TP63 and NOXA polypeptides or their encoding sequences may be generated synthetically.

If a polypeptide is to be expressed for use in cell-based screening assays, the appropriate nuclear targeting signal should be incorporated (see for a review, Pouton et ah, Adv. Drug Delivery. Reviews, 59: 698-717, 2007). If the polypeptide is secreted into the medium, the medium can be recovered in order to isolate said polypeptide.

Polypeptides can be recovered and purified from recombinant cell cultures or from other biological sources by well known methods including, ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, affinity chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, molecular sieving chromatography, centrifugation methods, electrophoresis methods, lectin chromatography, FPLC and HPLC. Combinations of these methods can be used as known by those skilled in the art. The present invention is further directed to compositions such as pharmaceutical compositions comprising a TP63 or PUMA or NOXA antagonist herein contemplated. The terms "antagonist", "modifier", "compound", "active agent", "moiety", "pharmacologically active agent", "medicament", "active" and "drug" are used interchangeably herein to refer to a molecule that induces a desired pharmacological and/or physiological effect and in particular antagonizes target activity or function or formation. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents contemplated herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "antagonist", "modifier", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "compound" is not to be construed narrowly as it extends to inorganic and organic molecules including genetic molecules, peptides, polypeptides and proteins and chemical analogs thereof. The terms include combinations of two or more actives such as one or more inhibitors of a target activity or complex formation or a compound therein. A "combination" also includes a two-part or more such as a multi-part pharmaceutical composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.

The subject agents are administered in an effective amount. The terms "effective amount" and "therapeutically effective amount" of an agent as used herein mean a sufficient amount of the agent to provide in the course the desired therapeutic or physiological effect in at least a statistically significant number of subjects. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount". The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation. In some 10 001299

- 54 - embodiments, an effective amount for a human subject lies in the range of about O. lng/kg body weight/dose to lg/kg body weight/dose. In some embodiments, the range is about ^g to lg, about lmg to lg, lmg to 500mg, lmg to 250mg, lmg to 50mg, or ^g to lmg/kg body weight/dose. Dosage regimes are adjusted to suit the exigencies of the situation and may be adjusted to produce the optimum therapeutic dose. For example, several doses may be provided daily, weekly, monthly or other appropriate time intervals. Thus, the time and conditions sufficient for conserving fertility can be determined by one skilled such as a medical practitioner who is able to specify a therapeutically or prophylactively effective amount.

By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

Similarly, a "pharmacologically acceptable" salt, ester, emide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.

Hence, the present invention provides a pharmaceutical composition comprising a target suppressive agent and one or more pharmaceutically acceptable carriers, diluents and/or excipients.

A medical protocol is also provided comprising the use of an agent which suppresses PUMA expression or PUMA activity one or more agents which suppress PUMA and NOXA expression or PUMA and NOXA activity and optionally combining this with cytotoxic cancer treatment, transfusion or another procedure. For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral, solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, International Patent Publication No. WO 96/11698. For parenteral administration, the compound may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.

The active agent is preferably administered in a therapeutically effective amount. The actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, (supra). Alternatively, targeting therapies may be used to deliver the active agent more specifically to reproductive tissues by the use of targeting systems such as antibody fragments or cell specific or cell penetrating ligands or vectors known in the art. Targeting may be desirable for a variety of reasons, e.g. to avoid targeting other areas or cells of the body (such as cancer cells), if the agent is unacceptably toxic or if it would otherwise require too high a dosage or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be produced in the target cell, e.g. in a viral vector such as those described above or in a cell based delivery system such as described in U.S. Patent No. 5,550,050 and International Patent Publication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635. The vector could be targeted to the target cells or expression of expression products could be limited to specific cells, stages of development or cell cycle stages. Cell based delivery system may be designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the target agent. Alternatively, the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See, for example, European Patent Application No. 0 425 731 A and International Patent Publication No. WO 90/07936.

In accordance with this aspect of the present invention, reproductive cells may be treated with a genetic composition encoding polynucleotide or peptide agents. Typically, the vector may combine with the host genome and be expressed therefrom.

Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman (In: Therapy for Genetic Disease, T. Friedman, Ed., Oxford University Press, pp. 105-121, 1991) or Culver {Gene Therapy: A Primer for Physicians, 2^nd Ed., Mary Ann Liebert, 1996). Suitable vectors are known, such as disclosed in U.S. Patent No. 5,252,479, International Patent Publication No. WO 93/07282 and U.S. Patent No. 5,691,198. Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and non- viral transfer methods. Non- viral gene transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes, direct DNA uptake, receptor-mediated DNA transfer and nucleofection. Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery. Expression vectors in the context of gene therapy are meant to include those constructs containing sequences sufficient to express a polynucleotide that has been cloned therein. In viral expression vectors, the construct contains viral sequences sufficient to support packaging of the construct. If the polynucleotide encodes a N-terminal deleted TP63 isoform, for example, expression will produce the RNA and ultimately the polypeptide. If the polynucleotide encodes a sense or antisense polynucleotide or a ribozyme or DNAzyme, expression will produce the sense or antisense polynucleotide or ribozyme or DNAzyme. Thus, to clarify, expression does not require that a protein product be synthesized. In addition to the polynucleotide cloned into the expression vector, the vector also contains a promoter functional in eukaryotic cells. The cloned polynucleotide sequence is under control of this promoter. Suitable eukaryotic promoters are routinely determined.

Receptor-mediated gene transfer may be achieved by conjugation of DNA to a protein ligand via polylysine. Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type. Receptors on the surface of liver cells may be advantageously targeted. These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs. To overcome the problem of intracellular destruction of DNA, co-infection with adenovirus can be included to disrupt endosome function.

Diagnosis of a transition to perimenopause or menopause in a female is routinely practised by the subject or a physician. Diagnosis typically relies upon on a consideration of the signs and symptoms of menopause or perimenopause and may include blood tests to determine levels or relative levels of hormones or factors normally associated with the transition to perimenopause or menopause. Recent research has suggested that it is possible to diagnose the risk of developing loss of fertility including that caused during menopause within a certain time range (or span) by assessing levels of one or more menopause associated hormones or factors in a sample from a subject and comparing the level or these levels with levels in a reference sample taken from a control subject. For example, International Publication No. WO 2007/125317 discloses an assay to determine fertility in a female by detecting the presence or concentration of polypeptides selected from Inhibin B, anti-Mullerian hormone and follicle stimulating hormone in a biological sample from the subject. The concentration of the respective hormone/s is used to provide a measure of fertility in terms of the number of oocytes retained by the female.

Accordingly, in some further embodiments a subject at risk or seeking to determine their risk of developing reduced fertility through menopause is tested by assessing the levels of one or more hormones or other factors associated with menopause in a sample from the subject. In some embodiments, the level or levels are compared with the level or levels of the respective hormone or factor in a reference sample obtained from a control subject. If the output from this analysis indicates that the subject is at risk of developing reduced fertility, i.e. is less than a predetermined period of time to entering perimenopause or entering menopause, then, an agent that suppresses PUMA expression or PUMA polypeptide activity is administered to the female. Alternatively, an agent that suppresses NOXA expression or NOXA polypeptide activity is administered. Alternatively, an agent that suppresses TP 63 expression or TP63 activity may be administered to the subject for a time and under conditions sufficient to delay onset or progression of perimenopause or menopause. In some embodiments the subject is retested in order to determine or monitor changes in the level or levels of one or more menopause associated hormones or factors post-treatment. In some embodiments the reference sample is a sample taken from the subject at an earlier time point. The age at which women start to lose fertility or enter perimenopause varies considerably. Accordingly, a reference sample could be obtained at several time points through the female subjects 20s, 30s, 40s and 50s, as required. Control subjects include groups of subjects falling with various categories such as non- menopausal, perimenopausal and menopausal.

In some embodiments the level of hormones or other factors associated with menopause from the test sample is compared to one or more threshold levels predetermined to distinguish between, for example non-menopause, perimenopause or menopause in a subject. Thresholds may be established by obtaining samples and determining a level or levels of analytes at an earlier development stage, to which later results may be compared. In these cases, the individual acts as their own control group. In some embodiments, a positive likelihood ratio is used to measure the ability of the method to predict menopause or perimenopause or treatment outcome.

In some embodiments, follicular development is assessed by assessing levels of anti- Miillerian hormone (AMH) in a female subject.

In another embodiment, one or more of the suppressive agents described herein is used ex vivo to conserve fertility by conserving oocytes that might otherwise undergo apoptosis, such as during in vitro fertilisation (IVF) procedures. Accordingly, in some embodiments, the present invention provides a method of preserving fertility during IVF treatment comprising contacting oocytes ex vivo with a herein described suppressive agent. In some embodiments, compositions comprising the suppressive agents are added to the fluid that is used to preserve eggs, such as for IVF treatment.

The following methods were used in developing the invention:

In vitro whole organ culture of ovaries

Ovaries were harvested from Pn5C57BL/6 females and cultured on floating filters in MEM/Ham's-F12 supplemented with 0.01% BASA, 0.01% Albumax II, 0.05mg/ml L- ascorbic acid, 5X ITS-X, 75 μg/mL penicillin-G and 50 μg/mL streptomycin sulfate (all supplied by Gibco), at 37°C in 5% C0₂. Ovaries were cultured for 2 h in the presence of Imatinib (10 μΜ) or vehicle (control) before being γ-irradiated (0.45 Gy), treated with cisplatin (20 μΜ) or being left untreated (control). Ovaries were collected for analyses after a further 6, 24 or 48 h of incubation. Cisplatin (Mayne Pharma) was diluted in saline. Imatinib and cisplatin doses were selected as disclosed by Gonfloni et al, Nat Med, 75:1179-1185, 2009.

Generation and genotyping of mice

The generation and genotyping of Puma^'1', Noxa^'1' (Villunger et al. 2003) (both generated on an inbred C57BL/6 background using C57BL/6-derived ES cells), Puma^'1' Noxa^"1' (Michalak et al. 2008) and Trp53^'!' mice (Jacks et al. 1994) (generated on a mixed C57BL/6xl29SV background but backcrossed with C57BL/6 >20 generations) have been described. Mice were housed in The Walter and Eliza Hall Institute (WEHI) mouse breeding facility (Parkville, VIC) under controlled conditions of 12 h light: 12 h dark, with free access to water and mouse chow in pathogen free conditions. Doses for γ-irradiation (0.45 or 4.5 Gy of γ-irradiation from a ⁶⁰Co source; Theratron Phoenix, Theratronics) were chosen because they have previously been shown to kill all primordial follicles in PN5 mice without causing significant morbidity or mortality (Suh et al., 2006 {supra)). Mice were killed and ovaries harvested at 3 or 6 h post treatment or at PN10. Alternatively, mice were observed until 7 weeks of age when breeding trials commenced. Follicle quantification

Representative primordial, primary and secondary follicle numbers (mean ± SEM) were expressed per 10⁴ μπι² ovarian tissue area. Randomly selected left or right ovaries from γ- irradiated cisplatin-treated or control (untreated) mice were fixed for 2 h in Bouin's fluid, processed into paraffin and 5 μηι serial sections of each ovary were stained with haematoxylin and eosin. From each set of serial sections, the middle section and two to three other sections located at 100 μιη intervals on either side of the middle section (to avoid counting follicles twice) were selected for semi-quantitative estimation of follicles using morphological criteria previously described (Myers et al, Reprod., 127: 569, 2004; Kerr et al, Reprod., 132: 95, 2006). Follicles with a morphologically normal oocyte nucleus were counted in the 3-5 selected sections per ovary and the area (in μηι²) of the ovarian sections was measured with image analysis software. Pyknotic oocytes showed U2010/001299

- 61 - densely-strained compacted nuclear chromatin as single or several clumps and intense eosinophilic cytoplasm. When the number of pkynotic bodies per ovary section exceeded 100, for quantitative data was expressed as > 5/10⁴ μηι² tissue area. In situ hybridisation and immunostaining

Puma probes (sense and antisense) were generated using IMAGE:6310857 clone as a template and ISH was performed as described (Hurt et ah, Biol Reprod., 75: 421, 2006). Probes were detected immuno-histochemically by using the DIG Nucleic Acid Detection Kit (Roche Diagnostics) according to the manufacturer's protocols. Positive staining was indicated by the development of a dark purple/brown color. Sections were counterstained with hematoxylin. Additional negative controls, were performed by in situ hybridization with Puma probes on ovaries from Puma-/- mice and with Noxa probes on ovaries from Noxa^'1' mice. Formalin or 4% PFA fixed ovaries (n=3 mice/group) were processed into paraffin and 5 μηι sections were cut. The identity of oocytes in untreated and irradiated ovaries was confirmed using antibodies specific for the germ cell-specific Y-box cytoplasmic protein marker MS Y2 (supplied by Dr Richard Schultz, University of Pennsylvania, Philadelphia, PA, USA) and antibodies specific for the nuclear marker germ cell nuclear antigen (GCNA, supplied by Dr George Enders, University of Kansas Medical Centre, Kansas City, KS, USA). Antigen retrieval was performed by microwaving for 10 min in 0.01 M citrate buffer pH 6. Endogenous peroxidases were quenched for 30 min in 0.3% H₂0₂ and non-specific binding blocked for 1 h with 10% normal goat serum. Sections were then incubated first with primary antibody, followed by biotinylated secondary antibody for 1 h at room temperature. Sections were incubated for a further 30 min with the avidin-biotin- peroxidase complex (Vector Laboratories) and peroxidase activity was visualised using 3,30-diaminobenzidine (DAB) as the substrate. Sections were counterstained with hematoxylin. Stainings with Ig isotype-matched control antibodies were used as negative controls. As an additional negative control, immuno-histochemical staining was performed with anti-PUMA antibodies on ovaries from Puma^'1" mice. The primary antibodies used for immunofluorescence staining were anti-yH2AX antibody (1 :500, Upstate) and anti-puma antibody (1 :200, Abeam). Antigen retrieval and blocking was performed as described above. After incubation with primary antibodies overnight at 4°C, sections were incubated at room temperature for 1 h with goat anti-mouse IgG-Cy3 (1 :400, Jackson ImmunoResearch Laboratories) and goat anti-rabbit IgG-Alexa 488 (1 :1000, Molecular Probes) antibodies. Slides were mounted with Fluorsave (Calbiochem) and analysed by fluorescent confocal microscopy using an Olympus 1X81 microscope. γ-irradiation induced DNA damage was confirmed and quantified by counting the number of γ-Η2ΑΧ foci within the nucleus of 50-100 primordial follicles for each group analysed.

TUNEL staining

TUNEL staining was performed using ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore) according to the manufacturer's protocol. Fertility trials

Female mice (PN5) were exposed to 0.45 Gy of γ-irradiation and then allowed to mature until 7 weeks of age before commencing breeding trials with nonirradiated proven males (wt C57BL/6 and/or Puma^'1"). Litters were inspected at 0800 on the day of birth, then twice weekly and at weaning. Some litters of Puma^"1" mothers were fostered at birth (including all in Figure 5 and Table 5). Adult mice were γ-irradiated (4.5 Gy) at 7 weeks of age, mated to vasectomized males for 7 weeks and then mated with non-irradiated proven males (wt C57BL/6).

Statistical analysis

Data are presented as means ± SEM and statistical analysis of follicle numbers was performed using GraphPad Prism software (GraphPad Software Inc, La Jolla, CA, USA). Follicle quantification data were analyzed by one-way ANOVA and the significance determined by the Tukey's post hoc multiple comparison test. Comparisons between breeding outcomes and comparisons of number of γ-Η2ΑΧ foci were compared using a Student's T-test (unequal variance for breeding trials). Differences were considered significant when PO.05. The present invention is further described by the following non-limiting Examples.

9

- 64 -

EXAMPLE 1: Puma is induced after irradiation of oocytes in mice

Post natal day 5 (PN5) C57BL/6 wild type mice were exposed to whole body γ-irradiation (0.45 Gy) and oocytes were isolated using laser capture microdissection 3 and 6 hours post γ-irradiation. γ-irradiation (0.45 Gy) induced puma mR A and Puma protein expression in oocytes from postnatal day (PN) 5 C57BL/6 mice (see Figures 1 and 6, corresponding to Figure 1 A and Figure 2 in the provisional application).

In situ hybridisation and anti-Puma immunofluorescence staining revealed expression of puma mRNA and puma protein, respectively, were negligible in untreated wild type oocytes, but both were induced within 3 hours of whole body irradiation (0.45 Gy) and persisted at 6 hours.

Expression of noxa mRNA was also induced in wt and puma^'1" primordial follicles 3 h following γ-irradiation (0.45 Gy) (see Figure 2, corresponding to Figure 3 in the provisional application). EXAMPLE 2: Follicles of puma or puma and noxa deficient mice are protected from apoptosis in γ-irradiated mice

In order to examine whether Puma and/or Noxa are the essential pro-apoptotic target genes for p63 gene deletion studies were conducted in mice. PN5 wild type mice lacking either Trp53, noxa or puma or puma and noxa were exposed to low (0.45 Gy) or high (4.5 Gy) dose γ-irradiation and their ovaries harvested at PN10. Follicle numbers were then determined. In the baseline non-irradiated state, mice deficient in puma or deficient in puma and noxa had significantly greater numbers of primordial follicles compared to wild type or Noxa^"7" mice (see Figure 3A) revealing that Puma- and Noxa-mediated apoptosis limits oocyte supply during the establishment of the primordial follicle pool in the developing ovary. γ-irradiation of wild type mice with 0.45 and 4.5 Gy resulted in complete destruction of the primordial follicle pool in wild type and Trp53^' mice as expected (see Suh et ah, 2006 {supra)) (Figure 3 A and B), In striking contrast, after 0.45 Gy γ-irradiation, 16% ± 3% (mean ± SEM, range 9-27%) of primordial follicles were protected from apoptosis in puma^"1' mice and 52% ± 6% (range 25-71%) in puma^'1' noxa^"1" mice. Following 4.5 Gy γ-irradiation 12 ± 1% (mean ± SEM, range 7-15%) of primordial follicles in Puma ^1' mice (pO.001) and 94 ± 8% in Puma^''^' Noxa^''^' mice (range 78-100%; Puma^'^Noxa^'1' vs wt: pO.001; Puma^'1' Noxa^"1' vs Puma^''^': pO.001) were protected from apoptosis. No protection was observed in mice lacking Noxa alone or in mice lacking Noxa and one allele of Puma {Puma ^'1" Noxa ^1' ; Figure 3 and data not shown).

EXAMPLE 3: DNA damage post irradiation Evidence of DNA damage post γ-irradiation was observed by staining for γ-Η2ΑΧ, although no difference was seen in the levels of damage observed, between either wild type, puma^'1' mice (see Figure 4 and 11 , corresponding to Figure 5 in the provisional application). The data shows that γ-irradiation causes a similar extent of DNA damage in oocytes from wt and Puma ^1" mice.

EXAMPLE 4: Reproductive potential of puma deficient mice subject to DNA damage by irradiation

Endogenous, environmental or anti-cancer therapy-induced DNA damage can cause oocyte depletion resulting in female sterility. It was determined whether resistance to DNA damage-induced apoptosis afforded to oocytes by loss of Puma could preserve fertility. Remarkably, 13 out of 13 γ-irradiated puma^1' females produced viable offspring at birth when mated with non-irradiated wt or puma'^' proven males, whereas all (5/5) γ-irradiated wt females proved to be infertile, as expected (see Figure 5, corresponding to Figure 6 in the provisional application). Twelve out of thirteen γ-irradiated puma'^' females produced >2 litters. All eleven 1^st and 2^nd litters from γ-irradiated puma^"/' females mated to wt males produced healthy mice at weaning, in similar proportions to those observed for non- irradiated litters (see Figure 5, corresponding to Figure 6 in the provisional application), confirming that irradiated primordial follicles could give rise to healthy pups. Four female puma ^1' offspring of γ-irradiated mothers, when themselves mated at 7 weeks of age, also produced live pups at birth, with three out of four producing similar numbers of healthy pups at weaning as those reported above (Table 5). These data indicate that the 16% on average of Puma ^1' oocytes rescued from γ-irradiation induced apoptosis (0.45 Gy) are functionally robust and are capable of giving rise to normal proportions of healthy offspring.

Accordingly, by way of general summary, these data establish that DNA damage-induced apoptosis of primordial follicle oocytes is dependent on TAp63 -mediated transcriptional induction of Puma and to a lesser extent, Noxa. Puma and Noxa are essential effectors for TAp63a-mediated apoptosis. These two BID -only proteins are responsible for apoptosis of oocytes that have sustained DNA damage postnatally. Whilst apoptosis might be the preferred mechanism for protecting germ line integrity, when apoptosis is blocked, DNA repair processes may be capable of rescuing oocyte quality. Thus, fertility may be prolonged during normal ageing and preserved during cytotoxic anti-cancer therapy by inhibition of TAp63a-mediated apoptosis in oocytes through blockade of Puma.

EXAMPLE 5: Puma and Noxa mediate p63 -dependent oocyte apoptosis

Experiments were conducted to identify the down-stream effectors that are critical for p63~ mediated oocyte apoptosis. The hypothesis that the pro-apoptotic BH3-only Bcl-2 family members, Puma and Noxa, may be critical for DNA damage-induced, p63 -mediated apoptosis in oocytes was supported by the finding herein that γ-irradiation (0.45 or 4.5 Gy) induced Puma as well as Noxa mRNA and PUMA protein expression in oocytes from postnatal day 5 (PN5) C57BL/6 mice (Figures 1, 2, 6 and 7) and Trp53-I- mice (Figure 8). EXAMPLE 6: PUMA and NOXA account for most, if not all apoptosis triggered by TAp63 in oocytes

To support the histological enumeration of oocytes in particular follicle types, the identity of oocytes in untreated and γ-irradiated ovaries was confirmed by staining with antibodies to the germ cell specific Y-box cytoplasmic protein marker, MSY2 and the nuclear marker, germ cell nuclear antigen, GCNA (Figure 3B and Figures 9 A, B). Growing follicles (primary and pre-antral), which are intrinsically resistant to γ-irradiation induced apoptosis were present after γ-irradiation in mice of all genotypes. In contrast, only remnants of empty primordial follicles in which no viable oocyte remained, were evident in γ-irradiated wt mice. Strikingly, primordial follicles that survived γ-irradiation were readily detectable in Puma^"1" and even more so in Puma^'1' Noxa^'1' mice (Figures 9A, B). TUNEL staining confirmed that γ-irradiation induced apoptosis in primordial follicle oocytes of wt mice, and this was substantially reduced in Puma^"1" mice (Figure 10). Histological examination and immuno-histochemical staining revealed a complete absence of normal primordial follicles in γ-irradiated wt, Trp53^''^' and Noxa^'1' ovaries (Figures 3 A, B and Figures 9 A, B). Remarkably, the protection of histologically normal primordial follicles observed at the higher dose of γ-irradiation in Puma^{"1 "}Noxa^"1' mice (Figure 3B) was comparable to that reported for loss of TAp63a (Suh et ah, 2006 (supra)), demonstrating that these two BH3- only proteins account for most, and perhaps all, of the death inducing activity of this transcription factor. The more potent killing activity of PUMA protein probably reflects its avid binding to all pro-survival BCL-2-like proteins, whereas NOXA displays selective binding activity to only MCL-1 and Al (Chen et al., Mol Cell 17: 393-403, 2005; uwana et al., Mol Cell 77(4): 525-535, 2005).

The improved rescue from apoptosis of oocytes observed post-higher dose γ-irradiation in Puma^'^Noxa^'1' mice (compared to the lower dose γ-irradiation) may reflect other, as yet undefined mechanisms of protection, such as a dose response in the balance between DNA damage, DNA repair, and apoptosis. Immunostaining for a marker of double-strand DNA breaks (γ-Η₂ΑΧ) at the early time-points (3-6 h), revealed that γ-irradiation induced comparable levels of DNA damage in oocytes from wt, Puma^"1" and Pumd^Noxa^"1' mice, with increased DNA damage being observed, at least initially, at the higher dose (Figure 4 and 11). Although counter-intuitive, it is possible that the higher dose of γ-irradiation might trigger more effective but as yet undefined DNA repair processes in PN5 oocytes that may be related to activation of Rad51 protein (Kujjo et ah, PLoS One 5(2): e9204, 2010) or spatial/cluster arrangements (Paap et al, Nucleic Acids Res 36(8): 2717-2727, 2008). Of note, oocytes from young mice which did not undergo apoptosis as readily because they lacked the pro-apoptotic protein, BAX, were shown to have more efficient DNA repair, associated with maintenance of activity of RAD51 (Kujjo et al., 2010 (supra)). Collectively, these results establish that DNA damage-induced apoptosis of primordial follicle oocytes is dependent on TAp63 -mediated transcriptional induction of Puma and, to a lesser extent, Noxa.

EXAMPLE 7: DNA damage-induced oocyte apoptosis and fertility loss requires p63- mediated induction of Puma and Noxa but not an Imatinib-sensitive kinase

Gonfloni et al. , 2009 (supra) have reported that inhibition of c-Abl with the tyrosine kinase inhibitor Imatinib protects oocytes from DNA damage, and therefore proposed that c-Abl is critical for p63-mediated apoptosis. They do not suggest that PUMA or NOXA and PUMA are the primary targets of p63 in ovaries as deslosed herein nor do they provide any suggestion to the skilled artisan that suppression of PUMA or NOXA and PUMA will be effective in conserving fertility. However, as the relationship between c-Abl and p63 isoforms is complex (Ongkeko et al, Laryngoscope, 6^'; 1390-1396, 2006, Leong et al, J Clin Invest, 7 7: 1370-1380, 2007) and as Imatinib itself is known to induce apoptosis in certain cell types (Kuribara et al, Mol Cell Biol, 24: 6172-6183, 2004, Kuroda et al. Proc Natl Acad Sci USA, 103: 14907-14912, 2006), the impact of Imatinib on the response of oocytes to DNA-damage was independently investigated as described herein.

Firstly, the ability of Imatinib to protect oocytes from γ-irradiation-induced death was investigated. For in vitro analysis, Imatinib (20 μΜ) was added to whole postnatal day (PN) 5 wt ovary cultures two hours prior to γ-irradiation (0.45 Gy). Quantification of follicle numbers after a further 48 h in culture showed that the number of surviving, histologically normal primordial oocytes did not increase when γ-irradiated ovaries were also treated with Imatinib (Figure 12a). 562 human Phi + (Bcr-ABL+) leukemia-derived cells were treated with the indicated concentrations of Imatinib for 72 h, showing the expected apoptotic response, confirming biologic activity of Imatinib. Viability of K562 cells was determined by the Cell Titre Glo assay, relative to no treatment control. For in vivo analysis, PN5 wt mice were treated with Imatinib (7.5 mg/kg i.p.) or vehicle and then 2 h later exposed to whole body γ-irradiation (0.45 Gy) or left non-irradiated. Ovaries were harvested at PN10 and ovarian follicles counted. Prior addition of Imatinib did not rescue primordial follicles from complete elimination due to γ-irradiation (Figure 12b). The ability of Imatinib to protect against cisplatin induced oocyte loss in vitro was also investigated. In contrast to data reported by Gonfloni et al, 2009 {supra), follicle numbers and TU EL staining showed that prior addition of Imatinib did not prevent cisplatin- induced elimination of oocytes after 24-48 h of whole ovary culture (Figure 12c and Figure 13). Interestingly, treatment with Imatinib alone increased by 2-fold or more the numbers of pyknotic bodies in ovaries both in vitro and in vivo (Figure 12a-c), indicating that the drug is active in this tissue and that an Imatinib-sensitive kinase, possibly c-Kit, is important for the survival of female germ cells (Hutt et al, Mol Hum Repod, 12: 61-69, 2006). Collectively, these findings demonstrate that Imatinib does not protect oocytes from apoptosis following γ-irradiation or cisplatin-induced DNA damage and thereby indicate that Imatinib-sensitive kinases are not required for 7>p<i3-mediated apoptosis in oocytes.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. TABLE 1: Summary of sequence identifiers

SEQUENCE ID NO: DESCRIPTION

1 nucleotide sequence of human PUMA isoform, transcript variant 1, mRNA (NCBI Reference Sequence:

NM_001127240.1)

2 nucleotide sequence of human PUMA isoform, transcript variant 2, mRNA (NCBI Reference Sequence:

NM_001127241.1)

3 nucleotide sequence of human PUMA isoform, transcript variant 3, mRNA (NCBI Reference Sequence:

NM_001127242.1)

4 nucleotide sequence of human PUMA isoform, transcript variant 4, mRNA (NCBI Reference Sequence:

NM_014417.3)

5 nucleotide sequence of mouse PUMA NM, mRNA (NCBI

Reference Sequence: NM_133234.2)

6 nucleotide sequence of mouse PUMA, variant 1, mRNA

(cDNA clone IMAGE:5133742), complete cds (GenBank

Accession No: BC044782.2)

7 nucleotide sequence of mouse PUMA, variant 2, mRNA, complete cds (GenBank Accession No: AF332560.1)

8 nucleotide sequence of mouse PUMA, variant 3, mRNA

(cDNA clone IMAGE:6310857), complete cds GenBank

Accession No: BC060370.1)

9 amino acid sequence encoded by SEQ ID NO: 1

10 amino acid sequence encoded by SEQ ID NO: 2

11 amino acid sequence encoded by SEQ ID NO: 3

12 amino acid sequence encoded by SEQ ID NO: 4

13 amino acid sequence encoded by SEQ ID NO: 5

14 amino acid sequence encoded by SEQ ID NO: 6

15 amino acid sequence encoded by SEQ ID NO: 7

16 amino acid sequence encoded by SEQ ID NO: 8

17 nucleotide sequence of human NOXA NM (NCBI Reference

Sequence: NM_021127.2)

18 nucleotide sequence of human NOXA, variant 1, mRNA

(cDNA clone MGC:45013 IMAGE:5529754), complete cds

(GenBank Accession No: BC032663.1) SEQUENCE ID NO: DESCRIPTION

19 nucleotide sequence of human NOXA, variant2, mRNA

(cDNA clone MGC:9202 IMAGE:3858958), complete cds

(GenBank Accession No: BC013120.1)

20 nucleotide sequence of human NOXA, variant3, mRNA

(GenBank Accession No: AK311943.1)

21 nucleotide sequence of mouse NOXA NM, mRNA (NCBI

Reference Sequence: NM_021451.2)

22 nucleotide sequence of mouse NOXA, variant 1 , mRNA, complete cds (GenBank Accession No: AB041230.1)

23 nucleotide sequence of mouse NOXA, variant 2, mRNA

(cDNA clone MGC:59280 IMAGE:6517820), complete cds

(GenBank Accession No: BC050821.1)

24 amino acid sequence encoded by SEQ ID NO: 17

25 amino acid sequence encoded by SEQ ID NO: 18

26 amino acid sequence encoded by SEQ ID NO: 19

27 amino acid sequence encoded by SEQ ID NO: 20

28 amino acid sequence encoded by SEQ ID NO: 21

29 amino acid sequence encoded by SEQ ID NO: 22

30 amino acid sequence encoded by SEQ ID NO: 23

31 nucleotide sequence of human tumor protein p63

(Tap63/TP63), alpha isoform, mRNA (NCBI Reference

Sequence: NM 003722.4)

32 nucleotide sequence of human tumor protein p63

(Tap63/TP63), beta isoform, mRNA (NCBI Reference

Sequence: NM_001114978.1)

33 nucleotide sequence of human tumor protein p63

(TAp63/TP63), gamma isoform, mRNA (NCBI Reference

Sequence: NM_001114979.1)

34 nucleotide sequence of mouse transformation related protein

63 (TAp63/TP63), alpha isoform, mRNA (NCBI Reference

Sequence: NM_001127259.1)

35 nucleotide sequence of mouse transformation related protein

63 (TAp63/TP63), beta isoform, mRNA (NCBI Reference

Sequence: NM_001127260.1)

36 nucleotide sequence of mouse transformation related protein

63 (TAp63/TP63), gamma isoform, mRNA (NCBI Reference Sequence: NM_001127261.1) SEQUENCE ID NO: DESCRIPTION

37 amino acid sequence encoded by SEQ ID NO: 31

38 amino acid sequence encoded by SEQ ID NO: 32

39 amino acid sequence encoded by SEQ ID NO: 33

40 amino acid sequence encoded by SEQ ID NO: 34

41 amino acid sequence encoded by SEQ ID NO: 35

42 amino acid sequence encoded by SEQ ID NO: 36

43 Human Puma nucleotide sequence transcript 1 -Exon 1

(Unspliced transcript sequence from the Ensembl genome browser version 55, Transcript ID: ENST00000300880)

44 Human Puma nucleotide sequence transcript 1 -Intron 1-2

45 Human Puma nucleotide sequence transcript 1 -Exon 2

46 Human Puma nucleotide sequence transcript 2 -Exon 1

(Unspliced transcript sequence from the Ensembl genome browser version 55, Transcript ID: ENST00000341983)

47 Human Puma nucleotide sequence transcript 2 -Intron 1-2

48 Human Puma nucleotide sequence transcript 2 -Exon 2

49 Human Puma nucleotide sequence transcript 2 -Intron 2-3

50 Human Puma nucleotide sequence transcript 2 -Exon 3

51 Human Puma nucleotide sequence transcript 3 -Exon 1

(Unspliced transcript sequence from the Ensembl genome browser version 55, Transcript ID: ENST00000359924)

52 Human Puma nucleotide sequence transcript 3 -Intron 1-2

(Unspliced transcript sequence from the Ensembl genome browser version 55, Transcript ID: ENST00000359924) P T/AU2010/001299

- 73 -

SEQUENCE ID NO: DESCRIPTION

53 Human Puma nucleotide sequence transcript 3 -Exon 2

54 Human Puma nucleotide sequence transcript 3 -Intron 2-3

55 Human Puma nucleotide sequence transcript 3 -Exon 3

56 Human Puma nucleotide sequence transcript 3 -Intron 3-4

57 Human Puma nucleotide sequence transcript 3 -Exon 4

58 Human Puma nucleotide sequence transcript 4 -Exon 1

(Unspliced transcript sequence from the Ensembl genome browser version 55, Transcript ID: ENST00000439096)

59 Human Puma nucleotide sequence transcript 4 -Intron 1-2

60 Human Puma nucleotide sequence transcript 4 -Exon 2

61 Human Puma nucleotide sequence transcript 4 -Intron 2-3

62 Human Puma nucleotide sequence transcript 4 -Exon 3

63 Human Puma nucleotide sequence transcript 4 -Intron 3-4

64 Human Puma nucleotide sequence transcript 4 -Exon 4

65 Human Puma nucleotide sequence transcript 5 -Exon 1

(Unspliced transcript sequence from the Ensembl genome . browser version 55, Transcript ID: ENST00000449228) SEQUENCE ID NO: DESCRIPTION

66 Human Puma nucleotide sequence transcript 5 -Intron 1-2

(Unspliced transcript sequence from the Ensembl genome browser version 55, Transcript ID: ENST00000449228)

67 Human Puma nucleotide sequence transcript 5 -Exon 2

68 Human Puma nucleotide sequence transcript 5 -Intron 2-3

69 Human Puma nucleotide sequence transcript 5 -Exon 3

70 Human Puma nucleotide sequence transcript 5 -Intron 3-4

71 Human Puma nucleotide sequence transcript 5 -Exon 4

72 Mouse Puma nucleotide sequence transcript 1 -Exon 1

(Unspliced transcript sequence from the Ensembl genome browser version 55, Transcript ID:

ENSMUST00000002152)

73 Mouse Puma nucleotide sequence transcript 1 -Intron 1-2

ENSMUST00000002152)

74 Mouse Puma nucleotide sequence transcript 1 -Exon 2

ENSMUST00000002152)

75 Mouse Puma nucleotide sequence transcript 1 -Intron 2-3

ENSMUST00000002152)

76 Mouse Puma nucleotide sequence transcript 1 -Exon 3

ENSMUST00000002152) SEQUENCE ID NO: DESCRIPTION

77 Mouse Puma nucleotide sequence transcript 1 -Intron 3-4

ENSMUST00000002152)

78 Mouse Puma nucleotide sequence transcript 1 -Exon 4

ENSMUST00000002152)

79 Human Noxa nucleotide sequence transcript 1 -Exon 1

(Unspliced transcript sequence from the Ensembl genome browser version 55, Transcript ID: ENST00000316660)

80 Human Noxa nucleotide sequence transcript 1 -Intron 1-2

81 Human Noxa nucleotide sequence transcript 1 -Exon 2

82 Human Noxa nucleotide sequence transcript 2 -Exon 1

(Unspliced transcript sequence from the Ensembl genome browser version 55, Transcript ID: ENST00000269518)

83 Human Noxa nucleotide sequence transcript 2 -Intron 1-2

84 Human Noxa nucleotide sequence transcript 2 -Exon 2

85 Human Noxa nucleotide sequence transcript 2 -Intron2-3

86 Human Noxa nucleotide sequence transcript 2 -Exon 3

87 Mouse Noxa nucleotide sequence transcript 1 -Exon 1

ENSMUST00000025399)

88 Mouse Noxa nucleotide sequence transcript 1 -Intron 1-2

ENSMUST00000025399) SEQUENCE ID NO: DESCRIPTION

89 Mouse Noxa nucleotide sequence transcript 1 -Exon 2

ENSMUST00000025399)

90 Mouse Noxa nucleotide sequence transcript 1 -Intron 2-3

ENSMUST00000025399)

91 Mouse Noxa nucleotide sequence transcript 1 -Exon 3

ENSMUST00000025399)

9

- 77 -

TABLE 2: Codes for non-conventional amino acids

Non-conventional amino acid Code Non-conventional amino acid Code cc-aminobutyric acid Abu L-N-methylalanine Nmala -amino- -methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet , L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr a-methyl-aminoisobutyrate Maib

D-valine Dval -methyl-y-aminobutyrate Mgabu

D-a-methylalanine Dmala -methylcyclohexylalanine Mchexa

D-a-methylarginine Dmarg a-methylcylcopentylalanine Mcpen

D-a-methylasparagine Dmasn a-methyl-a-napthylalanine Manap

D- -methylaspartate Dmasp a-methylpenicillamine Mpen

D- -methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D- -methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D- -methylhistidine Dmhis N-(3 -aminopropyl)glycine Nora

D-a-methylisoleucine Dmile N-amino-a-methylbutyrate Nmaabu

D-a-methylleucine Dmleu a-napthylalanine Anap

D- -methyllysine Dmlys N-benzylglycine Nphe

D-a-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-a-methylornithine Dmorn N-(carbamyImethyl)glycine Nasn

D-a-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-a-methylproline Dmpro N-(carboxyniethyl)glycine Nasp

D-a-methylserine Dmser N-cyclobutylglycine Ncbut. 10 001299

- 78 -

D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep

D- -methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-a-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-a-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methyl cysteine Dnmcys N-(3 ,3 -diphenylpropyl)glycine Nbhe

D-N-methyl glutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnnitrp N-( 1 -methylethyl)glycine Nval

D-N-methyltyrosine Dnnityr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(/?-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-ct-methylalanine Mala

L-a-methylarginine Marg L-a-methylasparagine Masn

L- -methylaspartate Masp L-a-methyl-t-butylglycine Mtbug

L-a-methylcysteine Mcys L-methyl ethyl glycine Metg

L-a-methylglutamine Mgln L- -methylglutamate Mglu

L-a-methylhistidine Mhis L-a-methylhomophenylalanine Mhphe

L-a-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-a-methylleucine Mleu L-a-methyllysine Mlys

L-a-methylmethionine Mmet L-a-methylnorleucine Mnle

L-a-methylnorvaline Mnva L-a-methylornithine Morn

L-a-methylphenylalanine Mphe L-a-methylproline Mpro

L-a-methylserine Mser L-a-methylthreonine Mthr

L-a-methyltryptophan Mtrp L-a-methyltyrosine Mtyr

L-a-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3 ,3 -diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine

1 -carboxy- 1 -(2,2-diphenyl- Nmbc

ethylamino)cyclopropane TABLE 3: Amino acid sub-classification

TABLE 4: Exemplary and Preferred Amino Acid Substitutions

Original Residue Exemplary Substitutions Preferred Substitutions

Ala Val, Leu, He Val

Arg Lys, Gin, Asn Lys

Asn Gin, His, Lys, Arg Gin

Asp Glu Glu

Cys Ser Ser

Gin Asn, His, Lys, Asn

Glu Asp, Lys Asp

Gly Pro Pro

His Asn, Gin, Lys, Arg Arg

He Leu, Val, Met, Ala, Phe, Norleu Leu

Leu Norleu, He, Val, Met, Ala, Phe He

Lys Arg, Gin, Asn Arg

Met Leu, He, Phe Leu

Phe Leu, Val, lie, Ala Leu

Pro Gly Gly

Ser Thr Thr

Thr Ser Ser

Trp Tyr Tyr

Tyr Trp, Phe, Thr, Ser Phe

Val He, Leu, Met, Phe, Ala, Norleu Leu Table 5: Loss of Puma or both Puma and Noxa protects fertility following y-irradiation

Untreated γ-irradiated

number number number live offspring number live offspring pups born at birth pups born at birth

4th litters from mothers: γ-irradiated as pups

mothers: offspring from females γ-irradiated as pups

Puma^'1'

6.0 ± 1.15 5.8 ± 1.5 N/A N/A n= 4 mothers

mothers: γ-irradiated as adults

wt

N/A N/A 0 0 n= 5 mothers

Puma^{' '}

N/A N/A 4.2 ± 3.2 3.7 ± 3.4 n= 6 mothers

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Claims

- 84 - CLAIMS

1. A method of conserving fertility in a female subject, the method comprising administering to the female an agent which suppresses PUMA expression or PUMA polypeptide activity.

2. The method of claim 1 wherein the female is undergoing or will undergo cancer treatment.

3. The method of claim 1 wherein the female is diagnosed as perimenopausal or menopausal or at risk of developing same within a predetermined time span.

4. The method of claim 1 wherein the female is undergoing in vitro fertilisation (IVF) treatment.

5. The method of claim 1 or 4 wherein administration to the female is administration ex vivo to oocytes.

6. The method of claim 1 wherein the agent binds to PUMA nucleic acid and suppresses PUMA expression.

7. The method of claim 6 wherein the agent comprises or encodes an antisense, siRNA, shRNA, miRNA, ribozyme, DNAzyme or other nucleic acid molecule.

8. The method of claim 1 wherein the agent binds to PUMA polypeptide and suppresses PUMA polypeptide activity.

9. The method of claim 8 wherein the agent comprises a small inhibitory molecule.

10. A method of conserving fertility in a female subject, the method comprising administering to the female one or more agents which suppress PUMA and NOXA expression or PUMA and NOXA polypeptide activity.

1 1. The method of claim 10 wherein the female is undergoing or will undergo cancer treatment. - 85 -

12. The method of claim 10 wherein the female is diagnosed as perimenopausal or menopausal or at risk of developing same within a predetermined time span.

13. The method of claim 10 wherein the female is undergoing in vitro fertilisation (IVF) treatment.

14. The method of claim 10 or 13 wherein administration to the female is administration ex vivo to oocytes.

15. The method of claim 10 wherein the agent or agents bind to PUMA and NOXA nucleic acid and suppresses PUMA and NOXA expression.

16. The method of claim 15 wherein the agent or agents comprise or encode an antisense, siRNA, shR A, miRNA, ribozyme, DNAzyme or other nucleic acid molecule.

17. The method of claim 10 wherein the agent or agents bind to PUMA and NOXA polypeptide and suppress PUMA and NOXA polypeptide activity.

18. The method of claim 17 wherein the agent comprises a small inhibitory molecule.

19. A method of conserving fertility in a female subject, the method comprising administering to the female an agent which suppresses NOXA expression or NOXA polypeptide activity.

20. The method of claim 19 wherein the female is undergoing or will undergo cancer treatment.

21. The method of claim 19 wherein the female is diagnosed as perimenopausal or menopausal or at risk of developing same within a predetermined time span.

22. The method of claim 19 wherein the female is undergoing in vitro fertilisation (IVF) treatment.

23. The method of claim 19 or 22 wherein administration to the female is administration ex vivo to oocytes. - 86 -

24. The method of claim 19 wherein the agent binds to NOXA nucleic acid and suppresses NOXA expression.

25. The method of claim 24 wherein the agent comprises or encodes an antisense, siRNA, shRNA, miRNA, ribozyme, DNAzyme or other nucleic acid molecule.

26. The method of claim 19 wherein the agent binds to NOXA polypeptide or a NOXA-binding agent and suppresses NOXA polypeptide activity.

27. The method of claim 26 wherein the agent comprises a small inhibitory molecule.

28. A method of conserving fertility in a female subject, the method comprising administering to the female an agent which suppresses TP63 expression or TP63 activity.

29. The method of claim 28 wherein the female subject undergoes cancer treatment.

30. The method of claim 28 wherein the female is diagnosed as perimenopausal or menopausal or at risk of developing same within a predetermined time span.

31. The method of claim 28 wherein the female is undergoing in vitro fertilisation (IVF) treatment.

32. The method of claim 28 or 31 wherein administration to the female is administration ex vivo to oocytes.

33. The method of claim 28 wherein the agent binds to TP 63 nucleic acid and directly suppresses TP63 expression.

34. The method of claim 33 wherein the agent comprises or encodes an antisense, siRNA, shRNA, miRNA, ribozyme, DNAzyme or other nucleic acid molecule.

35. The method of claim 28 wherein the agent binds to TP63 polypeptide or a TP63- binding agent and suppresses TP63 activity.

36. The method of claim 35 wherein the agent comprises a small inhibitory molecule.

37. The method of claim 1, 10, 19 or 28 wherein the agent binds to a complex comprising TP63 and PUMA or TP63 and NOXA and suppresses the formation or activity of the complex.

38. The method of any one of claims 28 to 36 wherein the TP63 is expressed in oocytes in the subject.

39. The method of any one of claims 28 to 36 or 38 wherein the TP63 is the a isoform of TP63.